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Quality Issues Arising from Post-translational Modification of Recombinant Antibodies

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Antibody Expression and Production

Part of the book series: Cell Engineering ((CEEN,volume 7))

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Abstract

This chapter explores the post translational modifications (PTMs) that can occur in recombinant monoclonal antibodies. The topic of glycosylation is covered in another chapter in this volume. These modifications can occur at each stage of bioprocessing i.e. cell expansion, fermentation, protein purification, formulation and long-term storage. The chapter focusses on the following PTMs: protein aggregation and misfolding, dimerization, oxidation (principally of methionine residues), and deamidation (principally of asparagine residues). It explores the mechanisms and possible causes of these PTMs, and also the assays used to track these changes. It is the responsibility of each manufacturer to define the limits of variation that exist for individual biopharmaceuticals through comprehensive analytics and submit these to the regulatory authorities. These limits, along with parameters defined during Quality by Design programs (QbD) using Process Analytical Technologies (PAT) are used to optimize the bioprocessing steps to minimize the presence of aberrant species.

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References

  • Andya JD, Hsu CC, Shire SJ. 2003. Mechanisms of aggregate formation and carbohydrate excipient stabilization of lyophilized humanized monoclonal antibody formulations. AAPS PharmSci 5(2):26–32.

    Article  Google Scholar 

  • Arakawa T, Philo JS, Tsumoto K, Yumioka R, Ejima D. 2004. Elution of antibodies from a Protein-A column by aqueous arginine solutions. Protein Expr Purif 36(2):244–8.

    Article  PubMed  CAS  Google Scholar 

  • Bahrami A, Shojaosadati SA, Khalilzadeh R, Mohammadian J, Vashghani Farahani E, Masoumian MR. 2008. Prevention of human G-CSF protein aggregation in recombinant Pichia pastoris fed-batch fermentation using additives. Biotechnol Appl Biochem 14:14.

    Google Scholar 

  • Brych SR, Gokarn YR, Hultgen H, Stevenson RJ, Rajan R, Matsumura M. 2010. Characterization of antibody aggregation: role of buried, unpaired cysteines in particle formation. J Pharm Sci 99(2):764–81.

    PubMed  CAS  Google Scholar 

  • Catak S, Monard G, Aviyente V, Ruiz-Lopez MF. 2008. Computational study on nonenzymatic peptide bond cleavage at asparagine and aspartic acid. J Phys Chem A 112(37):8752–61. Epub 2008 Aug 20.

    Article  PubMed  CAS  Google Scholar 

  • Chaderjian WB, Chin ET, Harris RJ, Etcheverry TM. 2005. Effect of copper sulfate on performance of a serum-free CHO cell culture process and the level of free thiol in the recombinant antibody expressed. Biotechnol Prog 21(2):550–53.

    Article  PubMed  CAS  Google Scholar 

  • Chakravarthi S, Jessop CE, Bulleid NJ. 2006. The role of glutathione in disulphide bond formation and endoplasmic-reticulum-generated oxidative stress. EMBO Rep 7(3):271–5.

    Article  PubMed  CAS  Google Scholar 

  • Chelius D, Rehder DS, Bondarenko PV. 2005. Identification and characterization of deamidation sites in the conserved regions of human immunoglobulin gamma antibodies. Anal Chem 77(18):6004–11.

    Article  PubMed  CAS  Google Scholar 

  • Chen B, Bautista R, Yu K, Zapata GA, Mulkerrin MG, Chamow SM. 2003. Influence of histidine on the stability and physical properties of a fully human antibody in aqueous and solid forms. Pharm Res. 20(12):1952–60.

    Article  PubMed  CAS  Google Scholar 

  • Chen S, Lau H, Brodsky Y, Kleemann GR, Latypov RF. 2010. The use of native cation-exchange chromatography to study aggregation and phase separation of monoclonal antibodies. Protein Sci 19(6):1191–204.

    Article  PubMed  CAS  Google Scholar 

  • Chen J, Tetrault J, Zhang Y, Wasserman A, Conley G, Dileo M, Haimes E, Nixon AE, Ley A. 2010. The distinctive separation attributes of mixed-mode resins and their application in monoclonal antibody downstream purification process. J Chromatogr A 1217(2):216–24.

    Article  PubMed  CAS  Google Scholar 

  • Chirino AJ, Mire-Sluis A. 2004. Characterizing biological products and assessing comparability following manufacturing changes. Nat Biotechnol 22(11):1383–91.

