PLoS ONE (Jan 2019)

Structural similarity, characterization of Poly Ethylene Glycol linkage and identification of product related variants in biosimilar pegfilgrastim.

  • Rakesh Shekhawat,
  • Chintan Kumar Shah,
  • Akash Patel,
  • Sankaranarayanan Srinivasan,
  • Poonam Kapoor,
  • Suvaskumar Patel,
  • Sharwan Kumar,
  • Sandeep Sonar,
  • Namrata More,
  • Manasvi Joshi,
  • Jatin Patel,
  • Milan Vachhani,
  • Bhargav Prasad Kodaganti,
  • Upasana Choavatiya,
  • Arabhi Pushpaja,
  • Shubhangi Argade,
  • Nidhi Nuwal,
  • Manish Kumar,
  • Sridevi Khambhampaty

DOI
https://doi.org/10.1371/journal.pone.0212622
Journal volume & issue
Vol. 14, no. 3
p. e0212622

Abstract

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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.