Physicochemical and functional comparability between the proposed biosimilar rituximab GP2013 and originator rituximab

Abstract

Background: Regulatory 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.

Objective: To compare GP2013 with originator rituximab using an extensive array of routine analytical and extended characterization methods.

Methods: Primary 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 (1)H 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.

Results: Intact 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.

Conclusion: GP2013 was shown to be physicochemically highly similar to originator rituximab at the level of primary and higher order structure, post-translational modifications and size variants. An extensive functional characterization package indicated that GP2013 has the same biological properties as originator rituximab.

Fig. 1

Comparison of UV chromatograms of Lys-C digested GP2013 and originator rituximab

Fig. 2

Comparison of 1D ¹H NMR spectra of GP2013 and originator rituximab. Blue line GP2013; red line originator rituximab. a Zoom of the amide region of the NMR spectra. b Zoom of the aliphatic region of the NMR spectra

Fig. 3

Super-positioned view on the complementarity determining regions (CDRs) of GP2013 and originator rituximab. a Ribbon representation of the CDRs. The heavy chain and light chain structures of Fab GP2013 are colored in blue and orange, respectively. The structure of Fab originator rituximab is colored in gray. b Ribbon representation of the CDRs with the side chains shown as a stick model, colored according to the chemical atom type (C GP2013 in blue and orange, C MabThera^® in gray, O in red, and N in blue). CDR loops are labeled

Fig. 4

Cation exchange chromatograms of GP2013 and originator rituximab (a) and after carboxypeptidase B (CPB) digest (b). As at one point in time changes in originator CEX-profiles were observed, both pre-shift and post-shift material is depicted

Fig. 5

Glycosylation pattern of GP2013 and originator rituximab—extended view (a) and zoomed view (b). As at one point in time changes in originator glycan profiles were observed, both pre-shift and post-shift material is depicted

Fig. 6

Electropherograms of non-reduced GP2013 and originator rituximab. Zoomed view of the electropherograms on the rituximab-related subunit corresponding to light chain (L), heavy chain (H), heavy-light chain (HL), heavy-heavy chain (HH), heavy-heavy-light chain (HHL) and high molecular weight variants (HMW)