    Article  PubMed  CAS  Google Scholar 

  • Chumsae C, Gaza-Bulseco G, Sun J, Liu H. 2007. Comparison of methionine oxidation in thermal stability and chemically stressed samples of a fully human monoclonal antibody. J Chromatogr B Analyt Technol Biomed Life Sci 850(1–2):285–94. Epub 2006 Dec 19.

    PubMed  CAS  Google Scholar 

  • Cromwell ME, Hilario E, Jacobson F. 2006. Protein aggregation and bioprocessing. Aaps J 8(3):E572–9.

    Article  PubMed  CAS  Google Scholar 

  • Dhingra V, Gupta M, Andacht T, Fu ZF. 2005. New frontiers in proteomics research: a perspective. Int J Pharm 299(1–2):1–18.

    Article  PubMed  CAS  Google Scholar 

  • Falini ML, Elli L, Caramanico R, Bardella MT, Terrani C, Roncoroni L, Doneda L, Forlani F. 2008. Immunoreactivity of antibodies against transglutaminase-deamidated gliadins in adult celiac disease. Dig Dis Sci 53(10):2697–701. Epub 2008 Feb 28.

    Article  PubMed  CAS  Google Scholar 

  • Gagnon P, Beam K. 2009. Antibody aggregate removal by hydroxyapatite chromatography. Curr Pharm Biotechnol 10(4):440–6.

    Article  PubMed  CAS  Google Scholar 

  • Gaza-Bulseco G, Faldu S, Hurkmans K, Chumsae C, Liu H. 2008. Effect of methionine oxidation of a recombinant monoclonal antibody on the binding affinity to protein A and protein G. J Chromatogr B Analyt Technol Biomed Life Sci 870(1):55–62. Epub 2008 Jun 5.

    Article  PubMed  CAS  Google Scholar 

  • Geiger T, Clarke S. 1987. Deamidation, isomerization, and racemization at asparaginyl and aspartyl residues in peptides. Succinimide-linked reactions that contribute to protein degradation. J Biol Chem 262(2):785–94.

    PubMed  CAS  Google Scholar 

  • Harris RJ. 2005. Heterogeneity of recombinant antibodies: linking structure to function. Dev Biol (Basel) 122:117–27.

    CAS  Google Scholar 

  • Harris RJ, Kabakoff B, Macchi FD, Shen FJ, Kwong M, Andya JD, Shire SJ, Bjork N, Totpal K, Chen AB. 2001. Identification of multiple sources of charge heterogeneity in a recombinant antibody. J Chromatogr B Biomed Sci Appl 752(2):233–45.

    Article  PubMed  CAS  Google Scholar 

  • Hermeling S, Schellekens H, Maas C, Gebbink MF, Crommelin DJ, Jiskoot W. 2006. Antibody response to aggregated human interferon alpha2b in wild-type and transgenic immune tolerant mice depends on type and level of aggregation. J Pharm Sci 95(5):1084–96.

    Article  PubMed  CAS  Google Scholar 

  • Hochuli E. 1997. Interferon immunogenicity: technical evaluation of interferon-alpha 2a. J Interferon Cytokine Res 17(Suppl 1):S15–21.

    PubMed  CAS  Google Scholar 

  • Houde D, Kauppinen P, Mhatre R, Lyubarskaya Y. 2006. Determination of protein oxidation by mass spectrometry and method transfer to quality control. J Chromatogr A 1123(2):189–98.

    Article  PubMed  CAS  Google Scholar 

  • Jenkins N. 2007. Modifications of therapeutic proteins: challenges and prospects. Cytotechnology 53:121–5.

    Article  PubMed  CAS  Google Scholar 

  • Jenkins N, Meleady P, Tyther R, Murphy L. 2009. Strategies for analysing and improving the expression and quality of recombinant proteins made in mammalian cells. Biotechnol Appl Biochem 53(Pt 2):73–83.

    Article  PubMed  CAS  Google Scholar 

  • Kroon DJ, Baldwin-Ferro A, Lalan P. 1992. Identification of sites of degradation in a therapeutic monoclonal antibody by peptide mapping. Pharm Res 9(11):1386–93.

    Article  PubMed  CAS  Google Scholar 

  • Lam XM, Yang JY, Cleland JL. 1997a. Antioxidants for prevention of methionine oxidation in recombinant monoclonal antibody HER2. J Pharm Sci 86(11):1250–5.

