Complementary MS Methods Assist Conformational Characterization of Antibodies with Altered S–S Bonding Networks

Abstract

As therapeutic monoclonal antibodies (mAbs) become a major focus in biotechnology and a source of the next-generation drugs, new analytical methods or combination methods are needed for monitoring changes in higher order structure and effects of post-translational modifications. The complexity of these molecules and their vulnerability to structural change provide a serious challenge. We describe here the use of complementary mass spectrometry methods that not only characterize mutant mAbs but also may provide a general framework for characterizing higher order structure of other protein therapeutics and biosimilars. To frame the challenge, we selected members of the IgG2 subclass that have distinct disulfide isomeric structures as a model to evaluate an overall approach that uses ion mobility, top-down MS sequencing, and protein footprinting in the form of fast photochemical oxidation of proteins (FPOP). These three methods are rapid, sensitive, respond to subtle changes in conformation of Cys → Ser mutants of an IgG2, each representing a single disulfide isoform, and may be used in series to probe higher order structure. The outcome suggests that this approach of using various methods in combination can assist the development and quality control of protein therapeutics.

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Acknowledgments

The work was supported by grants from the National Institute of General Medical Sciences (8 P41 GM103422-35) of the NIH, by a Pfizer Global Research and Development postdoctoral fellowship for L.M.J., and by the NSF (IBDR 0964199 to M.L.G.). Partial salary support for H.Z. and instrument support for the Waters Synapt G2 mass spectrometer were from the Photosynthetic Antenna Research Center, an Energy Frontier Research Center funded by the US DOE, Office of Basic Energy Sciences (grant no. DE-SC 0001035 to Robert E. Blankenship). The authors thank Professor Blankenship for providing instrument access, M. Martinez, Waters, for help with nanoESI, B. Ruotolo, University of Michigan, M. Plasencia, and H. Rohrs for helpful discussions, and B. Gau for help with data processing.

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Correspondence to Michael L. Gross.

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Lisa M. Jones and Hao Zhang contributed equally to this work.

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Jones, L.M., Zhang, H., Cui, W. et al. Complementary MS Methods Assist Conformational Characterization of Antibodies with Altered S–S Bonding Networks. J. Am. Soc. Mass Spectrom. 24, 835–845 (2013). https://doi.org/10.1007/s13361-013-0582-4

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Key words

  • Antibody characterization
  • Protein structure
  • Fast photochemical oxidation of proteins (FPOP)
  • Top-down
  • Ion mobility
  • Antibody mutants
  • IgG2