Structural Similarity, Characterization of Poly Ethylene Glycol Linkage and Identification of Product Related Variants in Biosimilar Pegfilgrastim

Overview

Journal PLoS One

Specialties General Medicine
Science

Date 2019 Mar 14

PMID 30865643

Citations 8

Authors

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

Affiliations

Soon will be listed here.

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.

Citing Articles

Demonstration of physicochemical and functional similarity between Stimufend (pegfilgrastim-fpgk) and Neulasta (pegfilgrastim): A comparative analytical assessment.

Sykes A, Ingram L, Kronthaler U, Chevalet L PLoS One. 2024; 19(10):e0309480.

PMID: 39446813 PMC: 11500887. DOI: 10.1371/journal.pone.0309480.

Demonstration of Physicochemical and Functional Similarity of Biosimilar Pegfilgrastim-cbqv to Pegfilgrastim.

Kuehne H, Davis J, Merewether L, McQueen M, Valentine E, Young G Drugs R D. 2024; 24(2):285-301.

PMID: 38958918 PMC: 11315836. DOI: 10.1007/s40268-024-00471-9.

Analytical sameness methodology for the evaluation of structural, physicochemical, and biological characteristics of Armlupeg: A pegfilgrastim biosimilar case study.

Deshmukh A, Goyal R, Sundaram K, Dange K, Lakhote T, Niranjan S PLoS One. 2023; 18(8):e0289745.

PMID: 37556495 PMC: 10411777. DOI: 10.1371/journal.pone.0289745.

Nupur N, Joshi S, Gulliarme D, Rathore A Front Bioeng Biotechnol. 2022; 10:832059.

PMID: 35223794 PMC: 8865741. DOI: 10.3389/fbioe.2022.832059.

Development of Site-Specific PEGylated Granulocyte Colony Stimulating Factor With Prolonged Biological Activity.

Kumari M, Sahni G, Datta S Front Bioeng Biotechnol. 2020; 8:572077.

PMID: 33330413 PMC: 7710547. DOI: 10.3389/fbioe.2020.572077.

References

Turecek P, Bossard M, Schoetens F, Ivens I . PEGylation of Biopharmaceuticals: A Review of Chemistry and Nonclinical Safety Information of Approved Drugs. J Pharm Sci. 2016; 105(2):460-475. DOI: 10.1016/j.xphs.2015.11.015. View

Shahbazi M, Tamaskany Zahedy E, Kiumarsi S, Hadi Soltanabad M, Shahbazi Azar S, Amini H . Determination of pegfilgrastim aggregates by size-exclusion high-performance liquid chromatography on a methacrylate-based column. Biologicals. 2017; 46:153-158. DOI: 10.1016/j.biologicals.2017.02.003. View

Caliceti P, Veronese F . Pharmacokinetic and biodistribution properties of poly(ethylene glycol)-protein conjugates. Adv Drug Deliv Rev. 2003; 55(10):1261-77. DOI: 10.1016/s0169-409x(03)00108-x. View

Kinstler O, Brems D, Lauren S, Paige A, Hamburger J, Treuheit M . Characterization and stability of N-terminally PEGylated rhG-CSF. Pharm Res. 1996; 13(7):996-1002. DOI: 10.1023/a:1016042220817. View

Holzmann J, Hausberger A, Rupprechter A, Toll H . Top-down MS for rapid methionine oxidation site assignment in filgrastim. Anal Bioanal Chem. 2013; 405(21):6667-74. PMC: 3730093. DOI: 10.1007/s00216-013-7138-0. View

Souza L, Boone T, Gabrilove J, Lai P, Zsebo K, Murdock D . Recombinant human granulocyte colony-stimulating factor: effects on normal and leukemic myeloid cells. Science. 1986; 232(4746):61-5. DOI: 10.1126/science.2420009. View

Hodgson D, Aubin Y . Assessment of the structure of pegylated-recombinant protein therapeutics by the NMR fingerprint assay. J Pharm Biomed Anal. 2017; 138:351-356. DOI: 10.1016/j.jpba.2017.01.058. View

Cindric M, Cepo T, Galic N, Bukvic-Krajacic M, Tomczyk N, Vissers J . Structural characterization of PEGylated rHuG-CSF and location of PEG attachment sites. J Pharm Biomed Anal. 2007; 44(2):388-95. DOI: 10.1016/j.jpba.2007.02.036. View

Levy M, Gucinski A, Sommers C, Ghasriani H, Wang B, Keire D . Analytical techniques and bioactivity assays to compare the structure and function of filgrastim (granulocyte-colony stimulating factor) therapeutics from different manufacturers. Anal Bioanal Chem. 2013; 406(26):6559-67. DOI: 10.1007/s00216-013-7469-x. View

10.

Lu X, Gough P, DeFelippis M, Huang L . Elucidation of PEGylation site with a combined approach of in-source fragmentation and CID MS/MS. J Am Soc Mass Spectrom. 2010; 21(5):810-8. DOI: 10.1016/j.jasms.2010.01.011. View

11.

Forstenlehner I, Holzmann J, Scheffler K, Wieder W, Toll H, Huber C . A direct-infusion- and HPLC-ESI-Orbitrap-MS approach for the characterization of intact PEGylated proteins. Anal Chem. 2013; 86(1):826-34. DOI: 10.1021/ac403390y. View

12.

Molineux G . The design and development of pegfilgrastim (PEG-rmetHuG-CSF, Neulasta). Curr Pharm Des. 2004; 10(11):1235-44. DOI: 10.2174/1381612043452613. View

13.

Roberts M, Bentley M, Harris J . Chemistry for peptide and protein PEGylation. Adv Drug Deliv Rev. 2002; 54(4):459-76. DOI: 10.1016/s0169-409x(02)00022-4. View

14.

Bagal D, Zhang H, Schnier P . Gas-phase proton-transfer chemistry coupled with TOF mass spectrometry and ion mobility-MS for the facile analysis of poly(ethylene glycols) and PEGylated polypeptide conjugates. Anal Chem. 2008; 80(7):2408-18. DOI: 10.1021/ac7020163. View

15.

Hill C, Osslund T, Eisenberg D . The structure of granulocyte-colony-stimulating factor and its relationship to other growth factors. Proc Natl Acad Sci U S A. 1993; 90(11):5167-71. PMC: 46676. DOI: 10.1073/pnas.90.11.5167. View

16.

Natalello A, Ami D, Collini M, DAlfonso L, Chirico G, Tonon G . Biophysical characterization of Met-G-CSF: effects of different site-specific mono-pegylations on protein stability and aggregation. PLoS One. 2012; 7(8):e42511. PMC: 3414461. DOI: 10.1371/journal.pone.0042511. View

17.

Brokx S, Scrocchi L, Shah N, Dowd J . A demonstration of analytical similarity comparing a proposed biosimilar pegfilgrastim and reference pegfilgrastim. Biologicals. 2017; 48:28-38. DOI: 10.1016/j.biologicals.2017.06.001. View

18.

Dozier J, Distefano M . Site-Specific PEGylation of Therapeutic Proteins. Int J Mol Sci. 2015; 16(10):25831-64. PMC: 4632829. DOI: 10.3390/ijms161025831. View

19.

Payne R, Murphy B, Manning M . Product development issues for PEGylated proteins. Pharm Dev Technol. 2010; 16(5):423-40. DOI: 10.3109/10837450.2010.513990. View