Forced degradation of recombinant monoclonal antibodies: A practical guide

Figures & data

Table 1. Purposes and rationale of forced degradation studies.

Purpose	Rational
Manufacturability evaluation	Evaluation of the propensity of multiple candidates to degrade under relevant process, formulation and storage conditions. Comparison of the intrinsic stability under various forced degradation conditions.
Formulation development	Identification of conditions such as buffers, excipients, pH, and/or temperature to provide appropriate long-term stability.
Method development	Using degraded samples to test method parameters such as resolution, limit of detection (LOD), limit of quantitation (LOQ) etc., and also to establish stability-indicating methods at early stage.
Method transfer	Using degraded materials to further ensure a successful transfer.
Method qualification and validation	Degraded samples used to qualify and validate method parameters and validate stability-indicating capabilities.
Product variants and impurities isolation and characterization	Generation of higher amounts of relevant product variants or impurities that are normally present at low levels in drug substance.
Generating materials for Critical Quality Attributes (CQA) assessment	Materials with specific modifications can be generated using forced degradation conditions at much higher abundance, facilitating identification.
Intrinsic stability of the products	Forced degradation can help define the boundary of instability under various environmental factors.
Degradation pathways	Characterization of the degradation products to define the major degradation pathways.
Comparability	Differences that cannot be detected by release and extended characterization can become detectable under forced degradation conditions in a relatively shorter time period. Same degradation pathways and comparable degradation kinetics further ensure comparability.
Excursion	Data from forced degradation conditions can be used to evaluate accidental exposure to extreme environmental conditions (pH, temperature, light, etc.) that are not covered by stability studies.

Figure 1. Major degradation pathways of recombinant monoclonal antibodies. Arrows indicate the main degradation sites. The commonly used methods to detect the degradation products are shown in the parenthesis.

Table 2. Recommended pre-screening conditions and methods.

Conditions	Suggested experiment design	Major degradation pathways	Methods to monitor
High temperature	Temperature: 40, 50°C	Aggregates (Precipitates, Particles, Soluble aggregates; covalent and non-covalent), fragments, oxidation, deamidation	SEC to monitor aggregates
	Time-points: Day 0, 1, 2, 3, 4		CEX or IEF to monitor charge
Freeze-thaw	Freeze to −80°C (or other temperature), thaw at a predefined temperature	Aggregates (Precipitates, Particles, Soluble aggregates)	SEC to monitor aggregates
	Repeat 3–8 cycles
Agitation	Shaking: 200 rmp	Aggregates (Precipitates, Particles, Soluble aggregates; covalent and non-covalent)	SEC to monitor aggregates
	Stirring: 100 rmp
	Temperatures: Room temperature
	Time points: Day 0, 3, 5
Low pH	pH 3, 4, 5	Aggregates (Precipitates, Particles, Soluble aggregates),aspartic acid isomerization	SEC to monitor aggregates
	Temperatures: 25, 30, 37°C
	Time points: Day 0, 1, 2, 3, 4
High pH	pH 9, 10	Aggregates (Precipitates, Particles, Soluble aggregates; covalent and non-covalent), deamidation	SEC to monitor aggregates
	Temperatures: 25, 30, 37°C		CEX or IEF to monitor charge
	Time points: Day 0, 1, 2, 3, 4		LC-MS peptide mapping to determine site-specific deamidation
Photo	Starting with 0.25x, 0.5X and 1x, ICH suggested exposure	Aggregates (Precipitates, Particles, Soluble aggregates; covalent and non-covalent), methionine and tryptophan oxidation	SEC or SEC-MALs to monitor aggregates
			CEX or IEF to monitor charge
Oxidation	Starting with incubation with hydrogen peroxide at a concentration of 0.02, 0.05, 0.1, 0.2, 0.5, 1% at predefined temperature for 1, 2, 3, and 4 hours	Oxidation of methionine and tryptophan residues	SEC to monitor aggregates
			RP-HPLC or HIC-HPLC to monitor hydrophobicity
			LC-MS of intact or reduced molecular weight analysis to monitor molecular weights
Glycation	Adding glucose to the samples using various molar ratios of glucose to antibody (or amine groups) and incubate at 37°C for different days	Modification of lysine residues	Boronate affinity chromatography to monitor overall glycation level
			CEX or IEF to monitor charge
			LC-MS of intact or reduced molecular weight analysis to monitor molecular weights

Table 3. Areas of applications, objectives and analytical tools of forced degradation studies throughout the lifecycle of mAb.

