Skip to main content
SpringerLink
Log in

Chemical and Biophysical Characteristics of Monoclonal Antibody Solutions Containing Aggregates Formed during Metal Catalyzed Oxidation

  • Research Paper
  • Published:
Pharmaceutical Research Aims and scope Submit manuscript

Abstract

Purpose

To physicochemically characterize and compare monoclonal antibody (mAb) solutions containing aggregates generated via metal catalyzed oxidation (MCO).

Methods

Two monoclonal IgG2s (mAb1 and mAb2) and one monoclonal IgG1 (rituximab) were exposed to MCO with the copper/ascorbic acid oxidative system, by using several different methods. The products obtained were characterized by complementary techniques for aggregate and particle analysis (from oligomers to micron sized species), and mass spectrometry methods to determine the residual copper content and chemical modifications of the proteins.

Results

The particle size distribution and the morphology of the protein aggregates generated were similar for all mAbs, independent of the MCO method used. There were differences in both residual copper content and in chemical modification of specific residues, which appear to be dependent on both the protein sequence and the protocol used. All products showed a significant increase in the levels of oxidized His, Trp, and Met residues, with differences in extent of modification and specific amino acid residues modified.

Conclusion

The extent of total oxidation and the amino acid residues with the greatest oxidation rate depend on a combination of the MCO method used and the protein sequence.

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

Similar content being viewed by others

Abbreviations

i.d.:

Inner diameter

ICP-MS:

Inductively coupled plasma-mass spectrometry

mAb:

Monoclonal antibody

MCO:

Metal catalyzed oxidation

MFI:

Micro-Flow Imaging

o.d.:

Outer diameter

PBMC:

Peripheral blood mononuclear cells

PDB:

Protein Data Bank

ppb:

Parts per billion

rCE-SDS:

Reduced capillary electrophoresis-sodium dodecyl sulfate

SAS:

Solvent accessible surface

References

  1. ICH Q3D Impurities: Guideline for elemental impurities. EMA/CHMP/ICH/353369/2013. 2009.

  2. ICH Q6B Test Procedures and Acceptance Criteria for Biotechnological/Biological Products. EMA/CHMP/ICH/365/96. 1999.

  3. Carpenter JF, Randolph TW, Jiskoot W, Crommelin DJ, Middaugh CR, Winter G, et al. Overlooking subvisible particles in therapeutic protein products: gaps that may compromise product quality. J Pharm Sci. 2009;4:1201–5.

    Article  Google Scholar 

  4. Joubert MK, Hokom M, Eakin C, Zhou L, Deshpande M, Baker MP, et al. Highly aggregated antibody therapeutics can enhance the in vitro innate and late-stage T-cell immune responses. J Biol Chem. 2012;30:25266–79.

    Article  Google Scholar 

  5. Bi V, Jawa V, Joubert MK, Kaliyaperumal A, Eakin C, Richmond K, et al. Development of a human antibody tolerant mouse model to assess the immunogenicity risk due to aggregated biotherapeutics. J Pharm Sci. 2013;102:3545–55.

    Article  CAS  Google Scholar 

  6. Bessa J, Boeckle S, Beck H, Buckel T, Schlicht S, Ebeling M, et al. The immunogenicity of antibody aggregates in a novel transgenic mouse model. Pharm Res. 2015;32:2344–59.

    Article  CAS  Google Scholar 

  7. Hermeling S, Aranha L, Damen JMA, Slijper M, Schellekens H, Crommelin DJA, et al. Structural characterization and immunogenicity in immune tolerant mice of recombinant human interferon alpha2b. Pharm Res. 2005;22:1997–2006.

    Article  CAS  Google Scholar 

  8. Van Beers MMC, Sauerborn M, Gilli F, Brinks V, Schellekens H, Jiskoot W. Oxidized and aggregated recombinant interferon beta is immunogenic in human interferon beta transgenic mice. Pharm Res. 2011;28:2393–402.

    Article  Google Scholar 

  9. Jiskoot W, Kijanka G, Randolph TW, Carpenter JF, Koulov AV, Mahler HC, et al. Mouse models for assessing protein immunogenicity: lessons and challenges. J Pharm Sci. 2016;105:1567–75.

    Article  CAS  Google Scholar 

  10. Torosantucci R, Schöneich C, Jiskoot W. Oxidation of therapeutic proteins and peptides: structural and biological consequences. Pharm Res. 2014;31:541–53.

    Article  CAS  Google Scholar 

  11. Hermeling S, Schellekens H, Maas C, Gebbink MFBG, Crommelin DJA, Jiskoot W. Antibody response to aggregated human interferon alpha2b in wildtype and transgenic immune tolerant mice depends on type and level of aggregation. J Pharm Sci. 2006;95:1084–96.

