Abstract
The 16th Workshop on Recent Issues in Bioanalysis (16th WRIB) took place in Atlanta, GA, USA on September 26–30, 2022. Over 1000 professionals representing pharma/biotech companies, CROs, and multiple regulatory agencies convened to actively discuss the most current topics of interest in bioanalysis. The 16th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on the ICH M10 BMV final guideline (focused on this guideline training, interpretation, adoption and transition); mass spectrometry innovation (focused on novel technologies, novel modalities, and novel challenges); and flow cytometry bioanalysis (rising of the 3rd most common/important technology in bioanalytical labs) were the special features of the 16th edition.
As in previous years, WRIB continued to gather a wide diversity of international, industry opinion leaders and regulatory authority experts working on both small and large molecules as well as gene, cell therapies and vaccines to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance, and achieving scientific excellence on bioanalytical issues. This 2022 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2022 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 1A) covers the recommendations on Mass Spectrometry and ICH M10. Part 1B covers the Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, Gene & Cell Therapy and Vaccine. Part 2 (LBA, Biomarkers/CDx and Cytometry) and Part 3 (Gene Therapy, Cell therapy, Vaccines and Biotherapeutics Immunogenicity) are published in volume 15 of Bioanalysis, issues 15 and 14 (2023), respectively.
| Abbreviations | ||
| A&P: | = | Accuracy and precision |
| AA: | = | Accelerated assessment |
| AAV: | = | Adeno-associated virus |
| ADA: | = | Anti-drug antibody |
| ADC: | = | Antibody drug conjugate |
| ADCC: | = | Antibody dependent cellular cytotoxicity |
| ADE: | = | Acoustic droplet ejection |
| ADME: | = | Absorption, distribution, metabolism, and excretion |
| API: | = | Active pharmaceutical ingredient |
| ASGPr: | = | Asialoglycoprotein receptor |
| AU: | = | Arbitrary unit |
| ASO: | = | Antisense oligonucleotide |
| B/R: | = | Benefit versus risk |
| BA: | = | Bioavailability |
| BAV: | = | Biomarker assay validation |
| BE: | = | Bioequivalence |
| BLA: | = | Biologics License Application |
| BLAST: | = | Basic Local Alignment Search Tool |
| BMV: | = | Bioanalytical method validation |
| BSA: | = | Bovine serum albumin |
| CAD: | = | Coronary artery disease |
| CAPA: | = | Corrective action/preventative action |
| CAR: | = | Chimeric antigen receptor |
| Cas protein: | = | CRISPR associated protein |
| CBA: | = | Cell-based assay |
| CCP: | = | Confirmatory cut point |
| CD: | = | Cluster of differentiation |
| CDR: | = | Complementarity-determining region |
| CDx: | = | Companion Diagnostics |
| CE: | = | Capillary electrophoresis |
| CIQ: | = | China Inspection and Quarantine |
| CLBA: | = | Competitive LBA |
| CLIA: | = | Clinical Laboratory Improvement Amendments |
| CMC: | = | Chemistry, manufacturing and controls |
| CMI: | = | Cell-mediated immunity |
| CNS: | = | Central nervous system |
| CoP: | = | Correlates of protection |
| COU: | = | Context of Use |
| CQA: | = | Critical quality attributes |
| CRISPR: | = | Clustered regularly interspaced short palindromic repeat |
| CRO: | = | Contract Research Organization |
| CSF: | = | Cerebrospinal fluid |
| CTA: | = | Clinical trial assay |
| CTD/eCTD: | = | Common technical document or electronic common technical document |
| CTIMP: | = | Clinical trial of an investigation medicinal product |
| DDI: | = | Drug-drug interaction |
| DIQC: | = | Dilution QC |
| DMPK: | = | Drug metabolism and pharmacokinetics |
| DNA: | = | Deoxyribonucleic acid |
| ECBS: | = | Expert Committee on Biological Standardization |
| ECLA: | = | Electrochemiluminescence assays |
| EDTA: | = | Ethylenediaminetetraacetic acid |
| ELISA: | = | Enzyme-linked immunosorbent assay |
| eQC: | = | endogenous QC |
| EU IVDD: | = | EU Directive 98/79/EC in vitro diagnostic medical devices |
| EUA: | = | Emergency Use Authorization |
| EV: | = | Extracellular vesicle |
| Fab: | = | Antigen binding fragment |
| Fc: | = | Crystallizable fragment |
| FFP: | = | Fit-for-purpose |
| FFPE: | = | Formalin-fixed, paraffin-embedded |
| FGP: | = | Full Qualification Package |
| GalNAc: | = | N-Acetylgalactosamine |
| GCLP: | = | Good Clinical Laboratory Practices |
| GCP: | = | Good Clinical Practices |
| GLP: | = | Good Laboratory Practices |
| GxP: | = | Good Practices |
| GTx: | = | Gene therapy |
| hELISA: | = | Hybridization ELISA |
| HGRAC: | = | Human Genetic Resources Administration of China |
| HILIC: | = | Hydrophilic interaction liquid chromatography |
| HPLC: | = | High performance liquid chromatography |
| HRMS: | = | High resolution mass spectrometry |
| IA: | = | Immunoaffinity |
| IBC: | = | Isobutyryl-L-carnitine |
| Ig: | = | Immunoglobulin |
| IIRMI: | = | Innate immune response modulating impurities |
| IMP: | = | Investigational medicinal product |
| IND: | = | Investigational new drug |
| IP: | = | Immunoaffinity purification |
| IS: | = | Internal standard |
| ISI: | = | Integrated Summary of Immunogenicity |
| ISR: | = | Incurred sample reproducibility |
| ISS: | = | Incurred sample stability |
| IU: | = | Intended use |
| IVD: | = | In vitro diagnostic device |
| IVDR: | = | EU In Vitro Diagnostic Medical Device Regulations |
| IVEB: | = | In vivo expressed biologic |
| KOL: | = | Key opinion leaders |
| LBA: | = | Ligand binding assay |
| LC: | = | Liquid chromatography |
| LCMS: | = | Liquid chromatography mass spectrometry |
| LIMS: | = | Laboratory information management system |
| LLE: | = | Liquid-liquid extraction |
| LLOQ: | = | Lower limit of quantitation |
| LNP: | = | Lipid nanoparticles |
| LOI: | = | Letter of Intent |
| LTS: | = | Long term stability |
| m1A: | = | N1-methyladenosine |
| MAA: | = | Marketing Authorization Application |
| mAb: | = | Monoclonal antibody |
| MAD: | = | Multiple ascending dose |
| MATE: | = | Multidrug and toxin extrusion protein |
| MFI: | = | Mean fluorescence intensity |
| MHC: | = | Major histocompatibility complex |
| miRNA: | = | MicroRNA |
| MOA: | = | Mechanism of action |
| MRD: | = | Minimum required dilution |
| MRM: | = | Multiple-reaction monitoring |
| mRNA: | = | Messenger RNA |
| MS: | = | Mass spectrometry |
| MSD: | = | MesoScale Discovery |
| NAb: | = | Neutralizing antibody |
| NMPA: | = | National Medical Products Administration |
| OCT: | = | Organic cation transporters |
| OSIS: | = | Office of Study Integrity and Surveillance |
| PASC: | = | Post-acute sequelae of SARS-COV2 |
| PBMC: | = | Peripheral blood mononuclear cell |
| PC: | = | Positive control |
| PCR: | = | Polymerase chain reaction |
| PD: | = | Pharmacodynamics |
| PI: | = | Phosphatidylinositols |
| PK: | = | Pharmacokinetics |
| POC: | = | Point of care |
| PoC: | = | Proof of concept |
| PRIME: | = | Priority Medicines |
| PRM: | = | Parallel reaction monitoring |
| QC: | = | Quality control |
| QFCM: | = | Quantitative flow cytometry |
| QP: | = | Qualification plan |
| qPCR: | = | Quantitative PCR |
| RISC: | = | RNA-induced silencing complex |
| RNA: | = | Ribonucleic acid |
| RNP: | = | Ribonucleic protein |
| RPLC: | = | Reversed-phased liquid chromatography |
| RT-qPCR: | = | Reverse transcription-quantitative PCR |
| Sa: | = | Staphylococcus aureus |
| SDS-PAGE: | = | Sodium dodecyl-sulfate polyacrylamide gel electrophoresis |
| SEC: | = | Size-exclusion chromatography |
| SERRF: | = | Systematic Error Removal using Random Forest |
| SFC: | = | Supercritical fluid chromatography |
| SIL: | = | Stable isotope label |
| siRNA: | = | Small interfering RNA |
| SLE: | = | Solid-liquid extraction |
| SNR: | = | Signal to noise ratio |
| SOP: | = | Standard operating procedure |
| Sp: | = | Streptococcus pyogenes |
| SPE: | = | Solid-phase extraction |
| t0: | = | Time zero |
| TCR: | = | T-cell receptor |
| TD: | = | Toxicodynamic |
| TDDS: | = | Transdermal drug delivery systems |
| TE: | = | Target engagement |
| TI: | = | Therapeutic Index |
| Transgene: | = | a gene that has been transformed to incorporate all appropriate elements critical for gene expression generally derived from a different species. |
| ULOQ: | = | Upper limit of quantitation |
| UPLC: | = | Ultra-performance liquid chromatography |
| WHO: | = | World Health Organization |
| WRIB: | = | Workshop on Recent Issues in Bioanalysis |
Index Part 1A
SECTION 1 – Mass Spectrometry, Chromatography and Sample Preparation
Hot Topics & Consolidated Questions Collected from the Global Bioanalytical Community
Discussions, Consensus and Conclusions
SECTION 2 – Mass Spectrometry Novel Technologies, Novel Modalities, and Novel Challenges
Hot Topics & Consolidated Questions Collected from the Global Bioanalytical Community
Discussions, Consensus and Conclusions
SECTION 3 – ICH M10 BMV Guideline & Global Harmonization
SECTION 3A – Impact of Global Harmonization on Regulated Bioanalysis
SECTION 3B - Common Mass Spectrometry and Ligand-binding Assays Issues
SECTION 3C – LBA Unique Challenges
Hot Topics & Consolidated Questions Collected from the Global Bioanalytical Community
Index Part 1B
SECTION 4 - Input from Regulatory Agencies on Regulated Bioanalysis/BMV and Biomarkers/CDx/BAV
SECTION 5 - Input from Regulatory Agencies on Immunogenicity, Gene & Cell Therapy and Vaccine
Introduction
The 16th Workshop on Recent Issues in Bioanalysis (16th WRIB) took place in Atlanta, GA, USA on September 26–30, 2022. Over 1000 professionals representing pharma/biotech companies, CROs, and multiple regulatory agencies convened to actively discuss the most current topics of interest in bioanalysis. The 16th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week to allow an exhaustive and thorough coverage of all major issues in bioanalysis of biomarkers, immunogenicity, gene therapy, cell therapy and vaccines.
Moreover, in-depth workshops on the ICH M10 BMV final guideline (focused on this guideline training, interpretation, adoption and transition); special features of the 16th edition included mass spectrometry innovation (focused on novel technologies, novel modalities, and novel challenges); and flow cytometry bioanalysis (rising of the 3rd most common/important technology in bioanalytical labs).
As in previous years, WRIB continued to gather a wide diversity of international, industry opinion leaders and regulatory authority experts working on both small and large molecules as well as gene, cell therapies and vaccines to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance, and achieving scientific excellence on bioanalytical issues.
The active contributing chairs included:
Dr. Chris Beaver (Syneos), Dr. Arindam Dasgupta (US FDA), Dr. Fabio Garofolo (BRI Frontage), Ms. Dina Goykhman (Merck), Dr. James Huleatt (Sanofi), Dr. Akiko Ishii-Watabe (Japan MHLW / ICH M10 EWG), Mr. Gregor Jordan (Roche), Dr. John Kamerud (Pfizer), Dr. Steve Keller (AbbVie), Dr. Lina Loo (Vertex), Mr. Fred McCush (Pfizer), Mr. Luis Mendez (Merck), Ms. Dulcyane Neiva Mendes Fernandes (Brazil ANVISA / ICH M10 EWG), Dr. Luying Pan (Takeda), Mr. Noah Post (Ionis), Dr. Mohsen Rajabi Abhari (US FDA), Dr. Yoshiro Saito (Japan MHLW / ICH M10 EWG), Dr. Daniel Spellman (Merck), Dr. Giane Sumner (Regeneron), Dr. Matthew Szapacs (Abbvie), Dr. Albert Torri (Regeneron), Dr. Montserrat Carrasco-Triguero (Sangamo), Dr. Elizabeth Verburg (Lilly), Dr. LaKenya Williams (BMS), Dr. Karl Walravens (GSK), Dr. Yongjun Xue (BMS)
The participation of major and influential regulatory agencies continued to grow at the 16th WRIB during its traditional Interactive Regulators' sessions including presentations and panel discussions on:
Regulated Bioanalysis and BMV Guidance/Guidelines: Dr. Chris Burns (UK MHRA), Dr. Seongeun Julia Cho (US FDA), Dr. Arindam Dasgupta (US FDA), Dr. Xiulian Du (US FDA), Dr. Akiko Ishii-Watabe (Japan MHLW / ICH M10 EWG), Dr. Elham Kossary (WHO), Dr. Yang Lu (US FDA), Ms. Dulcyane Neiva Mendes Fernandes (Brazil ANVISA / ICH M10 EWG), Dr. Mohsen Rajabi Abhari (US FDA), Dr. Yoshiro Saito (Japan MHLW / ICH M10 EWG), Mr. Stephen Vinter (UK MHRA / ICH M10 EWG), Dr. Yow-Ming Wang (US FDA), Dr. Li Yang (US FDA), Dr. Jinhui Zhang (US FDA)
Biotherapeutic Immunogenicity, Gene Therapy, Cell Therapy and Vaccines: Dr. Nirjal Bhattarai (US FDA), Dr. Eric Brodsky (US FDA), Dr. Isabelle Cludts (UK MHRA), Dr. Heba Degheidy (US FDA), Dr. Shirley Hopper (UK MHRA), Dr. Chad Irwin (Health Canada), Dr. Akiko Ishii-Watabe (Japan MHLW), Dr. Julie Joseph (Health Canada), Dr. Susan Kirshner (US FDA), Dr. Mohanraj Manangeeswaran (US FDA), Dr. Kimberly Maxfield (US FDA), Dr. Joao Pedras-Vasconcelos (US FDA), Dr. Mohsen Rajabi Abhari (US FDA), Dr. Zuben Sauna (US FDA), Dr. Vijaya Simhadri (US FDA), Dr. Therese Solstad (EU EMA/Norway NoMA), Dr. Seth Thacker (US FDA), Dr. Omar Tounekti (Health Canada), Dr. Daniela Verthelyi (US FDA), Dr. Meenu Wadhwa (UK MHRA), Ms. Leslie Wagner (US FDA), Dr. Joshua Xu (US FDA), Dr. Takenori Yamamoto (Japan MHLW), Dr. Lucia Zhang (Health Canada), Dr. Lin Zhou (US FDA).
Biomarkers/CDx and BAV Guidance/Guidelines: Mr. Abbas Bandukwala (US FDA), Dr. Shirley Hopper (UK MHRA), Dr. Kevin Maher (US FDA), Dr. Yoshiro Saito (Japan MHLW), Dr. Yow-Ming Wang (US FDA), Dr. Joshua Xu (US FDA)
The 16th WRIB included the traditional evening roundtables, which were attended by both industry key opinion leaders (KOL) and regulatory representatives. The extensive and fruitful discussions from these roundtables together with the lectures and open panel discussions amongst the presenters, regulators and attendees culminated in consensus and recommendations on items presented in this White Paper.
A total of 63 recent issues (‘hot’ topics) were addressed and distilled into a series of relevant recommendations. Presented in the current White Paper is the background on each issue, exchanges, discussions, consensus and resulting recommendations.
Due to its length, the 2022 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication covers Part 1A recommendations and Part 1B Regulatory Agencies' Inputs.
Part 1A – Volume 15 Issue 16 Month August 2023
Mass Spectrometry, Chromatography & Sample Preparation (4 Topics)
Hybrid Assays - Replacing Conventional Technologies
Hybrid Assays - New Applications/Approaches
Regulatory Challenges in Mass Spectrometry Bioanalysis
Innovation in Mass Spectrometry & Novel Challenges & Solutions
Mass Spectrometry Novel Technologies, Novel Modalities, & Novel Challenges (4 Topics)
Novel Applications & Novel Technologies in Bioanalysis
Oligonucleotides: Novel Modalities & Novel Method Development
ADC: Novel Modalities & Novel Method Development
Problem Solving for Non-Liquid and Rare Matrices
ICH M10 BMV Guideline & Global Harmonization (12 Topics)
Impact of Global Harmonization on Regulated Bioanalysis
Harmonization of Cross Validation in Regulated Bioanalysis
Patient Centric Sampling in Regulated Bioanalysis
Harmonization of Reference Standard Materials
Common Mass Spectrometry & Ligand-binding Assays Issues
Impact the 3Rs in Regulated Bioanalysis
Regulated Bioanalysis of Tissues & Secondary Matrices
Stability Issues in Regulated Bioanalysis
Harmonization of Endogenous Molecules Validation – Making the most of BMV & BAV Similarities
Novel/Alternative Technologies in Regulated Bioanalysis
LBA Unique Challenges
LBA Single Well Analysis (Singlicate) in Regulated Bioanalysis
Change of the Critical Reagents: “KISS - Keep It Simple & Straightforward”
LBA Carryover Assessment in Regulated Bioanalysis
Commercial, RUO & Diagnostic LBA Kits in Regulated Bioanalysis
Part 1B – Volume 15 Issue 16 Month August 2023
Input from Regulatory Agencies on Regulated Bioanalysis/BMV & Biomarkers/CDx/BAV
ICH M10
1: Introduction, 2: General principles, 4: Ligand Binding Assay
3: Chromatography, 5: Incurred Sample Reanalysis (ISR), 6: Partial and Cross Validation, 8: Documentation
7: Additional Considerations
Adoption by ANVISA
US FDA
Bioanalytical Considerations for Antibody-Drug Conjugates (ADC)
Recent Review Experience with Biosimilar Bioanalysis using LBA
Deficiency in Method Validation for Endogenous Analytes
General Considerations in Pharmacokinetic Bioequivalence Studies of Endogenous Compounds in ANDA Submission
Reflections on FDA Remote Evaluation Activities over the Past 2 Years
Regulatory Findings from Recent Inspections
Biomarkers for Biosimilars: US FDA perspective
CDRH CLIA Categorization Processes
Biomarker Qualification and Analytical Guidance
Next-Generation Sequencing (NGS) Panels for Precision Oncology Biomarkers
UK MHRA
Bioanalytical Observations, Findings and Data Integrity Issues
International Reference Standard Materials (RSM) for Biotherapeutics and Advanced Therapies
Japan MHLW
Recent Developments of Biomarker Assay Validation (BAV) in Japan for qPCR Assays
WHO
Inspection & Review of CROs' computerized systems validation
Input from Regulatory Agencies on Immunogenicity, Gene Therapy, Cell Therapy & Vaccines
Immunogenicity
US FDA
Immunogenicity Information in Prescription Drug Labeling
Assay Signal-to-Noise Ratio (S/N) as A Potential Alternative to Titer for An ADA Response
Preclinical tools for assessing the risk of innate immune response modulating impurities applied to biosimilars
Updates of the US FDA OCP Efforts on Evaluating Clinical Impact of Immunogenicity
Health Canada
Immunogenicity Labelling for Biologics in Health Canada Drug Submissions
UK MHRA
Development of reference material as positive controls for ADA assays
Gene & Cell Therapy & Vaccines
US FDA
Unique Scientific Challenges in the Immunogenicity Assessment of Novel Modalities
Understanding, Assessing and Managing Immune Responses to CAS-proteins
Perspective on Emerging Landscape of Gene Therapies
Application of Flow Cytometry in Cell Therapy; Current Perspective
Serology Assay Validation
EU EMA/ Norway NoMA
Regulatory Perspective on Vaccine Serological Assays- Validation as Clinical Endpoints
UK MHRA
Importance of immunobridging data for vaccine approval: recent experience with COVID-19 vaccines
Health Canada
Cell and Gene Therapies: Regulatory Challenges and Considerations
Authorization of new COVID-19 vaccines: The utility of immunobridging studies
Use of Functional Assays in the Development of Vaccines
Japan MHLW
Anti-SARS-CoV-2 Neutralizing Antibody Titer as a Clinical Endpoint of Vaccine Clinical Study in Japan
Two-Dimensional Droplet Digital PCR as a Tool for Titration and Integrity Evaluation of Recombinant Adeno-Associated Viral Vectors
Part 2 – Volume 15 Issue 15 Month August 2023
Biomarkers & CDx Development & Validation (8 Topics)
BAV for Primary/Secondary End Points in Clinical Studies
Method Development and BAV Strategies for Biomarker & CDx
Fit for Purpose Validation for Endogenous Analytes: BMV vs BAV for Mass Spectrometry and comparison with other Biomarker Assays
BAV for Vaccine Study Endpoints
Difficult Method Development and BAV: Tissues, Complex Matrices and ROS
Extracellular Vesicles Bioanalysis: Latest Developments and Next Steps
A decade of Free/Total Assays Discussions for Biomarker & PK Assays
Challenges with Multiplex Immunoassays for Biomarkers
Cytometry Validation & Innovation (6 Topics)
Vaccine Functional Assays
Cytometry in Tissue Bioanalysis
Innovation in Cytometry
Current Challenges with Cytometry Validation
Biomarkers, RO, Macrophage Polarization and Phagocytosis Measurements
Cytometry Conventional/Novel Technologies and Main Applications -
LBA, Enzyme Assays & Critical Reagents (5 Topics)
Novel Technologies & Automation in LBA
Novel Modalities, Novel Method Development/Validation Challenges
Rare Matrices
Problem Solving for Complex NAb Assays
Critical Reagents Deep Characterization
Part 3 – Volume 15 Issue 14 Month July 2023
Gene Therapy, Cell Therapy & Vaccines (14 Topics)
Immunogenicity
LNP Immunogenicity
Cell Therapy Immunogenicity Risk Assessment
Viral Vectors Immunogenicity
Bridging LBA to assess ADA response to CAR-T
Immunogenicity Assessment for Oligonucleotide-based Therapeutics
Lesson Learned on Cell & Gene Therapy Bioanalytical Strategy
Vaccine Immunogenicity Strategies
Vaccine Clinical Study Endpoints
Technologies
Guidance for Fit-for-Purpose NGS Assay Selection and Validation
NanoString Technology in Gene Expression
Novel Platform for Infectivity Assays
Bioanalytical PK Evaluation for siRNA using stem-loop RT-qPCR
qPCR and ddPCR Method Development and Validation
bDNA for for CRISPR-Cas9 Analysis of sgRNA
Immunogenicity of Biotherapeutics (10 Topics)
New FDA Draft Guidance on Including Immunogenicity Information in U.S. Prescribing Information
Immunogenicity & Bioanalysis for Drugs that have a Prolongation Effect in vivo
Affinity of ADA in Clinical Samples
Risk-based Approaches, Prediction and Mitigation
Characterization of “high” Incidence Clinical ADA beyond ADA and NAb Assay Testing
T-cell Engager (BiTE) Immunogenicity & Associated Cytokine Release
Target Interference on Screening Assays Cut Point & Importance of Risk Assessment for pH Sensitive Multi-domain Biotherapeutic (MDB)
Preclinical & Clinical Harmonization and Enhanced Tiered & Cut Point Approaches
NAb Assays Integrated Approach
ADA Assay Comparison & Monitoring
SECTION 1 – Mass Spectrometry, Chromatography & Sample Preparation
Matt Szapacs1, Wenying Jian5, Daniel Spellman2, Jennifer Cunliffe4, Elizabeth Verburg3, Surinder Kaur6, John Kellie7, Wenkui Li8, John Mehl7, Mark Qian9, Xiazi Qiu10, Federico Riccardi Sirtori11, Anton I. Rosenbaum12, Tim Sikorski7, Sekhar Surapaneni13, Jian Wang14, Amanda Wilson15 & Jinhui Zhang16
Authors are presented in alphabetical order of their last name, with the exception of the first 5 authors who were session chairs, working dinner facilitators and/or major contributors
Author affiliations can be found at the beginning of the article.
HOT TOPICS & CONSOLIDATED QUESTIONS COLLECTED FROM THE GLOBAL BIOANALYTICAL COMMUNITY
The topics detailed below were considered as the most relevant “hot topics” based on feedback collected from the 15th WRIB attendees. They were reviewed and consolidated by globally recognized opinion leaders before being submitted for discussion during the 16th WRIB. The background on each issue, discussions, consensus and conclusions are in the next section and a summary of the key recommendations is provided in the final section of this manuscript.
Mass Spectrometry – Replacing Conventional Technologies
How to apply innovative technologies in regulated bioanalysis to support regulatory filing? Does MIST guidance still apply for large molecules and novel modalities? What is the recommended platform for covalent inhibitors bioanalysis?
Hybrid Assays - New Applications/Approaches
Is microflow LC recommended to be used in biotherapeutics bioanalysis? What is the recommendation in performing MHC Associated Peptide Proteomics (MAPPs)? What is the latest common position on acceptance criteria for IA-MS methods? When and how to select the appropriate bioanalysis platform? What is considered as the critical reagents in IA-MS?
Regulatory Challenges in Mass Spectrometry Bioanalysis
What are the considerations for a pro-drug measurement for regulated studies? Should hybrid IA-MS methods be considered now routine for the regulated bioanalysis of biologics?
Innovation in Mass Spectrometry & Novel Challenges & Solutions
What is the consideration for bioanalysis of radio labeled therapies (RLT)? What information are we asking in native MS that traditional bioanalytical cannot be used to glean? What is needed for patient centric sampling to be ready for broad adoption in clinical development?
DISCUSSIONS, CONSENSUS & CONCLUSIONS
Mass Spectrometry – Replacing Conventional Technologies
Mass spectrometry often may be an alternative or preferred approach for the quantification of novel modalities and biotheraputics [Citation20,Citation28,Citation31]. The applications where mass spectrometry is replacing conventional technologies was discussed in 2021, and new case studies on oligonuclueotides, peptides, and receptor occupancy & target engagement measurements were discussed in 2022 to update previous recommendations.
Recommendations on the use of LCMS for oligonucleotide detection, specifically for ASO and siRNA therapeutics, were previously issued. Oligonucleotide therapeutics have been analyzed historically with LCMS, LC-fluorescence, hybridization ELISA, and step-loop RT-qPCR. It was previously recommended that bioanalytical assay selection is driven by the stage of development, assay development time, and need of critical reagents with qPCR having the least amount of experience for regulated work. Selectivity is one of the most difficult aspects for oligonucleotide assay development, even with the advantages of LCMS. If switching technologies is necessary, bridging experiments should be performed.
A case study of a sensitive LCMS assay for a GalNAc-siRNA in human plasma was shown. Combination of LLE and SPE extraction were used to achieve 1 ng/mL sensitivity. Hybridization using a complementary probe immobilized on magnetic beads is an alternative sample extraction that provides better sample cleaning efficiency than SPE or LLE. Hybridization LCMS assays have been previously reported for ASO analytes for example of a 16-mer ASO analysis using AB Sciex 4000 with an LLOQ of 10 ng/mL [Citation32] and a 17-mer ASO analysis using AB Sciex 6500+ with an LLOQ of 0.5 ng/mL. A case study of application of stem-loop RT-qPCR for quantitation of a siRNA in plasma for a non-GLP study was also presented. The sensitive stem-loop RT-qPCR assay achieved an LLOQ of 0.128 ng/mL in plasma with simple dilution as the sample pre-treatment. Based on the experiences, the advantages and challenges of LCMS methods in comparison to step-loop RT-qPCR for bioanalysis of ASO and siRNA are shown in . Overall stem-loop RT-qPCR enables superior sensitivity and has been applied for RISC (RNA-induced silencing complex)-PK assay and discovery bioanalysis, and shows promise for regulated bioanalysis.
Based on these experiences, application of innovative technologies in regulated bioanalysis to support regulatory filing was discussed. This presents challenges such as lack of regulatory guidance and limited industry experience but there was agreement that novel technologies, such as stem-loop qPCR, can be applied in regulated bioanalysis, as long as adequate assay performance is demonstrated and the difference between assay platforms is understood.
Another application of mass spectrometry is to replace LBA with a hybrid LCMS assay (IA-MS) when the traditional LBA does not meet requirements. A case study of this was discussed for peptides such as adrenocorticotropic hormone (ACTH or corticotropin). Measurement of plasma ACTH concentration provides useful information for PK modeling in clinical studies. Indirect or direct measurement of ACTH1-24 by immunoassays has contributed to significant variability in generated results, mainly due to interference from ACTH1-39 [Citation33,Citation34]. A hybrid assay was developed for the quantitation of ACTH1-24 with high selectivity and specificity to assist clinical study design and PK modeling. There was consensus from the results that the rapid, accurate and highly sensitive hybrid-LCMS/MS assay with 10 pg/mL LLOQ could be qualified as “fit-for-purpose” for the determination of ACTH1-24 in human plasma. Use of surrogate matrix calibration curve (5–400 pg/mL) showed acceptable accuracy, precision, and selectivity at all human plasma QC levels. Stability data indicated that study samples should be processed on ice.
