Skip to main content
SpringerLink
Log in

Analysis of deamidation artifacts induced by microwave-assisted tryptic digestion of a monoclonal antibody

  • Research Paper
  • Published:
Analytical and Bioanalytical Chemistry Aims and scope Submit manuscript

Abstract

The thorough characterization of biopharmaceuticals is essential for ensuring their quality and safety since many potential variations can cause changes to the properties of a drug that may be detrimental to the patient such as decreased efficacy, shorter half-life or increased immunogenicity. Prior to approval and release, protein-based drugs are subject to a battery of analyses to assess the nature of those parameters that are considered critical quality attributes. In some cases the analytical method used may itself cause modifications that are impossible to distinguish from those induced by the intended test conditions (e.g. storage time/temperature, light exposure) which are used to assess drug stability. It is therefore important to develop and utilize analytical methods which impose as few artifactual modifications as possible. Asparagine deamidation is a common protein modification and it is known to be induced during tryptic digestion. Therefore we examined common tryptic digestion protocols and compared their propensities towards asparagine modification. Since microwave assisted hydrolysis techniques are often used to shorten digestion times and the effect on deamidation is unknown we sought to compare this method against alternate digestion protocols.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

References

  1. Shields RL, Lai J, Keck R, O’Connell LY, Hong K, Meng YG, Weikert SH, Presta LG (2002) Lack of fucose on human IgG1 N-linked oligosaccharide improves binding to human FcγRIII and antibody-dependent cellular toxicity. J Biol Chem 277:26733–26740

    Article  CAS  Google Scholar 

  2. Sakurada M, Uchida Kanazawa H, Satoh M, Yamasaki M et al (2003) The absence of fucose but not the presence of galactose or bisecting N-acetylgucosamine of human IgG1 complex-type oligosaccharides shows the critical role of enhancing antibody-dependent cellular cytotoxicity. J Biol Chem 278:3466–3473

    Article  Google Scholar 

  3. Niwa R, Hatanaka S, Shoji-Hosaka E, Sakurada M, Kobayashi Y, Uehara A, Yokoi H, Nakamura K, Shitara K (2004) Enhancement of the antibody-dependent cellular cytotoxicity of low-fucose IgG1 is independent of FcγRIIIa functional polymorphism. Clin Cancer Res 10:6248–6255

    Article  CAS  Google Scholar 

  4. Niwa R, Shoji-HOsaka E, Sakurada M, Shinkawa T, Uchida K, Nakamura K, Matsushima K, Ueda R, Hanai N, Shitara K (2004) Defucosylated anti-CC chemokine receptor 4 IgG1 with enhanced antibody-dependent cellular cytotoxicity shows potent therapeutic activity to T cell leukemia and lymphoma. Cancer Res 64:2127–2133

    Article  CAS  Google Scholar 

  5. Niwa R, Natsume A, Uehara A, Wakitani M, Iida S, Uchida K, Satoh M, Shitara K (2005) IgG subclass-independent improvement of antibody-dependent cellular cytotoxicity by fucose removal from Asn297-linked oligosaccharides. J Immunol Methods 306:151–160

    Article  CAS  Google Scholar 

  6. Niwa R, Sakurada M, Kobayashi Y, Uehara A, Matsushima K, Ueda R, Nakamura K, Shitara K (2005) Enhanced natural killer cell binding and activation by low-fucose IgG1 antibody results in potent antibody-dependent cellular cytotoxicity induction at lower antigen density. Clin Cancer Res 11:2327–2336

    Article  CAS  Google Scholar 

  7. Iida S, Misaka H, Inoue M, Shibata M, Nakano R, Yamane-Ohnuki N, Wakitani M, Yano K, Shitara K, Satoh M (2006) Non-fucosylated therapeutic IgG1 antibody can evade the inhibitory effect of serum immunoglobulin G on antibody-dependent cellular cytotoxicity through its high binding to FcγRIIIa. Clin Cancer Res 12:2879–2887

    Article  CAS  Google Scholar 

  8. Kanda Y, Yamada T, Mori K, Okazaki A, Inoue M, Kitajima-Miyama K, Kuni-Kamochi R, Nakano R, Yano K, Kakita S, Shitara K, Satoh M (2006) Comparison of biological activity among nonfucosylated therapeutic IgG1 antibodies with three different N-linked Fc oligosaccharides: the high-mannose, hybrid, and complex types. Glycobiology 17:104–118

