Conference Report: Bioanalysis-Related Highlights from the 2011 AAPS National Biotechnology Conference

Abstract

The American Association of Pharmaceutical Scientists is a dynamic international forum for the exchange of knowledge among scientists to enhance their contributions to drug development. The annual National Biotechnology Conference, conducted and organized by the American Association of Pharmaceutical Scientists, is a forum dedicated to advancements in science and technology related to discovery, development and manufacture of medical biotechnology products. The 2011 National Biotechnology Conference meeting convened in San Francisco, CA, USA on 16–18 May. Over 300 abstracts were submitted and approximately 50 sessions examined topics pertaining to advances in drug development, emerging analytical technologies, bioanalysis-related issues, biosimilar therapies, updates on global regulatory documents and expectations, and other topics. The focus of this article is to highlight key developments relevant to immunogenicity and pharmacokinetic drug concentration bioanalysis.

Emerging technologies

Pharmacokinetic assessments

The landscape of bioanalytical technology platforms continues to advance. While ELISA or electrochemiluminescent immunoassays (ECLIA) still comprise the workhorse platforms for large-molecule pharmacokinetic and immunogenicity bioanalysis, newer technologies are proving to be viable alternatives, sometimes offering better sensitivity, increased throughput and lower matrix interference. A short course titled “Emerging Bioanalytical Techniques for Macromolecules (Platforms and Applications) – Technical and Regulatory Perspectives” hosted a series of speakers from the pharmaceutical and regulatory sectors who focused their talks on highlighting their experiences with new ligand-binding assay platforms as well as providing regulatory perspectives for submissions including bioanalysis using new platforms. Tatiana Plavina (Biogen Idec, USA) discussed the Imperacer^® (Chimera Biotec, Dortmund, Germany) Immuno-PCR platform, akin to a standard sandwich immunoassay format but utilizes a DNA-conjugated antibody for detection which, through PCR, amplifies the assay signal. Due to this format, Plavina cited improved sensitivity compared with typical ELISAs, in particular when testing complex matrices such as serum, plasma or cerebrospinal fluid. The enhanced sensitivity could allow options for various other assay parameter ‘trade-offs’ to be made, such as increased dilution to reduce matrix-based interferences. Furthermore, two contributing posters [1,2] also focused on the improved sensitivity obtained with the Immuno-PCR technology, with one group indicating a 100-fold improved sensitivity over their existing ELISA with a LLOD of 15 pg/ml [1].

Various hyphenated MS-based technologies represent an area of increased focus of current bioanalytical research. Traditionally applied for small-molecule bioanalysis, technological advancements and research focused on extending their application for the detection of peptides and proteins has already shown tremendous potential. Michael Zhou (Synta Pharmaceuticals, USA), focused on the use of LC–MS/MS to quantify proteins, especially those that typically have high levels of interference because of matrix issues. In his presentation titled “LC–MS/MS Applications for Macromolecule Therapeutics and Biomarkers Analysis”, advantages of the platform were discussed, including abbreviated method development timelines and improved dynamic range, whereas disadvantages included high cost, typically low-to-moderate assay throughput and inadequate sensitivity in comparison with ELISAs. Multiple contributing posters also weighed in on this topic, successes reported in the quantification of a relatively small protein in dog blood [3], to full-size therapeutic monoclonal antibodies in rat plasma samples [4] and therapeutic–Fc fusion proteins in rat and monkey sera [5].

A third platform that was highlighted in multiple contributing poster presentations was the Gyros Gyrolab™ (NJ, USA) platform. The platform utilizes centrifugal force and capillary action to create a sandwich immunoassay on streptavidin-coated beads. Jeffery Sailstad (Sailstad and Associates, Inc., USA) discussed some of the benefits of this platform, highlighting the nanoliter scale within the Bioaffy™ CD allowing for significantly reduced sample volume when compared with a typical ELISA method as well as reduced matrix effects. The manufacturer advertises a broader dynamic range and greater precision, theoretically minimizing the number of dilutions normally required for test samples and a reduced proportion of bioanalytical repeat analyses. Sailstad advised that is it imperative to fully understand the technology that any platform offers, and to verify the results obtained before bioanalytical implementation. Further corroborating the use of the Gyrolab platform were multiple contributing papers including a poster that demonstrated an extended range of quantification [6], but also highlighted were various areas of concern typically not seen with ELISA or MSD technologies including; carry-over, high coefficient of variation and software capabilities [7].

