Collision-Induced Unfolding Reveals Stability Differences in Infliximab Therapeutics under Native and Heat Stress Conditions.

Ion mobility-mass spectrometry (IM-MS) and collision-induced unfolding (CIU) assays of monoclonal antibody (mAb)-based biotherapeutics have proven sensitive to disulfide bridge structures, glycosylation patterns, and small molecule conjugation levels. Despite promising prior reports detailing the capabilities of IM-MS and CIU to differentiate biosimilars, generic mAb therapeutics, there remain questions surrounding the sensitivity of CIU to mAb structure changes that occur upon stress, the reproducibility of such measurements across IM-MS platforms, and the correlation between CIU and differential scanning calorimetry (DSC) datasets. In this report, we describe a comprehensive IM-MS and CIU dataset acquired for three Infliximabs: Remicade, Inflectra, and Renflexis. We subject each infliximab sample to forced degradation through heat stress and observe broadly similar yet subtly different stability patterns for these three biotherapeutics. We find that CIU is capable of tracking differences in mAb higher-order structure (HOS) imparted during forced heat stress degradation and that DSC is less sensitive to these alterations in comparison. Furthermore, we collected our comprehensive IM-MS and CIU data across two instrument platforms (Waters G2 and Agilent 6560), with both producing similar abilities to differentiate mAbs while also revealing minor differences between the results obtained on the two instruments. Finally, we demonstrate that CIU-based heatmaps and classification allow for rapid assessment of the most differentiating charge states for the analysis of infliximab, and using multiplexed classification, we conservatively estimate a 30-fold improvement in the time required to perform mAb stability and HOS measurements over standard DSC tools.

Research and Education Needs for Complex Generics.

Complex generics are generic versions of drug products that generally have complex active ingredients, complex formulations, complex routes of delivery, complex dosage forms, are complex drug-device combination products, or have other characteristics that can make it complex to demonstrate bioequivalence or to develop as generics. These complex products (i.e. complex generics) are an important element of the United States (U.S.) Food and Drug Administration's (FDA's) Generic Drug User Fee Amendments (GDUFA) II Commitment Letter. The Center for Research on Complex Generics (CRCG) was formed by a grant from the FDA to address challenges associated with the development of complex generics. To understand these challenges, the CRCG conducted a "Survey of Scientific Challenges in the Development of Complex Generics". The three main areas of questioning were directed toward which (types of) complex products, which methods of analysis to support a demonstration of bioequivalence, and which educational topics the CRCG should prioritize. The survey was open to the public on a website maintained by the CRCG. Regarding complex products, the top three selections were complex injectables, formulations, and nanomaterials; drug-device combination products; and inhalation and nasal products. Regarding methods of analysis, the top three selections were locally-acting physiologically-based pharmacokinetic modeling; oral absorption models and bioequivalence; and data analytics and machine learning. Regarding educational topics, the top three selections were complex injectables, formulations, and nanomaterials; drug-device combination products; and data analytics, including quantitative methods and modeling & simulation. These survey results will help prioritize the CRCG's initial research and educational initiatives.

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.

Overview of Humira® Biosimilars: Current European Landscape and Future Implications.

Humira® (adalimumab) by AbbVie has been the top-selling biologic drug product for the last few years - reaching nearly $20 billion in annual sales in 2018. Upon the October 2018 release of four adalimumab biosimilars into the European market, those sales began to shrink. By the end of 2019, the annual sales of Humira®, albeit still high, dipped closer to $19 billion as nearly 35% of European patients had been switched from Humira® to a biosimilar. Diminishing sales are expected to continue as the adoption of adalimumab biosimilars increases in Europe and Humira®'s patent protection is lost in the United States come 2023. In this review we discuss how impactful the availability of biosimilars has been to the European adalimumab market approximately two years after their release. We further analyze the marketed biosimilars with regards to differences in their formulation, delivery devices, biological activity, physicochemical properties, clinical trials data, and current financial foothold. More importantly, though, we highlight how "similar" these biosimilars are to Humira®. In doing so, we seek to educate the public on what they may be able to expect once adalimumab biosimilars enter the United States market in 2023.

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®.