The other use of hybrid assays replacing LBA methods was demonstrated for receptor occupancy & target engagement measurements. Updated recommendations and case studies were discussed based on recent publications [Citation28,Citation31]. Previous recommendations on this topic included using recombinant protein digestion and surrogate peptide selection, antibody screening with tissue homogenization/extraction optimization of sensitivity and recovery.
Clinical investigations of covalent inhibitors that selectively inhibit KRAS G12C were discussed. To understand target engagement at the site of action, two highly sensitive and targeted hybrid immunoaffinity 2D-LCMS/MS methods were developed to measure endogenous and drug-bound KRAS G12C in tissue biopsies, respectively. The immunoaffinity capture used commercially available reagents without the need for generating custom reagents for the KRAS G12C variant. The 2D LCMS/MS used high-pH RPLC as the first-dimension separation and low-pH RPLC as the second-dimension separation. The ratio of alkylated KRAS G12C to total KRAS G12C concentrations allowed a direct assessment of the KRAS G12C engagement with high sensitivity. Using as little as 4 μg of total tissue protein in a xenograft model, sub-fmol/μg LLOQ was achieved with acceptable precision and accuracy across three QC levels. Based on this experience, it was recommended to use targeted LCMS (active site vs total) platform for covalent inhibitor target engagement assessment.
Another discussion topic was whether metabolites in safety testing (MIST) guidance should be applied to large molecules and novel modalities such as oligonucleotides [Citation35,Citation36]. Currently there are no clear guidance documents on biotransformation studies for large molecules and novel modalities. There was agreement that biotransformation studies are necessary for large molecules and novel modalities and should be carefully investigated [Citation37]. MIST guidance can be used as a reference for biotransformation studies, though terminologies and guidance used for small molecules are not always applicable to large molecules and novel modalities. The primary concern for large molecules, oligonucleotides and other novel modalities is target-driven toxicity due to the specificity of the molecules, in contrast to off-target driven toxicity found in small molecules. There was alignment that it is important to characterize cross-species biotransformation profile.
Hybrid Assays - New Applications/Approaches
Aside from replacing conventional technologies, case studies and recommendations for the use of hybrid assays in new applications and approaches were also discussed. This includes its application in Cas protein measurement for gene editing, pepsin digestion in place of trypsin, micro flow LCMS, and LCMS for anti-drug antibodies (ADA).
Immunoaffinity extraction followed by chromatography coupled with tandem mass spectrometry had been used for the determination of insulin glargine and two metabolites M1 and M2 to support the regulatory submission of the first interchangeable biosimilar product approved in the US.
Genome editing using clustered regularly interspaced short palindromic repeats (CRISPR) has moved from bench to bedside into the world of therapeutics. Multi-technology bioanalytical strategies advance our understanding of CRISPR-Cas9 delivery efficiency, retention, and distribution. The Cas9 enzyme is the molecular scissor part of the CRISPR ribonucleoprotein (RNP) complex, and the guide directs the RNP complex to the target site and is required to bind to the double stranded DNA in order to make the cut and repair. Other components of interest for bioanalysis include the guide RNA that form the ribonucleic protein complex, the delivery vector distribution, the resulting genome edit and the resultant down-stream effect such as a transgene protein. There are multiple options for measuring the different components of interest. For Cas9 ligand binding, LCMS or hybrid approaches can be used. For the guide RNA there is PCR or the branched DNA format. For the vector, the lipid component of a lipid nanoparticle can be measured using LCMS, or for an AAV, PCR to monitor the viral vector.
Experiences developing a hybrid assay for Cas9 were discussed and compared to LBA. In one case study, a two-day workflow for tissue measurement of Cas9 with the N and C terminus (N terminal capture and C terminal monitoring) was able to achieve LLOQ of 0.087 ng/ mg mouse brain tissue compared to 0.018 ng/ mg tissue with the N-terminal MesoScale Discovery (MSD) immunoassay. This led to a discussion of when and how to select the appropriate bioanalysis platform. It was recommended to consider all assay types with strategy centered around the molecule and project needs. This should be done at both the stages of drug discovery and development. There was agreement that it is possible to change assay platform from drug discovery to drug development if the change can be justified (e.g., changes in the purpose of the assay).
Pepsin digestion for protein quantification was another new application and approach discussed for hybrid assays. The specificity and sensitivity of LCMS is primarily dependent on the selected signature peptide which was generated through digestions using single or multiple proteases to represents the protein. In the sample preparation workflow of “bottom-up” protein analysis, tryptic digestion has always been the gold standard approach due to the ubiquitous existence of basic amino acid residues in the protein structure and the high specificity of the trypsin enzyme. However, in support of biologics research in models involving multiple species and structure variants of targeted biologics, there is sometimes difficulty in using trypsin for generating signature peptides for differentiating variants with adequate specificity and sensitivity.
A case study demonstrated that pepsin, an endopeptidase, has the potential to serve as a promising alternative for hybrid assay protein quantification. The specific amino acid sequence mutations of LALA and/or LAGA of the IgG constant hinge-region have been widely used to mitigate antibody effector functions such as antibody-dependent cell-mediated cytotoxicity in antibody based biotherapeutics. Even at the early drug discovery stage, the mutations have been frequently incorporated to surrogate mouse antibodies and to evaluate chosen mouse models. Quantifying mutated mouse IgG isotypes in mouse pharmacokinetic studies imposes additional challenges to the bioanalytical method development especially when highly specific target antigens or anti-idiotypic antibodies are not available. The results demonstrated that a mutated mouse IgG2a was effectively digested by pepsin within 1 hour and a surrogate peptide AGAPSVF was produced under acidic conditions with good digestion reproducibility and an excellent LLOQ of 20 ng/mL. There was agreement that the reported strategy is potentially applicable to a broad range of bioanalytical applications.
Microflow was the next approach discussed. It is well recognized that as chromatographic flow rate decreases, LCMS signal response typically increases. This realization has fueled interest in adopting microflow (1–50 μL/min) LCMS (μLCMS) for quantitative bioanalysis and over the past decade, μLCMS has received considerable attention as a means for improving assay sensitivity. Numerous reports emphasize the soundness and advantages of integrating μLCMS into bioanalytical workflows, demonstrating sensitivity gains compared with conventional flow (200–800 μL/min). Instrument vendors have introduced μLCMS sources and chromatographic systems that enable easier implementation and improved ruggedness. Despite the enthusiasm and advances in μLCMS instrumentation, μLCMS has not been as widely adopted as anticipated.
The underlying reasons that some labs avoid implementing or have not embraced μLCMS and considerations for successful implementation were discussed. These considerations include instrument robustness, advances in sensitivity of detectors coupled with conventional flow sensitivity and the importance of sample preparation to fully take advantage of the sensitivity improvements provided by μLCMS. Not all sample preparation methodologies provide S/N gains and practitioners must focus on analyte-selective enrichment strategies that yield sufficient reduction in background. It was recommended to be mindful that μLCMS increases the response of analytes of interest, but also increases background signal response, providing no net gain in S/N if appropriate sample preparation methodologies are not used.
A case study was discussed showing μLCMS exhibits its own unique advantages (increased sensitivity) and disadvantages (increased stringency on sample preparation and instrument maintenance). There was consensus that μLCMS might be more suitable in discovery space (for more qualitative work) and might pose challenges in assay transferring to CRO partners and in the regulated bioanalysis space (more routine quantitative work). Decisions on whether to adopt μLCMS in bioanalysis workflows should be dependent on institutional and program needs and scientist's background.
ADA determination and immunogenicity prediction is another application of hybrid assay [Citation31], and new case studies were discussed to update the previous recommendations. Previously, it was confirmed that hybrid assays can be used successfully as an alternative/orthogonal approach to LBA for immunogenicity analysis. An additional benefit of these assays is for ADA isotyping, recommended in guidance by regulators. It was recommended previously that positive control selection is extremely important for hybrid assays. Using a positive control that has a high affinity may result in high extraction efficiency. Drug tolerance may be improved if the capture reagent is covalently bound to the beads; more stringent diluent conditions can be used.
Additional case studies were presented including discussion of the MAPPs (MHC-associated peptide proteomics) approach. This cell-based LCMS method is focused on the identification of potentially immunogenic peptides from protein and antibodies, that can interact with major histocompatibility complex class II (MHC II) on antigen-presenting cells (Dendritic cells). The protocol is focused on the identification of MHCII-bound peptides from peripheral blood mononuclear cells (PBMCs) of healthy donors treated with new biological candidates, using nano-ultra-performance liquid chromatography coupled to high-resolution mass spectrometry (nUHPLC–MS/MS). Peptides are extracted from cell lysates by an immunoaffinity step and next they are identified by nUHPLC–MS/MS. It was observed that a new bead material could improve sensitivity for identifying T cell epitopes for the immunoenrichment of MHCII eluted ligands. There was agreement from the data that the MAPPs platform could become a routine screening tool that can be considered for the preclinical and, potentially, clinical immunogenicity risk assessment of protein biotherapeutics.
Recommendations for the MAPPs method included using automation to reduce variability. It was suggested to use at least two million cells from at least 5–10 donors for MAPPs. Currently, no control experiment for MAPPs has been established. This assay is still emerging and can be served as a risk assessment for rank-ordering molecules during the drug discovery stage and for identifying potential immunogenicity liabilities in the early development phase.
Other general recommendations were provided for hybrid assay method development. First, it was discussed if there were updates to recommendations on acceptance criteria for IA-MS methods. There was consensus that 20/25% should be the acceptance criteria for IA-MS methods. The critical reagents that should be considered for IA-MS were also discussed. Critical reagents generation can take long time, so it was recommended to start reagent generation as early as feasible. The key difference between LBA and hybrid assays, is that immunocapture in IA-MS serves as an enrichment method rather than the quantitative purpose in conventional LBA assays. Therefore, capture reagents are typically less critical as those used in LBA assay. Nevertheless, capture reagents for a hybrid LCMS assay are recommended to be considered as critical reagents as they may significantly impact the key assay parameters such as capture efficiency, sensitivity, selectivity, parallelism, and so on. There was alignment that internal standards (i.e., SIL analytes) can be considered as a critical reagent in IA-MS and similar attention should be given to IS as in small molecule bioanalysis. The only exception is when an SIL protein is served as an IS. Obtaining characterization (certificate of testing) was recommended.
Regulatory Challenges in Mass Spectrometry Bioanalysis
Regulatory challenges in mass spectrometry were discussed for biotransformation studies, prodrugs, and special matrices. Biotransformation refers to a process of converting exogenous or endogenous compounds to products that are eliminated or excreted from the body or recycled. Pharmacokinetic analysis of biotherapeutic agents is routinely conducted in nonclinical and clinical research. Although mechanistic studies are performed occasionally to understand unusual pharmacokinetic behavior, currently biotransformation studies are not routinely carried out for development of conventional biotherapeutics such as monoclonal antibodies since their metabolism and clearance occurs via proteolytic mechanisms to peptides and individual amino acids which are recycled.
However, complex biologics such as antibody drug conjugates (ADC) or therapeutic protein containing unnatural amino acids warrant biotransformation studies for characterization of fate and understanding the pharmacokinetic and pharmacodynamics or toxicokinetics of the molecule [Citation38]. For antibody drugs, in addition to proteolytical cleavage, other modifications such as deglycation, oxidation, deamidation and conjugative reactions are possible. Each of these modifications can result in altered pharmacokinetic/toxicokinetic or pharmacodynamic profile. Bioanalytical methods typically consist of ligand binding based to measure biotherapeutic levels in the systemic circulation. However, currently employed bioanalytical assays lack specificity to distinguish metabolic variants from the intact dosed therapeutic. Although the current methodologies using a reference standard to quantitate drug in biological matrix are well accepted and routinely used for developing exposure-effect relationships, understanding biotransformation may help understand the stability of molecules and many variants that may be present in serum. Characterization of biotransformation of antibody drugs may help develop a better bioanalytical strategy and help better define the pharmacokinetic-pharmacodynamic strategies.
The discussion focused on biotherapeutic biotransformation process, technical approaches and challenges for biotransformation characterization and case studies. Given complexity of biotherapeutics, the process for biotransformation varies for each of the therapeutics described. For example, protein and mAb undergo proteolysis and hydrolysis of amide bonds leading to formation of smaller size peptides or amino acids which may be recycled. ASO and siRNA may undergo metabolism by exo and endonucleases leading to smaller nucleotides. Recent guidance also suggests biotransformation is case by case for modified proteins (example pegylated or conjugated proteins) [Citation39].
A case study was discussed of producing biotransformation data for a site-specific ADC using a multiple platform BA strategy. Total antibody was quantified with LBA, LCMS with immunocapture was used to quantitate ADC, HRMS evaluated DAR ratio over time, and LCMS/MS biotransformation studies identified catabolization of the cytotoxic payload. Another multiple platform strategy was shown for fusion proteins [Citation40].
Despite these successes, challenges and opportunities were discussed. LBA assays lack specificity beyond the epitope(s) used for capture/detection as reagents bind to only a small part of drug that is being quantitated. Any metabolic modifications in the absence of specific reagents may not be differentiated by reagents and will be quantitated as drug. Glycosylation changes (sialyation or afucosylation) to enhance PK or activity and any in vivo modification on the status of glycosylation may not be captured by LBAs. In addition, multiple functions need to work together to provide information for BA strategy for biotransformation because complex biologics require multiple assay formats to fully characterize PK and disposition. There was agreement that further discussion, case studies, and data will help establish regulatory use of biotransformation studies.
Recommendations were also discussed for regulated bioanalysis of pro-drugs. There are many factors which may prevent a small-molecule drug from reaching the desired exposure kinetics, such as low aqueous solubility, low lipid membrane permeability, short half-life in circulation, and inadequate distribution to the target organ. Pro-drugs are chemical derivatives of pharmacologically active molecules designed to overcome these barriers and be metabolized in vivo to the active parent drug. Esters and phosphates are among the most popular pro-drugs in drug development, but their lack of matrix stability can represent a great bioanalytical challenge due to ex vivo conversion. If pro-drug species are not stabilized ex vivo, the measured concentrations of the pro-drug and active drug analytes will be artificially skewed, thereby risking inaccurate calculations of exposure parameters during safety and efficacy studies.
A case study of an ester pro-drug was discussed to highlight overcoming these challenges in regulated bioanalysis. For example, the importance of considering how ex vivo pro-drug and acyl-glucuronide conversion may both lead to overestimations of the active acid moiety was demonstrated [Citation41,Citation42]. Thorough evaluation of the stability of the ester moiety throughout sample collection, processing, storage, and analysis was critical to assay design. A second case study of a phosphoramidate pro-drug with a chiral center was presented. A combination of pH adjustment, enzyme inhibitor, and temperature control was found to be necessary to stop degradation of the pro-drug to the active parent drug and prevent ex vivo isomerization. In addition, a chemical derivatization strategy was incorporated into the sample processing protocol to improve the chromatographic properties of this extremely hydrophilic pro-drug to allow for robust quantitative analysis.
In both case studies, there was an internal assessment on whether the pro-drug method needed to be fully validated or whether an exploratory assay was suitable to support preclinical Good Laboratory Practice toxicology studies. In cases where the pro-drug showed activity against the target (>50% of active entity) or was known to circulate at appreciable levels based on previous studies (>10% of active entity concentrations using profiling or qualified assays), a fully validated assay was used to support measurement of the pro-drug as a GLP endpoint. Incurred sample reanalysis was conducted and the percent difference between repeat and original results were within ±20%, thus confirming the repeatability of the assays. However, in cases where pro-drug showed low target potency and were present at negligible levels, measurement of the pro-drug was considered exploratory using a fit for purpose assay, which still included demonstration of pro-drug stability ex vivo.
Based on these case studies, it was recommended to look at the in vivo systemic exposure of the pro-drug in the matrix of interest. During such exposure assessment, appropriate stabilization should be used in sample treatment to ensure the pro-drug conversion into parent drug is not occurring ex vivo. However, there was also discussion on how stabilizing clinical samples can be logistically challenging, especially in late phase studies when samples are being collected in health care settings. Once sufficient evidence supports that there is low pro-drug exposure relative to active drug from either metabolic profiling, investigative, or regulated studies using samples that ensure pro-drug stability ex vivo, there's no need in continuing pro-drug measurement. However, if there is sustainable pro-drug exposure, pro-drug measurements should be continued in regulated studies whenever it is logistically possible. It was recommended to collaborate with CMC scientists to gain a fuller picture on the prodrug stability to help inform the bioanalytical strategy.
Another growing aspect of regulated bioanalysis is the use of patient centric rare and special matrices [Citation31]. Previously, there was consensus that miniaturizing bioanalytical methods can result in potential reduction in the use of rare matrices. However, challenges remain including ensuring homogeneous mixing of small sample volumes, liquid handling, transfer of small volumes, and assay sensitivity. Updated recommendations were provided on using LCMS for PK measurements in nasal lining fluid (NLF) addressing these challenges. To meet sensitivity requirements, a highly sensitive (5 ng/mL) and selective fit-for-purpose hybrid assay for quantification of two co-dosed antibodies was developed and qualified [Citation43]. To address high variability of sample collection, normalization with urea concentration was applied enabling quantification of antibody partitioning [Citation44] Low quantities of this rare matrix were addressed by utilizing a surrogate matrix and comprehensive matrix effect evaluations in convalescent and vaccinated patient sera. Furthermore, assay robustness has been carefully evaluated and tracked supporting sample analysis of numerous samples and batches.
One final regulatory discussion topic was if hybrid assays be considered now routine for the regulated bioanalysis of biologics. There was wide agreement that hybrid assays are suitable for regulated bioanalysis and clinical studies of biologics and have been widely accepted by health authorities. However, whether to choose hybrid assay or LBA is dependent on several factors, including specific program requirements, organizational operational resources, and CRO capabilities.
Innovation in Mass Spectrometry & Novel Challenges & Solutions
Innovative mass spectrometry approaches were shared for patient centric sampling, radioligand therapeutics, and native protein MS. First, the use of patient centric sampling is becoming a more accepted technique in clinical development programs for measuring drug exposure. Progress was shared for collection of samples by trial participants outside of traditional clinical sites for understanding both drug exposure and response to treatment. Dried blood samples were demonstrated to be successfully used in LCMS for metabolomics and lipidomics analysis. These analyses were combined bioinformatically with multi-omic approaches including DNA whole exome sequencing, RNA analysis using next generation sequencing and RNASeq, proteomics, metabolomics, lipidomics and qPCR. Clinical studies in oncology and infectious disease are implementing these approaches to enable a more personal and precision medicine approach in clinical trials.
There was agreement that these sampling techniques and multifaceted analytical platforms hold potential for better understanding pharmacokinetic and pharmacodynamic properties of therapeutics. However, challenges were discussed with these approaches that need to be addressed. Personalized clinical trial may improve the success rate of clinical studies thus controls coming from each patient are needed (e.g., DNA fingerprinting). Large sets of omics data are expected in such sampling, therefore there will be need for AI-driven data analysis.
Radioligand therapeutics (RLTs) are a targeted treatment modality being developed for cancer care. The RLT can be used for imaging or therapy, depending on which radioactive metal is present. Key components of RLTs include: 1) a molecule that binds with high affinity and specificity to the highly expressed target of interest on tumor cells, 2) a linker, 3) a chelating agent, and 4) a radioactive isotope that causes DNA damage and kills the tumor cell. RLTs are designed to be rapidly excreted as parent in urine to prevent unnecessary radiation exposure, therefore understanding the in vivo stability in circulation and clearance in early development is important to guide the design and selection of promising candidates. Specifically in the discovery phase, decision-making is accelerated by performing pivotal in vivo experiments with molecules containing non-radioactive metal to assess metabolism and excretion of both parent and metal containing metabolites. The bioanalytical methods used to analyze the in vivo samples are tailored for the question at hand to help guide the go or no-go decision to the next phase of development.
Case studies were discussed utilizing non-radioactive metals in the RLTs enabling use of ICP-MS to detect metal containing analytes. Depending on the configuration of the ICP-MS setup, quantitative and/or semi-quantitative detection of both total metal (via flow injection analysis, or FIA, ICP-MS) as well as individual parent and metabolite peaks (via LC-ICP-MS) can be performed. It was demonstrated that when combined with data from more traditional LCMS/MS approaches, a rich data set is acquired to guide prioritization of compounds with the desired ADME properties. For example, determination of the ratio of parent (from LCMS/MS) to total metal (from FIA-ICP-MS) can be used to assess the degree of RLT metabolism.
Other case studies with solutions to address LCMS/MS method development challenges were shown in bioanalytical support of preclinical development of RLT. The formulation of RLT in preclinical safety assessment must mimic the manufacturing conditions of the hot compound for human use, i.e., the RLT molecules without metal (free ligand) and RLT with cold metal (e.g.,175Lu-labelled, cold ligand) for preclinical toxicity studies in animals must be at the same ratio of the RLT molecules without metal (free ligand) and with radiolabeled metal (e.g., 177Lu-labelled, hot ligand) for clinical studies. LCMS/MS bioanalytical methods must be developed and validated for the simultaneous determination of the RLT molecules with and without the cold metal. Compared to many small molecule drug candidates, stable isotope (e.g., 2H, 13C) labeled internal standard (IS) for a RLT molecule is much more difficult to make, considering the complex structure of the molecule. Consequently, a RLT molecule labelled with a different cold metal (e.g., 66Ga) was employed as the IS in LCMS/MS bioanalysis. A challenge in developing robust LCMS/MS bioanalytical methods for RLT was the presence of matrix interference due to chelation of the free ligand with metal ions (e.g., K, Na) in matrix. The addition of EDTA to mobile phases significantly reduced this chelation. It was also recommended to add EDTA and formic acid to the IS working solution to minimize the instability of the IS and 2-propanol and propylene glycol to sample extract prior to drying down to further stabilize the IS. Finally, addition of acetone to the precipitant was recommended to help reduce IS response variability.
Based on the discussions, considerations for bioanalysis of radioligand therapies (RLTs) were given. There was agreement that both LCMS/MS and (LC)-ICP-MS can be used in determination of concentrations of RLT molecules in biological matrices. It was recommended to assess excretion of RLT in urine. As a key element of the bioanalytical strategies, stability of RLT molecules with metal (e.g., 175Lu) and without metal (free ligand) and with a different metal (internal standard, e.g., 66Ga) should be assessed thoroughly during method development and method validation prior to study sample analysis.
Finally, as protein mass spectrometry (MS) technologies advance, opportunities arise to turn a bespoke MS assay into a routine, platform assay. Native protein MS is a prime candidate for such development, as more research groups become comfortable with denatured intact protein analysis. In native protein MS, proteins are analyzed at a native pH which does not denature tertiary protein structure or quaternary protein structures in the form of non-covalent protein-protein interactions [Citation45,Citation46]. Limited examples of native MS can be found in the bioanalysis literature, an indicator of yet-untapped potential for native protein MS methods. Analytes may include antibodies (especially ADCs), antibody-complexes, or general protein complexes.
Factors required in development of a native MS platform include, MS instrumentation, immunocapture or other sample preparation considerations, front-end separations, data analysis, and in-house expertise. There was agreement this includes immunocapture with a specific capture reagent and MS-friendly concentrated elution for good peak shape. Optimization of the method is demonstrated by good S/N and mass differentiation.
The applications, advantages, and challenges of native MS were discussed. One application for native MS provides a direct observation and measurement of mAb-ligand complex in pre-clinical/ clinical studies [Citation47]. Yet another utility for native MS is the characterization of critical reagents. Depending on the reagent or antibody type/ format, native MS may provide the most accurate intact mass determination or label incorporation. Native MS for antibody characterization from serum samples is also a potential impact space (such as disulfide-bond scrambling or variants). Challenges of native MS include high concentration requirements and as a result assay sensitivity issues. Other challenges include how to develop a robust assay from both a separations and MS standpoint. Nevertheless, there was agreement that the technology is still emerging, and more discussion should be convened in the upcoming years.
RECOMMENDATIONS
Below is a summary of the recommendations made during the 16th WRIB:
Mass Spectrometry – Replacing Conventional Technologies
Mass spectrometry can replace conventional technologies such as LBA. Meanwhile, it adds value to have complementary technologies such as LCMS, LBA, RT-qPCR and flow cytometry in a bioanalytical lab to enable sensitive and selective quantitation of novel modalities. Novel methodology such as stem-loop qPCR for oligonucleotide can be used for regulated bioanalysis to support regulatory filing.
Bridging of the ‘new’ technology with conventional technology is the key to adopt innovative technologies in the regulated bioanalysis setting and can be done by comparing with what was being used previously and supporting with published literature.
Also, an understanding of what forms of the molecule are being quantitated is necessary in bridging two different technologies.
Currently there are no clear guidance documents on biotransformation studies for large molecules or novel modalities. MIST guidance can be used as a reference for biotransformation studies, though terminologies and guidance used for small molecules are not always applicable to large molecules and novel modalities.
Hybrid Assays – New Applications/ Approaches
To determine the most appropriate bioanalytical platform (e.g. hybrid assay vs. LBA) the strategy should be centered around the molecule and the project. It is recommended to consider all technology platforms at the beginning stages of drug discovery and development.
It is possible to change assay platform from drug discovery to drug development if the change can be justified (e.g., changes in the purpose of the assay).
A key difference between LBA and hybrid assays is that immunocapture in IA-MS serves as an enrichment method rather than the quantitative purpose in conventional LBA assays. Therefore, capture reagents selection criteria are typically different from those used in LBA assay. Nevertheless, capture reagent for a hybrid LCMS assay is recommended to be considered as a critical reagent as it may significantly impact the key
Internal standards (i.e., SIL analytes) can be considered as a critical reagent in IA-MS and similar attention should be given to IS in small molecule bioanalysis.
20/25% was the recommended acceptance criteria for hybrid assays.
Microflow LC is more suitable for discovery applications (for more qualitative work) and might pose challenges in assay transferring to CRO partners and in regulated bioanalysis (more routine quantitative work).
MHC associated peptide proteomics (MAPPs) is an emerging approach for immunogenicity assessment with hybrid IA-LCMS assays.
Highly sensitive and selective hybrid IA-MS methods can be developed for special, rare matrices such as NLF with variations in sample collection/processing being controlled via normalization using urea measurement
Hybrid assays are suitable for regulated bioanalysis and clinical studies of biologics and have been widely accepted by numerous health authorities
Regulatory Challenges in Mass Spectrometry Bioanalysis
Hybrid assays are suitable for regulated bioanalysis of biologics.
Choice of hybrid assays or LBA is dependent on specific program requirements, organizational operational resources, and CRO capabilities.
For measurement of pro-drugs in regulated studies, it is recommended to look at the in vivo stability of the pro-drug.
During such stability assessment, appropriate stabilization should be used in sample treatment to ensure the pro-drug conversion into parent drug is not occurring ex vivo.
If there is negligible pro-drug exposure relative to parent/active drug and the drug is less potent than the active entity, there's no need for bioanalytical pro-drug measurement. However, if there's sustainable pro-drug exposure, pro-drug measurements should be continued in regulated studies.
It is recommended to collaborate with CMC scientists to gain a more complete picture of the prodrug stability.
Innovation in Mass Spectrometry & Novel Challenges & Solutions
Patient centric sampling with dried blood is an innovative approach that can be applied to LCMS that may improve trial success rate with personalization. However patient specific controls are necessary.
Large sets of omics data are expected in such sampling, therefore there will be need for AI-driven data analysis.
Native MS is an emerging technology that is run under neutral pH so the antibody-ligand interaction can be preserved.
Native protein MS can be used in characterizing cysteine-based pay-load ADC, protein-ligand interaction, and critical reagents characterization.
Bioanalysis of radioligand therapies (RLTs) is challenging in drug discovery and preclinical development.
It was recommended to utilize LCMS/MS and LC-ICP-MS for fit-for-purpose bioanalysis of RLTs.
Urine was recommended for assessment of RLT excretion.
Stability of chelator-metal complex should be assessed thoroughly during LCMS/MS method development prior to method validation. Sample analysis should be initiated only after the stability of all analytes/IS are well-understood.
SECTION 2 – Mass Spectrometry Novel Technologies, Novel Modalities, & Novel Challenges
Yongjun Xue13, Noah Post17, Yue Huang12, Dina Goykhman2, Long Yuan25, Kasie Fang7, Kevin Bateman2, Ellen Casavant6, Linzhi Chen18, Yunlin Fu8, Ming Huang19, Allena Ji20, Jay Johnson21, Michael Lassman2, Jing Li22, Ola Saad6, Hetal Sarvaiya23, Lin Tao24, Yuting Wang10 & Naiyu Zheng13
Authors are presented in alphabetical order of their last name, with the exception of the first 5 authors who were session chairs, working dinner facilitators and/or major contributors.
The affiliations can be found at the beginning of the article.
HOT TOPICS & CONSOLIDATED QUESTIONS COLLECTED FROM THE GLOBAL BIOANALYTICAL COMMUNITY
The topics detailed below were considered as the most relevant “hot topics” based on feedback collected from the 15th WRIB attendees. They were reviewed and consolidated by globally recognized opinion leaders before being submitted for discussion during the 16th WRIB. The background on each issue, discussions, consensus and conclusions are in the next section and a summary of the key recommendations is provided in the final section of this manuscript.