Immunogenicity assessments

Several posters were presented highlighting new technologies for the detection of antidrug antibodies (ADAs) as well as technological improvements of existing immunoassay platforms.

A poster by Ray Yin (ANP Technologies, Inc., USA) demonstrated the application of a rapid lateral-flow immunogenicity assay to detect ADAs. The assay uses a proprietary process called Nano-Intelligent Detection System (NIDS^®), in which patient sera are incubated with a mixture of biotinylated and hapten-labeled drug to form a bridge complex. The complex is then introduced to a strip of antihapten antibodies. The biotinylated drug complex is then reacted with streptavidin-labeled gold particles and the assay signal is quantitatively measured with a handheld reader [8]. ANP claimed that this novel platform offers several advantages over standard immunoassays, including elimination of sample dilution and wash steps, reduced assay time, and most importantly, enhanced drug tolerance while maintaining equivalent assay sensitivity.

PEGylation (polyethyleneglycol) of biotherapeutics has become an increasingly popular strategy to increase the half-life of a biotherapeutic and also to potentially reduce its immunogenicity potential, despite the reported presence of pre-existing antibodies reactive with PEG [9]. Hence, one contribution at the conference examined the development of a double antigen bridging ELISA for the detection of anti-PEG antibodies [10]. In this method, hapten-labeled PEG polymer (40 kDa) is incubated with patient sera, and then incubated on a PEG-coated microplate to form a bridge complex. The plate is then washed and detection is achieved by incubation of horseradish peroxidase-labeled antihapten antibody, followed by another wash step, TMB incubation, and absorption measurement at 450/630 nm after addition of acid to stop the reaction. This method used a unique anti-PEG mouse monoclonal IgM antibody as a positive control that is capable of recognizing PEG of various lengths, and thus, presents a novel application in that it can be used as a universal positive control for any immunogenicity assay for PEGylated biotherapeutics.

Current US FDA guidance recommends the use of cell-based bioassays to detect neutralizing antibodies (NAbs) since they can be more representative of in vivo situations; however, these types of assays have many practical and technical limitations such as variability, limited dynamic range and long assay duration [11]. Two posters presented at the American Association of Pharmaceutical Scientists (AAPS) highlighted new and improved techniques applicable to cell-based assays. The first poster illustrated the development of a serum-free bioassay for the detection of NAbs [12]. In this method, biotinylated drug is incubated with patient sera in a streptavidin-coated plate, followed by a wash step to remove excess sera and thereafter the drug ligand. Free ligand is then measured through luciferase activity of A204pMARE-luc cells. The presence of NAbs prevents the ligand from binding to the biotinylated drug on streptavidin-coated plate, causing an increase in free ligand and increased luciferase activity in the cells. In the absence of NAbs, the ligand is immobilized by the biotinylated drug, causing a decrease in levels of free ligand and, as such, the luciferase activity. In this method, all protein–protein interactions occur in a solid phase rather than the traditional liquid phase. The removal of serum from the plate reduces its toxic and variable physiological effects on cells, provides better assay sensitivity, reduced variability and improved drug tolerance. Cell-based bioassays require continuous maintenance of cell cultures, which can be very labor intensive and require a broad variety of specialized materials and equipment. Thus, the second poster presented a simpler and practical approach to improve assay sensitivity, reduce variability, and increase operational efficiency and flexibility of all types of cell-based bioassays [13]. In order to overcome these problems, cryo-preserved cells were utilized. A CHO K1 cell line expressing the drug target receptor was stored in liquid nitrogen for a period of time, thawed before use and re-suspended in culture media before being added to the assay plate. Assay performance using frozen cells was compared with a validated assay that uses fresh cells. Results indicated that the use of frozen cells led to fourfold better assay sensitivity and precision nearly 200%, while also promoting increased operational efficiency due to the elimination of the need to maintain a living culture.

