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Characterization of the NISTmAb Reference Material using small-angle scattering and molecular simulation

Part I: Dilute protein solutions

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Abstract

Both conformational and colloidal stability of therapeutic proteins must be closely monitored and thoroughly characterized to assess the long-term viability of drug products. We characterized the IgG1 NISTmAb reference material in its histidine formulation buffer and report our findings on the higher order structure and interactions of NISTmAb under a range of conditions. In this paper we present the analysis of experimental small-angle scattering data with atomistic molecular simulations to characterize the monodisperse dilute solution of NISTmAb. In part II we describe the characterization of the NISTmAb at high protein concentration (Castellanos et al. 2018). The NISTmAb was found to be a flexible protein with a radius of gyration of 49.0 ± 1.2 Å in histidine formulation buffer using a variety of neutron and X-ray scattering measurements. Scattering data were then modeled using molecular simulation. After building and validating a starting NISTmAb structure from the Fc and Fab crystallographic coordinates, molecular dynamics and torsion-angle Monte Carlo simulations were performed to explore the configuration space sampled in the NISTmAb and obtain ensembles of structures with atomistic detail that are consistent with the experimental data. Our results indicate that the small-angle scattering profiles of the NISTmAb can be modeled using ensembles of flexible structures that explore a wide configuration space. The NISTmAb is flexible in solution with no single preferred orientation of Fc and Fab domains, but with some regions of configuration space that are more consistent with measured scattering profiles. Analysis of inter-domain atomistic contacts indicated that all ensembles contained configurations where residues between domains are ≤ 4 Å, although few contacts were observed for variable and C H 3 regions.

Heavy atom self contact maps of the NISTmAb indicate a highly-flexible structure.

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Notes

  1. Certain commercial equipment, instruments, materials, suppliers, or software are identified in this paper to foster understanding. 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.

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Acknowledgements

The authors acknowledge the following scientists for supporting this work: Emilie Poudevigne (previously affiliated with ESRF) for collecting SAXS data at the ESRF, Zhiyuan Wang (Tsinghua University) and Yun Liu (NIST, University of Delaware) for their help with SANS data collection, John Schiel (NIST, IBBR) for making the material available for this study and for helping with the SEC measurements, James Snyder (NIST) for performing the energy minimization of the resulting structures from the torsion-angle MC simulation. MMC acknowledges financial support from the NIST biomanufacturing initiative. SCH acknowledges financial support from the NIST NRC Postdoctoral Research Associateship Program. This work used CCP-SAS software developed through a joint EPSRC (EP/K039121/1) and NSF (CHE-1265821) grant.

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Authors and Affiliations

  1. NIST Center for Neutron Research, National Institute of Standards and Technology, 100 Bureau Drive, Mail Stop 6102, Gaithersburg, MD, 20899, USA

    Maria Monica Castellanos, Steven C. Howell & Joseph E. Curtis

  2. Institute for Bioscience and Biotechnology Research, University of Maryland, 9600 Gudelsky Drive, Rockville, MD, 20850, USA

    Maria Monica Castellanos

  3. Material Measurement Laboratory, National Institute of Standards and Technology, 100 Bureau Drive, Gaithersburg, MD, 20899, USA

    D. Travis Gallagher

  4. Institute for Bioscience and Biotechnology Research, 9600 Gudelsky Drive, Rockville, MD, 20850, USA

    D. Travis Gallagher

  5. Drug Product Development US, GSK Vaccines, 14200 Shady Grove Rd., Rockville, MD, 20850, USA

    Maria Monica Castellanos

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Castellanos, M.M., Howell, S.C., Gallagher, D.T. et al. Characterization of the NISTmAb Reference Material using small-angle scattering and molecular simulation. Anal Bioanal Chem 410, 2141–2159 (2018). https://doi.org/10.1007/s00216-018-0868-2

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