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Characterization of Therapeutic Monoclonal Antibodies Reveals Differences Between In Vitro and In Vivo Time-Course Studies

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ABSTRACT

Purpose

To examine and determine the sites and the kinetics of IgG1 mAb modifications from both in vitro (rat plasma and PBS) and in vivo (rat model) time-course studies.

Methods

A comprehensive set of protein characterization methods, including RPLC/MS, LC-MS/MS, iCIEF, capSEC, and CE-SDS were performed in this report.

Results

We demonstrate that plasma incubation and in vivo circulation increase the rate of C-terminal lysine removal, and the levels of deamidation, pyroglutamic acid (pyroE), and thioether-linked (lanthionine) heavy chain and light chain (HC-S-LC). In contrast, incubation in PBS shows no C-terminal lysine removal, and slower rates of deamidation, pyroE, and HC-S-LC formation. Other potential modifications such as oxidation, aggregation, and peptide bonds hydrolysis are not enhanced.

Conclusion

This study demonstrates that in vivo mAb modifications are not fully represented by in vitro PBS or plasma incubation. The differences in modifications and their rates reflect the dissimilarities of matrices and the impact of enzymes. These observations provide valuable evidence and knowledge in evaluating the criticality of modifications that occur naturally in vivo that might impact formulation design, therapeutic outcome, and critical quality attribute assessments for therapeutic mAb manufacturing and quality control.

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Abbreviations

capSEC:

capillary size exclusion chromatography

CE-SDS:

capillary electrophoresis-sodium dodecyl sulfate non-gel sieving

iCIEF:

imaged capillary iso-electric focusing

LC-MS/MS:

liquid chromatography couple with tandem mass spectrometry

RPLC/MS:

reverse phase liquid chromatography coupled with mass spectrometry

REFERENCES

  1. PhRMA. Biotechnology research continues to bolster arsenal against disease with 633 medicines in development. Med Dev, Biotechnol. 2008.

  2. Wingren C, Alkner U, Hansson U-B. Antibody classes. ELS, John Wiley & Sons, Ltd, 2001.

  3. Steinmeyer DE, McCormick EL. The art of antibody process development. Drug Discovery Today. 2008;13:613–8.

    Article  PubMed  CAS  Google Scholar 

  4. Jenkins N, Murphy L, Tyther R. Post-translational modifications of recombinant proteins: Significance for biopharmaceuticals. Mol Biotechnol. 2008;39:113–8.

    Article  PubMed  CAS  Google Scholar 

  5. Kozlowski S, Swann P. Current and future issues in the manufacturing and development of monoclonal antibodies. Adv Drug Delivery Rev. 2006;58:707–22.

    Article  CAS  Google Scholar 

  6. Correia IR. Stability of IgG isotypes in serum. mAbs. 2010;2:221–32.

    Article  PubMed  Google Scholar 

  7. Robinson NE, Robinson AB. Deamidation of human proteins. Proc Natl Acad Sci U S A. 2001;98:12409–13.

    Article  PubMed  CAS  Google Scholar 

  8. Li N, Kessler K, Bass L, Zeng D. Evaluation of the iCE280 analyzer as a potential high-throughput tool for formulation development. J Pharm Biomed Anal. 2007;43:963–72.

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  10. Vlasak J, Bussat MC, Wang S, Wagner-Rousset E, Schaefer M, Klinguer-Hamour C, et al. Identification and characterization of asparagine deamidation in the light chain cdr1 of a humanized IgG1 antibody. Anal Biochem. 2009;392:145–54.

    Article  PubMed  CAS  Google Scholar 

  11. Yu XC, Joe K, Zhang Y, Adriano A, Wang Y, Gazzano-Santoro H, et al. Accurate determination of succinimide degradation products using high fidelity trypsin digestion peptide map analysis. Anal Chem. 2011;83:5912–9.

    Article  PubMed  CAS  Google Scholar 

  12. Huang L, Lu J, Wroblewski VJ, Beals JM, Riggin RM. In vivo deamidation characterization of monoclonal antibody by LC/MS/MS. Anal Chem. 2005;77:1432–9.

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  14. Liu D, Ren D, Huang H, Dankberg J, Rosenfeld R, Cocco MJ, et al. Structure and stability changes of human IgG1 Fc as a consequence of methionine oxidation. Biochemistry. 2008;47:5088–100.

    Article  PubMed  CAS  Google Scholar 

  15. Lam XM, Yang JY, Cleland JL. Antioxidants for prevention of methionine oxidation in recombinant monoclonal antibody her2. J Pharm Sci. 1997;86:1250–5.