Plausible solutions to bioanalytical issues

Drug interference in immunogenicity assays

Sample pretreatment with acid is a common industry method used to dissociate drug–ADA complexes. This approach is widely believed to increase the drug tolerance of bridging immunoassays, thus improving sensitivity and tolerance. However, two posters [14,15] provided examples of studies demonstrating contrasting results that had the use of acid pretreatment of samples and produced adverse bioanalytical results. In the first example, acid pretreatment was used for an IR assay of a PEGylated peptide without an improvement in sensitivity or drug tolerance. Instead, improvements in sensitivity and drug tolerance were achieved by implementing a cocktail bridging assay. The cocktail consisted of two pairs of drug conjugates specifically labeled in areas where ADA binding sites are unhindered and more detectable [14]. The second example evaluated the effects of acid pretreatment on mAb drug bound target. Five distinct ADA bridging immunoassays were studied for drugs with mono- or di-meric soluble targets. This was done because monomeric drug targets usually produce false-negative data in bridging immunoassays, whereas homodimeric or higher order targets can produce false-positive data, which remain unidentified despite competitive inhibition-based specificity confirmation methods. Mock samples containing different combinations of biologic drug, its target and ADA were generated for each assay, then treated both with and without acid to determine whether interference due to soluble drug target existed. Results indicated that, in certain instances, acid treatment released dimeric soluble targets from the drug and caused a false-positive result. In other cases, acid treatment did not release drug target [15]. This leads one to conclude that acid pretreatment to enhance drug tolerance taken for granted as a method enhancement in the absence of sufficient research on the effects of the low pH conditions on other immune complexes (such as drug and target), which when also dissociated, could cause interferences in the method. Hence caution should be exercised and unintended consequences of such approaches be pondered.

Pharmacokinetic drug concentration assay ruggedness

While bioanalytical technology manufacturers continue to develop enhancements to tackle the nuances of immunoassays, there are still areas of persistent challenge. Method ruggedness for drug concentration assays is one of those challenging areas and inadequate ruggedness can lead to method transfer failures or complications with method-to-method comparisons. Two common means of testing method ruggedness is by exploring cross-validation and incurred sample reproducibility. There are multiple instances when cross-validation is required, nicely highlighted by Mark Ma (Amgen, USA) in his discussion titled, “When, What and How: The Issues in Method Cross-Validation”. Those instances include when two or more methods are used for the same study or when a single method is used in two laboratories. Appropriate study design for cross-validation conduct is critical and acceptance criteria must be set a priori, but those acceptance criteria vary from company to company, with most taking the mean ratio of the results and applying either a 90 [16] or 95% [17] confidence interval to the original result. As expected, failed cross-validations occur, but the sources for those failures vary due to reagent instability or disparate analyst performance [18] to inappropriate selection of incurred sample reproducibility samples used in the cross-validation [19].

Sample volume: dried blood spots

The need to ‘do less with more’ is more prominent than ever, particularly in regard to the volume of matrix samples obtained in nonclinical and clinical studies. Utilization of dried blood spots (DBS) has garnered significant attention over the last 3 to 5 years, as highlighted by Matthew Szapacs (GlaxoSmithKline, USA) in his discussion, “Method Development and Validation for the Quantitative Assessment of Biologics using Dried Blood Spots with LC–MS/MS and/or Immunoassay Detection Schemes”. Szapacs highlighted the feasibility of implementing DBS with large molecules and various challenges in method development including; spotting matrixes other than blood, the manual punching process, bridging historical data, spotting standard curve points and quality controls, as well as post-spotting card stability. Another key element to evaluate for quantification of the protein of interest is the development of a method for digestion of that protein. Some cards are pretreated allowing for immediate digestion of the protein, while other cards are untreated requiring additional steps which, as Szapacs described, can produce inconsistent digests especially when the samples are more complex. A contributing poster highlighted the feasibility of using DBS when measuring a therapeutic antibody in serum, but lack of long-term post-blotting stability continues to be a pivotal issue [20]. Until further experience is gained, the use of DBS remains a plausible sample collection tool and may be beneficial when sample volume is an issue. It is intriguing that sample volume is often cited as a reason for DBS application when assay miniaturization (i.e., use of 384-well microplate-based immunoassays) could adequately address the issue. In fact DBS may offer other benefits such as simplified collection procedures, shipping, storage conditions and lowering of associated costs.

