Skip to main content
SpringerLink
Log in

Fragmentation of a Monoclonal Antibody by Peroxotungstate

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

Abstract

Purpose

Tungsten and tungsten oxide leachates found in glass pre-filled syringes were identified to initiate protein precipitation and aggregation. Here, we tested the possibility of tungsten and tungsten oxide to induce the chemical degradation of proteins via reaction with hydrogen peroxide, a possible impurity present in protein formulations, to yield peroxotungstate.

Methods

A monoclonal antibody (mAb) was incubated with various concentrations of peroxotungstate and the reaction mixtures analyzed by SDS-PAGE and mass spectrometry.

Results

Exposure of a mAb to 1.07–1070 ppm peroxotungstate (based on tungsten content) at temperatures of 4°C and 22°C (pH 5–7) induced protein fragmentation. The extent of fragmentation increased with higher temperatures, lower pH and higher peroxotungstate concentrations. The mAb fragments were identified to contain different combinations of heavy chains (H) and light chains (L). Analogous mAb fragments were generated when the protein was exposed to H2O2 and orthotungstate at levels as low as 5 ppm. In addition, extracts from tungsten pins used to manufacture glass pre-filled syringes, in combination with H2O2 caused comparable fragmentation of the mAb. Mass spectrometric identification of the fragments suggests fragment generation by oxidative disulfide bond cleavage between the heavy and light chains, confirmed by mass spectrometry data on product formation. The mechanism of oxidative fragmentation was separately confirmed with insulin.

Conclusion

Fragmentation of the mAb by peroxotungstate is proposed to occur through inter-chain disulfide bond oxidation to form thiosulfinate (CyS(═O)SCy) and thiosulfonate [CyS(═O)2SCy], followed by hydrolysis.

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
Chart 1
Fig. 9
Fig. 10
Fig. 11
Scheme 1

Similar content being viewed by others

Notes

  1. The actual value given in the paper is ca. 250 mEq, but was expressed per kg of neat PS80 by multiplication by 5 (35). Therefore, the peroxide levels in 20% PS80 can be computed to be equivalent to ca. 50 mEq.

Abbreviations

ICP-MS:

Inductively coupled plasma mass spectrometry

mAb:

Monoclonal antibody

MS:

Mass spectrometry

SDS:

Sodium dodecyl sulfate

SDS-PAGE:

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

UPLC:

Ultra performance liquid chromatography

VHP:

Vaporized hydrogen peroxide

WO4 2− :

Orthotungstate

References

  1. Visiongain. Global biologics market, industry and R&D: Forecasts 2015–2025. Available from: https://www.visiongain.com/Report/1485/Global-Biologics-Market-Industry-and-R-D-Forecasts-2015-2025.

  2. Adler M. INJECTABLES-challenges in the development of pre-filled syringes for biologics from a formulation Scientist's point of view. Am Pharm Rev. 2012;15(1):96.

    CAS  Google Scholar 

  3. Jiang Y, Nashed-Samuel Y, Li C, Liu W, Pollastrini J, Mallard D, et al. Tungsten-induced protein aggregation: solution behavior. J Pharm Sci. 2009;98(12):4695–710.

    Article  CAS  Google Scholar 

  4. Bee JS, Nelson SA, Freund E, Carpenter JF, Randolph TW. Precipitation of a monoclonal antibody by soluble tungsten. J Pharm Sci. 2009;98(9):3290–301.

    Article  CAS  Google Scholar 

  5. Liu W, Swift R, Torraca G, Nashed-Samuel Y, Wen ZQ, Jiang Y, et al. Root cause analysis of tungsten-induced protein aggregation in pre-filled syringes. PDA J Pharm Sci Technol. 2010;64(1):11–9.

    CAS  PubMed  Google Scholar 

  6. Ronk M, Lee H, Fujimori K, Yeh P, Nashed-Samuel Y. Characterization of protein aggregating Tungstates: electrospray mass spectrometry analysis of extracts from prefilled syringes and from tungsten pins used in the manufacture of syringes. PDA J Pharm Sci Technol. 2016;70(1):51–61.

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

  8. Seidl A, Hainzl O, Richter M, Fischer R, Bohm S, Deutel B, et al. Tungsten-induced denaturation and aggregation of epoetin alfa during primary packaging as a cause of immunogenicity. Pharm Res. 2012;29(6):1454–67.

    Article  CAS  Google Scholar 

  9. Markovic I. Challenges associated with extractable and/or leachable substances in therapeutic biologic protein products. Am Pharm Rev. 2006;9(6):20–7.

    CAS  Google Scholar 

  10. Sacha G, Rogers JA, Miller RL. Pre-filled syringes: a review of the history, manufacturing and challenges. Pharm Dev Technol. 2015;20(1):1–11.