    Article  CAS  Google Scholar 

  12. Joubert MK, Luo Q, Nashed-Samuel Y, Wypych J, Narhi LO. Classification and characterization of therapeutic antibody aggregates. J Biol Chem. 2011;286:25118–33.

    Article  CAS  Google Scholar 

  13. Torosantucci R, Mozziconacci O, Sharov V, Schöneich C, Jiskoot W. Chemical modifications in aggregates of recombinant human insulin induced by metal-catalyzed oxidation: covalent cross-linking via Michael addition to tyrosine oxidation products. Pharm Res. 2012;29:2276–93.

    Article  CAS  Google Scholar 

  14. Torosantucci R, Sharov VS, van Beers M, Brinks V, Schöneich C, Jiskoot W. Identification of oxidation sites and covalent cross-links in metal catalyzed oxidized interferon Beta-1a: potential implications for protein aggregation and immunogenicity. Mol Pharm. 2013;10:2311–22.

    Article  CAS  Google Scholar 

  15. Luo Q, Joubert MK, Stevenson R, Ketchem RR, Narhi LO, Wypych J. Chemical Modifications in Therapeutic Protein Aggregates Generated under Different Stress Conditions. J Biol Chem. 2011;286:25134–44.

    Article  CAS  Google Scholar 

  16. Buettner GR, Jurkiewicz BA. Catalytic metals, ascorbate and free radicals: combinations to avoid. Radiat Res. 1996;145:532–41.

    Article  CAS  Google Scholar 

  17. Tadeo X, Pons M, Millet O. Influence of the Hofmeister anions on protein stability as studied by thermal denaturation and chemical shift perturbation. Biochemistry. 2007;46:917–23.

    Article  CAS  Google Scholar 

  18. Ren D, Pipes GD, Liu D, Shih LY, Nichols AC, Treuheit MJ, et al. An improved trypsin digestion method minimizes digestion-induced modifications on proteins. Anal Biochem. 2009;392:12–21.

    Article  CAS  Google Scholar 

  19. Zhang Z. Prediction of low-energy collision-induced dissociation spectra of peptides. Anal Chem. 2004;76:3908–22.

    Article  CAS  Google Scholar 

  20. Zhao F, Ghezzo-Schöneich E, Aced GI, Hong J, Milby T, Schöneich C. Metal-catalyzed oxidation of histidine in human growth hormone. Mechanism, isotope effects, and inhibition by a mild denaturing alcohol. J Biol Chem. 1997;272:9019–29.

    Article  CAS  Google Scholar 

  21. Hovorka SW, Hong J, Cleland JL, Schöneich C. Metal-catalyzed oxidation of human growth hormone: modulation by solvent-induced changes of protein conformation. J Pharm Sci. 2001;90:58–69.

    Article  CAS  Google Scholar 

  22. Zhou S, Mozziconacci O, Kerwin BA, Schöneich C. Fluorogenic tagging methodology applied to characterize oxidized tyrosine and phenylalanine in an immunoglobulin monoclonal antibody. Pharm Res. 2013;30:1311–27.

    Article  CAS  Google Scholar 

  23. Wang W, Singh S, Zeng DL, King K, Nema S. Antibody structure, instability, and formulation. J Pharm Sci. 2007;96:1–26.

    Article  CAS  Google Scholar 

  24. Xu J, Jordan RB. Kinetics and mechanism of the reaction of aqueous copper(II) with ascorbic acid. Inorg Chem. 1990;29:2933–6.

    Article  CAS  Google Scholar 

  25. Kato Y, Kitamoto N, Kawai Y, Osawa T. The hydrogen peroxide/copper ion system, but not other metal-catalyzed oxidation systems, produces protein-bound dityrosine. Free Radic Biol Med. 2001;31:624–32.

    Article  CAS  Google Scholar 

  26. Jansson, Patric J. Pro-oxidant activity of vitamin C in drinking water: role of copper, iron and bicarbonate. Department of Biochemistry and Pharmacy, Åbo Akademi University; 2006.

  27. Biaglow JE, Manevich Y, Uckun F, Held KD. Quantitation of hydroxyl radicals produced by radiation and copper-linked oxidation of ascorbate by 2-deoxy-D-ribose method. Free Radic Biol Med. 1997;22:1129–38.

    Article  CAS  Google Scholar 

  28. Ohta Y, Shiraishi N, Nishikawa T, Nishikimi M. Copper-catalyzed autoxidations of GSH and L-ascorbic acid: mutual inhibition of the respective oxidations by their coexistence. Biochim Biophys Acta. 2000;1474:378–82.

