Chemical Modifications in Therapeutic Protein Aggregates Generated under Different Stress Conditions

Abstract

In this study, we characterized the chemical modifications in the monoclonal antibody (IgG 2 ) aggregates generated under various conditions, including mechanical, chemical, and thermal stress treatment, to provide insight into the mechanism of protein aggregation and the types of aggregate produced by the different stresses. In a separate study, additional biophysical characterization was performed to arrange these aggregates into a classification system (Joubert, M. K., Luo, Q., Nashed-Samuel, Y., Wypych, J., and Narhi, L. O. (2011) J. Biol. Chem. 286, 25118 -25133). Here, we report that different aggregates possessed different types and levels of chemical modification. For chemically treated samples, metal-catalyzed oxidation using copper showed site-specific oxidation of Met 246 , His 304 , and His 427 in the Fc portion of the antibody, which might be attributed to a putative copper-binding site. For the hydrogen peroxidetreated sample, in contrast, four solvent-exposed Met residues in the Fc portion were completely oxidized. Met and/or Trp oxidation was observed in the mechanically stressed samples, which is in agreement with the proposed model of protein interaction at the air-liquid interface. Heat treatment resulted in significant deamidation but almost no oxidation, which is consistent with thermally induced aggregates being generated by a different pathway, primarily by perturbing conformational stability. These results demonstrate that chemical modifications are present in protein aggregates; furthermore, the type, locations, and severity of the modifications depend on the specific conditions that generated the aggregates.

Classification and Characterization of Therapeutic Antibody Aggregates

“…Results were used to differentiate aggregated solutions by percent aggregation, particle counts, size distribution, gross morphology, differences in secondary and tertiary structure, surface hydrophobicity, reversibility, and metal content. An in-depth investigation of chemical modifications introduced by the various stress conditions was also conducted in a separate study (41). The fact that aggregates from multiple IgG antibodies, mAb1, mAb2, and mAb3 (from both the IgG 1 and IgG 2 subclasses), and IVIG (containing all IgG subtypes) show the same attributes when subjected to the same treatment demonstrates that a similar pathway of aggregation may be used by distinct IgG molecules under the same applied stress.…”

Using extensional flow to reveal diverse aggregation landscapes for three IgG1 molecules

“…These developing in silico approaches complement established accelerated stress studies that are performed in vitro to predict the shelf‐life and stability of biologics (Jain et al, 2017; Yang et al, 2013). Various methods are employed to generate such data including heating (Cheng et al, 2012; Hamrang et al, 2015), stirring (Luo et al, 2011; Sediq, Van Duijvenvoorde, Jiskoot, & Nejadnik, 2016), shaking (Kiese, Papppenberger, Friess, & Mahler, 2008; Rudiuk, Cohen‐Tannoudji, Huille, & Tribet, 2012), and simulation of transportation (Fleischman, Chung, Paul, & Lewus, 2017). The extent of aggregation, however, can be heavily dependent on the type of accelerated stress employed (Fleischman et al, 2017; Joubert, Luo, Nashed‐Samuel, Wypych, & Narhi, 2011; Tamizi & Jouyban, 2016).…”

“…Differences in accelerated and innate aggregation propensities (Goldberg et al, 2017) may arise because the acceleration method increases the relative flux through certain pathways which are distinct to those traversed during production or upon storage (Chakroun, Hilton, Ahmad, Platt, & Dalby, 2016; Luo et al, 2011; Phillips et al, 2017; van der Kant et al, 2017). There is thus a need to develop stress tests that more closely replicate the conformational ensemble generated during processing and transport.…”

Media photo‐degradation in pharmaceutical biotechnology – impact of ambient light on media quality, cell physiology, and IgG production in CHO cultures

“…caused by alterations in protein expression, post‐translational modification and secretion) or in an increased IgG degradation rate in the irradiated medium (e.g. due to higher oxidative stress levels or direct IgG photolysis) 10, 18, 19. In this study, a bimodal analytical approach was taken to gain information on the extracellular, as well as on the intracellular IgG concentration.…”