Structural and functional comparability study of anti-CD20 monoclonal antibody with reference product

Abstract

BackgroundCell surface protein, CD20, is extensively expressed on the surface of B cells. Antibodies targeting CD20 protein are being used to treat B-cell malignancies and B-cell mediated autoimmune diseases. Considering the cost of therapy with innovator monoclonal antibodies for these diseases, development of biosimilar products for the treatment of such diseases provides affordable solution to rising healthcare costs.Materials and MethodsReference products of rituximab (six batches) were procured and stored as per manufacturer's instructions. Cell lines used in bioassay were procured from American Type Culture Collection and all other reagents used for analysis were of analytical grade. Primary structure was studied by intact mass analysis, peptide fingerprinting, peptide mass fingerprinting and sequence coverage analysis. Higher order structure was studied by circular dichroism, ultraviolet-visible spectroscopy, fluorescence spectroscopy, and disulfide bridge analysis. Different isoforms of reference product and SB-02 were identified using capillary isoelectric focusing and capillary zone electrophoresis. Glycosylation was studied by N-glycan mapping using LC-ESI-MS, point of glycosylation, released glycan analysis using ultra performance liquid chromatography (UPLC). Product related impurities such as oligomer content analysis and oxidized impurities were studied using size exclusion chromatography and reverse phase high performance liquid chromatography, respectively.Results and ConclusionHere, we report physicochemical and biological characterizations of Sun Pharma’s proposed biosimilar (SB-02) to rituximab, a monoclonal anti-CD20 antibody approved for the treatment of non-Hodgkin’s lymphoma and chronic lymphocytic leukemia. SB-02 and rituximab exhibited indistinguishable primary as well as higher-order structure upon analyzing with the array of analytical and extended characterization methods according to statistical methods. The molecule also displayed comparability to reference product in post-translational modifications and charge heterogeneity. In functional bioassays, SB-02 demonstrated comparable potency with respect to reference product. Our results indicate highly similar quality profile between SB-02 and rituximab.

Analytical Similarity Assessment of Biosimilars: Global Regulatory Landscape, Recent Studies and Major Advancements in Orthogonal Platforms

“…For intact mass analysis, there have been recent advancements in developing LC-ESI-[Native] MS-based methods that offer an orthogonal alternative to traditional denaturing RP-ESI-MS where other LC modalities (such as SEC, HIC or IEX) are explored to determine intact mass with possible sequence variants under non-denaturing (native) conditions to identify biologically active species and monitor protein dynamics. SEC-ESI-QTOF-MS has been employed in analytical biosimilarity assessment of biosimilar candidates of Amgen’s bevacizumab (ABP 215) ( Seo et al, 2018 ) and infliximab (ABP 710) ( Saleem et al, 2020 ), Sun Pharma’s rituximab (SB-02) ( Singh et al, 2018 ) and Celtrion’s infliximab (CT-P13) ( Hermosilla et al, 2019 ) compared to the respective innovators. The advantage of SEC-MS over RP-MS is that the native structure is preserved, and so the information gained is not only a comparison on exact mass, but also of the higher-order structure of the intended biosimilar.…”

“…Also, stable isotope labeling by amino acids in cell culture (SILAC) by dimethyl labeling [i.e., 2CH2 (rituximab) and 2CD2 (RNAi-mediated rituximab)] were used to detect sequence variants along with disulfide linkages, PTMs, and mutations ( Li et al, 2013 ). LC-QTOF has been used for the complete amino acid sequence analysis of Celtrion’s infliximab (Remsima ® ) ( Jung et al, 2014 ), trastuzumab (CT-P6) ( Lee J. et al, 2018 ), rituximab (CT-P10) ( Lee K. H. et al, 2018 ), Sun Pharma’s rituximab (SB-02) ( Singh et al, 2018 ) and Intas’s peg-filgrastim (INTP5) ( Shekhawat et al, 2019 ) biosimilars. Similarly, in recent years LC-Ion-Trap/Orbitrap Hybrid MS has been employed for characterization of certain biosimilars such as Amgen’s bevacizumab (ABP 215) ( Seo et al, 2018 ), rituximab (ABP 798) ( Seo et al, 2020 ), infliximab (ABP 710) ( Saleem et al, 2020 ), and Kyowa’s adalimumab (Hulio ® : FKB327) ( Schreiber et al, 2020 ) biosimilars, and LC-Q-Exactive-Orbitrap-MS was used for China’s bevacizumab (BVZ-BC) (company name not indicated in associated publication) ( Yu et al, 2020 ) and Amgen’s eculizumab (ABP 959) ( Hutterer et al, 2021 ) biosimilars, to determine similar amino acid sequence, PTM profiles, and disulfide linkages compared to the respective innovators.…”

“…It can quantify soluble aggregates/fragments (1–100 nm) and offers rapid analysis, excellent resolution, robustness, and reproducibility. More recently SEC has been augmented with multi-angle light scattering (MALS) for measuring the size, i.e., molar mass and root-mean-square (RMS) radius (also called the radius of gyration, R g ) of the different molecular species separated by SEC ( Flores-Ortiz et al, 2014 ; Lee K. H. et al, 2018 ; Singh et al, 2018 ; Seo et al, 2020 ).…”

“…Few similarity studies include Amgen’s bevacizumab (ABP 215) ( Seo et al, 2018 ), rituximab (ABP 798) ( Seo et al, 2020 ) and infliximab (ABP 710) ( Saleem et al, 2020 ). Similar to other chromatography based analysis, CE serves as a rapid, high-performance alternative tool orthogonal to CEX ( Moritz et al, 2015 ) and has been employed in biosimilarity assessments such as for Sun Pharma’s rhCG (SB005) ( Thennati et al, 2018 ) and rituximab (SB-02) ( Singh et al, 2018 ) ( Supplementary Table S4 ). Further, the application of different modes of CE and hyphenation of CEX or CE with MS, have introduced more orthogonal tools to the analytical armory with respect to charge variant analysis ( Gahoual et al, 2014 ; Haselberg et al, 2018 ).…”

Comparative biosimilar quality studies between a rituximab product and MabThera

“…This band is slightly broader than usual due to the presence of Nglycans in the C H 2 domain of the heavy chain of these mAbs imparting them some sort of microheterogeneity. However, under reducing conditions the single band should separate into two bands, one around 50 kDa and the second around 25 kDa due to the reduction of the disulfide bonds which are usually holding the heavy and light chains of the IgG homodimer together [28]. There is also a very faint band underneath the heavy chain major band which usually corresponds to the non-glycosylated heavy chain.…”