Comparative Assessment of Filgrastim Bio-similars via vertical gel electrophoresis (SDS-PAGE) and HPLC

Priyanka Chaudhary 1, Muthusamy Kalaivani 1, Kashish Chaudhary 2, Anubhuti Goyal 1, Meenakshi Dahiya1, Rajeev Singh Raghuvanshi 1

1 Department of Biologics, Indian Pharmacopoeia Commission, Ministry of Health and Family Welfare, Govt. India, Sector-23, Raj Nagar, Ghaziabad-201 002, India.

2 Department of Biotechnology, Bennett University, India


Background: Filgrastim bio-similar have emerged as cost-effective alternatives to the reference filgrastim, providing comparable efficacy and safety profiles. Objective: In this study, we aim to show physico-chemical similarity between filgrastim biosimilars using vertical gel electrophoresis & high-performance liquid chromatography. Materials & Methods: In the present study, we evaluate filgrastim biosimilars (grafeel, religrast, neukine, colstim, emigrast) via SDS-PAGE and HPLC thereby checking their molecular weights for similarity and their qualitative analysis. The same was compared with reference standard of filgrastim (Filgrastim, certified Reference Material). Results: In the scope of the data obtained, we found that the main band of filgrastim in filgrastim biosimilars has the same electrophoretic mobility as the reference standard under non-reduced circumstances in case of SDS-PAGE, the outcomes demonstrated that the position of the principal band in the electropherogram produced by the test solution and the principal band produced by the reference solution are similar. The retention time of the main peak obtained with the test solution and the peak obtained with the reference solution are similar. For HPLC, all the filgrastim biosimilars showed monomer peaks on the RP-HPLC chromatograms, which eluted at 12.4 minutes, matching the retention duration of the filgrastim reference standard. Conclusion: Filgrastim has transformed the management of neutropenia and has become an indispensable therapy in various clinical settings. The development and approval of filgrastim biosimilars have expanded treatment options and improved accessibility, offered cost-effective alternatives while maintained comparable safety and efficacy. .  

Keywords: Filgrastim, Bio-similar, Physicochemical, SDS-PAGE, HPLC.


