Skip to main content
U.S. flag

An official website of the United States government

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you’ve safely connected to the .gov website. Share sensitive information only on official, secure websites.

Structural Changes and Aggregation Mechanisms for Anti-Streptavidin IgG1 at Elevated Concentration

Published

Author(s)

Gregory V. Barnett, Wei Qi, Samiul Amin, E. Neil Lewis, Vladimir I. Razinkov, Bruce A. Kerwin, Yun Liu, Christopher J. Roberts

Abstract

Non-native protein aggregation may occur during manufacturing and storage of protein therapeutics, and this may decrease drug efficacy or jeopardize patient safety. From a regulatory perspective, changes in higher-order structure due to aggregation are of particular interest, but can be difficult to monitor directly at elevated protein concentrations. The present report focuses on non-native aggregation of anti-streptavidin (AS)IgG1 at 30 mg/mL under solution conditions that prior work at dilute concentrations (e.g. 1 mg/mL) indicated would result in different aggregation mechanisms. Time-dependent aggregation and structural changes were monitored in-situ with dynamic light scattering, small-angle neutron scattering, and Raman scattering; and ex-situ with far-UV circular dichroism and second Derivative UV spectroscopy. The effects of adding 0.15 M (5 w/w %) sucrose were also assessed. The addition of sucrose decreased monomer loss rates, but did not change protein-protein interactions, aggregation mechanism(s), or aggregate structure and morphology. Consistent with prior results, altering the pD or salt concentration had the primary effect of changing the aggregation mechanism. Overall, the results provide a comparison of aggregate structure and morphology created via different growth mechanisms using orthogonal techniques, and show that the techniques agree at least qualitatively. Interestingly, AS-IgG1 aggregate3s created at a pD 5.3 with no added salt formed the smallest aggregates, but had the largest structural changes compared to other solution conditions. The observation that the larger aggregates were also those with less structural perturbation compared to folded AS-IgG1 might be expected to extend to other proteins if the same strong electrostatic repulsions that mediate aggregate growth also mediate structural changes of the constituent proteins within aggregates.
Citation
Journal of Physical Chemistry B
Volume
119
Issue
49

Keywords

Protein Aggregation, Protein Structure, Aggregate Mechanism, Aggregate Morphology

Citation

Barnett, G. , Qi, W. , Amin, S. , Lewis, E. , Razinkov, V. , Kerwin, B. , Liu, Y. and Roberts, C. (2015), Structural Changes and Aggregation Mechanisms for Anti-Streptavidin IgG1 at Elevated Concentration, Journal of Physical Chemistry B, [online], https://tsapps.nist.gov/publication/get_pdf.cfm?pub_id=919844 (Accessed May 29, 2024)

Issues

If you have any questions about this publication or are having problems accessing it, please contact reflib@nist.gov.

Created December 9, 2015, Updated October 12, 2021