    Article  PubMed  CAS  Google Scholar 

  16. Liu H, Gaza-Bulseco G, Faldu D, Chumsae C, Sun J. Heterogeneity of monoclonal antibodies. J Pharm Sci. 2008;97:2426–47.

    Article  PubMed  CAS  Google Scholar 

  17. Martin WL, West Jr AP, Gan L, Bjorkman PJ. Crystal structure at 2.8 Å of an FcRn/heterodimeric Fc complex: Mechanism of ph-dependent binding. Mol Cell. 2001;7:867–77.

    Article  PubMed  CAS  Google Scholar 

  18. Ji JA, Zhang B, Cheng W, Wang YJ. Methionine, tryptophan, and histidine oxidation in a model protein, PTH: Mechanisms and stabilization. J Pharm Sci. 2009;98:4485–500.

    Article  PubMed  CAS  Google Scholar 

  19. Harris RJ. Processing of c-terminal lysine and arginine residues of proteins isolated from mammalian-cell culture. J Chromatogr, A. 1995;705:129–34.

    Article  CAS  Google Scholar 

  20. Cleland JL, Powell MF, Shire SJ. The development of stable protein formulations: A close look at protein aggregation, deamidation, and oxidation. Crit Rev Ther Drug Carrier Syst. 1993;10:307–77.

    PubMed  CAS  Google Scholar 

  21. Cromwell M, Hilario E, Jacobson F. Protein aggregation and bioprocessing. The AAPS J. 2006;8:E572–9.

    Article  CAS  Google Scholar 

  22. Shire S, Cromwell M, Liu J. Concluding summary: Proceedings of the AAPS biotec open forum on “aggregation of protein therapeutics”. The AAPS J. 2006;8:E729–30.

    Article  Google Scholar 

  23. Cohen SL, Price C, Vlasak J. Β-elimination and peptide bond hydrolysis: Two distinct mechanisms of human IgG1 hinge fragmentation upon storage. J Am Chem Soc. 2007;129:6976–7.

    Article  PubMed  CAS  Google Scholar 

  24. Cordoba AJ, Shyong B-J, Breen D, Harris RJ. Non-enzymatic hinge region fragmentation of antibodies in solution. J Chromatogr, A. 2005;818:115–21.

    CAS  Google Scholar 

  25. Gaza-Bulseco G, Liu H. Fragmentation of a recombinant monoclonal antibody at various ph. Pharm Res. 2008;25:1881–90.

    Article  PubMed  CAS  Google Scholar 

  26. Liu YD, van Enk JZ, Flynn GC. Human antibody Fc deamidation in vivo. Biologicals. 2009;37:313–22.

    Article  PubMed  CAS  Google Scholar 

  27. Khawli LA, Goswami S, Hutchinson R, Kwong ZW, Yang J, Wang X, et al. Charge variants in IgG1: Isolation, characterization, in vitro binding properties and pharmacokinetics in rats. mAbs. 2010;2:613–24.

    Article  PubMed  Google Scholar 

  28. Liu YD, Goetze AM, Bass RB, Flynn GC. N-terminal glutamate to pyroglutamate conversion in vivo for human IgG2 antibodies. J Biol Chem. 2011;286:11211–7.

    Article  PubMed  CAS  Google Scholar 

  29. Goetze AM, Liu YD, Arroll T, Chu L, Flynn GC. Rates and impact of human antibody glycation in vivo. Glycobiology. 2012;22:221–34.

    Article  PubMed  CAS  Google Scholar 

  30. Cai B, Pan H, Flynn GC. C-terminal lysine processing of human immunoglobulin G2 heavy chain in vivo. Biotechnol Bioeng. 2011;108:404–12.

    Article  PubMed  CAS  Google Scholar 

  31. Rea JC, Moreno GT, Lou Y, Farnan D. Validation of a pH gradient-based ion-exchange chromatography method for high-resolution monoclonal antibody charge variant separations. J Pharm Biomed Anal. 2011;54:317–23.

    Article  PubMed  CAS  Google Scholar 

  32. Michels DA, Brady LJ, Guo A, Balland A. Fluorescent derivatization method of proteins for characterization by capillary electrophoresis- sodium dodecyl sulfate with laser-induced fluorescence detection. Anal Chem. 2007;79:5963–71.

    Article  PubMed  CAS  Google Scholar 

  33. Gennaro LA, Salas-Solano O, Ma S. Capillary electrophoresis–mass spectrometry as a characterization tool for therapeutic proteins. Anal Biochem. 2006;355:249–58.