Novel Applications & Novel Technologies in Bioanalysis
Challenges with LCMS based protein panel assay (targeted proteomics) with 10, 50 or 100 proteins in one assay and what are the acceptance criteria? Is there an alternative affinity enrichment reagent to animal derived Abs? Is an aptamer a viable alternative? Use of DIA mass spectrometry to support discovery of fecal biomarkers: what are challenges in sample preparation method development and data analysis? Have HILIC method improvements made it an option for oligonucleotide bioanalysis? Is using ion-pairing agents still the preferred approach?
Oligonucleotides: Novel Modalities & Novel Method Development
Best practices for siRNA & ASO regulatory ADME package (assay selection & acceptance criteria, IS selection). “Active” drug quantitation requirements (active vs total). How much metabolite information is sufficient, does this amount vary vs mechanism of drug? What are pros and cons of different mass analyzers for oligo analysis, assay acceptance criteria, and metabolite profiling strategies?
Antibody Drug Conjugates (ADC): Novel Modalities & Novel Method Development
What are recent advances in LCMS analysis of ADCs: Selection of internal standards: total antibody assay and conjugated/free payload assay? What are current approaches for ADC PK evaluation? The importance of biotransformation of ADCs is becoming more evident. What is the right matrix for in vitro characterization of biologics?
Problem Solving for Non-Liquid & Rare Matrices
In addition to plasma/serum PK assays, is a PBMC or total cell assay necessary in drug development? What is the common practice for PBMC collection procedure? Is there a need to match the major biomarker isoform profile between authentic samples and the reference standard? What are the challenges for cross-validation between instruments or labs? What is the importance and significant challenges of bioanalysis in eye compartments with low volume, high sensitivity, and large dynamic range? What solutions exist for bioanalysis in non-liquid matrices for novel sample preparation, alternative digestion approaches, faster turnaround time, and higher sensitivity?
DISCUSSIONS, CONSENSUS & CONCLUSIONS
Novel Applications & Novel Technologies in Bioanalysis
The use of LCMS for novel applications and incorporating novel technologies was discussed for protein panels, aptamer affinity enrichment, collision and electron dissociation, stool sample preparation, and hydrophilic interaction liquid chromatography for oligonucleotides.
For drug discovery and development, mass spectrometry-based targeted proteomic approaches are needed to reliably quantify protein panels in biological samples with typically a panel of 10 - 200 proteins in one assay. These protein panels are essential to confirm the biomarker hypothesis or to better understand the biology of diseases and related pharmacological applications during the clinical trials for immuno-oncology, fibrosis, or other drug development programs. Despite extensive publications in targeted proteomics, many reported mass spectrometry-based methods for protein panels utilize a wide range of acceptance criteria and there is little understanding of the impact of variable criteria on the quality of the results generated. To ensure robustness of the assays and the quality of the data, a workshop previously held at the National Institutes of Health with experts from the academia and industry, led to a publication on the best practices for mass spectrometry-based assay development for quantitative analysis of protein panels [Citation48]. In the publication, “fit-for-purpose” approach was proposed for targeted proteomics by using three tiers of assays to distinguish the performance and extent of analytical characterization for each tier.
Case studies were given to highlight recent developments in mass spectrometry instrumentation and software on three major targeted proteomics platforms (multiple reaction monitoring - MRM, parallel reaction monitoring - PRM and data independence – DIA based platforms) [Citation49]. There are different targeted proteomics workflows for calibration depending on the data acquisition methods and context of use (COU): external calibration using synthetic peptide/recombinant proteins, one point calibration using the SIL-peptides (internal standards), no calibration curve for larger protein panels (relative quantitation) [Citation50]. There was agreement that LC-PRM based platforms have the advantage of high sensitivity but limited protein coverage (10–100). LC-DIA approaches have high protein coverage (100–200 or more) but requires bioinformatics experience.
It was agreed that fit for purpose validation is used with increasing requirements as the proteomics data is applied for more critical clinical trial uses. The complexity of calibration curve and QC sample preparation and implementation was discussed. It was recommended that since the nature of this assay is for exploratory (biomarker screening), these approaches may not need strict acceptance criteria. There is no current standard approach on defining the acceptance criteria in the pharma industry and no recommendations from agency.
The use of alternative and novel affinity enrichment reagents was also discussed. Commonly, anti-analyte antibodies are used as the immunoaffinity enrichment reagents in the hybrid assay workflow. The generation of anti-analyte (monoclonal or polyclonal) antibodies involves the use of animals. According to EU Directive 2010/63/EU, animals (or animal-derived products) should not be used, where a non-animal alternative exists, which provides the same or higher level of information. It is encouraged to use animal-free technologies to produce affinity reagents with equal or better quality (purity, activity, specificity, affinity, stability, reproducibility) than that offered by antibodies produced using conventional animal-based procedures. Aptamers are one alternative that was discussed which are single-stranded DNA or RNA molecules. They bind to their target molecules with high affinity and specificity by folding into distinct secondary and tertiary structures. Aptamers can be artificially generated via a procedure known as Systematic Evolution of Ligands by Exponential Enrichment (SELEX). The published research has shown that the aptamers generated by SELEX techniques can be chemically modified, making them more stable and suitable for recognition of the intended [Citation51]. Aptamers have some unique advantages over antibodies including easy to scale-up in vitro production, easy conjugation to functional groups, shorter production time (∼3 months), reduced lot-to-lot variability, and reduced interference for protein LCMS/MS analysis. In support of preclinical bioanalysis of therapeutic antibodies, a case study was discussed of an anti-human IgG aptamer as an alternative to an animal generated antibody as the affinity enrichment reagent in the hybrid assays workflow that employs streptavidin-coupled magnetic beads and a biotinylation step. The aptamer had high specificity to the human IgG Fc domain (KD 90.9 nM), no cross reactivity with other species IgGs (except primate), equivalent enrichment to Protein A, and ∼4 hour half-life in human serum at room temperature. Optimization was carried out for bead charging, affinity purification time and buffer conditions along with others. SILu™Mab was used as the internal standard. Four different human IgGs (IgG1, IgG2 and 2 different IgG4s) were individually tested using the optimized aptamer-based affinity enrichment workflow. Overall, the method met all conventional acceptance criteria. The study demonstrated that the aptamer had general humanized IgG specificity with no cross reactivity in rat, mouse, rabbit and dog. The workflow was cost effective (25–50%) compared to the one with the capture antibody generated using animals. There was consensus that the aptamer strategy is currently at the PoC stage, and challenges remain in method development for non-human IgG bioanalysis.
Fragmentation techniques for small molecules and their metabolites was the next novel methodology discussed. Improving the quality of the metabolism information, i.e. narrowing the possibilities of the Markush structure towards a definitive metabolite structure allows quickly testing hypotheses and prioritize syntheses. Mass Spectrometry with collisional-induced dissociation (CID) has been the main workhorse for small molecule metabolite structure elucidation. Software tools, such as Massmetasite, have been developed to assist in the interpretation of CID MS/MS spectra. A case study demonstrated that when looking at 180 compounds in liver microsomes from >10 programs and a multitude of chemical structural series, CID and software assisted metabolite identification using Massmetasite was able to narrow the site of oxidative metabolism to one third or less of the molecule for 90% of the 180 compounds examined. Alternative-CID fragmentation (i.e. electron activated dissociation; EAD) and/or metabolite information acquisition (e.g. collisional cross section; CCS) offers the potential to improve upon this and offers unique information for molecules in which CID does not offer key diagnostic fragments. Free electrons are captured by ions and form a radical state which then fragments [Citation52]. Electrons introduced with different energies will induce fragmentation in different molecule types. EAD also offers the potential to move closer to fully automated metabolite identification via more informative MS/MS spectra. Automated interpretation of EAD MS/MS spectra using existing algorithms needs to be tested, modified, and implemented. However, challenges were recognized such as incorporating and establishing different types of acquisition and software tools can be challenging in the current laboratory setting as vendor agnostic software and workflows are needed for mass spectrometers and laboratories.
The next novel application of LCMS is the development and qualification of novel sample preparation methods to support discovery of fecal biomarkers. Stool is a promising source of non-invasive biomarkers as it is proximal to the site of disease activity and it is already routinely collected in clinical trials and as part of clinical practice. However, stool is a challenging matrix to work with due to the high prevalence of lipids, metabolites, and other matrix artifacts that reduce sensitivity. A case study was discussed showing how protein extraction methods were optimized for LCMS/MS analysis and high-throughput sample handling methods were developed in order to evaluate the fecal proteome. Results from this study demonstrated that a bead-based protein clean up and tryptic digestion protocol provides efficient protein extraction and digestion and greatly improves the number of measurable proteins compared to existing methods. Over 700 human proteins consisting of 5190 human peptides were detected (>20% increase in identifications). This high-throughput stool proteomics method could be applied to interrogate the human or microbial response to disease or drug treatment and subsequently discover non-invasive biomarkers for gastrointestinal disease. Based on this case study, there was agreement that fecal matrix is very complex, and DIA MS proteomics can provide good alternative for both biomarker identification and quantitation. Extraction buffer selection is critical for sample clean-up to capture human secreted proteins. When doing normalization, use of total protein not mass was recommended. Bead-based sample clean-up strategy appears to work best with high reproducibility. DIA-MS based peptide quantitative data analysis method development is recommended to improve data quality.
The final methodology discussed was hydrophilic interaction liquid chromatography (HILIC) for oligonucleotides. Disadvantages of typical ion-pairing agents include strong signal suppression (including when switching to positive mode), significant MS cleaning & downtime, and higher cost for preparing fresh buffer before each run. HILIC has been implemented as a powerful tool for providing in-depth characterization of nucleic acid therapeutic modalities, such as anti-sense oligonucleotides and small interfering RNAs (siRNAs). A case study was discussed developing a generic hydrophilic interaction liquid chromatography (HILIC) hyphenated with tandem mass spectrometry method in the absence of ion-pairing reagents and demonstrated its capability as an attractive and robust alternative for oligonucleotide and siRNA analysis. HILIC separation of mixtures of unmodified and fully phosphorothioate-modified DNA oligonucleotides and their synthetic 3′ exonuclease-digested metabolites were also assessed. High-resolution mass spectrometric (HRMS) analysis was used to determine the deconvoluted masses of oligonucleotide and siRNA standards and their impurities. Though not yet as established as ion-pairing methods, HILIC is promising, and recent advancements including automated MS/MS annotation for unbiased sequence characterization, HRMS analysis resulting in deconvoluted mass spectrum, and the ability to analyze siRNA as single strands and duplex. However, limitations exist. There have been many attempts to replace ion-pairing system for a HILIC method, but rarely equivalent assay performance is seen. HILIC can achieve an LLOQ of 2.5 pmol on column, however this is not yet equivalent to ion-pairing. No HILIC evaluation is available for metabolites <12 nucleotides in length, and it is difficult for use with incurred samples (i.e. various matrix effects of different extractions).
Oligonucleotides: Novel Modalities & Novel Method Development
Until recently, LC/fluorescence or UV, hybridization-based ELISA and quantitative polymerase chain reaction have been the technology of choice for oligonucleotide analysis, but the changing face of the industry has brought high resolution MS to the fore [Citation31]. Additional case studies with novel method development led to updated recommendations. Previously, there was consensus that selectivity is one of the most difficult aspects for oligonucleotide assay development, even with the advantages of LCMS, particularly in cases of co-eluting minor metabolites without truncation of nucleotides (desulfurization, defluorination, deamination). One case study focused on the development of LC/HRMS technology for qualitative and quantitative analysis of parent drug and metabolite profiling/id of an GalNAc-conjugated siRNA. The metabolite profiling/id relies on the MS high-resolution power to distinguish metabolites from matrix peaks. As the LCMS responses of negatively charged oligonucleotides are primarily determined by the phosphate group, metabolite quantitation can be based on the back-calculations using parent calibration curve in the absence of metabolite reference materials. Mass spec was shown to now match or even exceed the sensitivity of fluorescence techniques, achieving up to 10 times more sensitivity using MS/MS analysis. At the same time, the run time was decreased from roughly 10 minutes a sample by LC-fluorescence to approximately five minutes by MS, increasing throughput significantly. LC-fluorescence requires a long gradient run to separate the parent drug and metabolites for individual quantification, whereas MS distinguishes between compounds based on their mass, so the gradient run time can be shorter.
There was agreement that even more of an advantage is that the HRMS allow quantification of the parent oligonucleotide and, simultaneously, perform metabolite profiling and quantification, which is difficult to achieve by LC-fluorescence, and not possible with ELISA or qPCR. However, drawbacks and challenges of the LCMS approach for oligonucleotide analysis were also discussed. Most oligonucleotide LCMS assays are based on RPLC and requires a stronger ion-pair (IP) reagent in order to achieve desired separation. As such, frequent cleaning of ion source is needed to minimize sensitivity loss over time. In addition, freshly prepared mobile phase, frequent cleaning of HPLC system are often needed while column life is shortened. Very recently, HILIC has been used as an alternate which eliminates these drawbacks. In the past decade, the metabolism of oligonucleotide drugs has also been studied extensively using LCMS methods because of the high specificity and sensitivity such methods can provide.
Two novel case studies were discussed focusing on understanding the in vivo metabolism of GalNAc-conjugated siRNAs and the bioanalysis of parent siRNAs and metabolites using high resolution mass spectrometry (HRMS). The first case study highlighted the discovery and characterization of a novel deaminated metabolite of a single-stranded oligonucleotide in vivo using LC-HRMS. The second case study discussed the identification and characterization of the deaminated metabolite on a double-stranded siRNA in vivo by LC-HRMS, and the strategies for bioanalysis of parent siRNA and deaminated metabolite. These case studies led to discussion of the pros and cons of different mass analyzers for oligo analysis, assay acceptance criteria, and metabolite profiling strategies. For example, for HRMS, extensive tuning of compounds is not necessary with similar LLOQ to triple quadrupole methods. There is also the addition of ID verification which can generate data on metabolites in same run as parent quantitation run. It is possible to calculate relative abundance of metabolites to parent due to using Q1 data (with caveats). On the other hand triple quadrupole methods have decreased run time, can add additional identity verification with precursor scan for non-endogenous components of drug, is easy to outsource, and typically has a lower LLOQ with higher s/n ratio. There was agreement that if/when possible, small molecule acceptance criteria should be used for LCMS assays of oligonucleotides. IS for oligos may be a stable isotope labeled drug (ideal), or a chemically similar non-interfering analog. The same IS can also be used for metabolite quantitation. There was also further discussion of bioanalysis of antisense oligonucleotides (ASO) which are short synthetic single-stranded oligonucleotides that can complementarily bind to mRNA via Watson–Crick base-pairing, and therefore modulate gene expression. ASO have been rapidly growing as a new class of therapeutics and are gaining increasing interests in drug research and development. Accurate and reliable bioanalysis of ASO in various biological matrices (plasma, cerebrospinal fluid, tissues etc.) is critical for understanding the pharmacokinetic (PK), toxicokinetic (TK), pharmacological and toxicological properties of ASO drug candidates.
Conventionally, hybridization ligand binding assay (LBA), e.g., hybridization enzyme linked immunosorbent assay (HELISA), has been the “gold standard” for the quantitative bioanalysis of ASO. Hybridization LBA has the advantages of high sensitivity and good assay performance. However, one major drawback of hybridization LBA is its lack of specificity. It is often challenging to differentiate the full-length ASO analyte from its truncated metabolites (e.g., N-1, N-2 metabolites). In addition, hybridization LBA has relatively narrow dynamic range and may be affected by anti-drug antibodies in the samples. LCMS/MS assay has its unique advantages of high specificity, wide dynamic range, and the ability to simultaneously quantify multiple analytes.
However, the significantly lower sensitivity of LCMS/MS assay compared to hybridization LBA limited its wider applications. Recently, a novel hybridization LCMS/MS methodology, which combines the advantages of hybridization LBA and LCMS/MS, was developed for the quantification of ASO in biological samples. This methodology utilized a DNA strand complementary to the targe ASO as the capture probe to specifically hybridize to the ASO analyte to achieve highly efficient sample extraction. As a result, hybridization LCMS/MS achieved greatly improved sensitivity that is comparable to hybridization LBA (below 1 ng/mL), and also maintained the advantage of high specificity of LCMS/MS [Citation53].
These case studies also led to discussion of fulfilling ADME regulatory package requirements for active drug quantitation. There was discussion of how much metabolite information is sufficient, does this amount vary vs mechanism of drug. Accurate PK data coupled with mechanism of action is needed to understand PD. Assays selected for BA need to be able to provide the appropriate BA data. For GalNAc-siRNA, after distribution, the main compartment with drug is liver. The antisense strand (AS) of siRNA is Argonaute 2 protein (Ago2) loaded and is the active drug. Remaining drug is in an intracellular depot that may eventually become Ago2 loaded [Citation54]. There was agreement that quantitation of AS in RNA-induced silencing complex (RISC) is nice to have in target tissues to derive the PK/PD relationship. Given very low AS concentrations in RISC, PCR assays appear to be the only option. Total tissue concentrations may be analyzed via LCMS. There was agreement that MIST guidance applies to all drug metabolites, including deaminated metabolites of siRNAs. The deaminated metabolites are active, but not found in circulation. Similar deamination biotransformation has not been reported in ASOs.
ADC: Novel Modalities & Novel Method Development
Hybrid assays are often preferred due to lack of specific reagent pairs to enable the ligand binding assay (LBA) at discovery or early-development stage. One of the challenges in LCMS bioanalysis of ADCs is the use of appropriate internal standards (IS) that could represent each of the three analytes being measured - total antibody (TAb), conjugated antibody (ADC), as well as free payload in circulation. In 2019, it was recommended to use intact labeled IS and stable isotype labeled (SIL) peptides. Later, it was recommended to use multiple lots of matrix for recovery if a surrogate peptide IS is spiked.
New case studies were discussed to provide updates to prior consensus on IS selection [Citation26,Citation30]. The first case study demonstrated how different IS affected accuracy, precision, and stability interpretation of PK data. Other case studies discussed automation to improve IS performance and using CDR peptides compared to RT matched generic peptide IS, especially in clinical bioanalysis to offer good selectivity and specificity over the matrix background. Overall, SIL-mAb can offer correction for capture and digestion efficiency in pre-clinical studies, where universal anti Fc capture and HuFc common peptide are used as detection. Galactic IS (GIS) and stable labeled IS (SLIS) were comparable for measuring payload.
There was also discussion on the overall bioanalytical PK strategy for ADCs. Due to the complexity of the molecular design and the nature for biotherapeutics, ADCs can be considered as a mixture of molecules that share structural similarities but may have differences such as post-translational modifications, drug antibody ratio, or specific conjugation sites. These differences may lead to variable pharmacokinetic behavior and may impact potency, efficacy, or safety. In addition to the heterogeneity of the drug substance, the ADC itself, especially the small molecule component and the linker chemistry may lead to potential biotransformation in vivo [Citation38]. Therefore, the ADC to be monitored from in vivo studies is a cluster of molecules with structural similarities and slight variations, considering the combined possibilities from the drug substance heterogeneity or potential in vivo biotransformations, or both.
When characterizing pharmacokinetic behavior of ADCs, it is critical to take into consideration the potential ADC species and have a clear understanding of what is been measured and why. Therefore, it is helpful to investigate which are the main species of ADCs in the biological matrices of interest. Thus, biotransformation characterization is similarly important as metabolite identification to understand small molecule drug metabolism. The methods used to investigate the in vivo ADC species can be generalized in to two main categories: 1) An in-silico prediction based on known structural information and known biotransformation pathways, or 2) A series of in vitro and in vivo experiments utilizing selective reagents and various analytical methods. There was agreement that the best approach to comprehensively understand ADC biotransformation species in vivo is usually a combination of the two methods.
Mass spectrometry when coupled with selective capture reagents can be very powerful both in the characterization and the quantification of ADC biotransformation. Among the multiple assay formats, there are two main categories of mass spectrometry-based assays for ADC biotransformation: the bottom-up approach and the top-down approach. The bottom-up approach monitors peptides or fragments specific to certain pre-specified region of the ADC. For example, through monitoring the concentrations using a light chain CDR peptide and comparing it to the corresponding heavy chain peptide derived concentrations, it is possible to infer if the light chain and heavy chain association is maintained after dosing [Citation55]. Similarly, monitoring the conjugated warhead and the protein backbone can provide complementary information of the in vivo deconjugation process [Citation56,Citation57]. Methods using bottom-up approach can be validated on triple quadrupole instruments and provides absolute quantification results. When monitoring multiple sections of an ADC, it is typically possible to multiplex the assay to increase operational efficiency.
The top-down approach is most commonly performed with a high-resolution mass spectrometer (time of flight, FTICR or orbitrap). The top-down approach has the advantage of identifying the various proteoforms of the molecule of interest [Citation58]. Structural information that may have been masked/lost by the enzymatic digestion in the bottom-up approach can be directly monitored in the top-down or middle down approach [Citation59], where the intact ADC or a sub-unit of ADC was analyzed. It is also possible to use this method to analyze the potential biotransformation that involved endogenous proteins, such as serum albumin [Citation60].
The challenge with this approach is obvious. It is difficult to provide absolute quantification results from this approach due to the challenge in acquiring the highly-purity reference standards for all the biotransformation species, and often MS is less sensitive, especially with bioconjugates analyzed in intact mode. Nonetheless, quantitate applications of intact mass spectrometry have been reported revealing differences in PK profiles when compared to bottom-up approaches [Citation38]. There have been various attempts to improve the sensitivity, including better capture steps or even multiple capture steps to reduce the background interference, analyzing the subunit, or application of advanced instrumentation.
Problem Solving for Non-Liquid & Rare Matrices
Typically, quantitative LCMS methods have been developed to measure circulating small molecules, peptides and proteins that can be directly measured from plasma or serum. However, there are many important pathways associated with disease that can only be measured in tissues or PBMCs. Recommendations on the use of PBMCs as a non-invasive matrix for measurement of biomarkers has been previously issued [Citation29], however their use in LCMS methods is emerging and case studies were discussed on their application and solutions to development challenges.
Although in principle, an LCMS method designed to measure protein in PBMC may not be fundamentally different than an LCMS method designed to measure circulating protein, there are important considerations while developing PBMC based LCMS assays. Importantly, PBMCs must be collected and isolated from blood using procedures that ideally yield high recovery and maintain the state of the protein to be measured. PBMC collection must be performed by trained personnel and with appropriate equipment that may not be available at all clinical sites.
A case study was discussed using PBMCs to measure PD-1 and GITR turnover rates [Citation61]. In this study, prior to LCMS, PBMCs were lysed twice with detergent to solubilize proteins. There was agreement that membrane proteins, which are most likely to be of interest such as for check point targets, can be separated from cytosolic and skeletal proteins to reduce the complexity of the matrix and decrease background noise in the mass spectrometer. Targeted methods can include antibody-based pulldowns, which further separates the protein of interest and decreases background signal. As an alternative to this workflow, when antibodies are unavailable or antibody-based pulldowns are impractical, such as for highly multiplexing assays, orthogonal approaches may be required to decrease the complexity of the sample prior to LCMS. In this case, samples can be so complex that high-flow LCMS may give better performance than low-flow LCMS.
Case studies were also shown for strategies for accurate determination of intracellular phosphorylated anabolites in PBMCs with LCMS/MS. Accurate quantitation of intracellular phosphorylated anabolites from nucleotide analogs (NA) is critical to understand NA drug exposure and efficacy. Strategies for selective and sensitive LCMS/MS assay development, robust PBMC collection and effective analyte stabilization were discussed through a case study. A chemical derivatization with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and hexylamine was used to reduce the polarity of the analytes and enables direct analysis on reversed phased LCMS/MS. Extensive efforts were spent to optimize the assay and minimize the analyte loss during PBMC isolation, processing, lysis, and storage. Treatment of PBMC with 30/70/2 (v/v/v) RPMI-1640/methanol/trichloroacetic acid was found effective for analyte stabilization and recovery. Robust and reliable PBMC isolation, counting and process procedures were critical to success.
Based on these case studies, recommendations were provided for the need for PBMC assays and best practices. There was agreement that it is important to first determine whether a PBMC or total cell assay is necessary in addition to plasma/serum one need to consider the cell uptake property and the action site of the drug. One application of using PBMCs is where there is potential endogenous nucleoside interference in a total cell assay. When using PBMCs it is critical to standardize a robust PBMC collection procedure across different clinical sites. Studies should consider separation of PBMC from RBCs and be careful to prevent lysing of PBMCs to stabilize drug/metabolites and minimize their loss.
In addition to PBMCs, there was also updated discussion and case studies on use of LCMS in tissues which has been discussed in previous white papers [Citation31]. Previous recommendations included using FFP methods for exploratory assays, assessing each tissue type separately, and utilizing mixed matrix approaches (e.g., standard in plasma and abundant tissue QCs, or homogenates diluted in plasma).
LCMS applications for ophthalmology and multiple ocular matrices was discussed. Bioanalysis of eye compartments (along with serum and other tissues) is important to determine exposure of the target organ to the drug and to help interpret local pharmacological or toxic effects as well as systemic exposure to demonstrate sustained release of the drug from devices. Based on the structural complexity of new modalities explored as well as the modes of delivery used to achieve long-acting delivery of these biotherapeutics, there are a number of key bioanalytical strategic considerations and challenges that must be overcome. There is need to develop multiple PK/PD assays to apply across various matrices and with very different dynamic ranges required for serum versus ocular tissues. In addition to highly sensitive serum assays, frequently there are also very low sample volumes available from ocular tissues such as aqueous/vitreous humor, which are also challenging to procure for assay development and validation purposes. With long residence times targeted for drugs to reduce dosing frequency, in vivo biotransformations and stability are important to understand. Hybrid immunoaffinity LCMS technologies have been used extensively to address many of these challenges and can provide foundational data, including PK, biomarker, and biotransformation information to enable our portfolio and drug development regulatory decision-making. Several example case studies were highlighted including Fab, pegylated hexamer-Fab, and other modalities.
There was agreement that there are challenges of bioanalysis in eye compartments with low volume, high sensitivity and large dynamic range assay requirements. It was recommended that there is a need to develop PK assays in multiple ocular matrices to evaluate target exposure. This includes fully validated assays in serum, and fit-for-purpose methods using surrogate matrices. Use of surrogate matrix or dilution of the ocular matrix with plasma is recommended. Consider use of a surrogate matrix for calibration and QCs. Matrix effects and IS behavior must be considered when selecting an appropriate surrogate matrix. Use authentic matrix only if unavoidable. It is important to avoid cross-contamination between ocular tissues during sampling.
Pushing the sensitivity limits in non-liquid matrices by novel sample preparation, chromatography, and instrumentations was discussed. The demand for high quality tissue target measurements utilizing quickly developed fit-for-purpose assays with varying quantitative ability are needed to support the development of many cutting-edge drug modalities such as protein degraders, gene therapy, mRNA/LNP, etc. In the past, protein level immunopurification (IP) has been used successfully to probe circulating and cytoplasmic located targets with great success. However, protein IP can suffer recovery issues when targeting membrane, structural, mechanical, or nuclear proteins requiring detergents such as SDS for release and solubilization, proteins with co-binders or in signaling complexes, as well as large proteins with multiple isoforms due to the selectivity of the IP reagent.
The case study showed successful deployment of SDS lysing and direct digestion of protein targets in non-liquid matrices using suspension trapping (STrap) plates combined with online peptide IA-LCMS/MS. The STrap method combines the advantage of efficient SDS-based protein extraction with rapid detergent removal, reactor-type protein digestion, and peptide cleanup into a plate format that has been heavily utilized for qualitative proteomics applications. However, the extension of these STrap devices to fully quantitative work streams has not been well documented. SDS lysis combined with STrap increased target signal for a lipid droplet associated protein target of interest in liver lysates ∼10x compared to Mammalian Protein Extraction Reagent lysis and protein IP. Furthermore, compared to the pellet digestion workflow, recovery relative to SIL spike increased an additional 20% for the STrap workflow likely due to the full removal of SDS while eliminating a full day of sample processing. More importantly, this workflow was well suited for quantification returning linear calibration curves This case study led to agreement that the STrap workflow highlighted here represents an off the shelf, default tissue preparation strategy that offers the highest target recovery, compatibility with the majority of protein targets inclusive of membrane and co-receptor bound target and is fully quantifiable with minimal analyst optimization.
Finally, there was discussion of cross-lab validation of assays developed in sphingolipid with multiple isomers. Sphingolipid biomarker assays such as sphingomyelin (18 isoforms), ceramide (11 isoforms), and globotriaosylceramide (16 isomers) were used as examples. In these bioanalytical LCMS/MS methods, animal lipids were often used as reference standard for total lipid level to quantify total lipid level in human plasma. The reference standard has significantly difference in isoform profiles (pattern) than the human study samples profile. Without adjusting instrument parameters, the results of total sphingolipid concentration could be different from two instruments of the same model, in the same lab or different labs when using the same bioanalytical method described instrument parameters. A general troubleshooting strategy was discussed including adjusting collision energy (CE), or decluttering potential (DP) to match the isoform distribution profile (pattern) from shared standards (spiked animal sphingolipid) and human samples from the sponsor's lab. For significant difference isoform profile between animal sphingolipid as standards and human study samples, the individual isoform needs to be synthesized to enrich the missing isoform in the original animal reference standard. then the new reference standard is generated to match to the isoform distribution profile(s) in human samples. In addition, it is strongly recommended to have at least one human endogenous QC level in duplicate besides spiked 6 QCs using animal lipid as reference standard. The human QC nominal value is the overall mean from 3 runs of accuracy and precision (n = 5) or ≥10 runs of inter-run data (n = 2 per run). The biomarker reference range should be determined at early stage of assay transferring and validation using shared healthy donor samples followed by one accuracy and precision run. If the normal range is not overlapping or shows significant difference between the two labs, the instrument parameters should be further adjusted until profiles matching and then re-start the validation A-P runs.
