Somayeh Vakili - Department of Medical Chemistry, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran
JamshidKhan Chamani - Department of Biology, Faculty of Sciences, Mashhad Branch, Islamic Azad University, Mashhad, Iran
Zeinab Amiri-Tehranizadeh - Department of Medical Chemistry, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran
Hossein Hosseinzadeh - Department of Pharmacodynamics and Toxicology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran
Fatemeh Mosaffa - Biotechnology Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical Sciences, Mashhad, Iran
Seyed-Mola Khatami - Department of Chemistry, Faculty of Samen Hojaj, Mashhad Branch, Technical and Vocational University (TVU), Tehran, Iran
Bahman Khameneh - Department of Pharmaceutical Control, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran
Mohammad Reza Saberi - Department of Medical Chemistry, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran

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 (۲ and ۵ 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 ۴ °C; a pronounced change in monomer degradation is observed when stored at ۲۵ °C and ۳۷ °C. hGH:Glu-mPEG ۲ kDa with a molar ratio of ۱:۱ 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.

Human Growth Hormone, Non-covalent PEGylation, Physical PEGylation, Protein formulation, Stability