Compensated Hydroxyl Radical Protein Footprinting Measures Buffer and Excipient Effects on Conformation and Aggregation in an Adalimumab Biosimilar
Unlike small molecule drugs, therapeutic proteins must maintain the proper higher-order structure (HOS) in order to maintain safety and efficacy. Due to the sensitivity of many protein systems, even small changes due to differences in protein expression or formulation can alter HOS. Previous work has demonstrated how hydroxyl radical protein footprinting (HRPF) can sensitively detect changes in protein HOS by measuring the average topography of the protein monomers, as well as identify specific regions of the therapeutic protein impacted by the conformational changes. However, HRPF is very sensitive to the radical scavenging capacity of the buffer; addition of organic buffers and/or excipients can dramatically alter the HRPF footprint without affecting protein HOS. By compensating for the radical scavenging effects of different adalimumab biosimilar formulations using real-time adenine dosimetry, we identify that sodium citrate buffer causes a modest decrease in average solvent accessibility compared to sodium phosphate buffer at the same pH. We find that the addition of polysorbate 80 does not alter the conformation of the biosimilar in either buffer, but it does provide substantial protection from protein conformational perturbation during short periods of exposure to high temperature. Compensated HRPF measurements are validated and contextualized by dynamic light scattering (DLS), which suggests that changes in adalimumab biosimilar aggregation are major drivers in measured changes in protein topography. Overall, compensated HRPF accurately measured conformational changes in adalimumab biosimilar that occurred during formulation changes and identified the effect of formulation changes on protection of HOS from temperature extremes.
KEY WORDSbiosimilars hydroxyl radical protein footprinting mass spectrometry protein conformations therapeutic proteins
J.S.S., R.O., and S.R.W. acknowledge support of the National Institute of General Medical Sciences (R43GM125420) to support commercial development of a benchtop FPOP device. J.S.S. and S.K.M. acknowledge support from the National Institute of General Medical Sciences for the development of compensation protocols for high radical scavenging environments (R01GM127267).
Compliance with Ethical Standards
Financial Conflict of Interest Disclosure
J.S.S., R.O., and S.R.W. disclose a significant financial interest in GenNext Technologies, Inc., an early-stage company seeking to commercialize technologies for protein higher-order structure analysis. This manuscript and all data were reviewed by S.K.M., who has no financial conflict of interest, in accordance with the University of Mississippi FCOI management practices.
- 1.Global biopharmaceuticals market growth, trends and forecasts (2016–2021). In: Current trends in biopharmaceuticals market. Hyderabad, India: Mordor Intelligence; 2016.Google Scholar
- 3.Giezen TJ, Mantel-Teeuwisse AK, Strauss S. Safety-related regulatory actions for biologicals approved in the United States and the European Union. J Am Med Soc. 2008;300(16):1887–96.Google Scholar
- 4.Giezen TJ, Schneider CK. Safety assessment of biosimilars in Europe: a regulatory perspective. Generics Biosimilars Initiat J. 2014;2014:1–8.Google Scholar
- 13.Huang W, Ravikumar KM, Chance MR, Yang S. Quantitative mapping of protein structure by hydroxyl radical footprinting-mediated structural mass spectrometry: a protection factor analysis. Biophys J. 2015;108(1):107–15. https://doi.org/10.1016/j.bpj.2014.11.013.CrossRefPubMedPubMedCentralGoogle Scholar
- 23.Lin M, Krawitz D, Callahan MD, Deperalta G, Wecksler AT. Characterization of ELISA antibody-antigen interaction using footprinting-mass spectrometry and negative staining transmission electron microscopy. J Am Soc Mass Spectrom. 2018;29(5):961–71. https://doi.org/10.1007/s13361-017-1883-9.CrossRefPubMedGoogle Scholar
- 25.Zhang Y, Wecksler AT, Molina P, Deperalta G, Gross ML. Mapping the binding interface of VEGF and a monoclonal antibody Fab-1 fragment with fast photochemical oxidation of proteins (FPOP) and mass spectrometry. J Am Soc Mass Spectrom. 2017;28(5):850–8. https://doi.org/10.1007/s13361-017-1601-7.CrossRefPubMedPubMedCentralGoogle Scholar
- 26.Li J, Wei H, Krystek SR Jr, Bond D, Brender TM, Cohen D, et al. Mapping the energetic epitope of an antibody/interleukin-23 interaction with hydrogen/deuterium exchange, fast photochemical oxidation of proteins mass spectrometry, and alanine shave mutagenesis. Anal Chem. 2017;89(4):2250–8. https://doi.org/10.1021/acs.analchem.6b03058.CrossRefPubMedPubMedCentralGoogle Scholar
- 29.Niu B, Mackness BC, Rempel DL, Zhang H, Cui W, Matthews CR, et al. Incorporation of a reporter peptide in FPOP compensates for adventitious scavengers and permits time-dependent measurements. J Am Soc Mass Spectrom. 2017;28(2):389–92. https://doi.org/10.1007/s13361-016-1552-4.CrossRefPubMedGoogle Scholar
- 35.Singh SM, Bandi S, Jones DNM, Mallela KMG. Effect of polysorbate 20 and polysorbate 80 on the higher-order structure of a monoclonal antibody and its Fab and Fc fragments probed using 2D nuclear magnetic resonance spectroscopy. J Pharm Sci. 2017;106(12):3486–98. https://doi.org/10.1016/j.xphs.2017.08.011.CrossRefPubMedPubMedCentralGoogle Scholar
- 36.Kaur P, Kiselar J, Yang S, Chance MR. Quantitative protein topography analysis and high-resolution structure prediction using hydroxyl radical labeling and tandem-ion mass spectrometry (MS). Mol Cell Proteomics. 2015;14(4):1159–68. https://doi.org/10.1074/mcp.O114.044362.CrossRefPubMedPubMedCentralGoogle Scholar