There was also agreement that matching the isoform profile is necessary for the CRO who will be running the on-going clinical trials for sample analysis and enable the ability to compare study results between labs. Otherwise, the CROs can use their own normal range for the entire clinical trials from the beginning to the end.
RECOMMENDATIONS
Below is a summary of the recommendations made during the 16th WRIB:
Novel Applications & Novel Technologies in Bioanalysis
There are multiple approaches to targeted proteomics with LCMS
The nature of this assay is exploratory (biomarker screening), which may not need strict acceptance criteria. There is no current standard approach on defining the acceptance criteria in the pharma industry and no recommendations from agency.
Alternative affinity enrichment reagents such as aptamers were discussed as alternatives to animal derived antibodies.
As single-stranded DNA or RNA molecules, aptamers bind to their target molecules with high affinity and specificity. The use of aptamers not only provides the same or higher level of information as obtained from animal procedures, but also help prevent a pitfall due to batch variability of animal-oriented capture antibody
The aptamer strategy is currently at the PoC stage, and challenges remain in method development for non-human IgG bioanalysis.
Use of DIA mass spectrometry and challenges in sample prep to support discovery of fecal biomarkers was discussed.
Fecal matrix is very complex, and DIA MS proteomics can provide good alternative for both biomarker identification and quantitation.
Extraction buffer selection is critical for sample clean-up to capture human secreted proteins. When doing normalization, use total protein not mass.
Bead-based sample clean-up strategy appears to work best with high reproducibility.
HILIC method improvements were discussed for use in oligonucleotide bioanalysis.
Cons of ion-pairing agents include strong signal suppression (including when switching to positive mode), significant MS cleaning & downtime, and higher cost for preparing fresh buffer before each run.
HILIC is promising with recent advancements including automated MS/MS annotation for unbiased sequence characterization, HRMS analysis resulting in deconvoluted mass spectrum, and the ability to analyze siRNA as single strands and duplexes
HILIC methods still do not see equivalent assay performance
Oligonucleotides: Novel Modalities & Novel Method Development
The pros and cons of different mass analyzers for oligo analysis
HRMS: extensive tuning of compounds not necessary, similar LLOQ to triple quadrupole mass spectrometry (QQQ) method, adds additional ID verification, can generate data on metabolites in same run as parent quantitation run, can calculate relative abundance of metabolites to parent due to using Q1 data (with caveats)
QQQ: decreased run time, can add additional identity verification with precursor scan for non-endogenous components of drug, easy to outsource, typically a lower LLOQ with higher s/n ratio
If/when possible, small molecule acceptance criteria should be used for LCMS assays of oligonucleotides.
IS for oligos may be a stable isotope labeled drug (ideal), or a chemically similar non-interfering analog.
The same IS can also be used for metabolite quantitation
For ADME packages for oligos, requirements for active drug quantitation and metabolites were discussed.
GalNAc-siRNA: After distribution, the main compartment with drug is liver. The AS of siRNA is Ago2 loaded and is the active drug. Remaining drug is in an intracellular depot that may eventually become Ago2 loaded.
Quantitation of AS in RISC complex is nice to have in target tissues to derive the PK/PD relationship.
Given very low AS concentrations in RISC, PCR assays appear to be the only option.
Total tissue concentrations may be analyzed via LCMS.
ADC: Novel Modalities & Novel Method Development
For internal standard selection, no guideline for ADC quantitation yet, but the assay accuracy affects ADC/Total antibody ratio that indicates the in vivo stability.
Multiple case studies of internal standards on mAb quantitation give mixed opinions in the past years (SIL-mAb, SIL-peptide, flank SIL-peptide).
Implementing automated sample prep workflow provides better precision and accuracy.
Using GIS as the IS for the payload assay is recommended when SIL-payload is not available in a non-reg BA setting.
There have been significant advances in qualitative characterization of ADCs using high-resolution mass spectrometry-based approaches. Intact LBA-LC-HRMS assay for ADC characterization helps understand the in vivo biotransformation and may provide relative quantification information as well.
For in vitro characterization of ADC biotransformation, serum, plasma or whole blood may show different results.
LCMS is a powerful analytical tool for the analysis of in vivo ADC biotransformation or payload metabolites.
Problem Solving for Non-Liquid & Rare Matrices
PBMCs are increasingly being applied to LCMS methods as a matrix for with applications for membrane protein detection and phosphorylation.
Determine whether a PBMC or total cell assay is necessary in addition to plasma/serum one need to consider the cell uptake property and the action site of the drug.
Standardize a robust PBMC collection procedure across different clinical sites. Other important considerations include separation of PBMC from RBCs and preventing lysing of PBMCs to stabilize drug/metabolites and minimize their loss.
To shorten sample preparation time and increase sensitivity in non-liquid matrices, it was recommended to apply novel workflows in tissue biomarker sample analysis that shortens assay development time (e.g., antibody-free LCMS assay).
Off the shelf preparation methods such as suspension trapping plate (S-trap) shows higher sensitivity and recovery when compared to pellet digest or in-solution digestion.
There was agreement that challenges of bioanalysis in eye compartments with low volume, high sensitivity, and large dynamic range. It was recommended that there is a need to develop PK assays in multiple ocular matrices to evaluate target exposure. This includes fully validated assays in serum, and fit-for-purpose methods using surrogate matrices. It is important to avoid cross-contamination between ocular tissues during sampling.
When developing an assay for multiple biomarker isoforms, it is necessary to match up the major isoform distribution profile between authentic samples and the reference standard by adding synthesized major isoforms which are significantly lower concentration.
When transferring such an assay, need to properly adjust instrument parameters to match the major isoform abundance between two instruments or two labs for more consistent data report ad comparison.
It is important to include one human QC which contained endogenous only in regular clinical sample testing run.
SECTION 3 – ICH M10 BMV Guideline & Global Harmonization
Arindam Dasgupta16, Mohsen Rajabi Abhari16, Akiko Ishii-Watabe26, Yoshiro Saito26, Dulcyane Neiva Mendes Fernandes27, Joseph Bower28, Chris Burns29, Kevin Carleton5, Seongeun (Julia) Cho16, Xiulian Du16, Marianne Fjording30, Fabio Garofolo31, Sumit Kar10, Olga Kavetska32, Elham Kossary33, Yang Lu16, Andrew Mayer7, Nisha Palackal19, Xiazi Qiu10, Danielle Salha34, Eric Thomas35, Tom Verhaeghe36, Stephen Vinter29, Katty Wan37, Yow-Ming Wang16, Yuting Wang10, Kathi Williams6, Eric Woolf2, Li Yang16, Eric Yang7 & Jinhui Zhang16
Authors are presented in alphabetical order of their last name, with the exception of the first 5 authors who were session chairs, working dinner facilitators or major contributors.
The affiliations can be found at the beginning of the article.
SECTION 3A – Impact of Global Harmonization on Regulated Bioanalysis
HOT TOPICS & CONSOLIDATED QUESTIONS COLLECTED FROM THE GLOBAL BIOANALYTICAL COMMUNITY
The topics detailed below were considered as the most relevant “hot topics” based on feedback collected from the 15th WRIB attendees. They were reviewed and consolidated by globally recognized opinion leaders before being submitted for discussion during the 16th WRIB. The background on each issue, discussions, consensus and conclusions are in the next section and a summary of the key recommendations is provided in the final section of this manuscript.
Harmonization of Cross Validation in Regulated Bioanalysis
Is a statistically powered cross validation approach preferred over the current industry practice of applying ISR acceptance criteria? Is there an acceptance range which should be targeted for these statistical analyses? With the issues of importing study samples into China, how do regulators feel about doing within-study cross validations of assays outside-inside of China with spiked QCs only (and not with incurred samples)?
Patient Centric Sampling in Regulated Bioanalysis
Are the current global regulatory requirements, including the 2022 ICH M10 BMV Guideline flexible enough to allow patient centric sampling? Is dry blood spot /matrix in section 7.6.1 Dried Matrix Methods provides sufficient guidance and/or flexibility? Special population matrix effect: how much /little is enough? Any alternative approaches to analytical testing?
Harmonization of Reference Standard Materials (RSMs)
High degree of inconsistency in the content of the Certificate of Analysis (CoA) for reference standards when purchased from vendors and manufacturers for use in regulated bioanalysis. What are the components labs may be required to assess and confirm and what are the best approaches for managing COAs and updates?
DISCUSSIONS, CONSENSUS & CONCLUSIONS
Harmonization of Cross Validation in Regulated Bioanalysis
Cross validation of bioanalytical assays, in particular pharmacokinetic assays, are important regulatory required assessments for control of assays which are performed when two different validated bioanalytical assays are used within the course of a single study, or when an assay is supported at two or more bioanalytical laboratories either within a study or between studies and data are to be combined or compared for regulatory decisions. The incurred sample reanalysis (ISR) criteria is often used (±20%/±30% for 2/3rds of samples analyzed) for PK assay cross-validations [Citation62]. These acceptance limits lack scientific basis and do not specifically call out systemic directional bias between the two methods or laboratories. There have been instances in which cross validations have met acceptance criteria however, the systemic bias may have led to an apparent PK difference. These systemic differences should be shared with clinical pharmacology colleagues to inform on the potential of assay related PK artifacts in data analysis for clinical studies that are going to be combined to support special dosing regimens. Hence, a question was raised whether a correction factor needs to be applied due to systemic directional bias between labs. While historically this assessment is often performed over a single analytical run in the originator and comparator laboratory, this approach does not allow for an understanding of between run precision and accuracy of a method. In high-risk situations, a more robust cross validation assessment may be considered, where multiple analytical runs are performed over multiple days to fully characterize the within and between day variability of the assay within and between laboratories [Citation24]. There was agreement that cross-validation between laboratories is important but difficult and thus regulators are sympathetic and may allow some latitude depending on what the results demonstrate in the submission if properly communicated with the agency. Based on the communication and discussion with the Agency, the proposed action is recommended to be documented in the pre-established standard operating procedure (SOP). Cross validation was agreed to be not necessary when multiple clinical studies are not compared within a submission. A case study discussed whether statistically powered cross validation is preferred over the current ISR based approach (2/3 should be within acceptance criteria). There was agreement that statistically powered driven cross validation is preferred over the “ISR-based approach”. The final ICH M10 has no acceptance range for these analyses, and the recommendation is to collaborate with clinical pharmacology department to determine the appropriate acceptance range with proper justification.
Another discussion topic was method cross validations between labs in China and labs outside China. Up until a few years ago the strategy for bioanalytical support of global multi-site clinical trials was to analyze all the samples from one trial in one lab. However, more recently this strategy has been pressure-tested, specifically for trials with clinical sites in China. Due to very strict China HGRAO (Human Genetic Resources Administration Office) regulation, export permits for shipping study samples to a lab outside of China are often not granted and samples need to be analyzed locally.
To avoid studies being delayed case studies were discussed implementing a bioanalysis strategy for studies conducted in China to expedite the transfer and validation of all bioanalytical methods for all modalities in China to support all human sample analyses locally. There are, however, some logistical hurdles that need to be tackled. The assays need to be cross validated between the lab in China and the originator lab if data are to be compared or are within the same clinical trial. When two different methods or labs are being used within the same clinical study (e.g., phase 3 trials), “cross validation with shared matrix QCs and non-pooled subject samples should be conducted at each site or laboratory to establish interlaboratory reliability”. The final ICH M10 states that study samples can be used if available.
The import of the cross-validation samples into China also requires a lot of documentation: proof is needed that samples originate from subjects free from Covid-19 and other infectious diseases (HIV, HCV, HBV, Syphilis. Etc.). For QC samples a certificate can usually be provided by the supplier of the blank material. For study samples however, obtaining such proof is more complicated or sometimes not feasible. Also, additional paperwork like site specific informed consent, ethics committee approval and CTA approval may be required for importing subject samples.
Because of these limitations, some are considering cross validating assays in China based labs with QCs only and no longer with incurred study samples. Regulators recognized the challenges of importing/exporting samples (study samples and QCs) into/out of China and recommended to mitigate the problem as best as possible. If the problem persists, reaching out to appropriate regulatory agencies for advice on the best strategy (e.g., QCs only) was preferred.
Patient Centric Sampling in Regulated Bioanalysis
Patient centric sampling is an important element of decentralized clinical trials (DCT), a novel approach taken by pharma industry to clinical trials conduct [Citation63]. The purpose of DCT is to reduce burden on patients, make study more accessible to the participants, increase diversity and equity of the trial, and improve patient's enrollment and retention rate. Patient centric sampling can be accomplished by collecting samples either at patient home or a diagnostics laboratory in the local patient community. At patient home sample collection could be done by visiting health professional, trained caregiver of as a self-collection, based upon the type of sample and sampling technique, and trial logistics. Self-collection is a rapidly developing and enabled by new technologies and devices. In combination with telemedicine and home visits by healthcare professionals, this approach is significantly reducing the number of visits to clinical sites and burden on trial participants.
The final ICH M10 recommends that if dry blood spot is used in addition to plasma samples in the same study, then cross-validation should be done. Considerations should be given to matrix effect in relevant patient populations or special populations when available. These points were discussed for updated recommendations. Cross validation vs. bridging multiple PK parameters for concordance was discussed. It was agreed that ICH M10 BMV does give the flexibility to allow patient centric sampling and for any innovative technologies including new microsampling plan. Decision making on cross-validation/ bridging strategy should be science based, and when necessary, micro sampling can be supported by statistics powered analysis similar to that used for a cross validation. For special population matrix effects, it was recommended to use matched matrix for validation.
Harmonization of Reference Standard Materials (RSMs)
Significant progress has been made over the last decade within the global regulated bioanalysis community working toward establishing one global regulatory standard governing Bioanalytical Method Validation (BMV), however, there continues to be a high degree of inconsistency found in the content of the Certificate of Analysis (CoA) for reference standards used for calibrators and QCs in regulated bioanalysis [Citation64]. While there is good agreement from regulatory agencies (e.g. FDA, EMA) of the requirements for the type of information which needs to be provided in CoA for reference materials; a lack of consistency existing in the quality and content of CoA provided by vendors and manufacturers of these critical reagents. Further complicating the issue is when a laboratory may not have a robust inventory system to ensure appropriate oversight of reference standard management processes.
Previous discussion on ICH M10 guidance of CoAs [Citation24] led to the recommendation that ICH M10 is helpful and adequate as a minimum standard, but additional white papers are needed to clarify and address specific challenges with CoAs for reference standards [Citation64].
Updated recommendations were provided. In addition, some of the most typical and most impactful gaps that the community continues to experience regarding common deficiencies encountered in CoA for reference material and critical reagents were discussed. Two case studies were discussed; the first case study focused on best practice of implementing a robust critical reagent inventory process, and the second case study focused on examples for new electronic tools available that provide a more robust and streamlined critical reagent inventory system.
There was agreement that the most important matter in CoA is content (powder formulation)/concentration (liquid formulation) data, purity, and bioactivity that related to the protein conformation in case of large molecule. The ideal source is the same batch of the drug substance that used for nonclinical/clinical studies (large molecule). The onus is on the receiver of the material to obtain as much information as possible. Bioanalytical evaluation is important for the bridging strategy from original material to new material. Robust internal SOPs, fully documented and with justification, are acceptable support.
RECOMMENDATIONS
Below is a summary of the recommendations made during the 16th WRIB.
Harmonization of Cross Validation in Regulated Bioanalysis
Statistically powered cross-validation is preferred over “ISR” criteria (which are 2/3 of incurred samples should be within acceptance criteria) for cross-validation,
Final ICH M10 has no acceptance range for these analyses, and the recommendation is to collaborate with clinical pharmacology department to determine the appropriate acceptance range with proper justification.
Some sponsors are considering doing within-study cross validations of assays inside of China with spiked QCs only (and not with incurred samples).
Regulators recognized the challenges of importing/exporting samples (study samples and QCs) into/out of China,and recommended to mitigate the problem as best as possible. If problems persist, reach out to appropriate regulatory agencies for advice.
Patient Centric Sampling in Regulated Bioanalysis
ICH M10 BMV does give the flexibility to allow patient centric sampling and for any innovative technologies including new microsampling plan.
Decision making on analytical testing strategy (cross validation vs. concordance of PK parameters) should be science based, and when necessary, microsampling can be supported by statistics powered analysis similar to cross validation.
For special population matrix effects, it is recommended to use matched matrix for validation.
Harmonization of Reference Standard Materials (RSMs)
High degree of inconsistency in the content of the Certificate of Analysis (CoA) for reference standards when purchased from vendors and manufacturers for use in regulated bioanalysis.
Labs may be required to assess and confirm:
Appropriate concentration and purity testing approaches
Expiration date/Recertification or re-establishment date
Instructions for reconstitution
Appropriate buffers
Post-reconstitution actions – filtering, additional testing, storage conditions, etc.
Most important is content (powder formulation)/concentration (liquid formulation) data, purity, and bioactivity that related to the protein conformation in case of large molecule
Bioanalytical evaluation is important for the bridging strategy from original material to new material. Robust internal SOPs, fully documented and with justification, are acceptable support
SECTION 3B - Common Mass Spectrometry & Ligand-binding Assays Issues
HOT TOPICS & CONSOLIDATED QUESTIONS COLLECTED FROM THE GLOBAL BIOANALYTICAL COMMUNITY
The topics detailed below were considered as the most relevant “hot topics” based on feedback collected from the 15th WRIB attendees. They were reviewed and consolidated by globally recognized opinion leaders before being submitted for discussion during the 16th WRIB. The background on each issue, discussions, consensus and conclusions are in the next section and a summary of the key recommendations is provided in the final section of this manuscript.
Impact the 3Rs in Regulated Bioanalysis
What are some examples of working through an obstacle on a bioanalytical question with a regulator during an inspection to compromise on a solution or approach?
Regulated Bioanalysis of Tissues & Secondary Matrices
What are examples where tissues have been the primary matrix for concentrations to support regulatory decisions? When are human urine assays validated per ICH M10 required for regulatory submissions? When are metabolite assays validated per ICH M10 required?
Stability Issues in Regulated Bioanalysis
What is the rationale for not allowing the additive exposure to count bench top stability from freeze-thaw (FT) experiments?
Can you reference stability data that is collected in another facility?
Can you omit high QC sample in stability validation when ULOQ QC is being used?
Harmonization of Endogenous Molecules Validation – Making the most of BMV & BAV Similarities
Why is endogenous compounds validation in regulated bioanalysis so important? How often is it necessary to use surrogate matrix working with endogenous molecules? What to do if selectivity fails in healthy matrix, do you then perform the selectivity experiment using the patient population matrix which are devoid of the endogenous molecule? Are standard addition and background subtraction methods often used in LBA and LCMS/MS assays? Can you use both surrogate matrix and background subtraction approach?
Novel/Alternative Technologies in Regulated Bioanalysis
New modalities (qPCR, flow cytometry, other modalities for cell and gene therapy) not specifically addressed in the ICH M10. Large molecule analysis via mass spectrometry using affinity capture; what is being measured - total, free, or active drug
DISCUSSIONS, CONSENSUS & CONCLUSIONS
Impact the 3Rs in Regulated Bioanalysis
The 3Rs (Replacement, Reduction, Refinement) means reducing the number of animals needed in any given study, replacing animals with other models whenever possible and refining procedures to involve the fewest number of animals while still giving valid results. It was discussed how bioanalysis can influence strategies to optimize the welfare of and number of animals used in pharmaceutical development and research.
Current and future bioanalytical efforts with respect to 3Rs were discussed. Examples included implementing liquid plasma microsampling for GLP rat studies. Liquid plasma microsampling has been widely adopted in preclinical toxicity studies in the pharmaceutical industry. With the advancement of LCMS instruments and microsampling technologies, serial bleeding has been gradually applied in industry for exploratory and GLP rodent studies. The use of microsampling techniques brings both ethical and scientific benefits including less invasive bleeding procedures, less animals required, and the ability to ascertain exposure levels and correlate toxicity to exposure on an individual animal basis.
A related discussion topic from the application of 3Rs was to provide examples of working through an obstacle on a bioanalytical question with a regulator during an inspection and compromising on a solution or approach. During inspections, most obstacles tend to be related to lack of transparency in study reports reflecting what happened during the study. So, discussions tend to be related to how best to report these issues, so reviewers have a clear picture of how the study was conducted. The recommendation was standardizing the reporting structures for method validation and sample analysis reports included in the submission for marketing applications. Global harmonization of the guidance serves as a great opportunity to start the conversation.
Regulated Bioanalysis of Tissues & Secondary Matrices
The pharmacokinetic characteristics of drug candidates are generally required to be assessed in order to characterize the molecule and ensure that the appropriate dose is administered to the target population at the appropriate frequency in order to achieve an optimal therapeutic effect while minimizing adverse reactions. Pharmacokinetic calculations are based on drug level concentrations from a primary matrix, typically plasma or serum. Nevertheless, often drug concentrations in “secondary” matrices are determined during the course of clinical programs. Examples of such secondary matrices include urine, tissues, PBMCs and cerebral spinal fluid (CSF). Concentrations in these matrices may be determined to study routes of drug elimination (urine), drug concentrations at site of action (tissue, PBMCs), or as a surrogate to brain concentration (CSF).
While regulatory expectations with respect to the validation of bioanalytical methods to determine drug concentrations in plasma/ serum are well known, expectations with respect to validation of methods to support analysis of secondary matrices are less clear. In addition to unclear regulatory expectations, solid as opposed to liquid secondary matrices have additional challenges. These include standard preparation methodology, the potential for blood contamination which may bias results, and the potential for inhomogeneity when biopsy samples require analysis.
The final ICH M10 guideline states secondary matrices should be validated “as necessary” and the sponsor can determine the level of assay qualification for studies not considered for regulatory decision-making or labeling (i.e., only used for internal decision-making).
Case studies were discussed showing the decision on validation assessments for the secondary matrix methods was based on what decisions/conclusions the associated results contribute to. For example, the degree of validation performed for assays generating data contributing to internal decision making may be less than that associated with product labeling content.
Recommendations were provided supporting and clarifying ICH M10 for examples where tissues have been the primary matrix for concentrations to support regulatory decisions. Topical and ocular therapeutics may be the examples. In the case where tissue is the primary matrix, the ICH M10 should be followed. Plasma and serum are still the most common matrices, other tissues can be justified based on plasma assay validation. Whether the tissue matrix needs validation depends on the ADME profile and what the data is used for. Urine assays would need some degree of validation if used in a secondary assay, but if it is the primary matrix, a full validation is needed. In the earlier days other approaches were acceptable, for example, when bisphosphonates were first marketed, urinary excretion was accepted to assess bioequivalence. This was based on the fact that these compounds are not metabolized, and full validation was required in such cases. There was an agreement that metabolite assays need to be validated per M10 if the metabolites contribute to activity or safety or are present at a significant amount.
Stability Issues in Regulated Bioanalysis
ICH M10 recommendations for stability evaluation, with the highlights of differences from previous guidance/guideline were reviewed. Recommendations on stability of high concentration samples were issued [Citation24] stating that QC stability evaluation above ULOQ does not need to be performed routinely. The ICH M10 recommendations specify that if the concentrations of the study samples are consistently higher than the ULOQ of the calibration range, the concentration of the high QC should be adjusted to reflect the higher concentration. The consistently higher concentrations were discussed and agreed that this was more applicable to later stage studies than in example FIH studies. If the samples are consistently higher than calibration range, then an alternative could be to change the calibration range.
There was agreement that if during method validation, the high QC was replaced with the dilution QC (if this level represents the study sample levels) the decision should be made based on the stage of the study. However, the dynamic range of standard curve needs to be adjusted when more information of the assay and study is obtained as the project progresses. For toxicity studies, ICH M10 recognizes that this high-level QC may not be possible to prepare due to solubility limitations. If high-level dilution QC stability point fails, there was consensus that having a high QC will help distinguish between a stability issue or a solubility issue when super high concentration QCs are used as dilution QC.
Another discussion was the use of a dilution QC during sample analysis and if stability of this dilution QC should be assessed. Updated discussions since the 2019 White Paper in Bioanalysis recommendations showed dilution QC stability was not routinely evaluated in method validation. When dilution linearity is evaluated, it is normally done in an accuracy and precision run with fresh calibration standards and QCs. As a result, stability issues of dilution QCs are discovered during sample analysis when aged and frozen dilution QCs are used. It is recommended that if using a frozen dilution QC during sample analysis to evaluate the stability of dilution QC early in method development.
The next stability topic was additive/cumulative stability evaluations which best represent how study samples are handled. For example, can the freeze/thaw and short-term bench top exposure stability be evaluated in a cumulative manner to include both the freeze/thaw cycles and the bench top exposure time for each freeze/thaw cycle which can be more meaningful than running separate stability evaluations from just FT cycles and an extended benchtop stability experiment alone. The cumulative effect can have the advantage of more accurately measured by simulating the actual freeze/thaw process (i.e., a thawing cycle + exposure time on the bench-top during thawing) of unknown samples. Additive/cumulative exposure approach is often used to perform impact assessment in the event of a temperature excursion. Consensus was reached upon discussion that stability testing should mimic sampling handling conditions. However, it should be noted that the total time for bench top should be concurrent, and it is not acceptable to use additive exposure to bench top condition (i.e., the time form each FT cycle cannot be added up).
The final stability topic was whether stability data collected in another facility can be referenced. When bioanalytical assays are transferred from lab A to lab B, it is not always clear what needs to be repeated in terms of stability assessment. It is noted that both scientific and operational considerations need to be considered when making decisions to reference stability data generated in another facility. If the same method is applied in both labs, then stability assessment does not need to be performed. There was agreement that it is acceptable to reference lab if the validation report has complete information. Access to raw data in case audit and review is needed as well.
Harmonization of Endogenous Molecules Validation – Making the most of BMV & BAV Similarities
When working with replacement therapies, it can be a challenge to quantitate the dosed drug when the method cannot distinguish between the therapeutic drug and the endogenous counterpart. Several approaches are outlined in the ICH M10 [Citation65].
The ICH M10 Section 7.1 endogenous compounds validation is important because there are many drug products or active drug metabolites that are endogenously present. ICH M10 does not cover quantitation of a biomarker, however, it was agreed that endogenous drug PK method validation principle and biomarker validation have many similarities.
Quantification of dosed drug is done by preparing calibration standard curves and QCs in the same matrix as the study samples, so that the matrix effect is the same in both the standard and study samples. The matrix should be free of matrix effect and interference. For endogenous analytes this can be a challenge as the level of the endogenous counterpart can be higher in the matrix used for the preparation calibrator standards and QCs than the dosed drug. When working with replacement therapies, one approach to circumvent matrix interference is to use surrogate matrix instead of authentic biological matrix. The surrogate matrix can be depleted matrix, protein buffer or from another species.
There was agreement that it is necessary to document efforts taken, and to test as many different lots of matrix as possible when authentic biological matrix with low level of endogenous drug is difficult to find.
Novel/Alternative Technologies in Regulated Bioanalysis
We have seen a significant expansion of the bioanalytical landscape in response to the continuing evolution of therapeutic strategies – and resulting broadening of analytical approaches to achieve optimal sensitivity, selectivity, and efficiency. While the approaches for pharmacokinetic analysis of small molecules via chromatographic assays and large molecules via ligand-binding assays have been well-characterized and governed by guidance from global regulatory agencies, there are many established workflows and platforms that are not specifically addressed by the finalized ICH M10. For over a decade, analysis of biologics or “large molecules” by mass spectrometry has become increasingly common. At least as far back as 2010, this topic was addressed in the White Paper in Bioanalysis [Citation3]. The 2014 White Paper in Bioanalysis specifically addressed the potential need for cross-validation of mass spectrometry assays with LBA [Citation9]. In the last few years, historical data were presented, demonstrating reliable performance of mass spec assays (hybrid assays and reagent-free mass spectrometry assays) in support of regulated PK bioanalysis.
The updated recommendation provided was that LBA and Mass Spectrometry technologies should be complementary. There is no universal “gold standard” – the platform should be chosen based on the specific needs for the program, to include discussion of required specificity, sensitivity, material availability, and timelines. Validation of an analytical method demonstrates that the assay is fit for purpose. The validation requires that the analyte, including in vivo processing of the analyte, is well understood. While it is important to perform cross-validation to support within-study comparisons (as would be also be required when changing reference material sources or mass spec platforms), there should be no a priori requirement to perform such comparison to establish that the platform itself is suitable. Even for newer technologies, cross-validation with existing platforms should not be required - as long as the means of detection is well understood.
