Amino acid-mPEGs: Promising excipients to stabilize human growth hormone against aggregation

Document Type : Original Article

Authors

1 Department of Medical Chemistry, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran

2 Department of Biology, Faculty of Sciences, Mashhad Branch, Islamic Azad University, Mashhad, Iran

3 Department of Pharmacodynamics and Toxicology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran

4 Pharmaceutical Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical Sciences, Mashhad, Iran

5 Biotechnology Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical Sciences, Mashhad, Iran

6 Department of Chemistry, Faculty of Samen Hojaj, Mashhad Branch, Technical and Vocational University (TVU), Tehran, Iran

7 Department of Pharmaceutical Control, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran

Abstract

Objective(s): Today, the non-covalent PEGylation methods of protein pharmaceuticals attract more attention and possess several advantages over the covalent approach. In the present study, Amino Acid-mPEGs (aa-mPEGs) were synthesized, and the human Growth Hormone (hGH) stability profile was assessed in their presence and absence.
Materials and Methods: aa-mPEGs were synthesized with different amino acids (Trp, Glu, Arg, Cys, and Leu) and molecular weights of polymers (2 and 5 KDa). The aa-mPEGs were analyzed with different methods. The physical and structural stabilities of hGH were analyzed by SEC and CD spectroscopy methods. Physical stability was assayed at different temperatures within certain intervals. Molecular dynamics (MD) simulation was used to realize the possible mode of interaction between protein and aa-mPEGs. The cell-based method was used to evaluate the cytotoxicity.
Results: HNMR and FTIR spectroscopy indicated that aa-mPEGs were successfully synthesized. hGH as a control group is known to be stable at 4 °C; a pronounced change in monomer degradation is observed when stored at 25 °C and 37 °C. hGH:Glu-mPEG 2 kDa with a molar ratio of 1:1 to the protein solution can significantly increase the physical stability. The CD spectroscopy method showed that the secondary structure of the protein was preserved during storage. aa-mPEGs did not show any cytotoxicity activities. The results of MD simulations were in line with experimental results.
Conclusion: This paper showed that aa-mPEGs are potent excipients in decreasing the aggregation of hGH. Glu-mPEG exhibited the best-stabilizing properties in a harsh environment among other aa-mPEGs.

