Structural and Functional Analysis of CEX Fractions Collected from a Novel Avastin® Biosimilar Candidate and Its Innovator: A Comparative Study

Abstract

Avastin® is a humanized recombinant monoclonal antibody used to treat cancer by targeting VEGF-A to inhibit angiogenesis. SIMAB054, an Avastin® biosimilar candidate developed in this study, showed a different charge variant profile than its innovator. Thus, it is fractionated into acidic, main, and basic isoforms and collected physically by Cation Exchange Chromatography (CEX) for a comprehensive structural and functional analysis. The innovator product, fractionated into the same species and collected by the same method, is used as a reference for comparative analysis. Ultra-Performance Liquid Chromatography (UPLC) ESI-QToF was used to analyze the modifications leading to charge heterogeneities at intact protein and peptide levels. The C-terminal lysine clipping and glycosylation profiles of the samples were monitored by intact mAb analysis. The post-translational modifications, including oxidation, deamidation, and N-terminal pyroglutamic acid formation, were determined by peptide mapping analysis in the selected signal peptides. The relative binding affinities of the fractionated charge isoforms against the antigen, VEGF-A, and the neonatal receptor, FcRn, were revealed by Surface Plasmon Resonance (SPR) studies. The results show that all CEX fractions from the innovator product and the SIMAB054 shared the same structural variants, albeit in different ratios. Common glycoforms and post-translational modifications were the same, but at different percentages for some samples. The dissimilarities were mostly originating from the presence of extra C-term Lysin residues, which are prone to enzymatic degradation in the body, and thus they were previously assessed as clinically irrelevant. Another critical finding was the presence of different glyco proteoforms in different charge species, such as increased galactosylation in the acidic and afucosylation in the basic species. SPR characterization of the isolated charge variants further confirmed that basic species found in the CEX analyses of the biosimilar candidate were also present in the innovator product, although at lower amounts. The charge variants’ in vitro antigen- and neonatal receptor-binding activities varied amongst the samples, which could be further investigated in vivo with a larger sample set to reveal the impact on the pharmacokinetics of drug candidates. Minor structural differences may explain antigen-binding differences in the isolated charge variants, which is a key parameter in a comparability exercise. Consequently, such a biosimilar candidate may not comply with high regulatory standards unless the binding differences observed are justified and demonstrated not to have any clinical impact.

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“…The assay setup was performed as stated above, except for the 270 nM TNF-α sample solution. Results were obtained with the double referencing method, where the presented data was subtracted from the zero-concentration sample and the blank surface [ 29 , 30 ].…”

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“…The chip surface was regenerated by injecting 10 mM glycine pH 1.5 buffer (GE Healthcare, Uppsala, Sweden) for 90 s. The results were found by subtracting responses from the blank flow cell and zero concentration analyte injection (running buffer) from the actual results. The equilibrium dissociation constants (K D ) were calculated by Biacore Evaluation Software using a 1:1 Langmuir binding model [35,36].…”

“…The components greater than 10% matched primary ions (b/y ions), and mass errors smaller than 10 ppm, but no insource fragments were allowed for identification. The percentages of modifications were calculated using the following equation: "%peptide = (Response of modified peptide/Total response of the modified and unmodified peptide) × 100" [35].…”