Study Type	Suggested forced degradation conditions	Suggested Analytical Toolkit	Development Stage
Manufacturability	Limited (prioritize heat; sequence-driven. e.g., if susceptible residues in CDR, include oxidation; if susceptible deamidation residues in CDR, include deamidation)	High throughput, low volume platform assays (e.g., SEC, cIEF)	Pre-candidate selection
Formulation development	Full Panel	Full panel of Release/characterization	IND-enabling^1 / Phase 1 to Phase 3^2
Method Development	Limited (prioritize conditions to generate realistic degradants and/or increase abundance of a single species, if possible)	Full panel of Release/characterization; Add assays based on observations	IND-enabling^1 / Phase 1 to Phase 3^2
Method transfer, Method Qualification and Validation	Limited (prioritize conditions to generate realistic degradants and/or increase abundance of a single species, if possible)	Full panel of Release/characterization; Add assays based on observations	Phase 1 to Phase 3; Post-launch
Product variants and impurities isolation and characterization	Limited (design based on release/stability assay for which peaks are being identified)	Full panel of release/characterization; LC-MS and peptide map or other ID methods as appropriate	BLA preparation
CQA Assessment	Full Panel	Full panel of Release/characterization; Add assays based on observations. LC-MS to identify degradation products	BLA preparation
Degradation pathways	Full Panel	Full panel of Release/characterization; Add assays based on observations. LC-MS to identify degradation products	BLA preparation
Comparability	Limited (based on prior knowledge)	Full panel of release and characterization assays; focus on assays that can potentially detect the differences if present and assays that can provide degradation trending	Last-stage and post-launch
Excursion	Limited (based on prior knowledge)	Release and selected characterization	As needed

¹ Initial development to support clinical

² Final development to support product launch

Table 4. Relevant guidance documents on forced degradation.

Documents	Forced degradation Application
ICH Q5C: Stability Testing of Biotechnological/Biological Products1995http://www.ich.org/products/guidelines/quality/article/quality-guidelines.htmlICH Q1B, Stability testing: Photostability testing of new drug substances and products	• Storage• Stability-indicating methods• Degradation pathways• Excursion• Supporting data to stability• Photo degradation pathways• Method development• Generation of information for product handling, packing, and labeling
1996
http://www.ich.org/products/guidelines/quality/article/quality-guidelines.html
ICH Q6B Specifications: Test procedures and acceptance criteria for biotechnological/biologic products	• Establishment of stability profiles, e.g. degradation pathways and stability-indicating methods
1999
https://www.fda.gov/downloads/drugs/guidancecomplianceregulatoryinformation/guidances/ucm073488.pdf
ICH M4Q(R1): The common technical document for the registration of pharmaceuticals for human use: Quality-M4Q(R1)	• Degradation pathways
Quality overall summary of module 2 module 3:quality
http://www.ich.org/products/ctd/ctdsingle/article/m4qr1-quality.html
ICH Q1A (R2): Stability Testing of New Drug Substances and Products	• Degradation pathways• Intrinsic stability• Stability-indicating methods
2003
http://www.ich.org/products/guidelines/quality/article/quality-guidelines.html
FDA Guidance for industry: INDs for phase II and III studies	• Degradation pathways• Intrinsic stability• Stability-indicating methods
2003
https://www.fda.gov/downloads/drugs/guidancecomplianceregulatoryinformation/guidances/ucm070567.pdf
ICH Q5E: Comparability of biotechnological/biologic products subject to changes in their manufacturing process	• Comparability
2004
http://www.ich.org/products/guidelines/quality/article/quality-guidelines.html
ICH Q2(R1) Validation of an`alytical procedures: text and methodology	• Specificity of analytical methods
2005
http://www.ich.org/products/guidelines/quality/article/quality-guidelines.html
FDA Analytical procedures and methods validation for drugs and biologics	• Specificity of stability-indicating methods
2015
https://www.fda.gov/downloads/drugs/guidancecomplianceregulatoryinformation/guidances/ucm386366.pdf
EMA Guideline on the requirements for quality documentation concerning biologic investigational medicinal products in clinical trials, Draft	• Degradation profiles• Stability indicating methods
2016
http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2016/07/WC500209618.pdf