RECOMMENDATIONS
Below is a summary of the recommendations made during the 16th WRIB:
Impact the 3Rs in Regulated Bioanalysis
During inspections, most obstacles tend to be related to lack of transparency in study reports reflecting what happened during the study. It is very important to report these issues, so reviewers have a clear picture of how the study was conducted.
Industry recommends standardizing the reporting structures for method validation and sample analysis reports included in the submission for marketing applications. Global harmonization of the guidance serves as a great opportunity to start the conversation
Liquid microsampling for preclinical toxicity studies are both ethically and scientifically beneficial.
It should be clear in the documentation that 3Rs and e.g. microsampling is used in validation and sample analysis so the inspectors and reviewers are well informed.
A global harmonized guideline on the topic is recommended.
Regulated Bioanalysis of Tissues & Secondary Matrices
ICH M10 guidance states secondary matrices should be validated “as necessary” and validation for studies not submitted for regulatory review should be determined by the sponsor for their intended use.
Topical and ocular tissues have been identified as primary matrix for regulatory decisions in some cases needing full validation.
Urine assays need to be validated only when this data is used as the basis for primary regulatory decisions (e.g., to demonstrate bioequivalence, as was the case when bisphosphonates were first marketed).
Metabolite assays need to be validated if metabolites contribute to activity or safety and are present at a significant amount
Stability Issues in Regulated Bioanalysis
The decision on whether to omit high QC samples when using an ULOQ QC should be made based on the stage of the
study. If ULOQ QC stability data fails, having a high QC will help distinguish between a stability issue or a solubility issue when super high concentration QCs are used as ULOQ QC.
Stability testing should mimic the sample handling conditions, and FT cycle should mimic the time a sample is stored on the bench top prior to next FT cycle.
It is acceptable to reference stability data from another facility as long as the validation report has complete information, and method is the same. It's recommended to have access to raw data in case review is needed.
Harmonization of Endogenous Molecules Validation – Making the most of BMV & BAV Similarities
Endogenous compound validation is important because many drug products or active drug metabolites are endogenously present.
ICH M10 Section 7.1 does not cover quantitation of biomarkers. However, it should be considered that the endogenous drug PK and biomarker validation bioanalysis have many similarities and several principles found in BMV guidelines can be applied also to BAV.
For endogenous molecules, parallelism should be shown in surrogate and authentic matrix in order to dilute authentic matrix with surrogate matrix.
Blank authentic matrix used in validation assay needs to be thoroughly screened for low basal concentration.
Must document efforts to try as many lots as possible when authentic biological matrix with low level of endogenous drug is difficult to find.
Novel/Alternative Technologies in Regulated Bioanalysis
New modalities bioanalysis using qPCR, flow cytometry, and specifically Hybrid assays are not addressed in the ICH M10.
Validation criteria are not different for ultrasensitive LBA (e.g. SIMOA) vs standard LBA.
Hybrid assays have been used for more than 10 years in Regulated Bioanalysis and plenty of data have demonstrated that they are robust and reliable assays.
SECTION 3C – LBA Unique Challenges
HOT TOPICS & CONSOLIDATED QUESTIONS COLLECTED FROM THE GLOBAL BIOANALYTICAL COMMUNITY
The topics detailed below were considered as the most relevant “hot topics” based on feedback collected from the 15th WRIB attendees. They were reviewed and consolidated by globally recognized opinion leaders before being submitted for discussion during the 16th WRIB. The background on each issue, discussions, consensus and conclusions are in the next section and a summary of the key recommendations is provided in the final section of this manuscript.
LBA Single Well Analysis (Singlicate) in Regulated Bioanalysis
Is singlicate analysis for LBA allowed under ICH M10 guidelines? What are strategies for implementation?
Change of the Critical Reagents: “KISS - Keep It Simple & Straightforward”
What guidelines should be followed for characterizing critical reagents and changes in lots of critical reagents?
LBA Carryover Assessment in Regulated Bioanalysis
What are approaches for minimizing carryover in LBA systems?
Commercial, RUO & Diagnostic LBA Kits in Regulated Bioanalysis
Are commercial kits appropriate for PK assays? What are guidelines for use?
DISCUSSIONS, CONSENSUS & CONCLUSIONS
LBA Single Well Analysis (Singlicate) in Regulated Bioanalysis
Single-well analysis has been the standard approach for small molecule pharmacokinetic bioanalysis for the past 30+ years. Large molecule bioanalysis has yet to truly adopt this practice and most assays are still conducted from duplicate well evaluations [Citation29]. Early immunoassays lacked the precision and accuracy of an LCMS/MS assay with a stable isotope internal standard. Therefore, it was conceivable that a duplicate measurement of a sample would yield a more accurate concentration of the drug product in systemic circulation. However, recent improvements in technology and science along with clear regulatory guidance have charted a pathway for large molecule bioanalysis to move in the direction of single-well analysis. In order to get safe and effective treatments to patients quicker and at more affordable prices, the bioanalytical community should make every effort possible to support single-well analysis for large molecule drugs. With the ability to produce higher quality reagents along with the improvement in instrument analysis of large molecule drug products, the science is clear – ligand binding assays are capable of (if not exceeding) the levels of precision and accuracy that a small molecule assay development could yield. However, still years removed from a preponderance of evidence, large molecule teams continue to operate under the assumption that a duplicate measurement of an individual sample produces a more accurate or precise result.
In 2019, discussion of the ICH M10 led to agreement that single well analysis is acceptable according to M10 BMV guidelines as long as the method was validated for singlicate use [Citation24]. Acceptance criteria are the same if using this approach.
Continued discussion on this subject highlighted concerns about inherent assay variability and reagent quality but reservations about the method are the same as they would be for small molecules. A gradual implementation was recommended, on a case-by-case basis, method specific, which can smooth the transition. Internal comparison of singlicate vs. duplicate processing can be helpful but is not required. It is expected that all ICH members will accept the approach when ICH M10 is implemented. Overall, the consensus was unchanged that singlicate analysis for LBA is acceptable if analytical performance criteria are satisfied and validation is performed with the singlicate analysis.
Change of the Critical Reagents: “KISS - Keep It Simple & Straightforward”
Robust and reproducible bioanalytical assay performance depends on the quality of critical protein reagents (CPR). Critical protein reagents are generally produced using a biological process that makes them inherently prone to variability. Maintaining lot-to-lot consistency can be challenging and can lead to variability in performance of bioanalytical assays. Therefore, understanding the unique characteristics of these reagents is crucial to assay performance, and require careful characterization. Final ICH M10 guidance states the criteria that should be provided on critical reagent data sheets and guidance for retesting and documenting stability as part of assay life cycle management [Citation65].
Thorough biochemical and biophysical characterization is performed concurrent with testing and selection of the reagent in the intended bioanalytical assay. Challenges associated with generation and characterization of these reagents were discussed with an emphasis on the importance of building a thorough understanding of the impact to the method performance from different lots of material. New modalities are challenging the established norms around the vast number of critical reagents and their uses. Even a “minor” change in a reagent can have a major impact on its performance in a PK assay. There was alignment that assay performance is the best measure of reagent quality. If performance drops/assay fails, and the cause should be investigated. There was consensus that the process governing the critical reagent changes should be kept as simple and straightforward as possible as per previously published recommendations [Citation66] and on a case by case basis.
In method development, it was recommended to test a minimum of two lots; establish and document a solid baseline characterization of the identified critical reagents. The characterization can include verification of purity and concentration determination using orthogonal methods. It is important to have robust internal SOPs for retesting/requalify the critical reagents which should include what to do if the performance tested in the assay fails. If purchasing reagents, qualify the vendors, issue questionnaires regarding their internal procedures for production of the reagents. When changing the lot, the performance should be evaluated by the bioanalytical method.
LBA Carryover Assessment in Regulated Bioanalysis
Enzyme-linked immunosorbent assay (ELISA) has been the ligand binding assay (LBA) gold standard for many protein quantitation methods used during preclinical and clinical biotherapeutic drug development. ELISAs are often used in the measurement of pharmacokinetic (PK), toxicokinetic (TK) drug studies, for biomarker evaluations and immunogenicity assessments. One of the technologies that has been able to overcome many of the disadvantages of ELISAs is the Gyrolab. The Gyrolab is essentially an ELISA on a compact disk, with all reagent and sample additions performed automatically by the instrument. In addition, the Gyrolab uses a microfluidic flow-through system, shortening incubation times, which has been shown to minimize matrix effects and the need for sample pretreatment. The system has good sensitivity, comparable to ELISA, when the same antibody reagents are used, but has a broader dynamic range. This feature of the technology minimizes time and error generated through sample dilutions, and the need for assay repeats. However, with the advantages of any automated LBA systems like the Gyrolab there maybe challenges such as carryover issues.
There was discussion of the advantages and disadvantages of using an automated LBA platform such as the Gyrolab and strategies to overcome carryover. Approaches for minimizing carry-over in automated systems are based on an analyte's properties. A full understanding of dilution, wash solution, and number of washes is important. Processing parameters must be established and followed. Concentration range may be truncated as appropriate or removing tips as a potential solution. Overall, it was recommended that the use of, and processes for, automated systems should be addressed and documented in method development, including an evaluation of carryover if necessary.
Commercial, RUO & Diagnostic LBA Kits in Regulated Bioanalysis
The interest in using off the shelf commercial kits for generation of pharmacokinetic (PK) and pharmacodynamic (PD) bioanalysis data to support drug development programs has increased over the years. However, many commercial kits are typically suited for early drug discovery and lack required specification criteria and standardization in critical reagent characterization to support regulated nonclinical and clinical PK and PD studies.
There was discussion on how commercial kits can be adapted for bioanalysis by performing early feasibility assessment with study matrix in order to choose the appropriate kit for the intended use. As some challenges may be encountered, optimization would be required to either increase the sensitivity of the method or to use appropriate matrix QCs as kits are often supplied with materials for testing in buffer only. The use of buffer instead of study matrix can also result in selectivity issues that would require resolution. The choice of reference standard provided by the kit is also not always representative of study samples and may not include sufficient number of standards across the calibration range as per guidance and therefore an appropriate adjustment to the reference standard is required. A fit for purpose validation is also required as several kit components may have been modified due to study and regulatory requirements. Any changes to a kit procedure or reagents will require a revalidation. Importantly, regulators will be interested in validation steps taken at the sponsor/CRO level.
Overall, it was recommended that commercial kits are most appropriate prior to the availability of reagents (preclinical toxicity, exploratory, very early phase), reagents in commercial kits mainly used for capture and detection. Use of kits for PK assays is uncommon. If necessary to use a kit, kits should be carefully selected based on study needy, tested, and validated for intended use and follow the ICH M10 guideline. It was recommended to switch to custom non-commercial reagents, with bridging, as soon as reagents become available,
RECOMMENDATIONS
Below is a summary of the recommendations made during the 16th WRIB:
LBA Single Well Analysis (Singlicate) in Regulated Bioanalysis
Highlighted concerns about inherent assay variability and reagent quality but reservations about the method are the same as they would be for small molecules.
A gradual implementation was recommended, on a case-by-case basis, method specific, which can smooth the transition.
Internal comparison of singlicate vs. duplicate processing can be helpful but is not required.
It is expected that all ICH members will accept the approach when ICH M10 is implemented.
Consensus was unchanged that singlicate analysis for LBA is acceptable under ICH M10 if analytical performance criteria are satisfied and validation is performed with the singlicate analysis.
Change of the Critical Reagents: “KISS - Keep It Simple & Straightforward”
The process governing the critical reagent changes should be kept as simple and straightforward as possible and case by case based.
Assay performance is the best measure of reagent quality. If performance drops/assay fails, the cause should be investigated.
In method development, establish and document a solid baseline characterization of the identified critical reagents.
The characterization can include verification of purity, concentration of reagent by using orthogonal methods.
It is important to have robust internal SOPs for retesting/requalify if an assay fails due to changes.
If purchasing reagents, qualify the vendors, issue questionnaires regarding their internal procedures for production and release of reagents.
LBA Carryover Assessment in Regulated Bioanalysis
The use of, and processes for, automated systems should be addressed and documented in method development, including an evaluation of carryover.
Approaches for minimizing carry-over in automated systems (gyros) based on an analyte's properties.
Understand properties of the wash solutions and number of washes and dilutions.
May truncate concentration range, use new tips.
Commercial, RUO & Diagnostic LBA Kits in Regulated Bioanalysis
Commercial kits are not common for PK assays and are most appropriate prior to the availability of de novo reagents.
To qualify for intended use, kits should be carefully selected, tested, and validated as needed.
Recommend to switch to reagents, with bridging, as soon as reagents become available.
SECTION 4 - Input from Regulatory Agencies on Regulated Bioanalysis/BMV & Biomarkers/CDx/BAV
Abbas Bandukwala16, Chris Burns29, Seongeun (Julia) Cho16, Arindam Dasgupta16, Xiulian Du16, Shirley Hopper29, Akiko Ishii-Watabe27, Elham Kossary33, Yang Lu16, Kevin Maher16, Dulcyane Neiva Mendes Fernandes33, Mohsen Rajabi Abhari16, Yoshiro Saito27, Stephen Vinter29, Yow-Ming Wang16, Joshua Xu38, Li Yang16 & Jinhui Zhang16
Authors are presented in alphabetical order of their last name.
The affiliations can be found at the beginning of the article.
The highlight of each WRIB conference is the annual input provided by international regulators on regulated bioanalysis and BMV. This year, regulators from US FDA, EU EMA, UK MHRA, Health Canada and Brazil ANVISA, Japan MHLW and WHO presented topics of interest to the Global Bioanalytical Community.
ICH M10
Introduction, General principles, Ligand Binding Assay
Differences between ICH M10 Draft versus final guidelines and QA/FAQ were highlighted for each section of the guidance [Citation65]. In the introduction, wording changes were made such as changing bioanalytical assay to method. New text was added saying this guideline intends to facilitate development of drugs in accordance with the principles of 3Rs (Reduce, Refine, Replace) for animal studies, where valid. In the FAQ, an example was given of non-clinical PK studies conducted as surrogates for clinical studies is rescue agents for acute radiation syndromes or anthrax etc., under the Animal Rule (FDA, United States) [Citation65].
In the final guideline, it was added under general principles that if a problem is encountered with the method during the analysis of nonclinical or clinical study samples that requires that the analysis be stopped, and any changes to the method and the rationale should be documented. New text in the full validation section mentions the assessments that are performed during validation should be relevant to the sample analysis workflow.
For LBA assays new wording was added for the specificity that it is related to the concept of cross-reactivity. It is important that the binding reagent specifically binds to the target analyte but does not cross-react with coexisting structurally related molecules (e.g., endogenous compounds, isoforms, or structurally related concomitant medication). For non-accuracy and precision validation runs, low, medium, and high QCs may be analyzed in duplicate. These QCs, along with the calibration standards, will provide the basis for the acceptance or rejection of the run.
For acceptance criteria of sample analysis, new wording was added that calibration standards in a failed batch cannot be used to support the acceptance of other batches within the analytical run. A Q&A was included on the ICH webpage to specify that the precision and accuracy of an additional QC concentration level should be demonstrated before use in study sample analysis. This can be documented either as a partial validation or as a note to the bioanalytical report.
Chromatography, Incurred Sample Reanalysis (ISR), Partial & Cross Validation, Documentation
New wording was added in section 3.2.8 for stability for chromatography validation saying One bulk QC should be prepared at each concentration level. For each concentration tested, the bulk sample should be divided into a minimum of 3 aliquots that will be stored, stressed, and analyzed. In addition, for fixed dose combination products and specifically labelled drug regimens, the freeze-thaw, bench-top and long-term stability tests of an analyte in matrix should be conducted with the matrix spiked with all the dosed compounds. New wording for acceptance criteria of sample analysis says if multiple dilution factors are used in one analytical run, then dilution QCs need only be diluted by the highest and lowest dilution factors. For study samples in which multiple analytes are being analyzed, a valid result for one analyte should not be rejected if the other analyte fails the acceptance criteria.
Additional Considerations
Text added for endogenous molecules states the standard addition approach is only applicable for analytical platforms with linear responses. Typically, the standard addition method is used to determine the concentration of the endogenous analyte in the authentic matrix to be used for preparation of standards and QCs. However, this approach can be employed for determination of study samples as well. The QCs should resemble study samples and should be prepared in the same matrix. In certain cases, dilution of the QCs with surrogate matrix may be necessary (e.g., for Background Subtraction methods) where the endogenous level is high and the LLOQ needs to be reduced. In these cases, the recovery and matrix effect experiments should be repeated with authentic biological matrices with endogenous concentrations between LLOQ and low QC, if available. An explanation of parallelism for LBA was also revised in 2022.
Adoption by ANVISA
Adoption of ICH M10 guideline by ANVISA involved identification of the national legal basis related to the theme, choosing the type of internalization of ICH guideline, and determining appropriate regulatory instrument for this internalization. It was determined there is a need to change some normative acts including RE 895/2003 that will need to revoke some items as tables and attachments of Validation and Bioanalytical Report and totally revoke the RDC 27/2012 that describes minimum requirements for the validation of bioanalytical methods used in studies for the purposes of medicine marketing authorization and post approval changes. Next steps are that the internalization plan must be submitted to DICOL's decision (fully approve, approve with reservations, or request diligences from the organizational unit to carry out complementation). After approval of the internalization plan, the ANVISA ICH coordinator must inform ICH secretariat of the way and deadlines defined by DICOL for internalization. The final step is elaboration of regulatory instrument (legal analysis, DICOL's deliberation, and publishing the final regulatory instrument).
US FDA
Bioanalytical Considerations for Antibody-Drug Conjugates (ADC) & Recent Review Experience with Biosimilar Bioanalysis using LBA
The draft guidance from FDA (Clinical Pharmacology considerations for Antibody-drug conjugates, Feb 2022) was presented, including which analyte to measure at the different development stages and for different studies. In early development it was recommended to measure the total antibody as well as unconjugated small-molecule or payload which both constitute the ADC. The bioanalytical method validation should follow the FDA BMV 2018. It was acknowledged that sponsor may need to distinguish ADC bound to target versus ADC not bound to target if shed target is in circulation. The guidance includes recommendations for which analytes to be measured by study type.
FDA perspective was shared on the review experience in biosimilar bioanalysis using LBA in particular the implementation of a single bioanalytical method for measuring the biosimilar and reference products. The 2016 FDA guidance [Citation67] recommends using a single bioanalytical method for measuring concentrations of the proposed biosimilar product and the reference product and validated for use with both products. A single bioanalytical method is defined as the one methodology and procedure using the same critical reagents, standards, and quality control materials prepared with one selected product throughout validation and analysis of the study samples.
Assessing bioanalytical method comparability among products is important in accuracy and precision evaluation of pre-study method validation. Method comparability builds the foundation to select the single product and mitigates the risk that method difference affects the PK similarity assessment [Citation68–70].
Recent observations in biosimilar review found inadequacy in method comparability assessment, which included missing one or two calibration curves (no comparison of calibration curves, no comparison of QC bias across products).
Deficiency in Method Validation for Endogenous Analytes
Validation of bioanalytical methods for endogenous analytes are challenging due to the endogenous concentrations. Selection of an appropriate matrix is fundamental during method development which requires additional efforts from both scientific and regulatory perspectives. In many cases, a surrogate matrix which is free of the target analyte(s) is used for the preparation of calibration standards. Per the US FDA's Center for Drug Evaluation and Research (CDER) current thinking, validation results demonstrating the feasibility of using surrogate matrix in endogenous methods are considered critical.
One strategy on matrix selection is to set the lower limit of quantitation (LLOQ) at least three times higher than endogenous level then treat authentic matrix as ‘interference free’. Acceptability of this approach should be evaluated based on drug concentration levels pre- and post-dose (if baseline correlation is needed). Common approaches for endogenous drug method validation when authentic matrices without interference are not available include the standard addition approach, background subtraction approach, surrogate matrix approach, and surrogate analyte approach. The QCs should always be prepared in authentic matrix whereas the calibrator standards can be in alternative matrix. Criteria for surrogate matrix selection are ‘Cleanness’ (i.e., not interfering LLOQ) and ‘Sameness’ (i.e., comparing to authentic matrix).
Deficiencies in validation documentations, when using surrogate matrix-related and potential validation approaches were shared. One example is when depleted matrix is considered a ‘surrogate’ matrix instead of an ‘analyte-free’ ‘authentic’ matrix. There was lack of clear documentation for matrix depletion procedures and lack of ‘cleanness’ and ‘sameness’ validation data. Potentially acceptable approaches for depleted matrix include parallelism, recovery comparison and matrix effect comparison before and after depletion, or a serial dilution of authentic matrix samples (i.e., authentic quality controls) with deleted matrix.
Several deficiencies in method validation for endogenous analytes examples were presented for assessing the selectivity when using surrogate vs. authentic matrix. These include demonstrating that the surrogate matrix is free of analyte, or the authentic matrix selectivity was only checked for internal standard only during validation. A third example was that the endogenous levels observed in blank matrix/baseline bioequivalence (BE) samples were significantly higher than usually seen, and the last example was the lack of evidence demonstrating ‘peak purity’. It was also acknowledged that it is difficult to prepare QC Low in authentic matrix. A potentially acceptable approach, was suggested, using a ‘confirming’ ion transition channel which can improve chromatographic resolution.
Internal standard variation not evaluated per FDA guidance [Citation70] is another matrix related deficiency observed during method validation. Potentially acceptable approaches are to 1) identify IS response ‘range’ using multiple sources of authentic matrix during validation and optimize the method, 2) identify IS response ‘variability’ with special matrix conditions (i.e., hemolyzed, lipemic) during validation, or 3) dilute ‘IS variable’ subject samples with surrogate matrix during repeat assay and compare to the original results.
General Considerations in Pharmacokinetic Bioequivalence Studies of Endogenous Compounds in ANDA Submission
Conduct of bioequivalence studies submitted in Abbreviated New Drug Applications (ANDAs) where an endogenous compound must be measured is challenging. Multiple factors such as timing of sample collection, acceptability of the analytical method used to measure the endogenous substance, appropriateness of criteria used for subject inclusion/exclusion in the bioequivalence statistical analysis, etc., could complicate the measurement. The FDA Guidance for Industry: Bioequivalence Studies with Pharmacokinetic Endpoints for Drugs Submitted Under an Abbreviated New Drug Application (August 2021) and Product Specific Guidances (PSGs) for certain drug products provide recommendations on studies where endogenous compounds are to be measured.
Study design considerations include the use of subjects from the general population (acceptable for most endogenous compounds) and general population with suppression of endogenous level (Hydrocortisone Acetate Rectal Aerosol Metered) where a 4 mg dose of dexamethasone should be administered 10 hours prior to drug administration as a pre-treatment to lower endogenous hydrocortisone levels. Special populations to consider are calcifediol extended-release capsules with baseline calcifediol concentration lower than 30 ng/mL to determine the effect of the test and reference products after correction for baseline calcifediol concentration. Another is for estradiol products non-smoking, postmenopausal women with no contraindication to estrogen therapy, and testosterone products in hypogonadal males with no other abnormalities.
In BE studies with PK endpoints, baseline-correction (BLC) is generally recommended for the drug product containing an active ingredient that is an analogue endogenous compound. Inclusion of endogenous levels in the concentration data can make the BE study less sensitive to formulation differences. More than 70 Product Specific Guidances (PSGs) recommends BLC. A majority of the above-mentioned PSGs recommend time averaged BLC. Baseline sampling durations are variable. Short duration is ≤1 hour prior to dosing (levothyroxine, Liothyronine, progesterone, epinephrine, leucovorin calcium, etc.). Intermediate duration is up to 10 hours or 18 hours prior to dosing (isotretinoin, calcitriol). Long duration is ≥24 hours prior to dosing (ergocalciferol, estrogens, phytonadione, ursodiol, etc.). And unspecified duration is (Testosterone Buccal Extended-Release Tablet, etc.). General recommendations related to BLC are that baseline concentrations should be measured for each subject and dosing period for multiple times. BLC should be subject and period specific. Any negative concentration for values should be set to “0” before calculation of PK parameters. PK and statistical analyses should be performed on both uncorrected and corrected data. Determination of BE is generally based on the BLC data. The timing of baseline sample collection should be selected with the consideration of circadian rhythms of the analytes.
Finally, baseline-correction is not needed for some endogenous drugs. Other parameters are more reliable to detect formulation difference than BLC conc. For example, for Iron Dextran Injectable, it is recommended to measure 1) total iron (TI) in serum and 2) transferrin-bound iron (TBI) in serum. BLC-TBI and BLC-TI are not sensitive to formulation changes due to possible transferrin saturation and BLC-TBI appears to be highly variable. Current PSG recommends BE based on maximum value of the difference in concentration between TI and TBI over all time points measured and difference in AUC between TI and TBI.
Data analysis considerations include the use of the 5% Cmax Rule in ANDAs, per FDA Draft Guidance for Industry: Bioequivalence Studies with Pharmacokinetic Endpoints for Drugs Submitted Under an ANDA. The main reason for this recommendation is to mitigate carryover effect. This reflects in the PSG recommendations for some endogenous drug products, such as Epinephrine metered aerosol inhalation, Testosterone gel, etc.
Reflections on FDA Remote Evaluation Activities over the Past 2 Years
In response to the COVID-19 public health emergency, FDA developed and implemented alternative tools to on-site inspections and continued to provide regulatory oversight despite the challenges due to travel restrictions. In Jun 2020, the Office of Study Integrity and Surveillance (OSIS) in CDER initiated Remote Regulatory Assessments (RRAs), which involve voluntary participation of study sites. Throughout the pandemic, RRAs allowed OSIS to continue its operation reviewing bioavailability, bioequivalence, and GLP studies supporting NDAs, ANDAs, BLAs, and INDs and sites' study conduct.
The process of an RRA includes FDA providing a list of requested documents to a site once receiving site's agreement on participation, site's submission of the information to the FDA-secured cloud repository, a virtual tour of a facility, and a series of meetings with site staff to conduct interviews, gather needed information, and evaluate site's overall operation and study conduct. An RRA is voluntary participation of a site and is not an inspection (not issuing Form FDA 482 (notice of inspection, domestic) and Form FDA 483 (inspectional observations)). Concerns identified during RRAs are communicated with site management at the daily wrap-up and the closeout meeting and sites have an opportunity to provide a written response to observations. RRAs have been a critical tool to allow FDA/OSIS to continue the office's mission of protecting study subjects and ensuring the quality, integrity, and regulatory compliance of bioavailability/bioequivalence, nonclinical, and animal rule studies throughout the pandemic.
The agency gained insight into logistical and technological challenges during this process. This included learnings on communication platforms, face-to-face vs. camaras on, quality of internet connection, communications across different time zones, review of PDF/scanned documents vs. electronic raw data, number of documents or files submitted by a site, and file structure/organization/naming of uploaded documents.
Despite the challenges, the Agency's experience with RRAs over the past 2 years and collective lessons gained from those evaluations suggest that RRA is a valuable tool that will have utility beyond the current pandemic and continue to be used as appropriate to enhance and complement FDA's inspection programs.
Regulatory Findings from Recent Inspections
The US FDA experienced unprecedented and unique challenges during the last two years due to the travel restrictions resulting from the Covid-19 pandemic. Consequently, FDA had to limit foreign and domestic travel for the purpose of performing on-site inspections, including those for Office of Study Integrity and Surveillance's (OSIS’) Bioresearch Monitoring Program (BIMO) programs. To maintain the continuity of operations and support the CDER review divisions, OSIS developed and implemented a Remote Regulatory Assessments (RRA) program to allow OSIS to evaluate BA/BE and GLP studies submitted to FDA in support of INDs, NDAs, BLAs and ANDAs. RRAs are voluntary and conducted remotely with significant interaction with the site and are not intended to replace or represent a US FDA onsite inspection. Using a combination of audio-visual tools and secure cloud-based system for document sharing, RRAs were used to audit source records and documentation, communicate with site personnel, visualize electronic systems, and virtually tour site's facility. Any findings were discussed during the virtual interactions at daily wrap-ups and at the conclusion of the remote regulatory assessments.
Recent findings include issues with reserve samples. 21 CFR 320.22 discusses the “Criteria for waiver of evidence of in vivo bioavailability or bioequivalence”. In vitro tests (e.g., dissolution, IVRT, IVPT, GSD, PSD, etc.) may be conducted for testing that are subject to different regulations (21 CFR 211-cGMP or 21 CRF 320-BE regulations). Testing sites should be aware if the testing was conducted for a BE study. Regulations for reserve samples apply to in vitro BE studies (21 CFR part 320.38 and 320.63). In this observation, reserve samples were not retained from all shipments provided for testing in an in vitro study. Specifically, 20 investigational product (IP) kits were received on March 1, 2020, from the IP supplier as the third shipment. However, all 20 IP kits were used for testing, and none were retained as reserve sample. Upon evaluation, the firm did not retain reserve samples of investigational product (IP) kits provided in the 3rd shipment. Consequently, OSIS could not verify the authenticity of drug products used in testing from the 3rd shipment. Because the firm did not retain reserve samples for reference and test product from the 3rd shipment, authenticity of the drug products used in part of the study that used drug products from the third shipment could not be verified. The firm agreed with the finding and indicated that the sponsor instructed them to not to retain reserve samples from the 3rd shipment. As a corrective action to prevent recurrence in future studies, the site plans to retain reserve samples from all shipments as required by 21 CFR 320.38 irrespective of the sponsor's instructions.
