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

• Published in: The journal of physical chemistry. B 2015-12, Vol.119 (49), p.15150-15163
• Main Authors: Barnett, Gregory V, Qi, Wei, Amin, Samiul, Lewis, E. Neil, Razinkov, Vladimir I, Kerwin, Bruce A, Liu, Yun, Roberts, Christopher J
• Format: Article
• Language: English
• Subjects: Circular Dichroism; Immunoglobulin G - chemistry; Immunoglobulin G - immunology; Kinetics; Protein Conformation; Spectrophotometry, Ultraviolet; Spectrum Analysis, Raman; Streptavidin - immunology
• ISSN: 1520-6106; 1520-5207
• DOI: 10.1021/acs.jpcb.5b08748

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 antistreptavidin (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 aggregates created at 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.