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

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.

The Effects of pH and Excipients on Exenatide Stability in Solution.

Exenatide, a glucagon-like peptide-1 receptor agonist, is the active pharmaceutical ingredient in Byetta® and Bydureon®, two type 2 diabetes drug products that have generics and multiple follow-up formulations currently in development. Even though exenatide is known to be chemically and physically unstable at pH 7.5, there lacks a systematic evaluation of the impact of pH and excipients on the peptide solution stability. In this study, we established analytical methods to measure the chemical and physical degradation of the peptide in solution. Exenatide remained relatively stable at pH 4.5 when incubated at 37 °C. At pH 5.5-6.5, degradation was driven by oxidation, while driven by deamidation at pH 7.5-8.5. Significant aggregation of exenatide at pH 7.5 and 8.5 was detected by size exclusion chromatography and dynamic light scattering. Each pH value greater than 4.5 exhibited unique profiles corresponding to a loss of α-helical content and an increase in unordered structures. The addition of sugars, including mannitol, sorbitol and sucrose, conferred small protective effects against peptide aggregation when incubating at pH 7.5 and 37 °C, as measured by size-exclusion chromatography and dynamic light scattering. The results of this study will be useful for investigators developing generic exenatide products, peptide analogs and novel exenatide drug delivery systems.

Characterization of attributes and in vitro performance of exenatide-loaded PLGA long-acting release microspheres.

Bydureon® (Bdn) is a once-weekly injectable long-acting release (LAR) product for adults with type 2 diabetes based on PLGA microspheres encapsulating the glucagon like peptide (GLP-1) analog, exenatide. Despite its widespread use in type 2 diabetes treatment, little information has been published concerning the physical-chemical aspects and exenatide stability in this product. Here, we developed and validated methods to evaluate attributes and performance of Bdn such as particle size/size distribution and residual levels of moisture and organic solvent(s). The reverse engineering of the exenatide LAR was also performed to identify and quantify principal components in the product. Stability-indicating UPLC and LC-MS methods were applied to characterize exenatide degradation (such as oxidation, deamidation and acylation products) during in vitro release evaluation. The 55-µm volume-median Bdn microspheres slowly released the exenatidein vitroover two months with a very low initial burst release to avoid unwanted side effects. Residual organic solvent levels (methylene chloride, ethanol, heptane, and silicon oil) also met the USP criteria. Peptide acylation was the most prominent peptide reaction during both encapsulation and in vitro release, and the acylated peptide steadily increased during release relative to parent exenatide, becoming the most abundant peptide species extracted from the microspheres at later release stages. The presence of peptide impurities during the release period, which are not extractable in the polymer and likely insoluble in water, might be one potential cause for immunogenicity. Further evaluation will be needed to confirm this hypothesis. Release of peptide was minimal over the first 2 weeks before the microspheres steadily released peptide for more than 28 days. The rigorous technical approach discussed in this paper may provide critical information for both companies and the FDA for developing generic exenatide-PLGA formulations and other important PLGA microsphere products.

Multifaceted assessment of rituximab biosimilarity: The impact of glycan microheterogeneity on Fc function.

Biosimilars are poised to reduce prices and increase patient access to expensive, but highly effective biologic products. However, questions still remain about the degree of similarity and scarcity of information on biosimilar products from outside of the US/EU in the public domain. Thus, as an independent entity, we performed a comparative analysis between the innovator, Rituxan® (manufactured by Genentech/Roche), and a Russian rituximab biosimilar, Acellbia® (manufactured by Biocad). We evaluated biosimilarity of these two products by a variety of state-of-the-art analytical mass spectrometry techniques, including tandem MS mapping, HX-MS, IM-MS, and intact MS. Both were found to be generally similar regarding primary and higher order structure, though differences were identified in terms of glycoform distribution levels of C-terminal Lys, N-terminal pyroGlu, charge variants and soluble aggregates. Notably, we confirmed that the biosimilar had a higher level of afucosylated glycans, resulting in a stronger FcγIIIa binding affinity and increased ADCC activity. Taken together, our work provides a comprehensive comparison of Rituxan® and Acellbia®.

Infliximab Biosimilars in the Age of Personalized Medicine.

Structural and functional differences between REMICADE and its two FDA-approved biosimilars appear to have clinical implications. We suggest a personalized biosimilar substitution approach based on prescribed indication, biosimilar afucosylation level, and a patient's FCGR3A polymorphism. We also advocate for establishing glycosylation variation limits for biosimilar approvals.

Biosimilarity under stress: A forced degradation study of Remicade® and Remsima™.

Remsima™ (infliximab) is the first biosimilar monoclonal antibody (mAb) approved by the European Medical Agency and the US Food and Drug Administration. Remsima™ is highly similar to its reference product, Remicade®, with identical formulation components. The 2 products, however, are not identical; Remsima™ has higher levels of soluble aggregates, C-terminal lysine truncation, and fucosylated glycans. To understand if these attribute differences could be amplified during forced degradation, solutions and lyophilized powders of the 2 products were subjected to stress at elevated temperature (40-60°C) and humidity (dry-97% relative humidity). Stress-induced aggregation and degradation profiles were similar for the 2 products and resulted in loss of infliximab binding to tumor necrosis factor and FcγRIIIa. Appearances of protein aggregates and hydrolysis products were time- and humidity-dependent, with similar degradation rates observed for the reference and biosimilar products. Protein powder incubations at 40°C/97% relative humidity resulted in partial mAb unfolding and increased asparagine deamidation. Minor differences in heat capacity, fluorescence, levels of subvisible particulates, deamidation and protein fragments were observed in the 2 stressed products, but these differences were not statistically significant. The protein solution instability at 60°C, although quite significant, was also similar for both products. Despite the small initial analytical differences, Remicade® and Remsima™ displayed similar degradation mechanisms and kinetics. Thus, our results show that the 2 products are highly similar and infliximab's primary sequence largely defines their protein instabilities compared with the limited influence of small initial purity and glycosylation differences in the 2 products.

A Multidimensional Analytical Comparison of Remicade and the Biosimilar Remsima.

In April 2016, the Food and Drug Administration approved the first biosimilar monoclonal antibody (mAb), Inflectra/Remsima (Celltrion), based off the original product Remicade (infliximab, Janssen). Biosimilars promise significant cost savings for patients, but the unavoidable differences between innovator and copycat biologics raise questions regarding product interchangeability. In this study, Remicade and Remsima were examined by native mass spectrometry, ion mobility, and quantitative peptide mapping. The levels of oxidation, deamidation, and mutation of individual amino acids were remarkably similar. We found different levels of C-terminal truncation, soluble protein aggregates, and glycation that all likely have a limited clinical impact. Importantly, we identified more than 25 glycoforms for each product and observed glycoform population differences, with afucosylated glycans accounting for 19.7% of Remicade and 13.2% of Remsima glycoforms, which translated into a 2-fold reduction in the level of FcγIIIa receptor binding for Remsima. While this difference was acknowledged in Remsima regulatory filings, our glycoform analysis and receptor binding results appear to be somewhat different from the published values, likely because of methodological differences between laboratories and improved glycoform identification by our laboratory using a peptide map-based method. Our mass spectrometry-based analysis provides rapid and robust analytical information vital for biosimilar development. We have demonstrated the utility of our multiple-attribute monitoring workflow using the model mAbs Remicade and Remsima and have provided a template for analysis of future mAb biosimilars.