A second finding related to non-physiologic pharmacokinetic data submitted to FDA for a PK endpoint BE study comparing two oral formulations. In an open label, crossover study with 24 subjects enrolled at one site, BE data submitted to FDA claimed bioequivalence was met between the test and reference formulations. Two drug products are said to be bioequivalent if the 90% confidence interval of the ratio of geometric means of the primary pharmacokinetic (PK) responses (after log-transformation) is within the bioequivalence limits of 80% and 125%. Subjects' PK study data appeared to separate into two distinct populations, with a change occurring after the midpoint of the study, which would not be expected based on normal subject physiological variability across a subject population. Bioequivalence assessment for Subjects 1–12 and Subjects 13–24 failed when compared to bioequivalence assessment of all study subjects. Presence of two distinct populations around the midpoint of the study, which would not be expected based on normal subject physiologic variability across a subject population. The inspected site could not provide a plausible explanation to resolve FDA's concerns regarding the validity of data.
There were other observations that may have an impact on reliability of pharmacokinetic data. The PK samples were centrifuged 1 day late. Whole blood stability data was established for 4 hours during method validation. PK samples had to be excluded from BA/BE analysis. In the next observation, sample processing worksheet was missing for number of PK timepoints. Sample processing times could not be verified, and PK concentration data was not reliable. Another observation occurred where the documented centrifuge time was earlier than the sample collection time in the source documents. Sample processing activities could not be verified and again PK concentration data was not reliable. In a final observation, the documented centrifuge time was earlier than the sample collection time in the source documents and again PK concentration data was not reliable.
The case studies presented showcase the importance of reviewing the source documentation to ensure data integrity, quality, protocol and regulatory compliance of the data before submission to any marketing applications. Unanticipated deviations from study plan may need additional validation experiments to be conducted post-study to evaluate the impact on data reliability. Industry needs to be aware of the intent of the study and applicable regulation. Industry needs to be vigilant and generate high quality data to ensure their products are safe, effective and of high quality to protect the public health.
Biomarkers for Biosimilars: US FDA perspective
The enactment of the Biologics Price Competition and Innovation Act of 2009 added the 351(k) Biologics License Application (BLA) pathway to the Public Health Service Act [Citation42 USC 262: Regulation of biological products, section (k)], which established an abbreviated approval pathway for the licensure of biosimilar and interchangeable biological products. The biosimilar BLA submissions are required to demonstrate biosimilarity by comparing the proposed biosimilar product to a reference product that is approved by the FDA, and the biosimilar development programs begin with extensive analytical similarity assessments. Clinical pharmacology data to support a demonstration of biosimilarity generally include pharmacokinetic (PK) data to show PK similarity and pharmacodynamic (PD) biomarker data to show PD similarity. Many approved biosimilar products have relied on data from comparative clinical studies with efficacy endpoints conducted in patients which can be costly and time consuming.
The FDA is conducting research to inform the Agency's thinking on critical aspects of the use of PD biomarkers to demonstrate biosimilarity, which can either streamline or eliminate the need for comparative clinical studies with efficacy endpoints. The goal is to leverage literature knowledge to find potential PD biomarkers for biosimilar programs. So far, approved biosimilars have used PD biomarkers that are tied to clinical efficacy. PD biomarkers for biosimilar development are not required to reflect clinical efficacy. It presents an opportunity to explore PD biomarkers previously showed a dose-response relationship. A good understanding of MoA may reveal opportunities for multiple PD biomarkers. The bioanalytical community expert knowledge in biomarker development and implementation in clinical trials will undoubtedly play a major role in increasing the reliance of PD biomarker data to support biosimilar approvals.
CDRH CLIA Categorization Processes
The Clinical Laboratory Improvement Amendments (CLIA) of 1988 were promulgated to ensure a high standard of accuracy and reliability of clinical test results from laboratories, regardless of location. The Act covers a range of regulations to certify laboratories and categorize in vitro diagnostic tests (IVDs). IVDs are CLIA categorized by level of complexity to ensure that testing is performed in laboratories that have the appropriate capabilities.
With regard to clinical laboratory testing, the Centers for Medicare and Medicaid Services (CMS) and FDA have complementary functions. CMS regulates clinical laboratories and certifies those laboratories to perform tests. FDA regulates in vitro diagnostic devices and performs CLIA test categorization. Under CLIA, tests are categorized into one of three complexity levels. Depending on how difficult they are to perform, IVDs are categorized as: Waived, Moderate Complexity, or High Complexity. The more complex the test, the more stringent the personnel and facility requirements are for the laboratory under CLIA. FDA/CDRH reviews two different types of CLIA categorization requests: CLIA Waiver by Application and CLIA Record. The CLIA Waiver by Application (CW), was codified under 42 U.S.C. § 263a(d)(3)(A) and includes the review of additional clinical and non-clinical studies and labeling.
The CLIA Record (CR), (defined in 42 CFR 493.17), involves the review of IVD labeling, following clearance/approval of the device by FDA (or as a standalone CR for certain IVDs that are exempt from premarket review), for the purpose of assigning the device category as waived moderate or high complexity. FDA categorizes IVDs, under CLIA, as test systems (42 CFR 493.2). Each instrument + analyte combination is categorized separately. Uncategorized test systems (e.g. those not on the lists of tests in the FDA CLIA database) and test systems used off-label are considered high complexity by default (42 CFR 493.17(c)(4)).
Some devices or components are not categorized. This includes devices that are used to test samples not taken from the human body, as well as separately cleared or approved IVDs that are not complete test systems, such as calibration materials, QC materials and sample collection kits. FDA/CDRH conducts CLIA categorizations under CRs following a cleared, approved or licensed premarket submission. CDRH also conducts CLIA categorization upon request by the manufacturer or premarket submission holder for legally marketed tests, including IVDs with name and/or distributor changes, IVDs exempt from premarket review, and new test systems (i.e. instrument & assay combinations) covered by the Replacement Reagent and Instrument Family Policy.
Biomarker Qualification & Analytical Guidance
The FDA's Biomarker Qualification Program provides a framework for the development and regulatory acceptance of biomarkers for use in drug development programs. Qualified biomarkers are publicly available and can be used in any drug development program for their qualified context of use. Qualification of a biomarker is based on evidence, both analytical and clinical, supporting its context of use. Accurate and reliable measurement of a biomarker (analytical validation) is critical to its use and qualification as a drug development tool. The goal of analytical validation is to establish that the performance characteristics of the measurement test or tool are sufficient to support the biomarker's COU.
An Analytical Considerations for Biomarkers guidance document is being drafted and will be or should be available for comments from the public. It will provide guidance for biomarker qualification, context of use (COU), and technologies, but does not replace requirements for FDA submissions, or other guidance documents. Selection of a method will be discussed including
pre-analytical Variables (Sample collection, handling, processing, storage, SOP), reference standards, measurement method description, performance characteristics, statistical considerations, and acceptable performance. Key analytical performance characteristics include accuracy/relative accuracy, measurement range, precision, repeatability, reproducibility, analytical specificity, and limits of detection/quantitation.
The FDA solicits public input on guidances prior to implementation. It publicizes by issuing a Notice of Availability (NOA) of the draft guidance in the Federal Register. The public has 60 days to provide comments to FDA on draft guidances. FDA reviews and considers the comments it has received, as it prepares the final guidance.
Next-Generation Sequencing (NGS) Panels for Precision Oncology Biomarkers
Precision oncology requires application of appropriate biomarker tests in order to individually customize treatment, improve outcomes, and at the same time advance our understanding of the role of genetics in cancer. An ineffective biomarker test is problematic as it could lead to the selection of an ineffective or harmful drug, so getting the biomarker test right is critical. Unmet needs to advance precision medicine include the development of reference samples, best practices, protocols, and quality metrics for NGS-based diagnostic assays.
The Sequencing Quality Control Phase 2 (SEQC2) consortium was formed to address these needs. SEQC2 is an international group composed of members from academia, government, and industry, and led by the U.S. Food and Drug Administration (FDA). In brief, SEQC2 [Citation72] developed a translational scientific infrastructure and applied it to common precision oncology situations. This includes the design and construction of a comprehensive and robust set of nucleic acid (DNA, RNA) reference materials [Citation73] which were then used to conduct a series of studies to assess the analytical performance of oncology panels: i) eight comprehensive solid tumor oncopanels, ii) five liquid biopsy assays, iii) the impacts of formalin fixed paraffin embedded (FFPE) sample processing and, iv) the utilities of spike-in standards [Citation74] for the detection of actionable mutations.
The multi-lab cross-oncopanel study reveals high sensitivity and reproducibility tailored to targeted regions and allele frequency ranges [Citation75]. Sensitivity was found to be high for variants previously verified to have variant allele frequency (VAF) greater than 5%. Four panels can reach VAF of 1%. The false positive rate (FPR) is small (less than 5 per million genome bases with VAF threshold at 2.5%) in certain high confidence coding regions but it can be noticeably and significantly larger (multiple folds) outside those regions, which leads to lower reproducibility. In applications where a minimal FPR is required (e.g., tumor mutational burden estimation), raising the VAF threshold was effective at reducing the FPR. The sensitivity for detecting indels is typically more variable and poorer than single nucleotide mutations, and this difference becomes more pronounced at low VAFs. Reference materials can be used to establish an optimal VAF threshold that reduces FPR and retains sensitivity. Utilizing a sample spiked-in at a specified amount can provide additional variants at known allele frequencies for analytical validation of oncopanels.
The liquid biopsy study [Citation76] involved evaluating the analytical validity of circulating tumor DNA sequencing assays for precision oncology. Mutations present above 0.5% VAF were detected with high sensitivity and reproducibility by all participating ctDNA assays, but performances were generally suboptimal below this VAF level and variable between assays. Fragment depth was a critical variable in ctDNA assays, with high coverage essential for sensitive detection of low-frequency mutations. In addition to depth, even coverage across target regions was important to ensure high sensitivity and reproducibility. Increasing DNA input quantity generally improved fragment depth, sensitivity, and reproducibility. UMIs enabled effective error correction, minimizing the detection of false positives. Low-frequency mutations (VAF <0.5%), mutations in exon edge regions, mutations in challenging genome sequence contexts, were detected with lower sensitivity.
Finally, in the FFPE sample processing effect study it was shown that multiple factors (from DNA extraction, library prep, to sequencing) can lead to a high false positive rate in FFPE samples [Citation77]. Quality check is crucial to exclude/identify the sample with high false positive calls that were not due to FFPE processing. Surface FFPE samples showed significantly more FFPE damage and artifacts due to hydrolytic deamination. The standardized FFPE procedure together with rigorous quality control can achieve an FPR close to fresh DNA samples. There was no impact of formalin fixation time on the FPRs of FFPE samples under 24 hours.
UK MHRA
Bioanalytical Observations, Findings & Data Integrity Issues
UK MHRA inspection findings raised during the inspection of bioanalytical laboratories were shared. The first category of findings was audit trails. In 2017, there was an example of entire instrument audit trail missing. In 2022, no missing audit trails were found but some issues identified included no suitable access to audit trail for quality personnel, no review of audit trial itself, or insufficient detail contained in audit trail.
In regard to internal standard response, in 2018 an example was found where not all assessment was always transparent, especially with respect to trends. Currently, there is wide adoption of review and typically done via a numerical assessment. Some laboratories have encountered some issues including numerical acceptance criteria errors and failure to document assessment and decision making.
For report transparency, previous deficiencies include knowingly not following guidance and not reporting, and details in report not matching what actually occurred. Other examples include repeated failures of selectivity experiments and subsequent investigations not discussed, selective reporting of results – the “best” results from analytical runs taken for the validation report, and no supporting information for ISR failure.
Data integrity issues (calibration and Quality Control (QC) standard injections) had been removed from the results summary tables and replaced with re-injected standards without explanation to justify these actions. In addition, consideration was not given to the security of data once processed within the software (no audits to check security of data, data stored in unsecure locations).
Inspections' findings include equipment in use that was not validated. Stability issues were found of several examples from inspections where stability samples have not been evaluated immediately after preparation. EMA and ICH M10 guidelines stipulate that stability samples are to be analysed immediately after preparation and after the applied storage conditions that are to be evaluated.
Other deficiencies were lack of temperature monitoring of incubators used for LBA, lack of an assessment of data integrity of new processes and software, and failure to ensure GLP requirements for test item characterisation are considered.
International Reference Standard Materials (RSM) for Biotherapeutics & Advanced Therapies
The expression of potency in units of bioactivity relative to an independent standard has, and continues to be, an essential regulatory tool to harmonize patient dosing for biological medicines. International Reference Standards have fulfilled this role for over a century and continue to be essential for blood products, vaccines, and newer medicines like monoclonal antibodies. This is particularly important for those monoclonal antibodies which are subject to intense biosimilar activity since this potentially leads to the coexistence of many approved versions. Although the biosimilar route to licensure increases patient accessibility to these important medicines, it also introduces a new regulatory challenge to ensure that there is consistency in the biological activities of these products over time and between products. International RSMs play an important role in the assessment of potential drift and divergence of different biological activities over time, which is important for the setting of specifications and extrapolation of indications for these biosimilars. In addition to the classical application of relative potency approaches to biological standardization, more recently there has been a requirement to develop novel classes of reference materials that help standardize methods or assays.
Nowhere is this more evident than in the field of Advanced Therapies, where cell and gene therapy approaches offer the promise of life-changing treatments for a variety of diseases. For instance, in vivo gene therapy approaches using Adeno-associated Virus (AAV) vectors are one of the fastest growing sectors of the gene therapy sector but face significant analytical and manufacturing challenges. A lack of standardization makes it difficult to compare data between different products and highlights a need for reference materials to assess a range of attributes such as empty/full particle ratio. As a result, recent developments in both gene and cell therapy standardization will also be presented.
The best example is for international standards for monoclonal antibodies to support new regulatory challenges (multiple complex bioactivities, multiple changes to manufacturing processes, different regulatory jurisdictions). The potential for important drifts and or shifts in bioactivities between products is significant. The regulatory framework cannot identify potential, cumulative, drifts in bioactivity (for innovator and authorized biosimilars), across jurisdictions and over time. International standards support the performance, calibration and monitoring of bioassays and in-house reference materials such as the 1st WHO IS for Rituximab [Citation78].
An upcoming need is international standards for new paradigms such as cell therapy with mesenchymal stromal cells and pluripotent stem cells, and reference reagents for lentiviral based therapies. The goal is to assess presence/ absence of specific populations of cells and variability between donors/ batches/ sources.
Another example of reacting at pace were WHO international standard for SARS-CoV-2 antibodies for serological responses, vaccine comparison, and immunological surveillance. A reference standard for mRNA vaccines include a stable lyophilized preparation with highly characterized attributes for lipid analysis, potency assays, and polyA tail assessments.
Japan MHLW
Recent Developments of Biomarker Assay Validation (BAV) in Japan for qPCR Assays
Quantitative PCR (qPCR) is a commonly used method for bioanalysis of DNA/RNAs as drugs (especially for gene therapy products) and biomarkers. In Japan, there is no published guideline on the bioanalysis by qPCR assay. For evaluation of nonclinical safety on gene therapy products, a guideline named “ensuring the quality and safety of gene therapy products” were released from MHLW in July 2019.
In this guideline, data on in vivo distribution, persistence and clearance of the products are requested in principle. In the analysis of biodistribution, tissue and blood should be collected at regular intervals after administration of the product, and the copy number of the vector should be measured using qPCR or other appropriate methods. Furthermore, by measuring changes in the copy number of the vector over time, information on its clearance can be obtained. In addition, “guideline for nonclinical studies of vaccines for the prevention of infectious diseases” is now revising in Japan. In principle, biodistribution studies should be conducted for nucleotide-based vaccines. DNA/RNA is also an attractive candidate for drug efficacy/safety biomarker.
The AMED Study Group previously published the points to consider document on analytical methods validation for protein and metabolite biomarkers analyzed by chromatography-based methods and ligand-binding assays [Citation79]. Now, the group has been developing the qPCR version. First, the group found an example of the RNA/DNA biomarker qualified by US FDA, and its validation parameters were investigated. Next, referable guidelines/white papers on qPCRs were also investigated for the validation parameters and their background thoughts.
There is now discussion among the group on each validation parameter of the biomarker qPCR assay, including standards, sensitivity, limit of detection, limit of quantification, specificity, calibration curve, linearity, accuracy, precision, stability, and recovery. There is discussion on these parameters for assay development and validation. The group is now drafting the points to consider document, which is scheduled to be finalized by the end of March 2023.
WHO
Inspection & Review of CROs' computerized systems validation
Computer system validation (CSV) is the documented process of assuring that a computerized system does exactly what it is designed to do in a consistent and reproducible manner. Documentation requested include an inventory list with validation master plan. The inventory must present a summary of information on each system, describing the name of the system; associated equipment or application; impact/criticality; category; ownership (sector, system owner, process owner); current version; provider; validation date and status. This inventory can be used for planning periodic reviews. This list will help the inspector identify the company's computerized systems.
During inspection, the process includes review of SOPs, review of qualification documentation, review of training documentation of relevant staff, and physical inspection of computerized systems. A CRO should have a validation master plan that reflects the key elements of validation. It should contain the validation policy, roles and responsibilities, selection & qualification of outsourced services, scope of qualification & validation, activities subject to validation, computerized systems, cleaning validation, revalidation, risk assessment, change control, and deviation management.
SOP for computerized system validation should outline and detail the validation of life cycle. It should include requirements for key areas such as intended use, validation master plan, user and functional requirements specification, system design specification, traceability matrix, installation qualification, operational qualification, performance qualification, validation summary report, system release memo, and revalidation provisions in case of change/modifications.
The SOP for security should outline the company's password policy, backup, access rights and role designation. The SOP for use and management of the software should include user instruction and training of relevant staff. Requirement specifications should be written to document user requirements, functional or operational requirements and performance requirements. Requirements may be documented in separate user requirements specification (URS) and functional requirements specifications (FRS) documents or a combined document.
Performance qualification is an essential component of the qualification process and comprises verifying and documenting the performance of the equipment. PQ should assess whether the equipment meets it operational requirements in a reproducible and consistent manner to ensure that the system can perform it intended function within the specified limits of its design and development. Prior to conducting of the PQ and UAT, and prior to release of the computerized system, there should be adequate written procedures and documents and training programs created defining system use and control. These may include supplier-provided user manuals as well as SOPs and training programs developed inhouse. Other issues to be verified include administrator's access to editing/deletion which should be justified and avoided if possible. There should be appropriate segregation of roles between personnel responsible for the business process and personnel for system administration and maintenance. A filtrated audit trail should be considered through a Risk Assessment. Filtrated audit trail increases the probability of detection in a risk assessment process. It is not acceptable to operate a computerized system without an audit trail, disabling the audit trail and backup. For more simple computerized systems, the audit trail function may be replaced by other options such as logbooks, third parties' solutions, etc.
SECTION 5 - Input from Regulatory Agencies on Immunogenicity, Gene & Cell Therapy & Vaccines
Eric Brodsky16✓, Isabelle Cludts29, Shirley Hopper29, Chad Irwin39, Akiko Ishii-Watabe26, Julie Joseph39, Susan Kirshner16, Mohanraj Manangeeswaran16, Kimberly Maxfield16, Joao Pedras-Vasconcelos16, Mohsen Rajabi Abhari16, Therese Solstad40, Seth Thacker16, Omar Tounekti39, Daniela Verthelyi16, Meenu Wadhwa29, Leslie Wagner16, Joshua Xu38, Takenori Yamamoto26, Lucia Zhang39 & Lin Zhou16
Authors are presented in alphabetical order of their last name.
The affiliations can be found at the beginning of the article.
✓Coauthor for the US FDA: Immunogenicity Information in Prescription Drug Labeling Section
The highlight of each WRIB conference is the annual input provided by regulators on immunogenicity, biomarkers, gene and cell therapy and vaccines. This year, regulators from US FDA, EU EMA, UK MHRA, Norway NoMA, Health Canada, Japan MHLW presented topics of interest to the Global Bioanalytical Community.
Immunogenicity
US FDA
Immunogenicity Information in Prescription Drug Labeling
FDA provided an overview of the recommendations in the FDA draft guidance for industry, Immunogenicity Information in Human Prescription Therapeutic Protein and Select Drug Product Labeling — Content and Format (February 2022).Footnote1 The goal of the guidance is to assist applicants with incorporating immunogenicity information into labeling of therapeutic proteins and select drug products that have immunogenicity assessments.
Presenting immunogenicity information in a consistent manner will enable health care practitioners to more easily identify and differentiate between products associated with clinically significant immunogenicity and products whose ADA are not associated with clinically significant effects on PK, PD, safety, or effectiveness.
In a review of 71 therapeutic proteins and drug products approved by CDER during a recent five-year period (2014–2018) with immunogenicity information in labeling, 98% of labeling included immunogenicity information in the ADVERSE REACTIONS section and 30% of labeling did not include any statements regarding the immunogenicity impact on safety or effectiveness [Citation80]. The FDA recommends a dedicated Immunogenicity subsection which allows for a consistent location for summarizing immunogenicity data and its PK and PD effects while reserving other sections for description of clinically significant effects of immunogenicity, as appropriate.
A new Logical Observation Identifiers Names and Codes (LOINC) was created for the Immunogenicity subsection to improve the ability of Structured Product Labeling (SPL) users to accurately search or extract immunogenicity data from the labeling.Footnote2
When new immunogenicity data/information could affect prescribing decisions or the clinical management, applicants should submit to FDA proposed revised labeling containing the updated immunogenicity information. When this guidance is final, FDA recommends that applicants propose labeling updates to be consistent with the format and organizational recommendations in this guidance (e.g., during the next planned prior approval supplement). Applicants can voluntarily update their labeling to be consistent with the recommendations in this draft guidance.
Assay Signal-to-Noise Ratio (S/N) as A Potential Alternative to Titer for An ADA Response
In the commonly used tiered immunogenicity testing strategy, all immunogenicity samples are first tested in a screening ADA assay if the S/N ratio is above the screening assay cut point, the sample is further tested in a confirmatory assay, which involves depletion of signal with unlabeled drug. If the S/N ratio is above the confirmatory assay cut point, the sample is considered ADA+ and is then tested in a neutralizing antibody assay and the magnitude of the ADA response is determined as titer using a serial dilution approach. The last dilution of a serum sample that tested positive in the ADA assay determines the titer. Current recommendations are that the titer calculations should take the minimum required dilution into account. Although titer is semi-quantitative, it is the parameter currently relied on for immunogenicity assessment, including determining immunogenicity incidence, evaluating the impact of ADA on PK/PD, efficacy, or safety.
There are limitations of replacing titer with S/N ratio from a clinical pharmacology perspective. First issue with using S/N ratio is how to determine if a subject is treatment-boosted ADA+ or not. Both treatment-induced ADA positive and treatment-boosted ADA positive subjects are considered ADA positive. In the commonly used method, treatment-boosted ADA positive is defined as “pre-existing ADA that were boosted to a higher level following biologic drug administration, i.e., any time after the initial drug administration the ADA titer is greater than the baseline titer by a scientifically reasonable margin such as four-fold or nine-fold…” [Citation81]. In addition, for interpolated titer, there is a more statistically stringent approach for titer assessment involves calculating the Minimum Significant Ratio to evaluate the relevance of titer changes between two time points as described in USP [Citation82]. Both methods rely on titer to determine whether a subject is treatment-boosted or not.
Second issue is a lack of clarity that S/N ratio data can accurately reflect change in ADA magnitude. Whether the amount of ADA is increasing, sustained, or decreasing over time in individual subjects is an important review consideration for evaluating the impact of ADA on efficacy or safety, especially for therapeutic proteins which have an endogenous counterpart. There are two types of saturation that can happen with S/N ratio. First, S/N ratio saturation due to limit of detection of the instrument. Second, depending on the assay characteristics, the same S/N values may represent widely separated titers, even when the sample signal falls below the saturation limit of the instrument.
In addition, other practical assay considerations can impact the usefulness of the S/N data such as drug tolerance, assay format, matrix effects, target tolerance, and intervals between sample testing (concurrent sample testing versus sequential sample testing). For labelling considerations, how changes in S/N can be reported in the labeling in a meaningful way to the health care practitioner (as recommended in the FDA 2022 draft Immunogenicity labelling guidance) is still unclear at this time.
In order to demonstrate that the S/N ratio is as accurate as titer for evaluating the clinical risk of immunogenicity it is important to clearly define criteria for a treatment-boosted positive sample using S/N ratio with reasonable scientific justification. It is also necessary to demonstrate that using either S/N ratio or titer leads to a similar conclusion aboutthe impact of immunogenicity on PK. A common approach is to conduct correlation analysis between PK vs. S/N, PK vs. titer, and S/N vs. titer. The data included in this type of analysis should be sufficiently sensitive to detect any impact of immunogenicity on PK. Another consideration is to apply this principle to similar analysis using PD data (e.g., PD vs. S/N, PD vs. titer, and S/N vs. titer). It would not be appropriate to use drug's target as a PD response biomarker.
Rigorous evaluations of the S/N approach versus the current titer approach with more biological products are needed to provide evidence-based support to establish a best practice for the use of the S/N approach in the future. Until the best practice is established, the use of titer data to assess clinical risk of immunogenicity is preferred. If sponsors opt to use S/N ratio in lieu of a formal titer assessment, we recommend that they discuss their choice with regulators during the development program. Part of that discussion may involve providing ADA assay development data comparing S/N versus titer using a suitable anti-drug antibody positive control. If limitations of replacing titer with S/N ratio can be reasonably addressed, S/N ratio may be acceptable on a case-by-case basis. Sponsors are further recommended to provide a suitable justification for the choice of S/N in the appropriate sections of the eCTD, such as eCTD 5.3.1.4 Reports of Bioanalytical and Analytical Methods for Human Studies, 2.7.1 Summary of Bioanalytical Methods, and 5.3.5.3 Integrated Summary of Immunogenicity of their marketing application to the FDA. Factors to consider in implementing the S/N approach would include, but not limited to, the immunogenicity risk of the product, the stage of development, and assay performance characteristics. Additional questions remain as to how changes in S/N ratio can be communicated effectively to the health care practitioner in a meaningful way as to potentially inform treatment decisions.
Preclinical tools for assessing the risk of innate immune response modulating impurities applied to biosimilars
Multiple product and process related impurities capable of activating innate immune receptors can modify the immunogenicity risk of peptides and proteins, thus assays that assess innate immune activation by drug products can contribute to their immunogenicity risk assessment. Currently immunogenicity cannot be predicted from product structure and formulation, therefore clinical studies are needed to assess product immunogenicity and its clinical consequences.
Pre-clinical bioanalytical tools to assess product and process related impurities can be used to inform an Immunogenicity risk assessment. This includes in silico and in vitro methods that examine innate immune activation by IIRMI, assess binding of product sequence variants or other product related impurities to MHC, and quantify subsequent T cell activation. For T cell epitopes, orthogonal approaches such as in silico and in vitro MHC binding assays are usually recommended. In silico methods have advantages of high throughput and covers multiple MHC including rare types. Importantly these assessments only consider primary sequence and have weak or no ability to assess the risk posed by unnatural amino acids or post-translational modifications such as oxidation or deamidation. Also, although most programs are based on an freely accessible epitope databases, the computational predictive algorithm superimposed onto this data and their validation is proprietary, making it difficult for regulatory agencies to evaluate some of the assumptions of the individual assessments. Application of the results of the in-silico assessment to donor selection in the in vitro studies (DC:T cell assays and MHC binding) can be useful.
For in vitro assays it is critical to provide clear experimental design and culture conditions. Justify method (size, MHC, target population), culture conditions and concentration of the product used. Confirm APC activation and presentation (MAPPS assay, DC activation markers) and justify readout selection (proliferation, cytokines, cell markers). It is also necessary to have suitability controls that confirm sensitivity for naïve T cell responses. Some commonly observed problems in DC:T cells assays relate to the number of screened T cells which may be too low to detect responder naïve T cells (∼1–10/1,000,000 ag-specific naïve T cells). Peptide loading can be inadequate to elicit response (<0.1 uM). Inadequate suitability controls are also an issue: LPS, PHA, or KLH can be used to ensure the presence of live APC and responsive cells in the culture but are not recommended as suitability controls for naïve T cell responses to specific antigens.
Innate immune response modulating Impurities (IIRMI) assays are designed to detect biological differences in impurities capable of inducing inflammation and/or acting like adjuvants. Changes in innate immune-related markers may indicate increased risk of eliciting immune responses. For many biologics a certain level of innate immune modulation is likely, so assays may need to distinguish impurity-driven differences in innate immune responses. Common deficiencies include inadequate demonstration that the assay is fit for purpose due to insufficient sensitivity or breadth or inappropriate suitability controls (negative, low (confirming LOD) and high positive controls). Another problem has been insufficient description of the critical assay parameters such as number of donors, donor selection criteria, cell numbers, duration and culture conditions used for the assay), or inadequate number, selection, or information of DP batches (e.g. dates of manufacturing, expiry and testing, DS lot used etc.). Excessive DP dilution can result in inadequate assay sensitivity. In general, highest concentration of minimally manipulated DP that does not decrease cell viability or metabolic activity needs to be tested in the assay. Calculations on the sensitivity of the assay should account for all dilutions and manipulations of the samples during the testing process.
