Sub Topic | Secondary Topic: Biotherapeutics and Biotechnology - Biosimilar | Biosimilarity Assessment
Authors: Alexander Benet, University of Michigan (Presenting Author); Karthik Pisupati, Eli Lilly and Company (Main Author); Yuwei Tian, University of Michigan; Solomon Okbazghi, University of Kansas; Jukyung Kang, University of Michigan; Michael Ford, MS Bioworks; Sergei Saveliev, Promega Corp.; K Sen, Protein Metrics; Eric Carlson, Protein Metrics; Thomas Tolbert, University of Kansas; Brandon Ruotolo, University of Michigan; Steven Schwendeman, University of Michigan; Anna Schwendeman, University of Michigan
Presenting Author: Alexander Benet
Purpose: Remsima (infliximab), a copy of Remicade, is the first biosimilar monoclonal antibody (mAb) approved by the US Food and Drug Administration (FDA). Remsima is highly similar to Remicade with identical formulation components. The two products however are not identical; Remsima has higher levels of soluble aggregates, C-terminal lysine truncation, and fucosylated glycans (Pisupati, 2017). By attempting to understand if these minor attribute differences could be amplified during forced degradation, we intend to investigate an analytical platform that can be applied to access future biosimilar antibody drug products.
Methods: For forced degradation studies, the powders of the two products were subjected to incubation at 40&[deg]C and various humidity levels (dry-97% RH) by applying desiccant or saturated solutions of NaBr and K2SO4 in desiccators. Vials were opened, and powder aliquoted into eppendorf tubes and incubated for 0, 1, 2 or 4 weeks. Following incubation, protein powders were reconstituted with water for injection (WFI). Levels of protein aggregation were examined by size exclusion chromatography (SEC). Protein aggregation was characterized using non-reducing and reducing condition SDS-PAGE and ion mobility-mass spectrometry (IM-MS). The antibodies were structurally characterized using intrinsic fluorescence, near UV circular dichroism (CD) (250-320 nm), and far UV CD (190-250 nm). LC-MS/MS (Tandem MS) analysis following trypsin digest was used to measure the levels of oxidation, deamidation and N-glycosylation. Two bioassays, ELISA and biolayer interferometry, were performed to test the effect of stress on antigen and Fc&[gamma]RIIIa binding affinity, respectively.
Results: Analysis of stressed mAbs revealed a time- and humidity-dependent loss of native monomer, and formation of soluble aggregate. However, the rates of monomer loss were similar for Remicade (0.42% monomer loss/day) and Remsima (0.44% monomer loss/day). Stressed samples were subjected to native and reduced condition SDS-PAGE. Due to the disappearance of aggregate bands in reduced condition SDS-PAGE, it is suggested that aggregates were likely due to disulfide bond rearrangements. In addition to aggregates, hydrolytic fragments were observed under native conditions by SDS-PAGE and IM-MS. Mass spectrometry studies revealed that protein incubations at elevated % RH resulted in partial mAb unfolding, increased asparagine (Asn) deamidation and methionine (Met) oxidation, while the level of N-glycosylation remained unchanged. Deamidation of Asn 57, located in the complementarity determining region (CDR), is expected to affect antigen binding, while deamidation of Asn 392 and oxidation of Met 252 and 431, located in the CH2 region of the Fc domain, are expected to affect Fc&[gamma]RIIIa binding. Infliximab TNF-&[alpha] binding affinity was reduced to 81.8% (Remicade) and 77.2% (Remsima) of the initial levels. Fc&[gamma]RIIIa binding affinity decreased with a KD increase from 173 to 545 nM for Remicade and an increase from 368 to 680 nM for Remsima. Stress-induced aggregation and degradation profiles were similar for the two products, further underlining product biosimilarity. Thus, the infliximab's degradation appears to be largely defined by its primary sequence, while the initial differences in purity and N-glycosylation in the two products appear to have only limited influence.
Conclusion: Our results show similar levels of aggregate formation, structural variation, and chemical modifications of Remicade and Remsima upon humidity/thermal stress. These results further support the notion that the products are biosimilar. We anticipate stress testing will be widely used for biosimilar assessment as more biologic products will come off patent and new biosimilar antibody products will permeate the markets.
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