EFFECT OF POLYSORBATE-80 CONCENTRATION ON G-CSF FORMULATION USING LIQUID CHROMATOGRAPHY

  • Amand Bhaskar Department of Bio-engineering Birla Institute of Technology, Mesra
  • Kunal Mukhopadhyay Department of Bio-engineering Birla Institute of Technology, Mesra
  • Manish Kumar Birla Institute of Technology, Mesra

Abstract

Objective: The aim of the study was to find the dual effects of polysorbate-80 concentration on oxidation and aggregation in the formulation of granulocyte colony stimulating factor.

Methods: The effect of polysorbate-80 at different concentration was monitored and analyzed using high performance liquid chromatography. Oxidative degradation and aggregate generation was studied using reverse phase and size exclusion chromatography method respectively.

Results: With increase in concentration of polysorbate-80 the amount of oxidation as well as aggregate formation increases in a concentration dependent manner. The aggregates present at higher concentration of polysorbate-80 formulation are not found with low concentration or without polysorbate-80. This result shows that higher concentration of Tween-80 force the formation of oligomers and leads to increased level of oxidation of granulocyte colony stimulating factor.

Conclusion: The dual effect of aggregation and oxidation of polysorbate-80 on the recombinant granulocyte colony stimulating factor indicate that during formulation development studies it is crucial to evaluate the amount of polysorbate-80 to be used in the formulation.

 

Keywords: Surfactants, Aggregates, Polysorbate-80, Oxidation, G-CSF.

Downloads

Download data is not yet available.

Author Biographies

Kunal Mukhopadhyay, Department of Bio-engineering Birla Institute of Technology, Mesra