    Article  PubMed  CAS  Google Scholar 

  34. Liu Y, Salas-Solano O, Gennaro LA. Investigation of sample preparation artifacts formed during the enzymatic release of n-linked glycans prior to analysis by capillary electrophoresis. Anal Chem. 2009;81:6823–9.

    Article  PubMed  CAS  Google Scholar 

  35. Ma S, Nashabeh W. Carbohydrate analysis of a chimeric recombinant monoclonal antibody by capillary electrophoresis with laser-induced fluorescence detection. Anal Chem. 1999;71:5185–92.

    Article  PubMed  CAS  Google Scholar 

  36. Nashef AS, Osuga DT, Lee HS, Ahmed AI, Whitaker JR, Feeney RE. Effects of alkali on proteins. Disulfides and their products. J Agric Food Chem. 1977;25:245–51.

    Article  PubMed  CAS  Google Scholar 

  37. Tous GI, Wei Z, Feng J, Bilbulian S, Bowen S, Smith J, et al. Characterization of a novel modification to monoclonal antibodies: Thioether cross-link of heavy and light chains. Anal Chem. 2005;77:2675–82.

    Article  PubMed  CAS  Google Scholar 

  38. Linetsky M, Hill JMW, LeGrand RD, Hu F. Dehydroalanine crosslinks in human lens. Exp Eye Res. 2004;79:499–512.

    Article  PubMed  CAS  Google Scholar 

  39. Tao MH, Morrison SL. Studies of aglycosylated chimeric mouse-human igg. Role of carbohydrate in the structure and effector functions mediated by the human IgG constant region. J Immunol. 1989;143:2595–601.

    PubMed  CAS  Google Scholar 

  40. Wawrzynczak EJ, Cumber AJ, Parnell GD, Jones PT, Winter G. Blood clearance in the rat of a recombinant mouse monoclonal antibody lacking the N-linked oligosaccharide side chains of the CH2 domains. Mol Immunol. 1992;29:213–20.

    Article  PubMed  CAS  Google Scholar 

  41. Goetze AM, Liu YD, Zhang Z, Shah B, Lee E, Bondarenko PV, et al. High-mannose glycans on the Fc region of therapeutic IgG antibodies increase serum clearance in humans. Glycobiology. 2011;21:949–59.

    Article  PubMed  CAS  Google Scholar 

  42. Hobbs SM, Jackson LE, Hoadley J. Interaction of aglycosyl immunoglobulins with the IgG Fc transport receptor from neonatal rat gut: Comparison of deglycosylation by tunicamycin treatment and genetic engineering. Mol Immunol. 1992;29:949–56.

    Article  PubMed  CAS  Google Scholar 

  43. Sinha S, Zhang L, Duan S, Williams TD, Vlasak J, Ionescu R, et al. Effect of protein structure on deamidation rate in the Fc fragment of an IgG1 monoclonal antibody. Protein Sci. 2009;18:1573–84.

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  45. Harris RJ, Kabakoff B, Macchi FD, Shen FJ, Kwong M, Andya JD, et al. Identification of multiple sources of charge heterogeneity in a recombinant antibody. J Chromatogr, B: Biomed Sci Appl. 2001;752:233–45.

    Article  CAS  Google Scholar 

  46. Wakankar AA, Borchardt RT, Eigenbrot C, Shia S, Wang YJ, Shire SJ, et al. Aspartate isomerization in the complementarity-determining regions of two closely related monoclonal antibodies. Biochemistry. 2007;46:1534–44.

    Article  PubMed  CAS  Google Scholar 

  47. Brennan TV, Clarke S. Spontaneous degradation of polypeptides at aspartyl and asparaginyl residues: Effects of the solvent dielectric. Protein Sci. 1993;2:331–8.

    Article  PubMed  CAS  Google Scholar 

  48. Robinson NE, Robinson AB. Molecular clocks. Proc Natl Acad Sci U S A. 2001;98:944–9.

    Article  PubMed  CAS  Google Scholar 

  49. DeLano WL, Ultsch MH. de AM, Vos, Wells JA. Convergent solutions to binding at a protein-protein interface. Science. 2000;287:1279–83.

    Article  PubMed  CAS  Google Scholar 

  50. Airaudo CB, Gayte-Sorbier A, Armand P. Stability of glutamine and pyroglutamic acid under model system conditions: Influence of physical and technological factors. J Food Sci. 1987;52:1750–2.