    Article  PubMed  CAS  Google Scholar 

  • Lam XM, Yang JY, Cleland JL. 1997b. Antioxidants for prevention of methionine oxidation in recombinant monoclonal antibody HER2. J Pharm Sci 86(11):1250–5.

    Article  PubMed  CAS  Google Scholar 

  • Levine RL, Moskovitz J, Stadtman ER. 2000. Oxidation of methionine in proteins: roles in antioxidant defense and cellular regulation. IUBMB Life 50(4–5):301–7.

    Article  PubMed  CAS  Google Scholar 

  • Lionberger RA, Lee SL, Lee L, Raw A, Yu LX. 2008. Quality by design: concepts for ANDAs. AAPS J 10(2):268–76.

    Article  PubMed  CAS  Google Scholar 

  • Liu DT. 1992. Deamidation: a source of microheterogeneity in pharmaceutical proteins. Trends Biotechnol 10(10):364–9.

    Article  PubMed  CAS  Google Scholar 

  • Liu J, Andya JD, Shire SJ. 2006b. A critical review of analytical ultracentrifugation and field flow fractionation methods for measuring protein aggregation. Aaps J 8(3):E580–9.

    Article  PubMed  CAS  Google Scholar 

  • Liu H, Gaza-Bulseco G, Sun J. 2006a. Characterization of the stability of a fully human monoclonal IgG after prolonged incubation at elevated temperature. J Chromatogr B Analyt Technol Biomed Life Sci 837(1–2):35–43.

    PubMed  CAS  Google Scholar 

  • Liu J, Nguyen MD, Andya JD, Shire SJ. 2005. Reversible self-association increases the viscosity of a concentrated monoclonal antibody in aqueous solution. J Pharm Sci 94(9):1928–40.

    Article  PubMed  CAS  Google Scholar 

  • Liu D, Ren D, Huang H, Dankberg J, Rosenfeld R, Cocco MJ, Li L, Brems DN, Remmele RL, Jr. 2008. Structure and stability changes of human IgG1 Fc as a consequence of methionine oxidation. Biochemistry 47(18):5088–100. Epub 2008 Apr 12.

    Article  PubMed  CAS  Google Scholar 

  • Lyubarskaya Y, Houde D, Woodard J, Murphy D, Mhatre R. 2006. Analysis of recombinant monoclonal antibody isoforms by electrospray ionization mass spectrometry as a strategy for streamlining characterization of recombinant monoclonal antibody charge heterogeneity. Anal Biochem 348(1):24–39.

    Article  PubMed  CAS  Google Scholar 

  • Maas C, Hermeling S, Bouma B, Jiskoot W, Gebbink MF. 2007. A role for protein misfolding in immunogenicity of biopharmaceuticals. J Biol Chem 282(4):2229–36. Epub 2006 Nov 29.

    Article  PubMed  CAS  Google Scholar 

  • Mahler HC, Friess W, Grauschopf U, Kiese S. 2009. Protein aggregation: pathways, induction factors and analysis. J Pharm Sci 98(9):2909–34.

    Article  PubMed  CAS  Google Scholar 

  • Mahler HC, Muller R, Friess W, Delille A, Matheus S. 2005. Induction and analysis of aggregates in a liquid IgG1-antibody formulation. Eur J Pharm Biopharm 59(3):407–17.

    Article  PubMed  CAS  Google Scholar 

  • Maislos M, Bialer M, Mead PM, Robbins DC. 1988. Pharmacokinetic model of circulating covalent aggregates of insulin. Diabetes 37(8):1059–63.

    Article  PubMed  CAS  Google Scholar 

  • Matamoros Fernandez LE, Kalume DE, Calvo L, Fernandez Mallo M, Vallin A, Roepstorff P. 2001. Characterization of a recombinant monoclonal antibody by mass spectrometry combined with liquid chromatography. J Chromatogr B Biomed Sci Appl 752(2):247–61.

    Article  PubMed  CAS  Google Scholar 

  • Mimura Y, Nakamura K, Tanaka T, Fujimoto M. 1998. Evidence of intra- and extracellular modifications of monoclonal IgG polypeptide chains generating charge heterogeneity. Electrophoresis 19(5):767–75.

    Article  PubMed  CAS  Google Scholar 

  • Moore JM, Patapoff TW, Cromwell ME. 1999. Kinetics and thermodynamics of dimer formation and dissociation for a recombinant humanized monoclonal antibody to vascular endothelial growth factor. Biochemistry 38(42):13960–7.

