Monoclonal antibodies (mAbs) are widely used as critical reagents in analytical assays. While regulatory guidelines exist for stability monitoring of biopharmaceutical antibodies, they do not apply directly to the stability of mAbs used as assay reagent. We investigated alternative approaches to real-time stability monitoring of assay reagents. We compared functional (ELISA and cell-based) and biochemical (aggregation, deamidation) assay results using temperature-stressed mAb reagents. Data from both assay groups were compared for indications of antibody degradation. Arrhenius model kinetics was used to further extrapolate stability trends. Changes detected by traditionally monitored biochemical changes were not directly predictive of assay function. Instead, monitoring of reportable results was a closer indication of changes in assay performance related to mAb degradation. Using Arrhenius kinetic modeling, we combined forced degradation of individual reagents with reportable assay results to classify reagents into risk groups with associated re-evaluation and monitoring plans. This combined approach mitigates risk by monitoring each mAb reagent individually under stressed conditions while streamlining expiry assignment through simplified Arrhenius kinetics with only limited real-time stability data.
This is a preview of subscription content, log in to check access.
Buy single article
Instant access to the full article PDF.
Tax calculation will be finalised during checkout.
Subscribe to journal
Immediate online access to all issues from 2019. Subscription will auto renew annually.
Tax calculation will be finalised during checkout.
Arturo C, Scharff MD. Return to the past: the case for antibody-based therapies in infectious diseases. Clin Infect Dis [Internet]. Clin Infect Dis; 1995 [cited 2020 Aug 16];21:150–61. Available from: https://pubmed.ncbi.nlm.nih.gov/7578724/
Nissim A, Chernajovsky Y. Historical development of monoclonal antibody therapeutics. Handb Exp Pharmacol [Internet]. Handb Exp Pharmacol; 2008 [cited 2020 Aug 16]. p. 3–18. Available from: https://pubmed.ncbi.nlm.nih.gov/18071939/
Köhler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature [Internet]. Nature; 1975 [cited 2020 Aug 16];256:495–7. Available from: https://pubmed.ncbi.nlm.nih.gov/1172191/
Nelson AL, Dhimolea E, Reichert JM. Development trends for human monoclonal antibody therapeutics [Internet]. Nat. Rev. Drug Discov. Nat Rev Drug Discov; 2010 [cited 2020 Aug 16]. p. 767–74. Available from: https://pubmed.ncbi.nlm.nih.gov/20811384/
Ecker DM, Jones SD, Levine HL. The therapeutic monoclonal antibody market [Internet]. MAbs. Landes Bioscience; 2015 [cited 2020 Aug 16]. p. 9–14. Available from: https://pubmed.ncbi.nlm.nih.gov/25529996/
ICH. International conference on harmonisation of technical requirements for registration of pharmaceuticals for human use ich harmonised tripartite guideline evaluation for stability data Q1E [Internet]. www.ich.org. 2003. p. 1–19. Available from: https://database.ich.org/sites/default/files/Q1E_Guideline.pdf
Nowak C, K. Cheung J, M. Dellatore S, Katiyar A, Bhat R, Sun J, et al. Forced degradation of recombinant monoclonal antibodies: a practical guide [Internet]. MAbs. Taylor and Francis Inc.; 2017 [cited 2020 Aug 16]. p. 1217–30. Available from: https://pubmed.ncbi.nlm.nih.gov/28853987/
Jaccoulet E, Daniel T, Prognon P, Caudron E. Forced degradation of monoclonal antibodies after compounding: impact on routine hospital quality control. J Pharm Sci [Internet]. Elsevier B.V.; 2019 [cited 2020 Aug 16];108:3252–61. Available from: https://pubmed.ncbi.nlm.nih.gov/31201907/
Halley J, Chou YR, Cicchino C, Huang M, Sharma V, Tan NC, et al. An industry perspective on forced degradation studies of biopharmaceuticals: survey outcome and recommendations [Internet]. J. Pharm. Sci. Elsevier B.V.; 2020 [cited 2020 Aug 16]. p. 6–21. Available from: https://pubmed.ncbi.nlm.nih.gov/31563512/
