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

Part II: Concentrated protein solutions

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Abstract

Protein-protein interactions in monoclonal antibody solutions are important for the stability of a therapeutic drug and directly influence viscosity in concentrated protein solutions. This study describes the use of small-angle scattering to estimate protein-protein interactions at high concentrations of the IgG1 NISTmAb reference material and validate colloidal models for interacting molecules. In particular, we studied the colloidal stability of the NISTmAb at high protein concentrations and analyzed protein-protein interactions upon adding sodium chloride and its effect on viscosity. Isotropic colloidal models for interacting molecules were combined with an ensemble of atomistic structures from molecular simulation to account for the flexibility of the NISTmAb in solution. In histidine formulation buffer, net repulsive electrostatic interactions are important for the colloidal stability of the NISTmAb at high concentrations. Addition of sodium chloride increased the viscosity of the NISTmAb and decreased the colloidal stability due to charge screening of the repulsive interactions. The interactions at high concentrations (up to ~ 250 mg/mL) were consistent with those from light scattering at low concentrations (below ~ 20 mg/mL). However, in the presence of sodium chloride, the screening of charges was less pronounced with increasing protein concentration and the interactions approached those of the repulsive hard-sphere models. Additionally, we studied the NISTmAb under frozen conditions using in situ neutron scattering to analyze the crowded state as proteins are excluded from the water-rich phase. In the frozen samples, where protein concentration can reach hundreds of mg/mL in the protein-rich phase, sodium chloride did not affect the molecular spacing and crowding of the NISTmAb.

Net repulsive interactions in concentrated NISTmAb solutions assessed by small-angle neutronscattering.

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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 their help and support to this work: 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 guiding the SEC measurements, Marco Blanco (NIST, IBBR) for discussions on G22, Samiul Amin (previously affiliated with Malvern Instruments) and Neil Lewis (Malvern Instruments) for starting and supporting a collaboration between Malvern and NIST. MMC acknowledges financial support from the NIST biomanufacturing initiative. This work used CCP-SAS software developed through a joint EPSRC (EP/K039121/1) and NSF (CHE-1265821) grant.

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  1. Maria Monica Castellanos

    Present address: Drug Product Development US, GSK Vaccines, 14200 Shady Grove Rd, Rockville, MD, 20850, USA

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, Susan Krueger & Joseph E. Curtis

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

    Maria Monica Castellanos

  3. Malvern Instruments, 117 Flanders Road, Westborough, MA, 01581, USA

    Kevin Mattison

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Correspondence to Joseph E. Curtis.

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Castellanos, M.M., Mattison, K., Krueger, S. et al. Characterization of the NISTmAb Reference Material using small-angle scattering and molecular simulation. Anal Bioanal Chem 410, 2161–2171 (2018). https://doi.org/10.1007/s00216-018-0869-1

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