Reagent stability

Two posters focused on the effects that variability between reagent vendors, or between lots (batches) of reagents from a single vendor, can have on pharmacokinetics and immunogenicity assays. One poster provided specific examples in which bovine serum albumin (BSA) obtained from a vendor, when used in assay diluent in an immunogenicity assay produced a dramatic increase in OD values for normal sera pool, high and low positive controls, and naive individual sera samples in stark contrast to BSA acquired from a second vendor. Similarly, dramatic differences in calibration curves were noted between two different lots of BSA produced by the same vendor in a drug concentration assay [21]. Another poster determined that poor recovery of the LLOQ in a drug concentration assay was directly attributed to a specific lot of Tween-20 [22]. These examples demonstrate that the source and lot of particular reagents can have an effect on both drug concentration and immunogenicity assays, emphasizing the value in using a variety of lots and suppliers when performing such analyses.

Automation

Automation of immunoassays is an increasingly applied strategy for increasing bioanalytical throughput as well as improving overall precision. Other benefits of automation include reduced headcount, mitigation of repetitive strain injuries among bioanalytical scientists and higher volume production. On the other hand, automation is still not cost effective in some cases due to the substantial capital investment required at the outset to procure equipment. Additional considerations of automated analysis devices include the increased consumption of reagents, the high cost of disposables and the regular validation, maintenance and calibration requirements of such equipment. In addition, automation startup requires significant time to develop and requires highly trained and experienced specialists who not only grasp the concepts of immunoassay development but also have a strong command of the programming structure and logic [23]. However, once implemented and optimized, as data in one poster demonstrated, automation using the Hamilton STARlet for reagent and sample additions can be as accurate, precise and robust as a bench analyst with data showing similar coefficient of variation and recovery [24].

Forging ahead: updates on global harmonization

The session “Updates on Global Harmonization of Bioanalytical Regulations, Global Bioanalytical Consortium (GBC) and Impact on Ligand-Binding Assays”, brought key experts in the field together to discuss global harmonization. Brian Booth (FDA, USA) discussed that an updated version of the FDA Bioanalytical Guidance [25] was to be expected by the end of the year. Fabio Garofolo (Algorithme Pharma, Inc., USA) proposed a harmonization process as outlined by the GBC in his talk titled “Updates on Global Harmonization of Bioanalysis Regulations and Activities of the Global Bioanalytical Consortium”, which focuses on obtaining key input from various scientific associations from around the world, reviewing the current status of the bioanalytical guidelines and then recommending the best practices. The GBC anticipates US and EU recommendations within the next 12 months.

Summary

The 2011 AAPS National Biotechnology Conference (NBC) meeting programming continues to focus on critical areas of scientific interest, bioanalytical trends and the current state of affairs in the biotechnology arena. While many different platforms were presented as viable options to assess immunogenicity and pharmacokinetic drug concentrations, it is imperative, regardless of the platform, to fully understand the underlying technology, methodologies, and analytical systems before implementation in bioanalysis. Several topics highlighting common bioanalytical issues pertaining to acid treatment of samples, reagent stability, automation and assay ruggedness were presented to emphasize their importance in immunogenicity and pharmacokinetic drug concentration assays. The NBC meeting has provided a valuable opportunity for scientists to exchange ideas amongst their peers. The next convention (2012 AAPS NBC) will be held on 21–23 May 2012 in San Diego, CA, USA.

Acknowledgements

The authors would like to acknowledge Gopi Shankar for guidance with writing this manuscript.

Financial & competing interests disclosure

The authors are employees of Centocor Research and Development, a division of Johnson & Johnson Pharmaceutical Research & Development, LLC and own stock in Johnson & Johnson, Inc. The authors have no other 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 apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.