Pihl S, Van Der Strate BWA, Golob M, Ryding J, Vermet L, Jaitner B, et al. EBF recommendation on practical management of critical reagents for antidrug antibody ligand-binding assays. Bioanalysis [Internet]. Future Medicine Ltd.; 2019 [cited 2020 Aug 16];11:1787–98. Available from: https://pubmed.ncbi.nlm.nih.gov/31657235/
King LE, Farley E, Imazato M, Keefe J, Khan M, Ma M, et al. Ligand binding assay critical reagents and their stability: recommendations and best practices from the global bioanalysis consortium harmonization team. AAPS J [Internet]. Springer New York LLC; 2014 [cited 2020 Aug 16];16:504–15. Available from: https://pubmed.ncbi.nlm.nih.gov/24687208/
Lequin RM. Enzyme immunoassay (EIA)/enzyme-linked immunosorbent assay (ELISA). Clin Chem [Internet]. Clin Chem; 2005 [cited 2020 Aug 16];51:2415–8. Available from: https://pubmed.ncbi.nlm.nih.gov/16179424/
Pepple J, Moxon ER, Yolken RH. Indirect enzyme-linked immunosorbent assay for the quantitation of the type-specific antigen of Haemophilus influenzae b: a preliminary report. J Pediatr [Internet]. J Pediatr; 1980 [cited 2020 Aug 16];97:233–7. Available from: https://pubmed.ncbi.nlm.nih.gov/6995567/
Pose AG, Rodríguez ER, Piñeiro MJ, Montesino R, Sánchez O, Toledo JR. Quantitative ELISA sandwich for a new vaccine against avian influenza virus H5N1. J Immunol Methods [Internet]. Elsevier B.V.; 2018 [cited 2020 Aug 16];459:70–5. Available from: https://pubmed.ncbi.nlm.nih.gov/29803776/
Zhang R, Ulery BD. Synthetic vaccine characterization and design. J Bionanoscience American Scientific Publishers; 2018. p. 1–11.
Florentinus-Mefailoski A, Safi F, Marshall JG. Enzyme linked immuno mass spectrometric assay (ELIMSA). J Proteomics [Internet]. Elsevier; 2014 [cited 2020 Aug 16];96:343–52. Available from: https://pubmed.ncbi.nlm.nih.gov/24316356/
Kulasingam V, Smith CR, Batruch I, Diamandis EP. Immuno-mass spectrometry: quantification of low-abundance proteins in biological fluids. Methods Mol Biol [Internet]. Methods Mol Biol; 2011 [cited 2020 Aug 16];728:207–18. Available from: https://pubmed.ncbi.nlm.nih.gov/21468950/
Levine MM, Sztein MB. Vaccine development strategies for improving immunization: the role of modern immunology [Internet]. Nat. Immunol. Nat Immunol; 2004 [cited 2020 Aug 16]. p. 460–4. Available from: https://pubmed.ncbi.nlm.nih.gov/15116108/
Vaccine Testing and Approval Process | CDC [Internet]. [cited 2020 Aug 16]. Available from: https://www.cdc.gov/vaccines/basics/test-approve.html
Kayser V, Chennamsetty N, Voynov V, Helk B, Forrer K, Trout BL. Evaluation of a non-arrhenius model for therapeutic monoclonal antibody aggregation. J Pharm Sci [Internet]. John Wiley and Sons Inc.; 2011 [cited 2020 Aug 16];100:2526–42. Available from: https://pubmed.ncbi.nlm.nih.gov/21268027/
Bajaj S, Sakhuja N, Singla D. Stability testing of pharmaceutical products. J Appl Pharm Sci. 2012:129–38.
Kommanaboyina B, Rhodes CT. Trends in stability testing, with emphasis on stability during distribution and storage [Internet]. Drug Dev. Ind. Pharm. Drug Dev Ind Pharm; 1999 [cited 2020 Aug 16]. p. 857–68. Available from: https://pubmed.ncbi.nlm.nih.gov/10459490/
Duddu SP, Dal Monte PR. Effect of glass transition temperature on the stability of lyophilized formulations containing a chimeric therapeutic monoclonal antibody. Pharm Res [Internet]. Pharm Res; 1997 [cited 2020 Aug 16];14:591–5. Available from: https://pubmed.ncbi.nlm.nih.gov/9165528/
Drake AC, Lee Y, Burgess EM, Karlsson JOM, Eroglu A, Higgins AZ. Effect of water content on the glass transition temperature of mixtures of sugars, polymers, and penetrating cryoprotectants in physiological buffer. PLoS One [Internet]. Public Library of Science; 2018 [cited 2020 Sep 2];13. Available from: /pmc/articles/PMC5755887/?report=abstract.