    Article  CAS  Google Scholar 

  11. Kishore RS, Pappenberger A, Dauphin IB, Ross A, Buergi B, Staempfli A, et al. Degradation of polysorbates 20 and 80: studies on thermal autoxidation and hydrolysis. J Pharm Sci. 2011;100(2):721–31.

    Article  CAS  Google Scholar 

  12. Agalloco JP, Akers JE. Overcoming limitations of vaporized hydrogen peroxide. Pharm Technol. 2013;37(9):46–56.

    CAS  Google Scholar 

  13. Ishimoto R, Kamata K, Mizuno N. A highly active protonated tetranuclear peroxotungstate for oxidation with hydrogen peroxide. Angew Chem Int Ed Engl. 2012;51(19):4662–5.

    Article  CAS  Google Scholar 

  14. Kudo T, Okamoto H, Matsumoto K, Sasaki Y. Peroxopolytungstic acids synthesized by direct reaction of tungsten or tungsten carbide with hydrogen peroxide. Inorg Chim Acta. 1986;111(2):L27–8.

    Article  CAS  Google Scholar 

  15. Nakajima H, Kudo T, Mizuno N. Reaction of metal, carbide, and nitride of tungsten with hydrogen peroxide characterized by 183W nuclear magnetic resonance and raman spectroscopy. Chem Mater. 1999;11(3):691–7.

    Article  CAS  Google Scholar 

  16. Okamoto A, Sugizaki K, Nakamura A, Yanagisawa H, Ikeda S. 5-Hydroxymethylcytosine-selective oxidation with peroxotungstate. Chem Commun. 2011;47(40):11231–3.

    Article  CAS  Google Scholar 

  17. Shi X-Y, Wei J-F. Selective oxidation of sulfide catalyzed by peroxotungstate immobilized on ionic liquid-modified silica with aqueous hydrogen peroxide. J Mol Catal A Chem. 2008;280(1–2):142–7.

    Article  CAS  Google Scholar 

  18. Usui Y, Sato K. A green method of adipic acid synthesis: organic solvent-and halide-free oxidation of cycloalkanones with 30% hydrogen peroxide. Green Chem. 2003;5(4):373–5.

    Article  CAS  Google Scholar 

  19. Howarth OW. Oxygen-17 NMR study of aqueous peroxotungstates. Dalton Trans. 2004;(3):476–81.

  20. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227(5259):680–5.

    Article  CAS  Google Scholar 

  21. Lassner E, Schubert W-D. Properties, chemistry, Technology of the Element, alloys, and chemical compounds. Vienna University of Technology, Vienna, Austria, Kluwer 1999:124–125.

  22. Scientific T-F. NanoDrop 2000/2000c Protein Assay Guide (T032 - Technical bulletin. Available from: https://assets.thermofisher.com/TFS-Assets/CAD/manuals/NanoDrop-2000-User-Manual-EN.pdf.

  23. Shevchenko A, Tomas H, Havli J, Olsen JV, Mann M. In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat Protoc. 2006;1(6):2856.

    Article  CAS  Google Scholar 

  24. Saphire EO, Parren PW, Pantophlet R, Zwick MB, Morris GM, Rudd PM, et al. Crystal structure of a neutralizing human IGG against HIV-1: a template for vaccine design. Science. 2001;293(5532):1155–9.

    Article  CAS  Google Scholar 

  25. Campbell NJ, Dengel AC, Edwards CJ, Griffith WP. Studies on transition-metal Peroxo complexes .8. The nature of Peroxomolybdates and Peroxotungstates in aqueous-solution. J Chem Soc Dalton Trans. 1989;(6):1203–8.

  26. Kamata K, Yamaguchi K, Mizuno N. Highly selective, recyclable epoxidation of allylic alcohols with hydrogen peroxide in water catalyzed by dinuclear peroxotungstate. Chem Eur J. 2004;10(19):4728–34.

    Article  CAS  Google Scholar 

  27. Kanner GS, Butt DP. Raman and electrochemical probes of the dissolution kinetics of tungsten in hydrogen peroxide. J Phys Chem A. 1998;102(47):9501–7.

    Article  CAS  Google Scholar 

  28. Ross-Medgaarden EI, Wachs IE. Structural determination of bulk and surface tungsten oxides with UV-vis diffuse reflectance spectroscopy and Raman spectroscopy. J Phys Chem C. 2007;111(41):15089–99.

    Article  CAS  Google Scholar 

  29. George B, Aruldhas G, Botto I. Vibrational spectra of sodium paratungstate 26 hydrate, Na 10 (H 2 W 12 O 42)·26H 2 O. J Mater Sci Lett. 1992;11(21):1421–3.