Assessing the risk of product and process related impurities is not sufficient to determine the immunogenicity risk of a new product but can support a risk assessment of “relative” immunogenicity risk as compared to the product that was used in clinical trials. Characterization of Product and Process-related Impurities can contribute to the totality of evidence used to assess the potential immunogenicity risk of new impurities following a manufacturing change. As methods progress, characterization of Product and Process-related Impurities could contribute to the assessment of immunogenicity risk for biosimilars & interchangeable products. Availability of well characterized standards will be critical to the development and validation of these assays.
Updates of the US FDA OCP Efforts on Evaluating Clinical Impact of Immunogenicity
Antidrug antibodies (ADAs) or neutralizing antibodies (NAbs) impact drug's PK by increasing exposure or clearance. PK is a sensitive metric compared to clinical endpoint to evaluate the clinical impact of immunogenicity. The OCP developed a tool called IS tool to enhance consistency and streamline review of immunogenicity data. The IS tool creates an integrated dataset for analysis from Demographic dataset (ADSL), PK dataset (ADPC), and immunogenicity dataset (ADIS). Multiple elements such as study design, sampling timepoint, number of subjects with “time-matched ADA and PK” data, the adequacy of ADA and PK assay will be assessed to interpret the results of the IS tool. From 2021 to present, 21 BLA were reviewed and 8 had IS impact on PK, 4 had no impact on PK, 9 were inconclusive.
OCP identified issues with datasets which include missing datasets, datasets submitted under incorrect domain, lack of standard variables, inconsistency in using variables (e.g. VISIT), missing final ADA status, and improper reporting. FDA is evaluating how to communicate these observations to sponsors and other stakeholders and anticipates this information will enhance the quality of immunogenicity data submitted by sponsors and improve review efficiency.
Health Canada
Immunogenicity Labelling for Biologics in Health Canada Drug Submissions
Unwanted immunogenicity is an immune response by an organism against a therapeutic antigen that has the potential to inactivate the therapeutic effects of the treatment and, in some cases, induce adverse effects. Given the potential risks associated with unwanted immunogenicity, drug submissions submitted to Health Canada should include an adequate characterization of the immunogenicity profile of a biologic drug in order to enable regulators to assess the clinical consequences of product immunogenicity. Regulatory decisions are made on a case-by-case basis depending on numerous considerations which affect the benefit/risk profile of a drug. Regulators also rely on the overarching life-cycle approach to mitigate risks associated with immunogenicity including product labelling.
A product monograph is a factual, scientific document on a drug product that, devoid of promotional material, describes the properties, claims, indications, and conditions of use for the drug, and that contains any other information that may be required for optimal, safe, and effective use of the drug. Unwanted immunogenicity findings are primarily labelled in three sections of the Product Monograph: 7 WARNINGS AND PRECAUTIONS, 8.2 Clinical Trial Adverse Reactions. And 14.4 Immunogenicity.
In warnings are precautions, it is necessary to include an “Immune” subheading, if applicable and include effects resulting from altered immune reactivity, clinically expressed as either immune activation, reactivation or immune suppression. Immunogenicity or allergenicity should be given special consideration if applicable. In adverse reactions, include an “Immunogenicity” subheading, if applicable. Include the proportion of participants that were positive for binding and neutralizing antibodies (expressed as % [positive participants/total participants]) in the pivotal trials as well as relevant study details (e.g. duration, time point[s], patient population[s], etc.). Also it is important to include a concise summary of the clinical impact of ADAs (i.e. on pharmacokinetics, efficacy, and safety) or if clinical significance is not known, a statement to that effect.
In the immunogenicity section, for biosimilars, include comparative immunogenicity results, if applicable, with a brief narrative describing the testing strategy for anti-drug antibodies (ADA) and the overall incidence of treatment-emergent or treatment-enhanced confirmed binding antibodies.
UK MHRA
Development of Reference Material as Positive Controls for ADA Assays
Positive controls play a central role in immunoassay development, validation and assay performance testing during drug development, but also post-licensure in some instances. For example, in the case of some TNF-antagonists, routine therapeutic drug monitoring (TDM) is being actively considered in clinical practice. TDM includes measuring trough drug levels and anti-drug antibody (ADA) levels in treated patients. Drug levels, and presence/levels of ADA in conjunction with clinical response to therapeutic are amongst the criteria considered in clinical decisions. Currently, timely and accurate assessment of results versus standardized validated methods, is challenging, due o different analytical techniques in use. As part of a WHO program on producing standards for assessment of immunogenicity of biotherapeutics, UK MHRA is developing reference materials for TDM and ADA assays for several therapeutics. It is anticipated that these reference antibody/panels would help in selection of suitable assays, benchmarking of in-house positive controls/standards where appropriate and assist in harmonizing ADA assays in routine clinical practice.
A WHO international collaborative study for producing reference standards for infliximab ADA assays has recently been completed. The study goal was to compare two lyophilized monoclonal antibodies (mAbs) A and B, originally isolated from an infliximab treated patient (provided by the Innovative Medicines Initiative, Europe, ABIRISK consortium) across available methods and assess their suitability for use as common standards/performance indicators. A further aim was to assign an arbitrary unitage, if feasible, for the lyophilized preparations to enable calibration of local standards and for assay harmonization. Study participants were sent the two lyophilized mAbs, a panel of anti-infliximab mAbs as well as some patients' sera diluted in appropriate matrix. Participants were requested to perform 3 independent assays using their own in-house methods, to test dilution series of samples if possible and to report quantitative results (titers or concentrations based on routine practice) for the samples, relative to in-house/kit standard and relative to the lyophilised material provided.
Binding assay data from clinical laboratories was mainly generated from ELISAs although electrochemiluminescence, total antibodies, chemiluminescent immunoassay and lateral flow were also used in some instances. For neutralization assays, competitive ligand binding assays and/or reporter gene assays were employed. The degree of recognition of samples varied depending on the assay type and the nature of the sample. Estimates for ADA levels varied among assays/labs, even when using same platform (and same IH/kit standard). As expected, ADA levels were more variable when quantified vs IH/kit standard than vs sample A. Use of sample A as the common standard narrows the range of calculated concentrations and greatly reduces the %GCV in all assay types, so it potentially harmonizes results across assays/platforms and laboratories. However, the degree of harmonization depends on assay type and sample. It was also noted that the use of A enabled detection of ADA in cases where they were missed using IH/kit standards. Detection of B is dependent on particular assay within labs using ELISA and is also platform dependent (higher levels noted in ECL as compared with ELISA). ATD stability studies have shown no loss in activity (binding and neutralization) of A and B following storage at elevated temperatures up to 20°C (tested up to 23 months). Post-reconstitution stability studies showed no decrease of the neutralizing activity of A and B after a week of storage either at 4°C or at RT; a slight decrease of binding activity was observed after storage at RT. Freeze-thaw stability studies showed no loss of activity (binding and neutralization) of A and B with repeated freeze-thaw cycles (up to 4).
The 1st WHO International Reference Panel for infliximab ADA comprising the lyophilized antibody preparations 19/234 and 19/232 was established by the WHO Expert Committee on Biological Standardization in October 2022. 19/234 (sample A) is intended as a ‘common standard’ for characterization and calibration of commercially available and in-house anti-infliximab ADAs and has been assigned an arbitrary unitage (50000 IU/ampoule of binding activity and 50000 IU/ampoule of neutralizing activity). 19/234 (sample B) is intended as a performance indicator for assessing suitability of ADA assays and has no unitage assigned to it.
Gene & Cell Therapy & Vaccines
US FDA
Unique Scientific Challenges in the Immunogenicity Assessment of Novel Modalities
In the context of gene & cell therapy, immunogenicity generally refers to immune responses to gene transfer vectors (both viral or non-viral), transgenes or other factors associated with these modalities. Immunogenicity is of concern during drug development and licensure as it can affect the safety and/or efficacy of drug products. Technological applications using novel modalities such as gene editing, gene therapy, cancer vaccines, immunotherapies etc. have begun to be approved and are also rapidly moving to clinical applications. For instance, technological success in mitigating safety risks associated with CRISPR-Cas mediated gene editing is contributing to move this technology to the clinic. As in vivo clinical applications of these technologies expand, immunogenicity is likely to be one of the major concerns. This is particularly true for Cas proteins used in gene editing, which are of bacterial origin. Moreover, studies have identified Cas9-specific T-cells and Cas9-specific antibodies in human populations. Thus, safety and efficacy of gene editing modalities in vivo using Cas protein needs to be carefully evaluated in preclinical and clinical studies.
Several methods are available for assessing product immunogenicity such as in silico (HLA peptide binding algorithms), in vitro (HLA peptide binding assay), ex vivo (MHC proteomics, TGEM, T cell amplification, cell-based assays), and in vivo methods (HLA transgenic mice). Some of these techniques have been used in other modalities such as recombinant protein therapy to assess immunogenicity. A post hoc study using an analog of recombinant FVIIa called Vatreptacog alfa (VA) demonstrated the applicability of in silico, in vitro and ex vivo tools to evaluate immunogenicity in a non-clinical setting [Citation83]. Wild type FVIIa has been used clinically for over two decades with no reports of immunogenicity. The variant, VA, which has three mutations, V158D, E296V, M298Q elicited immune responses in 11% of the patient population in the phase 3 clinical study leading to the development of the drug being discontinued. Examples of applying tools and algorithms include for Vatreptacog alfa {V158D, E296V, M298Q} for incidence of anti-FVIIa antibodies. These methods can determine if mutant peptides bind HLA-II molecules with high affinity (in silico), if mutant peptides presented on HLA-II molecules (MAPPs), and if mutant peptides that bind with high affinity elicit a T-cell response. While there is a large body of work demonstrating the use of these techniques in early stages of drug development, currently, it is not clear if such techniques can successfully assess immunogenicity risks for gene and cell therapies.
Technological approaches to reduce off target safety concerns have moved CRISPR-Cas mediated gene editing to the clinic. As in vivo clinical applications expand immunogenicity is likely to be a key concern since Cas proteins are of bacterial origin and likely to trigger host immune responses. Animal studies have identified Cas9-specific T-cells and Cas9-specific antibodies during CRISPR/Cas9 treatment. Cas9 is derived from Staphylococcus aureus or Streptococcus pyogenes which are common pathogens and T- and B-cell responses to Cas9 proteins have been identified in human subjects [Citation84,Citation85]. Cas-immunogenicity is also not the same as therapeutic proteins. Cas9 can be made intracellular & presented by MHC-I leading to peptide-MHC Class-I complexes if engaged by TCRs stimulate CD8+ T-cells. There are unmet needs of studying immune responses ranging from assays, model systems, and reagents. For instance, if studies do not have consistent and validated methods and statistical tools for determining a “cut-point” for identifying a subject as positive there will be wide variations in the results. Cells for ex vivo assays should be sourced from a cohort of donors that is representative (e.g., vis-à-vis HLA distribution) of the population that will receive the treatment. More characterization of reagents is necessary since many may procure laboratory grade Cas9 from numerous suppliers. These have varying amounts of endotoxin which affect responses in assays used to measure T-cell responses.
Understanding, Assessing & Managing Immune Responses to CAS-proteins
The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas system is a powerful gene editing tool and clinical applications are in the pipeline. In vivo delivery of CRISPR systems can induce immune reactions to the foreign Cas protein. Pre-existing immunity to Cas9 proteins derived from Staphylococcus aureus (Sa) and Streptococcus pyogenes (Sp) in human populations has been shown [Citation84–88]. As Cas proteins are of microbial origin it is not surprising that humans have preexisting immunity to these proteins. However, evaluating the immunogenicity risk of Cas proteins is a complex issue which is exacerbated by the fact that this is a technology undergoing rapid development and this includes diverse delivery systems. These differences can result in different pathways for the immune response as well as the potential safety risk associated with immunogenicity.
Another key aspect of the current state of the art of CRISPR/Cas mediated gene editing is that there are numerous naturally occurring alternatives to Cas9 and many of these are being leveraged for gene editing. Alternatives to Cas9 that have successfully been used in gene editing technologies include Cas12a (derived from Acidaminococcus sp), and Casφ (derived from a huge bacteriophage). It has been postulated that humans would not have pre-existing immunity to some of these Cas proteins. Although CasΦ is not a human pathogen, pre-existing antibody titers are comparable to SACas9. Therefore, careful evaluation of these molecules is needed before moving into the clinic.
Perspective on Emerging Landscape of Gene Therapies
Gene and Cell Therapy products have potential to treat or cure previously untreatable or incurable diseases. The field has rapidly grown in the last few years and is expected to continue to grow. This rapid development of cell and gene therapy products have also shown many scientific and regulatory challenges that need to be resolved for manufacturing and translating these therapeutic products. As it is anticipated that the cell and gene therapy products will continue to be more complex from product design standpoint to the manufacturing processes and quality control, the safety and efficacy of these newer products will need to be assessed rigorously. The field is also likely to encounter new unanticipated scientific and regulatory challenges, which will require novel strategies and innovations.
Special considerations needed for gene therapies are that they are novel products, manufactured using complex technologies. The in vivo mechanism of action is not always well understood and risks associated are not fully known and cannot be easily assessed especially since most therapies are in the first in human stage. Other challenges include the lack of reliable product attribute(s) to predict safety/ efficacy, lack of reliable tools for risk assessment (a wide variety of in vitro assays are used and animal models are not always predictive). Overall, there is a need for better strategies for risk mitigation. There are also regulatory challenges for manufacturing changes (scale up, automation, facility changes, comparability studies). If comparability cannot be demonstrated analytically, additional clinical studies may be required.
Early communication with CBER/OTP is highly recommended due to these challenges. Early opportunities include an INTERACT meeting (Informal Pre-Pre IND, Not PDUFA VI) and Pre-IND meeting. The OTAT website provides resources for stakeholders along with two new draft guidance documents in 2022.
Application of Flow Cytometry in Cell Therapy; Current Perspective
In the field of cell-based therapeutics, there is a great need for high-quality, robust, and validated measurements to ensure product safety. Flow cytometry has emerged as a critically important platform due to its high-throughput capability and its ability to simultaneously measure multiple parameters in the same sample. This technique is widely used in identifying critical quality attributes to characterize and release these products for clinical use (such as identity, potency, purity, viability etc.).
This field faces multiple challenges such as limited sample stability/ volume, inherent biological variability of the cells, reagent lot to lot variability, variabilities in fluorescence measurements between runs, days, and experiments, complexity of data output, and interpretation of results, lack of cellular reference standards, and consistency in measuring critical parameters of the final cellular product when performed in multiple laboratories, during scalable manufacturing processes and technology transfer.
Possible solutions are to identify samples stability (from collection to staining and between staining and acquisition), optimize staining panel and operating procedures, predefine gating strategy using the actual sample matrix. establish protocol for bridging studies to reduce variabilities between reagent lots, establish a robust process for daily instrument set-up and calibration, optimize gains (PMT) voltages to achieve equivalent fluorescence measurements between runs, days, experiments, and labs, establish internal and external quality control procedures, and select proper instrument standardization protocol to ensure consistency in measurements, standards in the areas of operator training, cell preparation, reagents, threshold and gate settings, instrument standardization, quantitation etc. are critical to ensure consistent delivery of safe and effective cellular product. As such, an effort has been initiated to develop standards for flow cytometry. NIST launched the Flow Cytometry Standards Consortium to accelerate the adoption of quantitative flow cytometry in biomanufacturing of cell and gene therapies.
The Flow Cytometry Standards Consortium has three active working groups in the areas of instrument calibration/standardization via Equivalent Number of Reference Fluorophores (ERF), development of standardized assay procedures and associated reference materials, and data analysis and repository. CBER has also published guidance documents for standards.
Serology Assay Validation
During product development, as the clinical program advances there is corresponding evolution of the assay, traditionally described as separate steps: Method development, qualification, validation, and life-cycle management. Apart from validation, these are not discreet steps, nor formally defined. Development and qualification are an iterative process of assay refinement to get to a point where the assay has the necessary characteristics to be used as intended to support your pivotal clinical study. Performance characteristics are defined prior to validation based on an assay's intended use. Validation is a “snapshot” of assay performance on that day - a demonstration of your assay's capability. Validation provides assurance that if following a defined procedure, use qualified reagents and acceptance criteria, that the assay validated today should perform as defined consistently over time.
While method development, qualification and life-cycle management do not have formal definitions, there are several points that should be considered as an assay evolves. For example, during early assay development, it is important to consider the right technology, relevant to what is being measured and has an appropriate dynamic range. Often forgotten at this early stage is the identification of critical reagents and their control. Assuming the right assay, it is important to get a preliminary estimate of the performance (i.e., precision) to determine if on the right path, this often leads to “adjustments” to assay design, reagents, etc. This is an iterative process, and shortcuts should not be taken. Data collected on the final version of an assay will form the basis of system suitability criteria and will be used to establish validation criteria. Method validation is a formal study to demonstrate the performance of the assay, including the procedure used during routine use and driven by validation criteria. Post-validation, it is important to monitor performance and have plans established to qualify new reagents, and re-validation if something changes that affects assay performance.
Biomarker assays can be used for many applications such as to estimate clinical efficacy as well as diagnostics. Efficacy studies are used in many situations such as novel products, new indications, comparability to licensed vaccines, co-administration, etc. All are defined by their clinical endpoints. Clinical endpoints can be described a number of ways including geometric mean titer, percent above threshold, and percent responder just to name a few. The assay will need to be designed and validated to support study endpoints. In addition to the endpoint possibilities, these will be driven by the types of assay that is selected. Serologic assays roughly fall into two categories: Ligand binding and functional assays. Functional assays tend to be a bit more challenging to conduct and control. Sometimes several assays are needed, and the assay may change as clinical development proceeds. Bottom line, whatever is chosen will be based on intended use. Once an assay is chosen, it is important to decide what to do with the output and how this becomes a reportable value. Reportable values are either absolute, such as a titer, or a relative value which is based on the performance of another material believed as truth such as a reference or control.
EU EMA/ Norway NoMA
Regulatory Perspective on Vaccine Serological Assays- Validation as Clinical Endpoints
From a regulatory point of view the bioanalytical/immunogenicity part of the marketing application dossier is of paramount importance and forms the basis for the reliability of the clinical data. For each type of vaccine, a different approach is needed on how to assess the different methods applied, methods for measuring immunogenicity in animals/humans and in the context of prophylactic vaccines, also methods for measuring correlates of protection as clinical endpoints. These assays are subsite endpoints which are defined as (WHO, WHO/IVB/13.01) a general term including correlates and surrogates of protection, or “intermediate endpoints” – in other words, quantities that may be measured instead of the clinical endpoint (i.e. disease) of interest.
Immune Correlate of Protection (CoP) is an immunological assay (either humoral or cellular immune response) that reliably predicts protection against disease or infection after vaccination or natural infection. CoP is of great importance because it can be used as a surrogate endpoint assessing vaccine efficacy without directly observing clinical endpoints. Compared with large-scale field efficacy trials, immunological trials are both time and cost effective. The availability of surrogate endpoints helps to avoid large-scale filed efficacy trials and facilitate getting vaccine candidates approved-fast approval. Seroprotection can be shown where a subject is said to be seroprotected if the antibody level is above a certain cut-off level. Since prophylactic vaccines do not have a PK profile, the bioanalytical methods in the MA dossier are placed under the PD/biomarker Section in Module 5.
UK MHRA
Importance of immunobridging data for vaccine approval: recent experience with COVID-19 vaccines
Six COVID-19 vaccines are currently approved by the UK MHRA. The first five vaccines (Pfizer/BioNTech, Moderna, Oxford/AstraZeneca, Janssen and Novavax) were approved based on randomised placebo-controlled clinical trials that were powered for vaccine efficacy. In contrast, the pivotal randomised active-controlled clinical trial of Valneva (Cov-Compare) was not powered for vaccine efficacy. Instead, the Valneva vaccine was approved in the UK based on an immuno-bridging approach. Extending the indications of some COVID-19 vaccines to the paediatric population has also been based on immuno-bridging.
In both scenarios, immunogenicity outcomes were pivotal. Humoral and cellular immune responses are likely to be important for COVID-19 vaccine efficacy, but there is no generally accepted immune correlate of protection. Currently, neutralising antibody titre is considered the best potential immune correlate when it is not feasible to demonstrate vaccine efficacy. It is essential that regulators have confidence in the robustness of SARS-CoV-2 immunogenicity assays used in clinical trials. This is especially true for the neutralization assays used to generate pivotal immuno-bridging data.
For Valneva using the immune bridging approach, the pivotal clinical immunogenicity and safety study had 3000 participants randomized 2:1 with co-primary endpoints: NAbs geometric mean titre (GMT) and seroconversion rate. The geometric mean ratio (GMR) was 1.39 (95% CI: 1.25, 1.56). Seroconversion rate was non-inferior. Results for IgG binding assays were like NAbs
Post-approval effectiveness studies are required in this approach.
The Spikevax (Moderna COVID-19 vaccine) extension of indication to children aged 6 to 11 years used based on immunobridging across studies. Immune response of ∼300 children aged 6 to 11 years was compared to ∼300 randomly selected adults aged 18 to 25 years from adult study. NAb GMT and seroresponse rate were non-inferior and GMR was 1.239 (95% CI: 1.072, 1.432). The clinical study database was larger (∼3000 children aged 6 to 11 years) for clinical safety evaluation.
For NAb assays, live virus or pseudovirus neutralisation assays are acceptable. The same assay should be used to compare immune responses across studies (e.g. extension of indication to paediatric population, introduction of booster dose). Validation reports should be submitted to regulators for each assay that is relied on for regulatory decision-making to check that methods are acceptable and fit for purpose. Laboratories that analyse clinical trial samples must comply with GCP and have an appropriate QMS in place.
Health Canada
Cell & Gene Therapies: Regulatory Challenges & Considerations
Gene therapy products and cell therapy products are regulated in Canada as Schedule D biological drugs under the Food and Drugs Act. Health Canada regulatory approach is sufficiently flexible to enable the development and authorization of novel cell and gene therapy products. The Food and Drugs Act enables a number of regulations, including the Food and Drug Regulations, the Safety of Human Cells, Tissues and Organs for Transplantation Regulations (CTO Regulations), and the Medical Devices Regulations, which accommodate a wide range of therapeutic products. These products are assigned to a regulatory pathway based on a formal classification process.
CTO Regulations apply in general to cells that are minimally manipulated, allogeneic, are intended for homologous use, and do not have a systemic effect. CTO Regulations focus on activities performed by establishments (importation, processing, distribution, and transplantation), on safety by mitigating risks of transmissible diseases, and require limited review of efficacy. There is no premarket review but establishment registration with Health Canada is required. On the other hand, cell therapies (cells excluded from the CTO Regulations) and gene therapies are considered drugs with a requirement of pre-market approval; establishment license; good manufacturing practices; lot release testing; and supporting evidence of quality, safety, and efficacy data. Equipment used to manufacture cell and gene therapies is regulated in different ways under the Food and Drug Act. For combination products, where the device and drug are integrated into a singular product, Food and Drug Regulations apply when the primary mechanism of action is through the drug. For devices used in the manufacture of a cell or gene therapy product, the output remains a drug regulated under the Food and Drug Regulations. For co-packaged products where drug and device can be used separately, both the Medical Devices Regulations and Food and Drug Regulations apply.
The diversity and complexity of cell and gene therapy products pose challenges to the assessment of product characterization and control strategy. For example, process and product should be well characterized. Manufacturing is often done on a small scale or in patient-specific lots where there may be considerable lot-to-lot heterogeneity. Also, products often have a limited shelf-life, which makes strategies for product testing, storage, and shipping highly product-specific.
Authorization of new COVID-19 vaccines: The utility of immunobridging studies
Health Canada and UK MHRA are aligned on acceptability of well-justified and appropriately designed immunobridging studies to authorize new COVID vaccines. The expectation is to show non-inferiority and/or superiority based on the comparator vaccine, and the viral strains being targeted (new vaccine vs. modified vaccine). There is a need for measures to ensure the clinical trial cohorts are well-balanced. Proof of concept studies can be shown in relevant animal models.
For SARS-CoV2, there is a strong association with induction of neutralizing antibodies (nAb). A cut-off for protection not identified. Immunobridging endpoints can be primary: (Neutralizing antibody titers against target VOC) and secondary (Seroresponse rates against target VOC). GMTs and seroresponse rates against ancestral strain and circulating VOI/VOC should be described. Given the evolving variant landscape, breadth of coverage is important.
In regard to the bioanalytical assays used, neutralizing antibodies, binding antibodies, and at least in a subset of each cohort, T-cell responses should be evaluated. Assays linked to primary and secondary analyses to be fully validated for COVID-19 and be fit for purpose, prior to use. Centralized testing, use of standardized assays, use of international standards/reference materials are recommended to facilitate cross-trial and cross-platform comparisons
Use of Functional Assays in the Development of Vaccines
Assay design and validation considerations for the use of functional assays in the support of vaccine regulatory approval were discussed. Functional assays play an important role throughout a vaccines lifecycle. During pre-clinical and clinical development, functional assays can play a key role in making important formulation decisions (e.g. adjuvants, antigen content) or determining correlatives of protection. Well-designed and appropriately validated functional assays are also important for routine manufacturing control, stability monitoring, and comparability assessments performed to support manufacturing changes of vaccines.
In early development, in vivo assays can be important for proof of concept studies and in making decisions on formulation (e.g. adjuvant, selection of antigen). Clinical Development may use both in vivo and in vitro assays to characterize immune responses (e.g. humoral, cell-mediated), characterize vaccine antigenicity, verify suitability of in vitro assay for stability monitoring, and establish potency specifications. For routine manufacturing/post-approval changes, and in vitro assay is preferred for routine manufacturing, stability monitoring and to support changes. It is possible to use an in vivo assay to support additional characterization studies performed as comparability assessments following manufacturing changes. Examples of in vivo assays are immunogenicity (e.g. GMT) and cell immune cell responses, protection against challenge, virus neutralization, and bacterial opsonization. In vitro assays include antibody/antigen binding (e.g. Sandwich ELISA), antigen expression and characterization (e.g. receptor binding inhibition), and cytokine production.
An example of functional assays for pre-clinical formulation decisions is the COVID-19 mRNA vaccines [Citation89]. Upon entry into cells unmodified foreign mRNAs can activate immune signalling pathways (e.g. TLRs) leading to immune response against the introduced mRNA. COVID-19 mRNA vaccines contain mRNA modifications that serve to improve stability, improve translation efficiency and reduce this undesired cellular defense immune response. One example is the substitution of uridine to pseudouridine (ψ)/ N1-methylpseudouridine (m1ψ). Modified mRNAs have been shown to be less immunogenic when transfected into dendritic cells. Modified mRNAs have demonstrated higher expression and persistence when administered to mice [Citation90]
Another example for formulations decisions are pneumococcal vaccines [Citation91]. Another example of functional assays as part of routine manufacturing is the use of MAT assays. Traditional assays that assess pyrogenicity (e.g. rabbit pyrogenicity test, bacterial endotoxin) were developed for the analysis of products that are usually free of pyrogenic components. These assays are highly variable (RPT) or only focus on limited pyrogens (e.g. LPS). MAT assays can be used to evaluate pyrogenicity to non-LPS components of vaccines, but require consideration of reference selection and cells. Donor cells can have different sensitivities and responses curves and for complex products, the use of a reference vaccine is usually preferable [Citation92]. Primary cells may be useful for characterization but donor-to-donor variability may present challenges for routine testing. Strategies to reduce variability can include use of pooled donors and use of a well-characterized cell line.
Japan MHLW
Anti-SARS-CoV-2 Neutralizing Antibody Titer as a Clinical Endpoint of Vaccine Clinical Study in Japan
As a regulatory consideration for evaluating SARS-CoV-2 vaccines in Japan, a guidance document was released on September 2, 2020 in alignment with the discussion of ICMRA. Subsequently, Addendum 1: On the evaluation of vaccines against mutant strains (April 5, 2021), Addendum 2: On the ethical considerations for subjects in placebo-controlled trials (June 11, 2021), and Addendum 3: Concept of evaluation of vaccine based on immunogenicity (October 22, 2021) were released from PMDA.
In the Addendum 3, it is described that the geometric mean of neutralizing antibody titer against SARS-CoV-2 can be used as a primary endpoint of SARS-CoV-2 vaccine clinical trials. It is necessary to use the reliable neutralizing antibody assay method having sufficient analytical performance (e.g., precision and linearity) as much as possible. The neutralizing antibody titer should be determined by calibration using the anti-SARS-CoV-2 international standard immunoglobulin provided by WHO. From these statements, the bioanalytical method used for evaluating the neutralizing antibody plays an important role in the evaluation of SRAS-CoV-2 vaccine in Japan.
These concepts have been implemented in evaluation of SARS-CoV-2 vaccine products approved in Japan, i.e., two types of mRNA vaccines, two types of adenoviral vector vaccines, and a recombinant spike protein vaccine. Although the methods for evaluating anti-SARS-CoV-2 antibody titer in clinical trials are developed by each company, many antibody assay kits are available for research and clinical settings so far. In Japan, antibody assay kits are not categorized as diagnostic kits approved under the Pharmaceutical Affairs Law, however, it is important to use the reliable methods even in such situations. Therefore, an original reference standard by pooling the COVID-19 patient serum in Japan has been prepared and compared in the response of antibody assay kits available in Japan [Citation93]. WHO international standard was also used.