    Article  PubMed  CAS  Google Scholar 

  • Oliva A, Llabres M, Farina JB. 2004. Applications of multi-angle laser light-scattering detection in the analysis of peptides and proteins. Curr Drug Discov Technol 1(3):229–42.

    Article  PubMed  CAS  Google Scholar 

  • Pan H, Chen K, Chu L, Kinderman F, Apostol I, Huang G. 2009. Methionine oxidation in human IgG2 Fc decreases binding affinities to protein A and FcRn. Protein Sci 18(2):424–33.

    Article  PubMed  CAS  Google Scholar 

  • Pekar A, Sukumar M. 2007. Quantitation of aggregates in therapeutic proteins using sedimentation velocity analytical ultracentrifugation: practical considerations that affect precision and accuracy. Anal Biochem 367(2):225–37.

    Article  PubMed  CAS  Google Scholar 

  • Rathore AS. 2009. Roadmap for implementation of quality by design (QbD) for biotechnology products. Trends Biotechnol 27(9):546–53.

    Article  PubMed  CAS  Google Scholar 

  • Remmele RL, Jr., Callahan WJ, Krishnan S, Zhou L, Bondarenko PV, Nichols AC, Kleemann GR, Pipes GD, Park S, Fodor S, et al. 2006. Active dimer of Epratuzumab provides insight into the complex nature of an antibody aggregate. J Pharm Sci 95(1):126–45.

    Article  PubMed  CAS  Google Scholar 

  • Robinson DK, Chan CP, Yu Lp C, Tsai PK, Tung J, Seamans TC, Lenny AB, Lee DK, Irwin J, Silberklang M. 1994. Characterization of a recombinant antibody produced in the course of a high yield fed-batch process. Biotechnol Bioeng 44(6):727–35.

    Article  PubMed  CAS  Google Scholar 

  • Rosenberg AS. 2006. Effects of protein aggregates: an immunologic perspective. Aaps J 8(3):E501–7.

    Article  PubMed  Google Scholar 

  • Schroder M, Friedl P. 1997. Overexpression of recombinant human antithrombin III in Chinese hamster ovary cells results in malformation and decreased secretion of the recombinant protein. Biotechnol Bioeng 53:547–59.

    Article  PubMed  CAS  Google Scholar 

  • Shire SJ, Shahrokh Z, Liu J. 2004. Challenges in the development of high protein concentration formulations. J Pharm Sci 93(6):1390–402.

    Article  PubMed  CAS  Google Scholar 

  • Shukla AA, Gupta P, Han X. 2007a. Protein aggregation kinetics during Protein A chromatography. Case study for an Fc fusion protein. J Chromatogr A 1171(1–2):22–8. Epub 2007 Sep 22.

    Article  PubMed  CAS  Google Scholar 

  • Shukla AA, Hubbard B, Tressel T, Guhan S, Low D. 2007b. Downstream processing of monoclonal antibodies – application of platform approaches. J Chromatogr B Analyt Technol Biomed Life Sci 848(1):28–39.

    Article  PubMed  CAS  Google Scholar 

  • Silveira JR, Hughson AG, Caughey B. 2006. Fractionation of prion protein aggregates by asymmetrical flow field-flow fractionation. Methods Enzymol 412:21–33.

    Article  PubMed  CAS  Google Scholar 

  • Sinha S, Zhang L, Duan S, Williams TD, Vlasak J, Ionescu R, Topp EM. 2009. Effect of protein structure on deamidation rate in the Fc fragment of an IgG1 monoclonal antibody. Protein Sci 18(8):1573–84.

    Article  PubMed  CAS  Google Scholar 

  • Soenderkaer S, Carpenter JF, van de Weert M, Hansen LL, Flink J, Frokjaer S. 2004. Effects of sucrose on rFVIIa aggregation and methionine oxidation. Eur J Pharm Sci 21(5):597–606.

    Article  PubMed  CAS  Google Scholar 

  • Taggart C, Cervantes-Laurean D, Kim G, McElvaney NG, Wehr N, Moss J, Levine RL. 2000. Oxidation of either methionine 351 or methionine 358 in alpha 1-antitrypsin causes loss of anti-neutrophil elastase activity. J Biol Chem 275(35):27258–65.

