Streamlining the Characterization of Disulfide Bond Shuffling and Protein Degradation in IgG1 Biopharmaceuticals Under Native and Stressed Conditions

Abstract

Post translational modifications (PTMs) have been shown to negatively impact protein efficacy and safety by altering its native conformation, stability, target binding and/or pharmacokinetics. One PTM in particular, shuffled disulfide bonds, has been linked to decreased potency and increased immunogenicity of protein therapeutics. In an effort to gain more insights into the effects of shuffled disulfide bonds on protein therapeutics’ safety and efficacy, we designed and further optimized a semi-automated LC-MS/MS method for disulfide bond characterization on two IgG1 protein therapeutics—rituximab and bevacizumab. We also compared originator vs. biosimilar versions of the two therapeutics to determine if there were notable variations in the disulfide shuffling and overall degradation between originator and biosimilar drug products. From our resulting data, we noticed differences in how the two proteins degraded. Bevacizumab had a general upward trend in shuffled disulfide bond levels over the course of a 4-week incubation (0.58 ± 0.08% to 1.46 ± 1.10% for originator) whereas rituximab maintained similar levels throughout the incubation (0.24 ± 0.21% to 0.51 ± 0.11% for originator). When we measured degradation by SEC and SDS-PAGE, we observed trends that correlated with the LC-MS/MS data. Across all methods, we observed that the originator and biosimilar drugs performed similarly. The results from this study will help provide groundwork for comparative disulfide shuffling analysis by LC-MS/MS and standard analytical methodology implementation for the development and regulatory approval of biosimilars.

Fast and accurate disulfide bridge detection

“…In this paper, we present an efficient and precise sample preparation, data acquisition and analysis approach for the accurate detection of disulfide bridges. In earlier studies, researchers attempted to identify disulfide bridges through indirect means like differential alkylation or direct detection as 'crosslinked' peptides [41][42][43][44][45][46][47][48]. Typically, these methods involved analyzing samples under unfavorable conditions.…”

Monoclonal antibody and protein therapeutic formulations for subcutaneous delivery: high-concentration, low-volume vs. low-concentration, high-volume

“… 36 Lower pH ranges can provide improvements in disulfide shuffling and decrease deamidation rates. 60 Engineered protein therapeutics and proteins with weak disulfide bonds that are prone to disulfide shuffling can lead to decreased potency and increased immunogenicity. 60 However, higher solution pH is associated with decreased electrostatic interactions, increased mAb reversible self-associations, and greater solution viscosity, possibly due to decreased electrostatic repulsive interactions.…”

Biosimilars Adoption: Recognizing and Removing the RoadBlocks

“…19 Also significant is the correlation of glycosylation microheterogeneity with Fc-receptor binding and ADCC for mAbs, and the examination of initial levels of aggregates (non-covalent and covalent) and correlation with clinical immunogenicity and differences found in the structure. 20 The FDA has recently disclosed a plan to ensure that protein aggregation that contributes to immunogenicity can be monitored using flow imaging microscopy (FIM), and to ensure that the images are correctly interpreted, using an artificial intelligence/machine learning approach (AI/ML) of convolutional neural networks (CNNs or ConvNets). 21 The FDA has also disclosed a suitable software, ParticleSentry AI, 22 to analyze these data during development and commercial manufacturing.…”

“…Also significant is the correlation of glycosylation microheterogeneity with Fc-receptor binding and ADCC for mAbs, and the examination of initial levels of aggregates (non-covalent and covalent) and correlation with clinical immunogenicity and differences found in the structure. 20 …”