Open AccessStructural similarity, characterization of Poly Ethylene Glycol linkage and identification of product related variants in biosimilar pegfilgrastim
Author(s) -
Rakesh Shekhawat,
Chintan Kumar Shah,
Akash J. Patel,
Sankaranarayanan Srinivasan,
Poonam Malhotra Kapoor,
Suvaskumar Patel,
Sanjay Kumar,
Sandeep Sonar,
Namrata More,
Manasvi Joshi,
Jatin H. Patel,
Milan Vachhani,
Bhargav Prasad Kodaganti,
Upasana Choavatiya,
Arabhi Pushpaja,
Shubhangi Argade,
Nidhi Nuwal,
Manish Kumar,
Sridevi Khambhampaty
Publication year - 2019
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0212622
Subject(s) - biosimilar , pegfilgrastim , pegylation , differential scanning calorimetry , chemistry , high performance liquid chromatography , circular dichroism , chromatography , combinatorial chemistry , biochemistry , polyethylene glycol , filgrastim , organic chemistry , medicine , physics , toxicity , neutropenia , thermodynamics
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.