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

Part I: Dilute protein solutions
  • Maria Monica Castellanos
  • Steven C. Howell
  • D. Travis Gallagher
  • Joseph E. Curtis
Research Paper
  • 102 Downloads

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.

Graphical Abstract

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

Keywords

Small-angle scattering NISTmAb reference material Antibody structure Protein conformation Antibody flexibility Higher order structure 

Notes

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.

Compliance with Ethical Standards

Conflict of interests

All authors of this article declare no conflict of interest.

Supplementary material

216_2018_868_MOESM1_ESM.pdf (1.3 mb)
(PDF 1.25 MB )

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Copyright information

© This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply 2018

Authors and Affiliations

  1. 1.NIST Center for Neutron ResearchNational Institute of Standards and TechnologyGaithersburgUSA
  2. 2.Institute for Bioscience and Biotechnology ResearchUniversity of MarylandRockvilleUSA
  3. 3.Material Measurement LaboratoryNational Institute of Standards and TechnologyGaithersburgUSA
  4. 4.Institute for Bioscience and Biotechnology ResearchRockvilleUSA
  5. 5.Drug Product Development US, GSK VaccinesRockvilleUSA

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