    Article  Google Scholar 

  9. Ghaderi D, Taylor RE, Padler-Karavani V, Diaz S, Varki A (2010) Implications of the presence of N-glycolylneuraminic acid in recombinant therapeutic glycoproteins. Nat Biotechnol 28:863–867

    Article  CAS  Google Scholar 

  10. Maeda E, Kita S, Kinoshita M, Urakami K, Hayakawa T, Kakehi K (2012) Analysis of nonhuman N-glycans as the minor constituents in recombinant monoclonal antibody pharmaceuticals. Anal Chem 84:2373–2379

    Article  CAS  Google Scholar 

  11. Pan H, Chen K, Chu L, Kinderman F, Apostol I, Huang G (2008) Methionine oxidation in human IgG2 Fc decreases binding affinities to protein A and FcRn. Protein Sci 18:424–433

    Article  Google Scholar 

  12. Wang W, Vlasak J, Li Y, Pristatsky P, Fang Y, Pittman T, Roman J, Wang Y, Prueksaritanont T, Ionescu R (2011) Impact of methionine oxidation in human IgG1 Fc on serum half-life of monoclonal antibodies. Mol Immunol 48:860–866

    Article  CAS  Google Scholar 

  13. Cacia J, Keck R, Presta LG, Frenz J (1996) Isomerizaation of an aspartic acid residue in the complementarity-determining regions of a recombinant antibody to human IgG: identification and effect on binding affinity. Biochemistry 35:1897–1903

    Article  CAS  Google Scholar 

  14. 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 Chromotogr B 752:233–245

    Article  CAS  Google Scholar 

  15. Yu XC, Joe K, Zhang Y, Adriano A, Wang Y, Gazzano-Santoro H, Keck RG, Deperalta G, Ling V (2011) Accurate determination of succinimide degradation products using high fidelity trypsin digestion peptide map analysis. Anal Chem 83:5912–5919

    Article  CAS  Google Scholar 

  16. 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 262:785–794

    CAS  Google Scholar 

  17. Robinson AB, McKerrow JH, Legaz M (1974) Sequence dependent deamidation rates for model peptides of cytochrome-c. Int J Pept Protein Res 6:31–35

    Article  CAS  Google Scholar 

  18. Patel K, Borchardt RT (1990) Chemical pathways of peptide degradation. III. Effect of primary sequence on the pathways of deamidation of asparaginyl residues in hexapeptides. Pharm Res 7:787–793

    Article  CAS  Google Scholar 

  19. Xie M, Schowen RL (1999) Secondary structure and protein deamidation. J Pharm Sci 88:8–13

    Article  CAS  Google Scholar 

  20. Robinson NE, Robinson AB (2001) Prediction of protein deamidation rates from primary and three-dimensional structure. Proc Natl Acad Sci U S A 98:4367–4372

    Article  CAS  Google Scholar 

  21. Robinson NE, Robinson ZW, Robinson BR, Robinson AL, Robinson JA, Robinson ML, Robinson AB (2004) Structure-dependent nonenzymatic deamidation of glutaminyl and asparaginyl pentapeptides. J Pept Res 63:426–436

    Article  CAS  Google Scholar 

  22. Scotchler JW, Robinson AB (1974) Deamidation of glutamyl residues: dependence on pH, temperature, and ionic strength. Anal Biochem 59:319–322

    Article  CAS  Google Scholar 

  23. Song Y, Schowen RL, Borchardt RT, Topp EM (2001) Effect of ‘pH’ on the rate of asparagine deamidation in polymeric formulations: ‘pH’-rate profile. J Pharm Sci 90:141–156

    Article  CAS  Google Scholar 

  24. Stratton LP, Kelly RM, Rowe J, Shively JE, Sith DD, Carpenter JF, Manning MC (2001) Controlling deamidation rates in a model peptide: effects of temperature, peptide concentration, and additives. J Pharm Sci 90:2141–2148

    Article  CAS  Google Scholar 

  25. Wanaker A, Borchardt R (2006) Formulation considerations for proteins susceptible to asparagine deamidation and aspartate isomerization. J Pharm Sci 95:2321–2336

    Article  Google Scholar 

  26. Pace AL, Wong RL, Zhang YT, Kao Y-H, Wang YJ (2013) Asparagine deamidation dependence on buffer type, pH, and temperature. J Pharm Sci 102:1712–1723