1.    Sekhon B S: Biopharmaceuticals: An overview, Thai J. pharm Sci. 2010; 34:1-19
2.    Subbiah, V. The next generation of evidence-based medicine. Nat Med 29, 2023; 49-58. 
3.    Kirchhoff CF, Wang XM, Conlon HD, Anderson S, Ryan AM, Bose A. Biosimilars: Key regulatory considerations and similarity assessment tools. Biotechnol Bioeng. 2017;114(12):2696-705.
4.    Kim H, Alten R, Avedano L et al The Future of Biosimilars: Maximizing Benefits Across Immune-Mediated Inflammatory Diseases. Drugs. 2020; 80:99-113
5.    Halim L A, Marquez M, Maas-Bakker R F et al, Quality comparison of biosimilar and copy filgrastim products with the innovator product; Pharm Res 2018;35:226
6.    Hoglund M. Glycosylated and non-glycosylated recombinant human granulocyte colony-stimulating factor (rhG-CSF)–what is the difference? Med Oncol. 1998;15(4):229–33
7.    Herman AC, Boone TC, Lu HS. Characterization, formulation, and stability of Neupogen (Filgrastim), a recombinant human granulocyte-colony stimulating factor. Pharma Biotech. 1996;9: 303–28
8.    Hollingshead LM, Goa KL. Recombinant Granulocyte Colony-Stimulating Factor (rG-CSF). 1991; Drugs 42, 300–30 
9.    Panopoulos AD, Watowich SS. Granulocyte colony-stimulating factor: molecular mechanisms of action during steady state and 'emergency' hematopoiesis. Cytokine. 2008; 42(3):277-8
10.    Segal AW. How neutrophils kill microbes. Annu Rev Immunol. 2005; 23:197-223
11.    Lyman GH, Kuderer NM. Filgrastim in patients with neutropenia: potential effects on quality of life. Drugs. 2002;62 Suppl 1:65-78.
12.    Bendall L J, Bradstock K F, G-CSF: From granulopoietic stimulant to bone marrow stem cell mobilizing agent, Cytokine & Growth Factor Reviews, 2014; 4: 25. 
13.    Awad M, Singh P, Hilas O. Zarxio (Filgrastim-sndz): The First Biosimilar Approved by the FDA. P T. 2017;42(1):19-23.
14.    Kraj L, Krawczyk-Lipiec J, Gorniewska J et al. Efficacy and safety of biosimilar filgrastim in primary and secondary prevention of febrile neutropenia. Biomed Rep. 2017; 2:143-47.
15.    Meher BR, Balan S, Mohanty RR, Jena M, Das S. Biosimilars in India; Current Status and Future Perspectives. J Pharm Bioallied Sci. 2019;11(1):12-15
16.    Joshi D, Khursheed R, Gupta S et al Biosimilars in Oncology: Latest Trends and Regulatory Status. Pharmaceutics. 2022;14(12):2721
17.    Sörgel, Fritz L, Lauber HT. Physicochemical and Biologic Comparability of a Biosimilar Granulocyte Colony-Stimulating Factor with Its Reference Product. BioDrugs: clinical immunotherapeutics, biopharmaceuticals and gene therapy.  2010;24: 347-57.
18.    Qureshi H, Veeresham C. and Srinivas C. Analytical Method Development and Validation of Filgrastim by UV and RP-UPLC Methods. American Journal of Analytical Chemistry 2021;12: 333-46.
19.    Hannah R, Eyers, Claire- Analysis of Post-translational Modifications by LC-MS/MS. Methods in molecular biology (Clifton, N.J.). 2010;658: 93-108.
20.    Jadaun GPS, Dixit S, Saklani V et al HPLC for Peptides and Proteins: Principles, Methods and Applications Pharm Methods. 2017; 8(1): 1-6
21.    Kirley TL, Norman AB. Unfolding of IgG domains detected by non-reducing SDS-PAGE. Biochem Biophys Res Commun. 2018;503(2):944-49.
22.    Mantovani M, Caruso C S, Antonio D F et al, Pharmacokinetic and pharmacodynamic properties of a new biosimilar filgrastim TPI G-CSF in comparison to the marketed reference filgrastim Neupogen: a double blind, single dose, two period cross over trial Dove press 2017;166:90.
23.    Macedo M K, Romero-Diaz A J, Miranda Hernandez M P, Duran K M, Flores-ortiz L F, Rivero E M; Characterization and comparability of biosimilars: a filgrastim case of study and regulatory perspectives for Latin America; Electronic J Biotech. 2016; 19:63-69. 
24.    Skrlin A, Radic I, Vuletic M et al Comparison of physicochemical properties of filgrastim; Science Direct 2010; 5:557-66. 
25.    Cory.E. Muraco; a fast and reliable method for purity analysis of filgrastim; reporter US 34.2. 
26.    Rathore AS, Bhambure R. Establishing analytical comparability for “biosimilars”: filgrastim as a case study. Anal Bioanal Chem. 2014; 406: 6569–76.  
27.    Tekdemir B, Seckin Z, Kacar AI et al. Evaluation of Structural, Biological, and Functional Similarity of Biosimilar Granulocyte Colony Stimulating Factor to its Reference Product. Pharm Res; 2020: 37:215
28.    Skrlin A, Radic I, Schwinke, D et al. Comparison of the physicochemical properties of a biosimilar filgrastim with those of reference filgrastim. Biologicals: Journal of the International Association of Biological Standardization. 2010; 38:557-66.
29.    Sörgel F, Schwebig A, Holzmann J, Prasch S, Singh P, Kinzig M. Comparability of biosimilar filgrastim with originator filgrastim: protein characterization, pharmacodynamics, and pharmacokinetics. Bio Drugs. 2015; 29:123-31.

Article ID: O0601027 RA Preprint 
Received: 05/06/2024
Accepted: 19/06/2024
Published: 05/07/2024

P. Chaudhary et al. Comparative Assessment of Filgrastim Bio-similars via vertical gel electrophoresis (SDS-PAGE) and HPLC. Journal of Allbiosolution, 2024, 6(1):271-277
P. Chaudhary et al. Comparative Assessment of Filgrastim Bio-similars via vertical gel electrophoresis (SDS-PAGE) and HPLC. Allbiosolution, 2024, 6(1):271-277