Antibody assay kits distributed in Japan can generally detect antibodies against SARS-CoV-2. Although concerns were not identified in precision of the results obtained by all kit, the maximum dilution factor of the reference standard that gives a positive result differed for each kit, suggesting that the positive judgment criteria differ depending on the kit. Standardization of antibody titer and clarification of target analytical performance can improve the reliability of antibody tests to be developed.
Two-Dimensional Droplet Digital PCR as a Tool for Titration & Integrity Evaluation of Recombinant Adeno-Associated Viral Vectors
There has been active research on the development of adeno-associated virus (AAV) vectors that can safely introduce therapeutic genes in vivo. The dose definition of an AAV vector requires precise quantification through both preclinical and clinical studies. In the quantification of AAV vectors, the AAV vector genome copy numbers are measured in most cases, for which quantitative PCR (qPCR) has been used to date. However, such qPCR-based measurements of the AAV vector genome are influenced by the selection of the qPCR standard and the amplification target sites.
A two-dimensional droplet digital PCR (2D ddPCR) was developed for the absolute quantitation of genome copy numbers and for evaluating the integrity of the AAV vector genome [Citation94]. When plasmid DNAs treated by restriction enzymes were used as standard DNAs with different conformation, there was a considerable change in the AAV vector genome copy numbers measured by qPCR, suggest ing that qPCR amplification is significantly influenced by the secondary structure of the standard DNA. In contrast, there was no effect on the AAV vector genome copy numbers measured by ddPCR by the conformation of the vector genome or the primer-probe sets for different positions of the standard DNA. Furthermore, the integrity of the AAV genome could be evaluated by 2D ddPCR with different fluorescent-labeled probes, which targeted different positions in the same AAV genome. The genomic integrities obtained using this method highly correlated with the infectious activities of the AAV vector. Therefore, the 2D ddPCR method is a useful tool for the quantification and quality evaluation of AAV vectors.
Table 1. Comparison of LCMS and Stem Loop qPCR for bioanalysis of oligonucleotide therapeutics.
Acknowledgments
US FDA, Europe EMA, UK MHRA, Norway NoMA, Brazil ANVISA, Health Canada, Japan MHLW and WHO for supporting this workshop
All Session Chairs & Working Dinner Facilitators for chairing the workshop and the White Paper discussions: Dr. Chris Beaver (Syneos), Dr. Arindam Dasgupta (US FDA), Dr. Fabio Garofolo (BRI Frontage), Ms. Dina Goykhman (Merck), Dr. James Huleatt (Sanofi), Dr. Akiko Ishii-Watabe (Japan MHLW / ICH M10 EWG), Mr. Gregor Jordan (Roche), Dr. John Kamerud (Pfizer), Dr. Steve Keller (AbbVie), Dr. Lina Loo (Vertex), Mr. Fred McCush (Pfizer), Mr. Luis Mendez (Merck), Ms. Dulcyane Neiva Mendes Fernandes (Brazil ANVISA / ICH M10 EWG), Dr. Luying Pan (Takeda), Mr. Noah Post (Ionis), Dr. Mohsen Rajabi Abhari (US FDA), Dr. Yoshiro Saito (Japan MHLW / ICH M10 EWG), Dr. Daniel Spellman (Merck), Dr. Giane Sumner (Regeneron), Dr. Matthew Szapacs (Abbvie), Dr. Albert Torri (Regeneron), Dr. Montserrat Carrasco-Triguero (Sangamo), Dr. Elizabeth Verburg (Lilly), Dr. LaKenya Williams (BMS), Dr. Karl Walravens (GSK), Dr. Yongjun Xue (BMS)
All the workshop attendees and members of the Global Bioanalytical Community who have sent comments and suggestions to the workshop to complete this White Paper
Future Science Group as a trusted partner
Financial & competing interests disclosure
The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.
No writing assistance was utilized in the production of this manuscript.
Notes
1 When final, this guidance will represent the FDA's current thinking on this topic.
2 See the Logical Observation Identifiers Names and Codes (LOINC) for Human Prescription Drug and Biological Product Labeling at https://www.fda.gov/media/114007/download
References
- Savoie N , BoothBP, BradleyTet al. The 2nd Calibration and Validation Group Workshop on Recent Issues in Good Laboratory Practice Bioanalysis. Bioanalysis (2009).
- Savoie N , GarofoloF, Van AmsterdamPet al. 2009 White Paper on Recent Issues in Regulated Bioanalysis from the 3rd Calibration and Validation Group Workshop. Bioanalysis.2(1), (2010).
- Savoie N , GarofoloF, Van AmsterdamPet al. 2010 White Paper on Recent Issues in Regulated Bioanalysis & Global Harmonization of Bioanalytical Guidance. Bioanalysis.2(12), (2010).
- Garofolo F , RocciML, DumontIet al. 2011 White Paper on Recent Issues in Bioanalysis and Regulatory Findings From Audits and Inspections. Bioanalysis (2011).
- Desilva B , GarofoloF, RocciMet al. 2012 White Paper on Recent Issues in Bioanalysis and Alignment of Multiple Guidelines. Bioanalysis.4(18), (2012).
- Stevenson L , GarofoloF, DesilvaBet al. 2013 White Paper on Recent Issues in Bioanalysis: “Hybrid” – The Best of LBA and LCMS. Bioanalysis.5(23), (2013).
- Fluhler E , HayesR, GarofoloFet al. 2014 White Paper on Recent Issues in Bioanalysis: A Full Immersion in Bioanalysis (Part 1-Small Molecules by LCMS). In: Bioanalysis (2014).
- Dufield D , NeubertH, GarofoloFet al. 2014 White Paper on Recent Issues in Bioanalysis: A Full Immersion in Bioanalysis (Part 2-Hybrid LBA/LCMS, ELN & Regulatory Agencies' Input). In: Bioanalysis (2014).
- Stevenson L , AmaravadiL, MylerHet al. 2014 White Paper on Recent Issues in Bioanalysis: A Full Immersion in Bioanalysis (Part 3-LBA and Immunogenicity). Bioanalysis.6(24), (2014).
- Welink J , FluhlerE, HughesNet al. 2015 White Paper on Recent Issues in Bioanalysis: Focus on New Technologies and Biomarkers (Part 1-Small Molecules by LCMS). Bioanalysis.7(22), (2015).
- Ackermann B , NeubertH, HughesNet al. 2015 White Paper on Recent Issues in Bioanalysis: Focus on New Technologies and Biomarkers (Part 2 - Hybrid LBA/LCMS and Input from Regulatory Agencies). Bioanalysis.7(23), (2015).
- Amaravadi L , SongA, MylerHet al. 2015 White Paper on Recent Issues in Bioanalysis: Focus on New Technologies and Biomarkers (Part 3-LBA, Biomarkers and Immunogenicity). In: Bioanalysis (2015).
- Yang E , WelinkJ, CapeSet al. 2016 White Paper on Recent Issues in Bioanalysis: Focus on Biomarker Assay Validation (BAV) (Part 1 - Small Molecules, Peptides and Small Molecule Biomarkers by LCMS). Bioanalysis8(22), (2016).
- Song A , LeeA, GarofoloFet al. 2016 White Paper on Recent Issues in Bioanalysis: Focus on Biomarker Assay Validation (BAV): (Part 2 - Hybrid LBA/LCMS and Input From Regulatory Agencies). Bioanalysis.8(23), (2016).
- Richards S , AmaravadiL, PillutlaRet al. 2016 White Paper on Recent Issues in Bioanalysis: Focus on Biomarker Assay Validation (BAV): (Part 3 - LBA, Biomarkers and Immunogenicity). Bioanalysis.8(23), (2016).
- Welink J , YangE, HughesNet al. 2017 White Paper on Recent Issues in Bioanalysis: Aren't BMV Guidance/Guidelines “Scientific”? (Part 1 - LCMS: Small Molecules, Peptides and Small Molecule Biomarkers). Bioanalysis.9(22), (2017).
- Neubert H , SongA, LeeAet al. 2017 White Paper on Recent Issues in Bioanalysis: Rise of Hybrid LBA/LCMS Immunogenicity Assays (part 2: Hybrid LBA/LCMS Biotherapeutics, Biomarkers & Immunogenicity Assays and Regulatory Agencies' Inputs). Bioanalysis.9(23), (2017).
- Gupta S , RichardsS, AmaravadiLet al. 2017 White Paper on Recent Issues in Bioanalysis: A Global Perspective on Immunogenicity Guidelines & Biomarker Assay Performance (Part 3-LBA: Immunogenicity, Biomarkers and PK Assays). In: Bioanalysis (2017).
- Welink J , XuY, YangEet al. 2018 White Paper on Recent Issues in Bioanalysis: “A Global Bioanalytical Community Perspective on Last Decade of Incurred Samples Reanalysis (ISR)” (Part 1-Small Molecule Regulated Bioanalysis, Small Molecule Biomarkers, Peptides & Oligonucleotide Bioanalysis). Bioanalysis.10(22), (2018).
- Neubert H , OlahT, LeeAet al. 2018 White Paper on Recent Issues in Bioanalysis: Focus on Immunogenicity Assays by Hybrid LBA/LCMS and Regulatory Feedback Part 2 - PK, PD & ADA Assays by Hybrid LBA/LCMS & Regulatory Agencies' Inputs on Bioanalysis, Biomarkers and Immunogenicity. In: Bioanalysis (2018).
- Stevenson L , RichardsS, PillutlaRet al. 2018 White Paper on Recent Issues in Bioanalysis: Focus on Flow Cytometry, Gene Therapy, Cut Points and Key Clarifications on BAV (Part 3 – LBA/Cell-Based Assays: Immunogenicity, Biomarkers and PK Assays). Bioanalysis.10(24), 1973–2001 (2018).
- Garofolo W , SavoieN. The Decennial Index of the White Papers in Bioanalysis: ‘A Decade of Recommendations (2007–2016)’. Bioanalysis [ Internet]. 9(21), 1681–1704 (2017). Available from: 10.4155/bio-2017-4979
- Fandozzi C , EvansC, WilsonAet al. 2019 White Paper on Recent Issues in Bioanalysis: Chromatographic Assays (part 1 - Innovation in Small Molecules and Oligonucleotides & Mass Spectrometric Method Development Strategies for Large Molecule Bioanalysis). In: Bioanalysis (2019).
- Booth B , StevensonL, PillutlaRet al. 2019 White Paper on Recent Issues in Bioanalysis: FDA BMV Guidance, ICH M10 BMV Guideline and Regulatory Inputs (Part 2-Recommendations on 2018 FDA BMV Guidance, 2019 ICH M10 BMV Draft Guideline and Regulatory Agencies' Input on Bioanalysis, Biomarkers and Immunogenicity). In: Bioanalysis (2019).
- Piccoli S , MehtaD, VitalitiAet al. 2019 White Paper on Recent Issues in Bioanalysis: FDA Immunogenicity Guidance, Gene Therapy, Critical Reagents, Biomarkers and Flow Cytometry Validation (Part 3-Recommendations on 2019 FDA Immunogenicity Guidance, Gene Therapy Bioanalytical Challenges, Strategies for Critical Reagent Management, Biomarker Assay Validation, Flow Cytometry Validation & CLSI H62). In: Bioanalysis. Future Medicine Ltd., 2207–2244 (2019).
- Spitz S , ZhangY, FischerSet al. 2020 White Paper on Recent Issues in Bioanalysis: BAV Guidance, CLSI H62, Biotherapeutics Stability, Parallelism Testing, CyTOF and Regulatory Feedback (Part 2A - Recommendations on Biotherapeutics Stability, PK LBA Regulated Bioanalysis, Biomarkers Assays, Cytometry Validation & Innovation Part 2B - Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, Gene & Cell Therapy and Vaccine). Bioanalysis.13(5), (2021).
- Corsaro B , YangT, MurphyRet al. 2020 White Paper on Recent Issues in Bioanalysis: Vaccine Assay Validation, qPCR Assay Validation, QC for CAR-T Flow Cytometry, NAb Assay Harmonization and ELISpot Validation (Part 3 – Recommendations on Immunogenicity Assay Strategies, NAb Assays, Biosimilars and FDA/EMA Immunogenicity Guidance/Guideline, Gene & Cell Therapy and Vaccine Assays). Bioanalysis.13(6), 415–463 (2021).
- Neubert H , AlleySC, LeeAet al. 2020 White Paper on Recent Issues in Bioanalysis: BMV of Hybrid Assays, Acoustic MS, HRMS, Data Integrity, Endogenous Compounds, Microsampling and Microbiome (Part 1 - Recommendations on Industry/Regulators Consensus on BMV of Biotherapeutics by LCMS, Advanced Application in Hybrid Assays, Regulatory Challenges in Mass Spec, Innovation in Small Molecules, Peptides and Oligos). Bioanalysis.13(4), (2021).
- Hersey S , KellerS, MathewsJet al. 2021 White Paper on Recent Issues in Bioanalysis: ISR for Biomarkers, Liquid Biopsies, Spectral Cytometry, Inhalation/Oral & Multispecific Biotherapeutics, Accuracy/LLOQ for Flow Cytometry (Part 2 – Recommendations on Biomarkers/CDx Assays Development & Validation, Cytometry Validation & Innovation, Biotherapeutics PK LBA Regulated Bioanalysis, Critical Reagents & Positive Controls Generation). Bioanalysis.14(10), 627–692 (2022).
- Loo L , HarrisS, MiltonMet al. 2021 White Paper on Recent Issues in Bioanalysis: TAb/NAb, Viral Vector CDx, Shedding Assays; CRISPR/Cas9 & CAR-T Immunogenicity; PCR & Vaccine Assay Performance; ADA Assay Comparability & Cut Point Appropriateness (Part 3 – Recommendations on Gene Therapy, Cell Therapy, Vaccine Assays; Immunogenicity of Biotherapeutics and Novel Modalities; Integrated Summary of Immunogenicity Harmonization). Bioanalysis.14(11), 737–793 (2022).
- Kaur S , AlleySC, SzapacsMet al. 2021 White Paper on Recent Issues in Bioanalysis: Mass Spec of Proteins, Extracellular Vesicles, CRISPR, Chiral Assays, Oligos; Nanomedicines Bioanalysis; ICH M10 Section 7.1; Non-Liquid & Rare Matrices; Regulatory Inputs (Part 1A – Recommendations on Endogenous Compounds, Small Molecules, Complex Methods, Regulated Mass Spec of Large Molecules, Small Molecule, PoC & Part 1B - Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, Gene & C. Bioanalysis.14(9), 505–580 (2022).
- Sips L , EdiageEN, IngelseB, VerhaegheT, DillenL. LCMS Quantification of Oligonucleotides in Biological Matrices with SPE or Hybridization Extraction. Bioanalysis.11(21), (2019).
- Darmon P , DadounF, FracheboisCet al. On the Meaning of Low-Dose ACTH(1-24) Tests to Assess Functionality of the Hypothalamic-Pituitary-Adrenal axis. Eur J Endocrinol.140(1), (1999).
- Alía P , VillabonaC, GiménezO, SospedraE, SolerJ, NavarroMA. Profile, Mean Residence Time of ACTH and Cortisol Responses after Low and Standard ACTH Tests in Healthy Volunteers. Clin Endocrinol (Oxf).65(3), (2006).
- U.S. Department of Health and Human Services Food and Drug Administration Center for Drug Evaluation and Research (CDER) . Safety Testing of Drug Metabolites Guidance for Industry. https://www.fda.gov/media/72279/download (2020).
- International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use . Guidance on Nonclinical Safety Studies for the Conduct of Human Clinical Trials and Marketing Authorization for Pharmaceuticals M3(R2). https://database.ich.org/sites/default/files/M3_R2__Guideline.pdf (2009).
- Mu R , YuanJ, HuangYet al. Bioanalytical Methods and Strategic Perspectives Addressing the Rising Complexity of Novel Bioconjugates and Delivery Routes for Biotherapeutics. Bio Drugs36(2), (2022).
- Huang Y , MouS, WangY, MuR, LiangM, RosenbaumAI. Characterization of Antibody-Drug Conjugate Pharmacokinetics and in Vivo Biotransformation Using Quantitative Intact LC-HRMS and Surrogate Analyte LC-MRM. Anal Chem.93(15), (2021).
- Baillie TA , DalvieD, RietjensIMCM, CyrusKhojasteh S. Biotransformation and Bioactivation Reactions – 2015 Literature Highlights. Drug Metab Rev48(2), (2016).
- Hall MP , GeggC, WalkerKet al. Ligand-Binding Mass Spectrometry to Study Biotransformation of Fusion Protein Drugs and Guide Immunoassay Development: Strategic Approach and Application to Peptibodies Targeting the Thrombopoietin Receptor. AAPS Journal.12(4), (2010).
- Rautio J , KumpulainenH, HeimbachTet al. Prodrugs: Design and Clinical Applications. Nat Rev Drug Discov7(3), (2008).
- Perez HL , KnechtD, BuszM. Overcoming Challenges Associated with the Bioanalysis of an Ester Prodrug and its Active Acid Metabolite. Bioanalysis.9(20), (2017).
- Mu R , HuangY, BouquetJet al. Multiplex Hybrid Antigen-Capture LC-MRM Quantification in Sera and Nasal Lining Fluid of AZD7442, a SARS-CoV-2-Targeting Antibody Combination. Anal Chem.94(43), (2022).
- Loo YM , McTamneyPM, ArendsRHet al. The SARS-CoV-2 Monoclonal Antibody Combination, AZD7442, is Protective in Nonhuman Primates and has an Extended Half-Life in Humans. Sci Transl Med.14(635), (2022).
- Leney AC , HeckAJR. Native Mass Spectrometry: What is in the Name?J Am Soc Mass Spectrom.28(1), (2017).
- Zhang L , VasicekLA, HsiehS, ZhangS, BatemanKP, HenionJ. Top-down LCMS Quantitation of Intact Denatured and Native Monoclonal Antibodies in Biological Samples. Bioanalysis.10(13), (2018).
- Carle K , KellieJF, GunnGR, JiangY. Determination of Label Efficiency and Label Degree of Critical Reagents by LCMS and Native MS. Anal Biochem.664 (2023).
- Carr SA , AbbatielloSE, AckermannBLet al. Targeted Peptide Measurements in Biology and Medicine: Best Practices for Mass Spectrometry-Based Assay Development Using a Fit-For-Purpose Approach. Molecular and Cellular Proteomics.13(3), (2014).
- She X , ZouC, ZhengZ. Differences in Vitreous Protein Profiles in Patients With Proliferative Diabetic Retinopathy Before and After Ranibizumab Treatment. Front Med (Lausanne).9 (2022).
- Ogata S , MasudaT, ItoS, OhtsukiS. Targeted Proteomics for Cancer Biomarker Verification and Validation. In: Cancer Biomarkers (2022).
- Bognár Z , GyurcsányiRE. Aptamers Against Immunoglobulins: Design, Selection and Bioanalytical Applications. Int J Mol Sci21(16), (2020).
- Baba T , CampbellJL, LeBlanc JCY, HagerJW, ThomsonBA. Electron Capture Dissociation in a Branched Radio-Frequency Ion Trap. Anal Chem.87(1), (2015).
- Li P , DupuisJF, VrionisV, MekhssianK, MageeT, YuanL. Validation and Application of Hybridization Liquid Chromatography-Tandem Mass Spectrometry Methods for Quantitative Bioanalysis of Antisense Oligonucleotides. Bioanalysis.14(9), (2022).
- Brown CR , GuptaS, QinJet al. Investigating the Pharmacodynamic Durability of GalNAc-siRNA Conjugates. Nucleic Acids Res.48(21), (2021).
- Huang Y , DelNagro CJ, BalicKet al. Multifaceted Bioanalytical Methods for the Comprehensive Pharmacokinetic and Catabolic Assessment of MEDI3726, an Anti-Prostate-Specific Membrane Antigen Pyrrolobenzodiazepine Antibody-Drug Conjugate. Anal Chem.92(16), (2020).
- Faria M , PeayM, LamBet al. Multiplex LCMS/MS Assays for Clinical Bioanalysis of MEDI4276, an Antibody-Drug Conjugate of Tubulysin analogue Attached via Cleavable Linker to a Biparatopic Humanized Antibody Against HER-2. Antibodies.8(1), (2019).
- Lanshoeft C , StutzG, ElbastWet al. Analysis of Small Molecule Antibody-Drug Conjugate Catabolites in Rat Liver and Tumor Tissue by Liquid Extraction Surface Analysis Micro-Capillary Liquid Chromatography/Tandem Mass Spectrometry. Rapid Communications in Mass Spectrometry.30(7), (2016).
- Toby TK , FornelliL, KelleherNL. Progress in Top-Down Proteomics and the Analysis of Proteoforms. Annual Review of Analytical Chemistry9 (2016).
- Kotapati S , DeshpandeM, JashnaniA, ThakkarD, XuH, DollingerG. The Role of Ligand-Binding Assay and LCMS in the Bioanalysis of Complex Protein and Oligonucleotide Therapeutics. Bioanalysis13(11), (2021).
- Wei C , ZhangG, ClarkTet al. Where Did the Linker-Payload Go? A Quantitative Investigation on the Destination of the Released Linker-Payload from an Antibody-Drug Conjugate with a Maleimide Linker in Plasma. Anal Chem.88(9), (2016).
- Lassman ME , ChappellDL, McAvoyTet al. Experimental Medicine Study to Measure Immune Checkpoint Receptors PD-1 and GITR Turnover Rates In Vivo in Humans. Clin Pharmacol Ther.109(6), (2021).
- Briggs RJ , NicholsonR, VazvaeiFet al. Method Transfer, Partial Validation, and Cross Validation: Recommendations for Best Practices and Harmonization from the Global Bioanalysis Consortium Harmonization Team. AAPS Journal.16(6), (2014).
- Stegemann S , SheehanL, RossiAet al. Rational and Practical Considerations to Guide a Target Product Profile for Patient-Centric Drug Product Development with Measurable Patient Outcomes – A Proposed Roadmap. European Journal of Pharmaceutics and Biopharmaceutics177 (2022).
- Bower J , ZimmerJ, McCownSet al. Recommendations for the Content and Management of Certificates of Analysis for Reference Standards from the GCC for Bioanalysis. Bioanalysis. bio-2021-0046 (2021).
- international Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use . Bioanalytical Method Validation and Study Sample AnalysiS M10. https://database.ich.org/sites/default/files/M10_Guideline_Step4_2022_0524.pdf (2022).
- Nehls C , BuonaratiM, CapeSet al. GCC Consolidated Feedback to ICH on the 2019 ICH M10 Bioanalytical Method Validation Draft Guideline. Bioanalysis.11(18s), 1–228 (2019).
- U.S. Department of Health and Human Services Food and Drug Administration Center for Drug Evaluation and Research (CDER) Center for Biologics Evaluation and Research (CBER) . Clinical Pharmacology Data to Support a Demonstration of Biosimilarity to a Reference Product Guidance for Industry. https://www.fda.gov/media/88622/download (2016).
- Obianom ON , OkusanyaOO, EarpJ, ThwayTM. A Simulation Approach to Evaluate the Impact of Patterns of Bioanalytical Bias Difference on the Outcome of Pharmacokinetic Similarity Studies. Clin Pharmacol Ther.108(1), (2020).
- Obianom ON , ThwayTM, SchrieberSJet al. Retrospective Analysis of Bioanalytical Method Validation Approaches in Biosimilar Biological Product Development. AAPS Journal.21(6), (2019).
- Thway TM , WangYM, BoothBP, MaxfieldK, HuangSM, ZinehI. Current Perspectives on Ligand-Binding Assay Practices in the Quantification of Circulating Therapeutic Proteins for Biosimilar Biological Product Development. AAPS Journal.22(1), (2020).
- U.S. Department of Health and Human ServicesFood and Drug AdministrationCenter for Drug Evaluation and Research (CDER) . Evaluation of Internal Standard Responses During Chromatographic Bioanalysis: Questions and Answers. https://collections.nlm.nih.gov/catalog/nlm:nlmuid-101760391-pdf (2019).
- Mercer TR , XuJ, MasonCE, TongW. The Sequencing Quality Control 2 Study: Establishing Community Standards for Sequencing in Precision Medicine. Genome Biol22(1), (2021).
- Jones W , GongB, NovoradovskayaNet al. A Verified Genomic Reference Sample for Assessing Performance of Cancer Panels Detecting Small Variants of Low Allele Frequency. Genome Biol.22(1), (2021).
- Willey JC , MorrisonTB, AustermillerBet al. Advancing NGS Quality Control to Enable Measurement of Actionable Mutations in Circulating Tumor DNA. Cell Reports Methods.1(7), (2021).
- Gong B , LiD, KuskoRet al. Cross-oncopanel Study Reveals High Sensitivity and Accuracy with Overall Analytical Performance Depending on Genomic Regions. Genome Biol.22(1), (2021).
- Deveson IW , GongB, LaiKet al. Evaluating the Analytical Validity of Circulating Tumor DNA Sequencing Assays for Precision Oncology. Nat Biotechnol.39(9), (2021).
- Zhang Y , BlomquistTM, KuskoRet al. Deep Oncopanel Sequencing Reveals Within Block Position-Dependent Quality Degradation in FFPE Processed Samples. Genome Biol.23(1), (2022).
- Prior S , MetcalfeC, HuftonSE, WadhwaM, SchneiderCK, BurnsC. Maintaining ‘Standards’ for Biosimilar Monoclonal Antibodies. Nat Biotechnol39(3), (2021).
- Ohtsu Y , TanakaS, IgarashiHet al. Analytical Method Validation for Biomarkers as a Drug Development Tool: Points to Consider. Bioanalysis.13(18), (2021).
- Guinn D , MadabushiR, WangYM, BrodskyE, ZinehI, MaxfieldK. Communicating Immunogenicity-Associated Risk in Current U.S. FDA Prescription Drug Labeling: A Systematic Evaluation. Ther Innov Regul Sci.54(6), (2020).
- Shankar G , ArkinS, CoceaLet al. Assessment and Reporting of the Clinical Immunogenicity of Therapeutic Proteins and Peptides - Harmonized Terminology and Tactical Recommendations. AAPS Journal16(4), (2014).
- Myler H , Pedras-VasconcelosJ, PhillipsKet al. Anti-drug Antibody Validation Testing and Reporting Harmonization. AAPS Journal.24(1), (2022).
- Lamberth K , Reedtz-RungeSL, SimonJet al. Post Hoc Assessment of the Immunogenicity of Bioengineered Factor VIIa Demonstrates the use of Preclinical Tools. Sci Transl Med.9(372), (2017).
- Simhadri VL , McGillJ, McMahonS, WangJ, JiangH, SaunaZE. Prevalence of Pre-existing Antibodies to CRISPR-Associated Nuclease Cas9 in the USA Population. Mol Ther Methods Clin Dev.10 (2018).
- Simhadri VL , HopkinsL, McGillJRet al. Cas9-Derived Peptides Presented by MHC Class II that Elicit Proliferation of CD4+ T-cells. Nat Commun.12(1), (2021).
- Tang XZE , TanSX, HoonS, YeoGW. Pre-Existing Adaptive Immunity to the RNA-editing Enzyme Cas13d in Humans. Nat Med.28(7), (2022).
- Charlesworth CT , DeshpandePS, DeverDPet al. Identification of preexisting adaptive immunity to Cas9 proteins in humans. Nat Med.25(2), (2019).
- Wagner DL , PeterL, Schmueck-HenneresseM. Cas9-Directed Immune Tolerance in Humans—A Model to Evaluate Regulatory T cells in Gene Therapy?Gene Ther28(9), (2021).
- Morais P , AdachiH, YuYT. The Critical Contribution of Pseudouridine to mRNA COVID-19 Vaccines. Front Cell Dev Biol9 (2021).
- Andries O , McCafferty S, DeSmedt SC, WeissR, SandersNN, KitadaT. N1-methylpseudouridine-incorporated mRNA Outperforms Pseudouridine-incorporated mRNA by Providing Enhanced Protein Expression and Reduced Immunogenicity in Mammalian Cell Lines and Mice. Journal of Controlled Release.217 (2015).
- Poolman J , FraschC, NurkkaA, KäyhtyH, BiemansR, SchuermanL. Impact of the Conjugation Method on the Immunogenicity of Streptococcus Pneumoniae Serotype 19F Polysaccharide in Conjugate Vaccines. Clinical and Vaccine Immunology.18(2), (2011).
- Valentini S , SantoroG, BaffettaFet al. Monocyte-activation Test to Reliably Measure the Pyrogenic Content of a Vaccine: An In Vitro Pyrogen Test to Overcome In Vivo Limitations. Vaccine.37(29), (2019).
- Shibata H , NishimuraK, MaedaTet al. Evaluation of the Analytical Performance of Anti-SARS-CoV-2 Antibody Test Kits Distributed or Developed in Japan. Bioanalysis.14(6), (2022).
- Furuta-Hanawa B , YamaguchiT, UchidaE. Two-Dimensional Droplet Digital PCR as a Tool for Titration and Integrity Evaluation of Recombinant Adeno-Associated Viral Vectors. Hum Gene Ther Methods.30(4), (2019).