    Article  CAS  Google Scholar 

  27. Stroop SD (2007) A modified peptide mapping strategy for quantifying site-specific deamidation by electrospray time-of-flight mass spectrometry. Rapid Commun Mass Spectrom 21:830–836

    Article  CAS  Google Scholar 

  28. Ren D, Pipes GD, Liu D, Shih L-Y, Nichols AC, Treuheit MJ, Brems DN, Bondarenko PV (2009) An improved trypsin digestion method minimizes digestion-induced modifications on proteins. Anal Biochem 392:12–21

    Article  CAS  Google Scholar 

  29. Gentleman R, Ihaka R et al The R Project for Statistical Computing. http://www.r-project.org/

  30. Filliben JJ, Heckert A, Lipman RR. NIST Dataplot. http://www.itl.nist.gov/div898/software/dataplot/

  31. Walmsley SJ, Rudnick PA, Liang Y, Dong Q, Stein SE, Nesvizhskii AI (2013) Comprehensive analysis of protein digestion using six trypsins reveals the origin of trypsin as a significant source of variability in proteomics. J Proteome Res 12(12):5666–5680

    Article  CAS  Google Scholar 

  32. Proc JL, Kuzyk MA, Hardie DB, Yang J, Smith DS, Jackson AM, Parker CE, Borchers CH (2010) A quantitative study of the effects of chaotropic agents, surfactants, and solvents on the digestion efficiency of human plasma proteins by tryptic digestions. J Proteome Res 9(10):5422–5437

    Article  CAS  Google Scholar 

  33. Robinson NE (2002) Protein deamidation. Proc Natl Acad Sci U S A 99:5283–8288

    Article  CAS  Google Scholar 

  34. Kossiakoff AA (1988) Tertiary structure is a principal determinant to protein deamidation. Science 240(4849):191–194

    Article  CAS  Google Scholar 

  35. Johnson BA, Shirokawa JM, Hancock WS, Spellman MW, Basa LJ, Aswad DW (1989) Formation of isoaspartate at two distinct sites during the in vitro aging of human growth hormone. J Biol Chem 264(24):14262–14271

    CAS  Google Scholar 

  36. NIST/SEMATECH e-Handbook of Statistical Methods, http://www.itl.nist.gov/div898/handbook/eda/section3/eda33c.htm, April 2012

  37. Tyler-Cross R, Schirch V (1991) Effects of amino acid sequence, buffers, and ionic strength on the rate and mechanism of deamidation of asparagine residues in small peptides. J Biol Chem 266(33):22549–22556

    CAS  Google Scholar 

  38. NIST/SEMATECH e-Handbook of Statistical Methods, http://www.itl.nist.gov/div898/handbook/prc/section4/prc43.htm, April 2012

  39. NIST/SEMATECH e-Handbook of Statistical Methods, http://www.itl.nist.gov/div898/handbook/prc/section4/prc437.htm, April 2012

  40. NIST/SEMATECH e-Handbook of Statistical Methods, http://www.itl.nist.gov/div898/handbook/prc/section4/prc471.htm, April 2012

Download references

Disclaimer

Certain commercial equipment, instruments, or materials are identified in this article to specify adequately the experimental procedure. Such identification does not imply recommendation or endorsement by the National Institute of Standards and Technology, nor does it imply that the materials or equipment identified are necessarily the best available for the purpose.

Author information

Authors and Affiliations

  1. Material Measurement Laboratory, Biomolecular Measurement Division, National Institute of Standards and Technology, 100 Bureau Drive, Stop 8314, Gaithersburg, MD, 20899, USA

    Trina Formolo & Karen W. Phinney

  2. Information Technology Laboratory, Statistical Engineering Division, National Institute of Standards and Technology, 100 Bureau Drive, Stop 8980, Gaithersburg, MD, 20899, USA

    Alan Heckert

Authors
  1. Trina Formolo
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Alan Heckert
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Karen W. Phinney
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Trina Formolo.

Additional information

Published in the topical collection Analysis of Biological Therapeutic Agents and Biosimilars with guest editor Karen Phinney.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(PDF 302 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Formolo, T., Heckert, A. & Phinney, K.W. Analysis of deamidation artifacts induced by microwave-assisted tryptic digestion of a monoclonal antibody. Anal Bioanal Chem 406, 6587–6598 (2014). https://doi.org/10.1007/s00216-014-8043-x

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-014-8043-x

Keywords

Search

Navigation