    PubMed  CAS  Google Scholar 

  • Takata T, Oxford JT, Demeler B, Lampi KJ. 2008. Deamidation destabilizes and triggers aggregation of a lens protein, betaA3-crystallin. Protein Sci 17(9):1565–75. Epub 2008 Jun 20.

    Article  PubMed  CAS  Google Scholar 

  • Tsai PK, Bruner MW, Irwin JI, Ip CC, Oliver CN, Nelson RW, Volkin DB, Middaugh CR. 1993. Origin of the isoelectric heterogeneity of monoclonal immunoglobulin h1B4. Pharm Res 10(11):1580–6.

    Article  PubMed  CAS  Google Scholar 

  • Venter JC, Adams MD, Myers EW, Li PW, Mural RJ, Sutton GG, Smith HO, Yandell M, Evans CA, Holt RA, et al. 2001. The sequence of the human genome. Science 291(5507):1304–51.

    Article  PubMed  CAS  Google Scholar 

  • Vlasak J, Bussat MC, Wang S, Wagner-Rousset E, Schaefer M, Klinguer-Hamour C, Kirchmeier M, Corvaia N, Ionescu R, Beck A. 2009. Identification and characterization of asparagine deamidation in the light chain CDR1 of a humanized IgG1 antibody. Anal Biochem 392(2):145–54.

    Article  PubMed  CAS  Google Scholar 

  • Vlasak J, Ionescu R. 2008. Heterogeneity of monoclonal antibodies revealed by charge-sensitive methods. Curr Pharm Biotechnol 9(6):468–81.

    Article  PubMed  CAS  Google Scholar 

  • Wei Z, Feng J, Lin HY, Mullapudi S, Bishop E, Tous GI, Casas-Finet J, Hakki F, Strouse R, Schenerman MA. 2007. Identification of a single tryptophan residue as critical for binding activity in a humanized monoclonal antibody against respiratory syncytial virus. Anal Chem 79(7):2797–805.

    Article  PubMed  CAS  Google Scholar 

  • Weintraub SJ, Deverman BE. 2007. Chronoregulation by asparagine deamidation. Sci STKE 2007(409):re7.

    Article  PubMed  Google Scholar 

  • Weintraub SJ, Manson SR. 2004. Asparagine deamidation: a regulatory hourglass. Mech Ageing Dev 125(4):255–7.

    Article  PubMed  CAS  Google Scholar 

  • Yang J, Wang S, Liu J, Raghani A. 2007. Determination of tryptophan oxidation of monoclonal antibody by reversed phase high performance liquid chromatography. J Chromatogr A 1156(1–2):174–82.

    Article  PubMed  CAS  Google Scholar 

  • Ye H. 2006. Simultaneous determination of protein aggregation, degradation, and absolute molecular weight by size exclusion chromatography-multiangle laser light scattering. Anal Biochem 356(1):76–85. Epub 2006 Jun 9.

    Article  PubMed  CAS  Google Scholar 

  • Zhang W, Czupryn MJ. 2002. Free sulfhydryl in recombinant monoclonal antibodies. Biotechnol Prog 18(3):509–13.

    Article  PubMed  Google Scholar 

  • Zhang Y, Martinez T, Woodruff B, Goetze A, Bailey R, Pettit D, Balland A. 2008. Hydrophobic interaction chromatography of soluble interleukin I receptor type II to reveal chemical degradations resulting in loss of potency. Anal Chem 80(18):7022–8. Epub 2008 Aug 16.

    Article  PubMed  CAS  Google Scholar 

  • Zhou JX, Solamo F, Hong T, Shearer M, Tressel T. 2008. Viral clearance using disposable systems in monoclonal antibody commercial downstream processing. Biotechnol Bioeng 100(3):488–96.

    Article  PubMed  CAS  Google Scholar 

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  1. National Institute for Bioprocessing Reasearch and Training, University College Dublin, Dublin, Ireland

    Raymond Tyther & Nigel Jenkins

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  1. Raymond Tyther
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  2. Nigel Jenkins
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Correspondence to Nigel Jenkins .

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  1. Dept. Chemical & Biochemical, Engineering, University College, Dublin, Belfield, Dublin 4, Ireland

    Mohamed Al-Rubeai

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Tyther, R., Jenkins, N. (2011). Quality Issues Arising from Post-translational Modification of Recombinant Antibodies. In: Al-Rubeai, M. (eds) Antibody Expression and Production. Cell Engineering, vol 7. Springer, Dordrecht. https://doi.org/10.1007/978-94-007-1257-7_13

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