Comparative Evaluation of Well-Defined IgG1 Fc Glycoforms as a Model System for Biosimilar Comparability Analysis

The patents of several best-selling biologic therapeutic products are expiring soon. Consequently, the interest of developing biosimilar products is growing. A biosimilar product is developed if there are no clinically meaningful differences in terms of safety, efficacy, and purity after evaluating side-by-side with the originator. Biosimilar products are anticipated to be accessible to healthcare providers and patients at a lower cost compared to the originators. Unlike small-molecule generic drug products, which are structurally replicable and well-defined, that guarantee the safety and efficacy, biologic products are structurally complex, larger in size and often contain mixtures of various posttranslational modifications. Consequently, demonstrating the similarity of a biosimilar molecule with the reference product requires extensive characterization to ensure safety and efficay. One of the challenges in developing a biosimilar product is the lack of knowledge about the reference product’s manufacturing process, which is not accessible to the public because it is a proprietary knowledge. Therefore, the biosimilar sponsor needs to develop a process by extensive characterization of a biosimilar candidate side-by-side with a reference product. This is an iterative process aimed at developing a biosimilar molecule similar to the reference product. The first step in biosimilarity assessment is to establish structural similarity by extensive characterization using several analytical techniques. Therefore, analytical tools play a vital role in demonstrating structural similarity as well as process development of a biosimilar candidate. In addition, the level of similarity established by the analytical tools guides the type of non-clinical and clinical data packages required for regulatory approval. If a high degree of similarity is demonstrated using analytical techniques, then phase II clinical trials are not required for registration of a biosimilar candidate. Consequently, this will lower the cost of developing a biosimilar product. Hence, developing sensitive and robust analytical techniques is vital in a biosimilar development process. In this dissertation, four homogeneous glycoforms of IgG1 Fc (HM-Fc, GlcNAc-Fc, Man5-Fc, and N297Q-Fc) were produced using recombinant protein expression combined with in-vitro enzymatic reactions to be utilized as a model for biosimilar comparability analysis. These glycoforms were characterized by mass spectrometry, CIUE, SDS-PAGE, and cIEF. The main focus of the project was to produce homogeneous glycoforms of IgG1 Fc and to utilize them to develop new biolayer interferometry (BLItz) assay methods. Two biolayer interferometry methods with different immobilization techniques were developed to measure the binding affinity of IgG1 Fc glycoforms to FcRIIIa and FcRIIb. In addition, these four glycoforms were mixed in pre-defined composition to examine the characteristics of mixtures of glycoforms and to study important biological and physicochemical features of protein drugs in a biosimilar analysis. For the mixture samples, differences in binding were observed when the two immobilization formats were employed. Furthermore, these glycoforms were incubated at low and elevated temperatures for an elongated time, and a trend of decreasing binding affinity was observed with the increasing incubation period

Physical stability comparisons of IgG1-Fc variants: effects of N-glycosylation site occupancy and Asp/Gln residues at site Asn 297

This is the author's accepted manuscript. Made available by the permission of the publisher.The structural integrity and conformational stability of various IgG1-Fc proteins produced from the yeast Pichia pastoris with different glycosylation site occupancy (di-, mono-, and non- glycosylated) was determined. In addition, the physical stability profiles of three different forms of non-glycosylated Fc molecules (varying amino acid residues at site 297 in the CH2 domain due to point mutations and enzymatic digestion of the Fc glycoforms) were also examined. The physical stability of these IgG1-Fc glycoproteins was examined as a function of pH and temperature by high throughput biophysical analysis using multiple techniques combined with data visualization tools (three index empirical phase diagrams and radar charts). Across the pH range of 4.0 to 6.0, the di- and mono- glycosylated forms of the IgG1-Fc showed the highest and lowest levels of physical stability respectively, with the non-glycosylated forms showing intermediate stability depending on solution pH. In the aglycosylated Fc proteins, the introduction of Asp (D) residues at site 297 (QQ vs. DN vs. DD forms) resulted in more subtle changes in structural integrity and physical stability depending on solution pH. The utility of evaluating the conformational stability profile differences between the various IgG1-Fc glycoproteins is discussed in the context of analytical comparability studies

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 over 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 FcγRIIIa 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 due to 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