Professor

Department of Bio-engineering

Manish Kumar, Birla Institute of Technology, Mesra

Assistant Professor

Department of Bio-engineering

References

1. Metcalf D. Granulocyte-macrophage colony-stimulating factors. Sci 1985;229:16-22.
2. Beekman R, Ivo PT. G-CSF and its receptor in myeloid malignancy. Blood 2010;115:5131-6.
3. Aapro MS, Cameron DA, Pettengell R, Bohlius J, Crawford J, Ellis M, et al. European Organisation for Research and Treatment of Cancer (EORTC) Granulocyte Colony-Stimulating Factor. Guidelines Working Party EORTC guidelines for the use of granulocyte-colony stimulating factor to reduce the incidence of chemotherapy-induced febrile neutropenia in adult patients with lymphomas and solid tumours. Eur J Cancer 2006;42:2433–53.
4. Bondos SE, Bicknell A. Detection and prevention of protein aggregation before, during and after purification. Anal Biochem 2003;316:223-31.
5. Duncan MR, Lee JM, Warchol MP. Influence of surfactants upon protein/peptide adsorption to glass and polypropylene. Int J Pharm 1995;120(2):179–88.
6. Mahler HC, Huber F, Ravuri SKK, Reindl J, Ruckert P, Muller R. Adsorption behaviour of a surfactant and a monoclonal antibody to sterilizing-grade filters. J Pharm Sci 2010;99(6):2620-7
7. Hillgren A, Lindgren J, Alden M. Protection mechanism of Tween 80 during freeze-thawing of a model protein, LDH. Int J Pharm 2002;237:57–69.
8. Kiese S, Pappenberger A, Friess W, Mahler HC. Shaken, not stirred: mechanical stress testing of an IgG1 antibody. J Pharm Sci 2008;97:4347–66.
9. Carpenter JF, Chang BS, Garzon RW, Randolph TW. Rational design of stable lyophilized protein formulations: theory and practice. Pharm Biotechnol 2002;13:109–33.
10. Carpenter JF, Pikal MJ, Chang BS, Randolph TW. Rational design of stable lyophilized protein formulations: some practical advice. Pharm Res 1997;14:969–75.
11. Katakam M, Bell LN, Banga AK. Effect of surfactants on the physical stability of recombinant human growth hormone. J Pharm Sci 1995;84(6):713-6.
12. Wei G, Lu LF, Lu WY. Stabilization of recombinant human growth hormone against emulsification-induced aggregation by Pluronic surfactants during microencapsulation. Int J Pharmaceutics 2007;338(1-2):125-32.
13. Hawe A, Filipe V, Jiskoot W. Fluorescent molecular rotors as dyes to characterize polysorbate-containing IgG formulations. Pharm Res 2010;27(2):314-26.
14. Maa Y-F, Hsu CC. Investigation on fouling mechanisms for recombinant human growth hormone sterile filtration. J Pharm Sci 1998;87(7):808–12.
15. Cromwell MEM, Hilario E, Jacobson F. Protein aggregation and bioprocessing. AAPS J 2006;8(3):E572–E9.
16. Eckhardt BM, Oeswein JQ, Bewley TA. Effect of freezing on aggregation of human growth hormone. Pharm Res 1991;8(11):1360–4.
17. Mahler HC, Friess W, Grauschopf U, Kiese S. Protein aggregation: pathways, induction factors and analysis. J Pharm Sci 2009;98(9):2909–34.
18. Kappos L, Clanet M, Sandberg WM, Radue EW, Hartung HP, Hohlfeld R, et al. Neutralizing antibodies and efficacy of interferon β-1a: A 4-year controlled study. Neurology 2005;65:40–7.
19. Jefferis R. Aggregation, immune complexes and immunogenicity. MAbs 2011;3(6):503–4.
20. Casadevall N, Nataf J, Viron B, Kolta A, Kiladjian JJ, Martin DP, et al. Pure red-cell aplasia and antierythropoietin antibodies in patients treated with recombinant erythropoietin. New Engl J Med 2002;346:469–75.
21. Vartanian T, Sorensen PS, Rice G. Impact of neutralizing antibodies on the clinical efficacy of interferon beta in multiple sclerosis. J Neurol 2004;251:1125–30.
22. Donbrow M, Azaz E, Pillersdorf A. Autoxidation of polysorbates. J Pharm Sci 1978;67:1676–81.
23. Jaeger J, Sorensen K, Wolff SP. Peroxide accumulation in detergents. J Biochem Biophys Methods 1994;29:77–81.
24. Ha E, Wang W, Wang YJ. Peroxide formation in polysorbate-80 and protein stability. J Pharm Sci 2002;91(10):2252–64.
25. Kishore RS, Kiese S, Fischer S, Pappenberger A, Grauschopf U, Mahler HC. The degradation of polysorbates 20 and 80 and its potential impact on the stability of biotherapeutics. Pharm Res 2011;28(5):1194-210.
26. Van Beers MMC, Sauerborn M, Gilli F, Brinks V, Schellekens H, Jiskoot W. Oxidized and aggregated recombinant human interferon beta is immunogenic in human interferon beta transgenic mice. Pharm Res 2011;28(10):2393-402.
27. Knepp VM, Whatley JL, Muchnik A, Calderwood TS. Identification of antioxidants for prevention of peroxide-mediated oxidation of recombination human ciliary neurotrophic factor and recombinant human nerve growth factor. J Pharm Sci Technol 1996;50:163–71.
28. Azaz E, Donbrow M, Hamburger R. Incompatibility of non-ionic surfactants with oxidisable drugs. Pharm J 1975;211:5.
29. Simat TJ, Steinhart H. Oxidation of free tryptophan and tryptophan residues in peptides and proteins. J Agric Food Chem 1998;46(2):490–8.
30. Milzani A, Rossi R, Di Simplicio P, Giustarini D, Colombo R, Dalledonne I. The oxidation produced by hydrogen peroxide on Ca-ATP-G-actin. Protein Sci 2001;9:1774–82.
31. Van Beers MMC, Sauerborn M, Gilli F, Brinks V, Schellekens H, Jiskoot W. Oxidized and aggregated recombinant human interferon beta is immunogenic in human interferon beta transgenic mice. Pharm Res 2011;28(10):2393-02.
32. Wang W, Wang YJ, Wang DQ. Duel effects of Tween 80 on protein stability. Intl J Pharmaceut 2008;347:31-8.
33. Katakam M, Bell LN, Banga AK. Effect of surfactants on the physical stability of recombinant human growth hormone. J Pharm Sci 1995;84(6):713-6.
34. Maggio ET. Polysorbates, peroxides, protein aggregation and immunogenicity: a growing concern. J Excip Food Chem 2012;3:45–53.
35. http: //www. fda. gov/ Drugs/ Guidance Compliance Regulatory Information/Guidances/default. html.
36. Jahn EM, Schneider CK. How to systematically evaluate immunogenicity of therapeutic proteins regulatory considerations. N Biotechnol 2009;25(5):280-6.
37. Buss NA, Henderson SJ, McFarlane M, Shenton JM, de Haan L. Monoclonal antibody therapeutics: history and future. Curr Opin Pharmacol 2012;12(5):615–22.
Statistics
910 Views | 4788 Downloads
How to Cite
Bhaskar, A., K. Mukhopadhyay, and M. Kumar. “EFFECT OF POLYSORBATE-80 CONCENTRATION ON G-CSF FORMULATION USING LIQUID CHROMATOGRAPHY”. International Journal of Pharmacy and Pharmaceutical Sciences, Vol. 6, no. 9, 1, pp. 299-02, https://innovareacademics.in/journals/index.php/ijpps/article/view/2257.
Section
Original Article(s)