    Article  CAS  Google Scholar 

  30. Zhu ZC, Chen Y, Ackerman MS, Wang B, Wu W, Li B, et al. Investigation of monoclonal antibody fragmentation artifacts in non-reducing SDS-PAGE. J Pharm Biomed Anal. 2013;83:89–95.

    Article  CAS  Google Scholar 

  31. Fradkin AH, Mozziconacci O, Schoneich C, Carpenter JF, Randolph TW. UV photodegradation of murine growth hormone: chemical analysis and immunogenicity consequences. Eur J Pharm Biopharm. 2014;87(2):395–402.

    Article  CAS  Google Scholar 

  32. Sreedhara A, Lau K, Li C, Hosken B, Macchi F, Zhan D, et al. Role of surface exposed tryptophan as substrate generators for the antibody catalyzed water oxidation pathway. Mol Pharm. 2013;10(1):278–88.

    Article  CAS  Google Scholar 

  33. Ghiron AF, Thompson RC. Kinetic study of the oxygen-transfer reactions from the oxo diperoxo complexes of molybdenum (VI) and tungsten (VI) to (thiolato)-and (sulfenato) cobalt (III) complexes. Inorg Chem. 1988;27(26):4766–71.

    Article  CAS  Google Scholar 

  34. Nagy P, Ashby MT. Reactive sulfur species: kinetics and mechanism of the hydrolysis of cysteine thiosulfinate ester. Chem Res Toxicol. 2007;20(9):1364–72.

    Article  CAS  Google Scholar 

  35. Ha E, Wang W, Wang YJ. Peroxide formation in polysorbate 80 and protein stability. J Pharm Sci. 2002;91(10):2252–64.

    Article  CAS  Google Scholar 

  36. Wasylaschuk WR, Harmon PA, Wagner G, Harman AB, Templeton AC, Xu H, et al. Evaluation of hydroperoxides in common pharmaceutical excipients. J Pharm Sci. 2007;96(1):106–16.

    Article  CAS  Google Scholar 

  37. Lam XM, Lai WG, Chan EK, Ling V, Hsu CC. Site-specific tryptophan oxidation induced by autocatalytic reaction of polysorbate 20 in protein formulation. Pharm Res. 2011;28(10):2543–55.

    Article  CAS  Google Scholar 

  38. Wu Y, Levons J, Narang AS, Raghavan K, Rao VM. Reactive impurities in excipients: profiling, identification and mitigation of drug-excipient incompatibility. AAPS PharmSciTech. 2011;12(4):1248–63.

    Article  CAS  Google Scholar 

  39. Ding SL. Quantitation of Hydroperoxides in the aqueous-solutions of nonionic surfactants using Polysorbate-80 as the model surfactant. J Pharm Biomed Anal. 1993;11(2):95–101.

    Article  CAS  Google Scholar 

  40. Skliri E, Lykakis IN, Armatas GS. Ordered mesoporous V2O5/WO3 composite catalysts for efficient oxidation of aryl alcohols. RSC Adv. 2014;4(86):46170–8.

    Article  CAS  Google Scholar 

  41. Khaksar M, Boghaei DM, Amini M. Synthesis, structural characterization and reactivity of manganese tungstate nanoparticles in the oxidative degradation of methylene blue. Cr Chim. 2015;18(2):199–203.

    Article  CAS  Google Scholar 

Download references

ACKNOWLEDGMENTS AND DISCLOSURES

We would like to thank Genentech for support of this study, and Drs. G. Macpherson and C. Marshall at the Department of Geology, The University of Kansas, for their help with the ICP-MS measurements and Raman spectroscopy studies.

Author information

Author notes

  1. Olivier Mozziconacci

    Present address: Merck Research Laboratories, Rahway, NJ, 07065, USA

Authors and Affiliations

  1. Department of Pharmaceutical Chemistry, University of Kansas, 2095 Constant Ave, Lawrence, Kansas, 66047, USA

    Hasitha Rathnayaka, Olivier Mozziconacci & Christian Schöneich

  2. Department of Chemistry, University of Kansas, Lawrence, Kansas, 66045, USA

    Hasitha Rathnayaka

  3. Late Stage Pharmaceutical Development, Genentech Inc., S. San Francisco, California, 94080, USA

    Alavattam Sreedhara

Authors
  1. Hasitha Rathnayaka
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Olivier Mozziconacci
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Alavattam Sreedhara
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Christian Schöneich
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Christian Schöneich.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Rathnayaka, H., Mozziconacci, O., Sreedhara, A. et al. Fragmentation of a Monoclonal Antibody by Peroxotungstate. Pharm Res 35, 219 (2018). https://doi.org/10.1007/s11095-018-2496-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1007/s11095-018-2496-0

Key Words

Search

Navigation