Lazar KL, Patapoff TW, Sharma VK. Cold denaturation of monoclonal antibodies. MAbs [Internet]. MAbs; 2010 [cited 2020 Aug 16];2:42–52. Available from: https://pubmed.ncbi.nlm.nih.gov/20093856/
Ertel ’ KD, Carstensen JT. Examination of a modified Arrhenius relationship for pharmaceutical stability prediction. Int. J. Phar~ace~zics. 1990.
Kirkwood TBL. Predicting the stability of biological standards and products. Biometrics [Internet] 1977;33:736–742. Available from: https://search.ebscohost.com/login.aspx?direct=true&db=edselc&AN=edselc.2-52.0-0017658328&site=eds-live&scope=site
Rauk AP, Guo K, Hu Y, Cahya S, Weiss WF. Arrhenius time-scaled least squares: a simple, robust approach to accelerated stability data analysis for bioproducts. J Pharm Sci [Internet]. John Wiley and Sons Inc.; 2014 [cited 2020 Aug 16];103:2278–86. Available from: https://pubmed.ncbi.nlm.nih.gov/24974956/
Wang W, Roberts CJ. Non-arrhenius protein aggregation [Internet]. AAPS J. AAPS J; 2013 [cited 2020 Aug 16]. p. 840–51. Available from: https://pubmed.ncbi.nlm.nih.gov/23615748/
Chi EY, Krishnan S, Randolph TW, Carpenter JF. Physical stability of proteins in aqueous solution: mechanism and driving forces in nonnative protein aggregation [Internet]. Pharm. Res. Pharm Res; 2003 [cited 2020 Aug 16]. p. 1325–36. Available from: https://pubmed.ncbi.nlm.nih.gov/14567625/
Arakawa T, Kita Y, Carpenter JF. Protein–solvent interactions in pharmaceutical formulations [Internet]. Pharm. Res. An Off. J. Am. Assoc. Pharm. Sci. Pharm Res; 1991 [cited 2020 Aug 16]. p. 285–91. Available from: https://pubmed.ncbi.nlm.nih.gov/2052513/
Duhamel L, Gu Y, Barnett G, Tao Y, Voronov S, Ding J, et al. Therapeutic protein purity and fragmented species characterization by capillary electrophoresis sodium dodecyl sulfate using systematic hybrid cleavage and forced degradation. Anal Bioanal Chem [Internet]. Springer Verlag; 2019 [cited 2020 Aug 16];411:5617–29. Available from: https://pubmed.ncbi.nlm.nih.gov/31214752/
Thiagarajan G, Semple A, James JK, Cheung JK, Shameem M. A comparison of biophysical characterization techniques in predicting monoclonal antibody stability. MAbs [Internet]. Taylor and Francis Inc.; 2016 [cited 2020 Aug 26];8:1088–97. Available from: https://pubmed.ncbi.nlm.nih.gov/27210456/
Wagner E, Colas O, Chenu S, Goyon A, Murisier A, Cianferani S, et al. Determination of size variants by CE-SDS for approved therapeutic antibodies: key implications of subclasses and light chain specificities. J Pharm Biomed Anal [Internet]. Elsevier B.V.; 2020 [cited 2020 Aug 16];184. Available from: https://pubmed.ncbi.nlm.nih.gov/32113118/
Geiger T, Clarke S. Deamidation, isomerization, and racemization at asparaginyl and aspartyl residues in peptides succinimide-linked reactions that contribute to protein degradation*. J Bloloclcal Chem.
