Physicochemical and Functional Comparability Between the Proposed Biosimilar Rituximab GP2013 and Originator Rituximab

Abstract

BackgroundRegulatory approval for a biosimilar product is provided on the basis of its comparability to an originator product. A thorough physicochemical and functional comparability exercise is a key element in demonstrating biosimilarity. Here we report the characterization of a proposed biosimilar rituximab (GP2013) and originator rituximab.ObjectiveTo compare GP2013 with originator rituximab using an extensive array of routine analytical and extended characterization methods.MethodsPrimary and higher order protein structures were analyzed using a variety of methods that included high-performance liquid chromatography electrospray ionization mass spectrometry (HPLC-ESI-MS), peptide mapping with UV and MS detection, circular dichroism (CD), Fourier transform infrared (FTIR) spectroscopy, hydrogen deuterium exchange (HDX) MS, 1D 1H nuclear magnetic resonance (NMR) spectroscopy, X-ray crystallography and differential scanning calorimetry (DSC). Charge and amino acid modifications were assessed using cation exchange chromatography (CEX) and peptide mapping using reversed-phase (RP) HPLC. Boronate affinity chromatography was used to determine the relative amount of glycation. Glycans were identified and quantified after 2-aminobenzamide (2-AB) labeling and separation using normal phase HPLC with fluorescence and MS detection, respectively. Glycan site occupancy was determined using reducing capillary electrophoresis with sodium dodecyl sulfate (CE-SDS). Size heterogeneity was determined using reducing and non-reducing CE-SDS, size exclusion chromatography (SEC) and asymmetric flow field flow fractionation (AF4). Biological characterization included a series of bioassays (in vitro target binding, antibody-dependent cell-mediated cytotoxicity [ADCC], complement-dependent cytotoxicity [CDC] and apoptosis) and surface plasmon resonance (SPR) Fc receptor binding assays.ResultsIntact mass analysis of GP2013 and the heavy and light chains using RP HPLC–ESI–MS revealed the expected molecular mass of rituximab. The amino acid sequence was shown to be identical between GP2013 and the originator rituximab. Further sequence confirmation using RP-HPLC-UV/MS peptide mapping showed non-distinguishable chromatograms for Lys-C digested GP2013 and originator rituximab. The higher order structure of GP2013 was shown to be indistinguishable from originator rituximab using a large panel of redundant and orthogonal methods. GP2013 and originator rituximab were comparable with regard to charge variants, specific amino acid modifications and the glycan pattern. GP2013 was also shown to have similar purity, aggregate and particle levels when compared with the originator. Functionally, and by using a comprehensive set of bioassays and binding assays covering a broad range of rituximab’s functional activities, GP2013 could not be distinguished from originator rituximab.ConclusionGP2013 was shown to be physicochemically highly similar to originator rituximab at the level of primary and higher order structure, post-translational modifications ...

Biosimilars in Oncology

“…36 An example of physicochemical similarity between the originator rituximab and its biosimilar has been published, including primary and higher order structure, post-translational modifications and size variants, as well as an extensive functional characterization package. 37 Interestingly, in this analysis the biosimilar GP2013 was analytically more similar to US manufactured Rituxan (rituximab; Genentech, Inc., South San Francisco, CA, US) than Rituxan was to European manufactured MabThera (rituximab;…”

A Method for Mapping Glycosylation Sites in Proteins

“…7 The peptides 2:V306-K 321 (VVSVLTVLHQDWLNGK), containing N319, and 2:G375-K396 (GFYPSDIAVEWESNGQPENNYK), containing N388, are both located within the Fc region of the heavy chain, which shares the same sequence with other human or humanized mAbs. More than one asparagine is present in the sequences, but the asparagines highlighted in bold are identified as deamidation hot spots.…”

“…More than one asparagine is present in the sequences, but the asparagines highlighted in bold are identified as deamidation hot spots. 7 The second peptide is known as the "PENNY peptide", but both peptides are a decent indicator for induced deamidation of mAbs. Figure 8 shows the TIC chromatogram for the SMART Digest sample ( Figure 8A) and extracted ion current (XIC) chromatograms with a 5 ppm mass extraction window for the different samples ( Figure 8B).…”

A chemical and computational approach to comprehensive glycation characterization on antibodies

“…This will be particularly relevant in the biosimilar space where these high resolution analyses can assess similarity of complex post-translational modifications in a rapid and comprehensive manner. 28,29 Glycation derivatization can be completed in very few steps and essentially adds only a one hour incubation to the entire protocol. This can easily be executed by analysts as part of routine peptide mapping protein characterization.…”