Abstract
The approval of biosimilars requires demonstration of biosimilarity, which rests on the base of thorough analytical characterization of the biosimilar product. In addition to demonstration of biosimilarity, the product related impurities need to be thoroughly characterized and controlled at minimal levels. Pegylation of peptides and proteins creates significant challenges for detailed structural characterization, such as PEG (Poly Ethylene Glycol) heterogeneity, site of addition and number of attached pegylated moieties. A combination of several methods including circular dichroism, FTIR (Fourier-transform Infrared Spectroscopy), fluorescence spectroscopy, DSC (Differential Scanning Calorimetry), 1D and 2D NMR (Nuclear Magnetic Resonance), Edman degradation and peptide mapping by LC-MS (Liquid Chromatography Mass Spectrometry) were used for characterization of N-terminally pegylated filgrastim. Product related impurities such as oxidized, reduced, deamidated, dipegylated variants and monopegylated positional isomers have been characterized in detail using various HPLC (High Performance Liquid Chromatography) based methods and LC-MS techniques. The functional characterization in terms of receptor binding and cell proliferation assay was conducted for the similarity assessment and the potential impact of the product variants on the in vitro biological activity has also been assessed. In summary, this study presents, for the first time, a detailed structural and molecular level characterization of a biosimilar pegfilgrastim providing a strong base for the demonstration of overall biosimilarity of the product with the innovator product.
Highlights
A biosimilar product is defined as the biological product which is highly similar to the reference product not withstanding minor differences in clinically inactive components and that there are no clinically meaningful differences between the biological product and the reference product in terms of the safety, purity and potency of the product [1]
The results of N-terminal sequencing provided in Table A in S1 Appendix shows that the INTP5, EU sourced Neulasta and US sourced Neulasta contain the same N-terminal amino acid sequence up to the first 20 residues
N-terminus is identical for all products tested and consistent with the expected amino acid sequence for a pegfilgrastim molecule
Summary
A biosimilar product is defined as the biological product which is highly similar to the reference product not withstanding minor differences in clinically inactive components and that there are no clinically meaningful differences between the biological product and the reference product in terms of the safety, purity and potency of the product [1]. EMA guidance suggests that demonstration of similarity of a biological drug to the reference medicinal product in terms of pharmaceutical quality, structure, functional activity, efficacy, immunogenicity profile and safety based on the comparability exercise which is entailed to be established [2]. The demonstration of biosimilarity involves extensive physicochemical and biological characterization of the product in comparison to the innovator product followed by clinical studies required to demonstrate complete biosimilarity. Intas has successfully developed a biosimilar filgrastim (Grastofil/Accofil) which was approved in EU in 2015. The quality of Intas Filgrastim has been compared in independent studies [3, 4] and found to be highly similar to the innovator product, with very low levels of product variants. A limited biosimilarity assessment of this product was demonstrated in comparison to the US sourced Neulasta [5]. A comprehensive assessment of the Intas product in comparison to both EU and US sourced innovator products is discussed in this research article
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