    Article  CAS  Google Scholar 

  29. Davies MJ. Protein oxidation and peroxidation. Biochem J. 2016;473(Pt 7):805–25.

    Article  CAS  Google Scholar 

  30. Schöneich C. Mechanisms of metal-catalyzed oxidation of histidine to 2-oxo-histidine in peptides and proteins. J Pharm Biomed Anal. 2000;21:1093–7.

    Article  Google Scholar 

  31. Glover ZK, Basa L, Moore B, Laurence JS, Sreedhara A. Metal ion interactions with mAbs: pH and conformation modulate copper-mediated site-specific fragmentation of the IgG1 hinge region. Part 1. MAbs. 2015;7:901–11.

    Article  CAS  Google Scholar 

  32. Telikepalli S, Shinogle HE, Thapa PS, Kim JH, Deshpande M, Jawa V, et al. Physical characterization and in vitro biological impact of highly aggregated antibodies separated into size-enriched populations by fluorescence-activated cell sorting. J Pharm Sci. 2015;104:1575–91.

    Article  CAS  Google Scholar 

  33. Björn B, Juliana B, Emilien F. Quiroz Anacelia Ríos, Schmidt Roland, Bulau Patrick, Finkler Christof, Mahler Hanns-Christian, Huwyler Jörg, Iglesias Antonio, and Koulov Atanas V. Extensive chemical modifications in the primary protein structure of IgG1 subvisible particles are necessary for breaking immune tolerance. Mol Pharm. 2017;14:1292–12992017.

    Article  Google Scholar 

Download references

ACKNOWLEDGMENTS AND DISCLOSURES

The authors wish to thank Diana Woehle for the preparation of mAb2, and Daniel Weinbuch for the preparation of MCO treated rituximab.

Author information

Authors and Affiliations

  1. Attribute Sciences, Amgen Inc., One Amgen Center Dr, M/S 30-1-B, Thousand Oaks, California, 91320, USA

    Linda O. Narhi, Quanzhou Luo, Jette Wypych, Kiyoshi Fujimori, Yasser Nashed-Samuel & Marisa K. Joubert

  2. Coriolis Pharma, Martinsried, Munich, Germany

    Riccardo Torosantucci, Andrea Hawe & Wim Jiskoot

  3. Medical Sciences, Amgen Inc., Thousand Oaks, California, 91320, USA

    Vibha Jawa

  4. Currently at Merck, Kenilworth, NJ, USA

    Vibha Jawa

  5. Division of Drug Delivery Technology, Cluster BioTherapeutics, Leiden Academic Centre for Drug Research (LACDR), Leiden University, Leiden, The Netherlands

    Wim Jiskoot

Authors
  1. Linda O. Narhi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Quanzhou Luo
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jette Wypych
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Riccardo Torosantucci
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Andrea Hawe
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Kiyoshi Fujimori
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Yasser Nashed-Samuel
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Vibha Jawa
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Marisa K. Joubert
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Wim Jiskoot
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Linda O. Narhi.

Electronic supplementary material

Supplemental Figure 1

Comparison of particle number and size distribution of micron particles generated by MCO via different detection techniques. mAb1, mAb2, and rituximab untreated and MCO samples were examined by A) HIAC/light obscuration and B) MFI to determine the number and size range of particles present. For each sample, the differential particle counts per ml for each size range is shown. (JPG 132 kb)

High Resolution Image (TIFF 3.79 mb)

Supplemental Figure 2

Comparison of the biophysical and chemical properties of the mAb2 antibody treated by three different MCO methods. MCO methods 1, 2 and 3 are described in detail in the materials and methods section. A) mAb2 untreated and MCO samples were examined by HIAC to determine the number and size range of particles present. For each sample, the differential particle counts per ml for each size range is shown. B) Particle images were captured on a MFI system. Representative images of the largest particles detected are shown. The size threshold (equivalent circular diameter) indicates the lower size limit of the particles that were used for comparison. C) The level of elemental copper (ppb) in each sample was determined by ICP-MS. The ratio shown represents the calculated ratio of the molecules of antibody (mAb) to molecules of copper. D) The percent oxidation of each amino acid of mAb2 was determined by peptide map. (JPG 192 kb)

High Resolution Image (TIFF 5.32 mb)

Supplemental Figure 3

HCD MS/MS spectrum of the His 272 oxidized peptide H20 (A), His272 oxidized peptide H21 (B) and His 314 oxidized peptide H24 (C). (JPG 186 kb)

High Resolution Image (TIFF 6.00 mb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Narhi, L.O., Luo, Q., Wypych, J. et al. Chemical and Biophysical Characteristics of Monoclonal Antibody Solutions Containing Aggregates Formed during Metal Catalyzed Oxidation. Pharm Res 34, 2817–2828 (2017). https://doi.org/10.1007/s11095-017-2262-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11095-017-2262-8

Key words

Search

Navigation