Duddu SP, Zhang G, Dal Monte PR. The relationship between protein aggregation and molecular mobility below the glass transition temperature of lyophilized formulations containing a monoclonal antibody. Pharm Res [Internet]. Pharm Res; 1997 [cited 2020 Aug 16];14:596–600. Available from: https://pubmed.ncbi.nlm.nih.gov/9165529/
Rustandi RR, Wang Y. Use of CE-SDS gel for characterization of monoclonal antibody hinge region clipping due to copper and high pH stress. Electrophoresis [Internet]. Electrophoresis; 2011 [cited 2020 Aug 26];32:3078–84. Available from: https://pubmed.ncbi.nlm.nih.gov/22145164/
Rustandi RR, Washabaugh MW, Wang Y. Applications of CE SDS gel in development of biopharmaceutical antibody-based products. Electrophoresis [Internet]. Electrophoresis; 2008 [cited 2020 Aug 26];29:3612–20. Available from: https://pubmed.ncbi.nlm.nih.gov/18803223/
Kerwin BA, Remmele RL. Protect from light: photodegradation and protein biologics. J Pharm Sci [Internet]. John Wiley and Sons Inc.; 2007 [cited 2020 Aug 26];96:1468–79. Available from: https://pubmed.ncbi.nlm.nih.gov/17230445/
Development issues: antibody stability, developability, immunogenicity, and comparability. Ther Antib Eng. Elsevier; 2012. p. 377–595.
Wang Y, Lu Q, Wu SL, Karger BL, Hancock WS. Characterization and comparison of disulfide linkages and scrambling patterns in therapeutic monoclonal antibodies: using LC-MS with electron transfer dissociation. Anal Chem [Internet]. Anal Chem; 2011 [cited 2020 Aug 26];83:3133–40. Available from: https://pubmed.ncbi.nlm.nih.gov/21428412/
Goyon A, Excoffier M, Janin-Bussat MC, Bobaly B, Fekete S, Guillarme D, et al. Determination of isoelectric points and relative charge variants of 23 therapeutic monoclonal antibodies. J Chromatogr B Anal Technol Biomed Life Sci [Internet]. Elsevier B.V.; 2017 [cited 2020 Aug 16];1065–1066:119–28. Available from: https://pubmed.ncbi.nlm.nih.gov/28961486/
Liu H, Caza-Bulseco G, Faldu D, Chumsae C, Sun J. Heterogeneity of monoclonal antibodies. J. Pharm. Sci. John Wiley and Sons Inc.; 2008. p. 2426–47.
Wu G, Yu C, Wang W, Wang L. Interlaboratory method validation of icIEF methodology for analysis of monoclonal antibodies. Electrophoresis [Internet]. Wiley-VCH Verlag; 2018 [cited 2020 Aug 16];39:2091–8. Available from: https://pubmed.ncbi.nlm.nih.gov/29797663/
Robinson NE. Protein deamidation. Proc Natl Acad Sci U S A [Internet]. National Academy of Sciences; 2002 [cited 2020 Aug 26];99:5283–8. Available from https://doi.org/10.1073/pnas.082102799
Anderson CL, Wang Y, Rustandi RR. Applications of imaged capillary isoelectric focussing technique in development of biopharmaceutical glycoprotein-based products. Electrophoresis [Internet]. Electrophoresis; 2012 [cited 2020 Aug 26];33:1538–44. Available from: https://pubmed.ncbi.nlm.nih.gov/22736354/
Navarrete del Toro MA, García-Carreño FL. Evaluation of the progress of protein hydrolysis. Curr Protoc Food Anal Chem [Internet]. Wiley; 2003 [cited 2020 Aug 26];10:B2.2.1-B2.2.14. Available from: http://doi.wiley.com/10.1002/0471142913.fab0202s10
Shacter E. Quantification and significance of protein oxidation in biological samples. Drug Metab Rev [Internet]. Drug Metab Rev; 2000 [cited 2020 Aug 26]. p. 307–26. Available from: https://pubmed.ncbi.nlm.nih.gov/11139131/
Yang R, Jain T, Lynaugh H, Nobrega RP, Lu X, Boland T, et al. Rapid assessment of oxidation via middle-down LCMS correlates with methionine side-chain solvent-accessible surface area for 121 clinical stage monoclonal antibodies. MAbs [Internet]. Taylor and Francis Inc.; 2017 [cited 2020 Aug 26];9:646–53. Available from: https://pubmed.ncbi.nlm.nih.gov/28281887/
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
About this article
Cite this article
Fink, M., Cannon, E.M., Hofmann, C. et al. Monoclonal Antibody Reagent Stability and Expiry Recommendation Combining Experimental Data with Mathematical Modeling. AAPS J 22, 145 (2020). https://doi.org/10.1208/s12248-020-00521-5
- Arrhenius kinetics
- monoclonal antibody
- Q10 rule
- reagent stability