J Appl Biomed 14:257-264, 2016 | DOI: 10.1016/j.jab.2016.06.002

Evaluation of physicochemical and stability properties of human growth hormone upon enzymatic PEGylation

Bahman Khameneha, Mohammad Reza Saberib,*, Mohammad Hassanzadeh-Khayyatc, Hamid Mohammadpanahd, Morteza Ghandadie, Mehrdad Iranshahif, Ali Baratiang, Mahmoud Reza Jaafarid,*
a Department of Pharmaceutical Control, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran
b Medical Chemistry Department, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran
c Pharmaceutical Research Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran
d Nanotechnology Research Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran
e Department of Pharmaceutical Biotechnology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran
f Biotechnology Research Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran
g Department of Pharmaceutics, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran

Background
PEGylation is an increasingly important strategy for improving stability, pharmacokinetic and pharmacodynamic properties of proteins.
Objective
In this study, site-specific PEGylated human growth hormone (hGH) was prepared by microbial transglutaminase and physicochemical and stability properties of bioconjugates were tested.
Method
hGH was PEGylated by 20 kDa mPEG-NH2. PEG-hGH was purified by size exclusion chromatography method and analyzed by SDS-PAGE, IEF gel and CD spectroscopy methods. Physicochemical properties, size and zeta potentials, of native and PEGylated hGH were evaluated by Dynamic Light Scattering (DLS) method. Physical and chemical stabilities were assayed at different temperatures (37, 25 and 4 °C) within three weeks by SEC- and RP-HPLC methods, respectively.
Results
By this preparation method, mono-PEG-hGH was obtained and the secondary structure was unchanged. The DLS results indicated that by PEGylation the size and zeta potentials of the protein were increased and decreased, respectively. These data indicated that PEG chain covered the protein surface. The isoelectric point (pI) of protein was not altered following PEGylation. The results of stability indicated that, the mono-PEG-hGH was considerably more stable especially in elevated temperatures as compared with the non-PEGylated one.
Conclusion
PEGylation changed the physicochemical properties of hGH and also enhanced the stability of the protein.

Keywords: Human growth hormone; PEGylation; Microbial transglutaminase; Physicochemical properties; Physical stability; Chemical stability

Received: March 6, 2016; Revised: June 8, 2016; Accepted: June 16, 2016; Published: November 1, 2016  Show citation

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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 Appl Biomed. 2016;14(4):257-264. doi: 10.1016/j.jab.2016.06.002.
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