Assessment of structural and functional similarity of biosimilar products: Rituximab as a case study

Abstract

Biosimilars are products that are similar in terms of quality, safety, and efficacy to an already licensed reference/ innovator product and are expected to offer improved affordability. The most significant source of reduction in the cost of development of a biosimilar is the reduced clinical examination that it is expected to undergo as compared to the innovator product. However, this clinical relief is predicated on the assumption that there is analytical similarity between the biosimilar and the innovator product. As a result, establishing analytical similarity is arguably the most important step towards successful development of a biosimilar. Here, we present results from an analytical similarity exercise that was performed with five biosimilars of rituximab (Ristova®, Roche), a chimeric mouse/ human monoclonal antibody biotherapeutic, that are available on the Indian market. The results show that, while the biosimilars exhibited similarity with respect to protein structure and function, there were significant differences with respect to size heterogeneity, charge heterogeneity and glycosylation pattern.

Keywords: analytical similarity; biosimilars; critical quality attributes; indian market; rituximab.

Figure 1.

Comparison of deconvoluted ESI-TOF mass spectra for (A) intact and (B) reduced (LC – light chain, HC – heavy chain) Rituximab biosimilars and Ristova®, (C) Total compound chromatogram (TCC) mirror plots of peptide mapping for reduced Rituximab biosimilars (lower) and Ristova® (upper) obtained from ESI-TOF mass spectra upon trypsin digestion.

Figure 2.

Comparison of higher order structure: overlay of (A) Far UV CD spectra, (B). Near UV CD spectra, (C) Secondary derivative FTIR spectra, (D) Intrinsic fluorescence spectra, and (E) Extrinsic fluorescence spectra for Rituximab biosimilars and Ristova®.

Figure 3.

Comparison of size and charge heterogeneity of Rituximab biosimilars and Ristova®: (A) Non reducing SDS-PAGE profile, (B) SEC chromatogram (insert shows a zoomed-in view), (C) Particle size distribution by DLS (insert shows a zoomed-in view), (D). CEX chromatogram before CpB digestion, (E) CEX chromatogram after CpB digestion.

Figure 4.

Comparison of in-vitro binding assays to receptors on CD20+ B cells through (A) ADCC, (B) CDC, (C) FACS for Rituximab biosimilars and Ristova® (Green line represents the range for Ristova®).