    Article  CAS  Google Scholar 

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ACKNOWLEDGMENTS AND DISCLOSURES

The authors would like to thank colleagues at Genentech for their support and scientific discussions during this project, especially, Jeanne Kwong, Betty Chan, Will McElroy, Monica Parker, Jennifer Rea, George (Tony) Moreno, Mellisa Alvarez, Oleg Borisov, Jennifer Zhang, Hongbin Liu, David Michels, Dell Farnan, Keyang Xu, Randy Dere, Surinder Kaur, and Tom Patapoff. They also express appreciation to Jose Imperio and Sheila Ulufato from In Vivo Study Group for excellent animal studies support. All authors are employees of Genentech, a member of the Roche Group, and hold a financial interest in Roche.

Author information

Authors and Affiliations

  1. Protein Analytical Chemistry, Genentech, Inc., South San Francisco, California, USA

    Sheng Yin & John T. Stults

  2. Department of Pharmacokinetic and Pharmacodynamic Sciences, Genentech, Inc., South San Francisco, California, USA

    Cinthia V. Pastuskovas & Leslie A. Khawli

  3. 1 DNA way, MS 62, South San Francisco, California, 94080, USA

    John T. Stults

Authors
  1. Sheng Yin
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  2. Cinthia V. Pastuskovas
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  3. Leslie A. Khawli
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Corresponding author

Correspondence to John T. Stults.

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Table SI

Observed HMWS determined by capSEC for mAb-1 during in vivo circulation, and mAb-1 (a), -2 (b), and -3 (c) during PBS and plasma incubation. For plasma- and PBS-incubated mAbs, each time point is prepared and analyzed three times. Averaged value and standard deviation (stdev) are reported. For in vivo-circulated mAb-1, one set of samples are prepared and analyzed in duplicate, with the average value reported. For PBS-incubated mAbs, the level of aggregation is maintained at a low level during incubation. For plasma-incubated mAbs, aggregation appears to increase at very low rate (<0.1%/day). For in vivo-circulated mAb-1, aggregation level does not appear to change and is maintained around 0.3%. (DOC 43.5 kb)

Table SII

(a)Total of three peptides with one or two deamidation sites (bolded amino acids) for plasma-incubated mAb-1 are reported. (b) Two peptides for plasma-incubated mAb-2 are deamidated. (c) Only the deamidated PENNY peptide was observed for mAb-3. The PENNY peptide contributes to the majority percentage of total deamidation. Data for all time points are averaged value of three experiments, standard deviations are also included. (DOC 47 kb)

Figure S1

Capillary SEC chromatogram of 0, 2, 6, and 10 days plasma-incubated mAb-1. Chromatograms are normalized based on mAb monomer peak, two areas are assigned as High Molecular Weight Species (HWMS) show a slight increase during plasma incubation. (DOC 170 kb)

Figure S2

The HC-S-LC is observed in plasma-incubated mAb-1(●), mAb-2 (■) and mAb3 (▲). In mAb-1, HC-S-LC is formed at a rate of 0.7% per day, and in mAb-2 and mAb-3, HC-S-LC is formed at a rate of 0.2% per day. Each data point represents three sets of plasma-incubated mAb, and the error bars represent standard deviations. (DOC 38 kb)

Figure S3

iCIEF results of plasma-incubated mAb-1(●), mAb-2 (■) and mAb3 (▲) in which each data point represents three repeated incubation and analysis, and error bars represent standard deviations. Acidic variants increase with rate of 2.9%, 2.1% and 3.2% per day, for plasma-incubated mAb-1, mAb-2 and mAb-3, respectively. (DOC 46.0 kb)

Figure S4

Deamidation rate of PENNY peptides for plasma-incubated mAb-1(●), mAb-2 (■) and mAb-3 (▲). At each time point, the average value of triplicate measurements is reported. Upward error bars represent standard deviations. The deamidation rate of the PENNY peptide for plasma-incubated mAb-1 is 1.7% per day, for mAb-2 is 1.8% per day, and for mAb-3 is 1.5% per day. (DOC 38.5 kb)

Figure S5

LC-MS/MS data provide the rate of pyroE formation for plasma-incubated mAb-1(●), mAb-2(■), and mAb-3(▲). At each time point, average value of triplicate measurements is reported. Upward error bars represent standard deviations. The rate of pyroE formation for mAb-1 and mAb-2 is 0.6% per day, and for mAb-3 is 0.5% per day. (DOC 43 kb)

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Yin, S., Pastuskovas, C.V., Khawli, L.A. et al. Characterization of Therapeutic Monoclonal Antibodies Reveals Differences Between In Vitro and In Vivo Time-Course Studies. Pharm Res 30, 167–178 (2013). https://doi.org/10.1007/s11095-012-0860-z

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  • DOI: https://doi.org/10.1007/s11095-012-0860-z

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