Keywords

Main Subjects

References

1. Asoodeh A, Sepahi S, Ghorani-Azam A. Purification and modeling amphipathic alpha helical antimicrobial peptides from skin secretions of Euphlyctis cyanophlyctis. Chem Biol Drug Des 2014;83:411-417.
2. Fazly Bazzaz BS, Seyedi S, Hoseini Goki N, Khameneh B. Human antimicrobial peptides: Spectrum, mode of action and resistance mechanisms. Int J Pept Res Ther 2021; 27:801-816.
3. Khodaverdi E, Javan M, Tabassi SAS, Khameneh B, Kamali H, Hadizadeh F. Sustained drug delivery system for insulin using supramolecular hydrogels composed of tri-block copolymers. J Pharma Invest 2017;47:263-273.
4. Pisal DS, Kosloski MP, Balu-Iyer SV. Delivery of therapeutic proteins. J Pharm Sci 2010;99:2557-2575.
5. Khameneh B, Saberi MR, Hassanzadeh-Khayyat M, Mohammadpanah H, Ghandadi M, Iranshahi M, et al. Evaluation of physicochemical and stability properties of human growth hormone upon enzymatic PEGylation. J App Biomed 2016;14:257-264.
6. Platts L, Darby SJ, Falconer RJ. Control of globular protein thermal stability in aqueous formulations by the positively charged amino acid excipients. J Pharm Sci 2016;105:3532-3536.
7. Kamerzell TJ, Esfandiary R, Joshi SB, Middaugh CR, Volkin DB. Protein-excipient interactions: Mechanisms and biophysical characterization applied to protein formulation development. Adv Drug Deliv Rev 2011;63:1118-1159.
8. Ghorani-Azam A, Balali-Mood M, Aryan E, Karimi G, Riahi-Zanjani B. Effect of amino acid substitution on biological activity of cyanophlyctin-β and brevinin-2R. J Mol Struct 2018;1158:8-14.
9. Chi EY, Krishnan S, Randolph TW, Carpenter JF. Physical stability of proteins in aqueous solution: Mechanism and driving forces in nonnative protein aggregation. Pharm Res 2003;20:1325-1336.
10. Grigoletto A, Maso K, Pasut G. 13-Enzymatic approaches to new protein conjugates. In: Pasut G, Zalipsky S, Editors. Polymer-Protein Conjugates: Elsevier; 2020. p. 271-295.
11. Reichert C, Borchard G. Noncovalent PEGylation, an innovative subchapter in the field of protein modification. J Pharm Sci 2016;105:386-390.
12. Khameneh B, Jaafari MR, Hassanzadeh-Khayyat M, Varasteh A, Chamani J, Iranshahi M, et al. Preparation, characterization and molecular modeling of PEGylated human growth hormone with agonist activity. Int J Biol Macromol 2015;80:400-409.
13. Ambrosio E, Barattin M, Bersani S, Shubber S, Uddin S, Van Der Walle CF, et al. A novel combined strategy for the physical PEGylation of polypeptides. J Control Release 2016;226:35-46.
14. Mueller C, Capelle MA, Arvinte T, Seyrek E, Borchard G. Tryptophan-mPEGs: Novel excipients that stabilize salmon calcitonin against aggregation by non-covalent PEGylation. Eur J Pharm Biopharm 2011;79:646-657.
15. Mueller C, Capelle MA, Seyrek E, Martel S, Carrupt P-A, Arvinte T, et al. Noncovalent PEGylation: Different effects of dansyl-, l-tryptophan-, phenylbutylamino-, benzyl-and cholesteryl-PEGs on the aggregation of salmon calcitonin and lysozyme. J Pharm Sci 2012;101:1995-2008.
16. Mueller C, Capelle MAH, Arvinte T, Seyrek E, Borchard G. Tryptophan-mPEGs: Novel excipients that stabilize salmon calcitonin against aggregation by non-covalent PEGylation. Eur J Pharm Biopharm 2011;79:646-657.
17. Mero A, Campisi M, Caputo M, Cuppari C, Rosato A, Schiavon O, et al. Hyaluronic acid as a protein polymeric carrier: An overview and a report on human growth hormone. Curr Drug Targets 2015;16:1503-1511.
18. Ciorba MA, Heinemann SH, Weissbach H, Brot N, Hoshi T. Modulation of potassium channel function by methionine oxidation and reduction. Proc Natl Acad Sci 1997;94:9932-9937.
19. Xu AY, Castellanos MM, Mattison K, Krueger S, Curtis JE. Studying excipient modulated physical stability and viscosity of monoclonal antibody formulations using small-angle scattering. Mol Pharm. 2019;16:4319-4338.
20. Wu H, Truncali K, Ritchie J, Kroe-Barrett R, Singh S, Robinson AS, et al. Weak protein interactions and pH-and temperature-dependent aggregation of human Fc1. MAbs 2015;7:1072-1083
21. Garidel P, Hegyi M, Bassarab S, Weichel M. A rapid, sensitive and economical assessment of monoclonal antibody conformational stability by intrinsic tryptophan fluorescence spectroscopy. Biotechnol J 2008;3:1201-1211.
22. Roberts CJ. Therapeutic protein aggregation: Mechanisms, design, and control. Trend Biotechnol 2014;32:372-380.
23. Riahi-Zanjani B, Balali-Mood M, Asoodeh A, Es’haghi Z, Ghorani-Azam A. Potential application of amino acids in analytical toxicology. Talanta 2019;197:168-174.
24. Zapadka KL, Becher FJ, Gomes dos Santos A, Jackson SE. Factors affecting the physical stability (aggregation) of peptide therapeutics. Interface Focus 2017;7:20170030.
25. Bian S, Pagan C, Andrianova “Artemyeva” AA, Du G. Synthesis of polycarbonates and poly (ether carbonate) s directly from carbon dioxide and diols promoted by a Cs2CO3/CH2Cl2 system. ACS omega 2016;1:1049-1057.
26. Gu Z, Wang F, Lu H, Wang X, Zheng Z. Trypsin-inspired poly (urethane-urea) s based on poly-lysine oligomer segment. J Biomater Sci Polym Ed 2015;26:311-321.
27. Reslan M, Demir YK, Trout BL, Chan H-K, Kayser V. Lack of a synergistic effect of arginine-glutamic acid on the physical stability of spray-dried bovine serum albumin. Pharm Dev Technol 2017;22:785-791.
28. Poole LB. The basics of thiols and cysteines in redox biology and chemistry. Free Radic Biol Med 2015;80:148-157.
29. Mastrotto F, Bellato F, Andretto V, Malfanti A, Garofalo M, Salmaso S, et al. Physical PEGylation to prevent insulin fibrillation. J Pharm Sci 2020;109:900-910.
30. Maikawa CL, Smith AAA, Appel EA. Supramolecular pegylation as an innovative approach to biopharmaceutical formulation and delivery. Soc Biomaterials.
31. Martinez AP, Qamar B, Marin A, Fuerst TR, Muro S, Andrianov AK. Biodegradable “scaffold” Polyphosphazenes for Non-Covalent PEGylation of Proteins. In: Allcock HR, Andrianov AK, Editors. ACS Symposium Series: American Chemical Society; 2018. p. 121-141.
32. Kheddo P, Tracka M, Armer J, Dearman RJ, Uddin S, Van Der Walle CF, et al. The effect of arginine glutamate on the stability of monoclonal antibodies in solution. Int J Pharm 2014;473:126-133.
33. Nieto-Orellana A, Di Antonio M, Conte C, Falcone FH, Bosquillon C, Childerhouse N, et al. Effect of polymer topology on non-covalent polymer-protein complexation: Miktoarm versus linear mPEG-poly(glutamic acid) copolymers. Polym Chem 2017;8:2210-2220.
34. Asayama S, Nagashima K, Kawakami H. Facile Method of Protein PEGylation by a Mono-Ion Complex. ACS Omega 2017;2:2382-2386.
35. Jevsevar S, Kunstelj M, Porekar VG. PEGylation of therapeutic proteins. Biotechnol J 2010;5:113-128.
36. Kurinomaru T, Shiraki K. Noncovalent PEGylation of L-asparaginase using PEGylated polyelectrolyte. J Pharm Sci 2015;104:587-592.
37. Appel EA, Maikawa CL, Smith AAA, Agmon G, editors. Supramolecular Pegylation as an Innovative Approach to Biopharmaceutical Formulation and Delivery 2019: Society for Biomaterials.
38. Kurinomaru T, Kuwada K, Tomita S, Kameda T, Shiraki K. Noncovalent PEGylation through protein-polyelectrolyte interaction: Kinetic experiment and molecular dynamics simulation. J Physic Chem B. 2017;121:6785-6791.
 
Iranian Journal of Basic Medical Sciences
Volume 26, Issue 6
June 2023
Pages 635-644

Files

Share

How to cite

Statistics