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

Abstract 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.

[1]  P. Kingshott,et al.  Characterisation and physical stability of PEGylated glucagon. , 2007, International journal of pharmaceutics.

[2]  Walter Keller,et al.  Effect of protamine on the solubility and deamidation of human growth hormone. , 2012, International journal of pharmaceutics.

[3]  G. Pasut,et al.  Chemical and enzymatic site specific PEGylation of hGH. , 2013, Bioconjugate chemistry.

[4]  H. Rastegar,et al.  Effects of Different Buffers and pH on the Stability of Recombinant Human Growth Hormone , 2013 .

[5]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[6]  C. R. Middaugh,et al.  Improving the stability of the EC1 domain of E-cadherin by thiol alkylation of the cysteine residue. , 2012, International journal of pharmaceutics.

[7]  D. Urry,et al.  Nonenzymatic deamidation of asparaginyl and glutaminyl residues in proteins. , 1991, Critical reviews in biochemistry and molecular biology.

[8]  Prasad Sarangapani,et al.  Protein aggregation, particle formation, characterization & rheology , 2014 .

[9]  D. Brems,et al.  Modulation of protein aggregation by polyethylene glycol conjugation: GCSF as a case study , 2006, Protein science : a publication of the Protein Society.

[10]  F. Veronese,et al.  Transglutaminase-mediated PEGylation of proteins: direct identification of the sites of protein modification by mass spectrometry using a novel monodisperse PEG. , 2009, Bioconjugate chemistry.

[11]  A. Madadkar-Sobhani,et al.  Design, modeling, expression, and chemoselective PEGylation of a new nanosize cysteine analog of erythropoietin , 2011, International journal of nanomedicine.

[12]  Brian M. Murphy,et al.  Stability of Protein Pharmaceuticals: An Update , 2010, Pharmaceutical Research.

[13]  H. Sato,et al.  Enzymatic procedure for site-specific pegylation of proteins. , 2002, Advanced drug delivery reviews.

[14]  R T Borchardt,et al.  Chemical instability of protein pharmaceuticals: Mechanisms of oxidation and strategies for stabilization. , 1995, Biotechnology and bioengineering.

[15]  Yatin R. Gokarn,et al.  Effect of PEGylation on protein hydrodynamics. , 2012, Molecular pharmaceutics.

[16]  R. Xie,et al.  PEGylation of somatropin (recombinant human growth hormone): Impact on its clearance in humans , 2008, Xenobiotica; the fate of foreign compounds in biological systems.

[17]  S. Doglia,et al.  Biophysical Characterization of Met-G-CSF: Effects of Different Site-Specific Mono-Pegylations on Protein Stability and Aggregation , 2012, PloS one.

[18]  N. Brot,et al.  Modulation of potassium channel function by methionine oxidation and reduction. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[19]  K. Abnous,et al.  Preparation, characterization and molecular modeling of PEGylated human growth hormone with agonist activity. , 2015, International journal of biological macromolecules.

[20]  R. Duncan,et al.  Effect of PEG molecular weight and linking chemistry on the biological activity and thermal stability of PEGylated trypsin. , 2008, International journal of pharmaceutics.

[21]  Anna Mero,et al.  A new method to increase selectivity of transglutaminase mediated PEGylation of salmon calcitonin and human growth hormone. , 2011, Journal of controlled release : official journal of the Controlled Release Society.

[22]  Gerhard Winter,et al.  Head to head comparison of the formulation and stability of concentrated solutions of HESylated versus PEGylated anakinra. , 2015, Journal of pharmaceutical sciences.

[23]  Chih-Chuan Chang,et al.  A Novel High-Throughput Screening Method for Microbial Transglutaminases with High Specificity toward Gln141 of Human Growth Hormone , 2010, Journal of biomolecular screening.

[24]  Hong Zhao,et al.  Structure-function engineering of interferon-beta-1b for improving stability, solubility, potency, immunogenicity, and pharmacokinetic properties by site-selective mono-PEGylation. , 2006, Bioconjugate chemistry.

[25]  M. Al-Ghobashy,et al.  Assessment of the Degradation Pattern and Extent of PEG Interferon α-2b Using a Stability-Indicating SE-HPLC Assay , 2014, Chromatographia.

[26]  Aarti Sharma,et al.  Recent trends in protein and peptide drug delivery systems , 2009 .

[27]  P. Caliceti,et al.  A new site-specific monoPEGylated filgrastim derivative prepared by enzymatic conjugation: Production and physicochemical characterization. , 2012, Journal of controlled release : official journal of the Controlled Release Society.

[28]  M. Alibolandi,et al.  Injectable Supramolecular Hydrogel from Insulin-Loaded Triblock PCL-PEG-PCL Copolymer and γ-Cyclodextrin with Sustained-Release Property , 2014, AAPS PharmSciTech.

[29]  C. Glaser,et al.  Reaction of bovine growth hormone with hydrogen peroxide. , 1974, Biochemistry.

[30]  G. Pasut.  Pegylation of Biological Molecules and Potential Benefits: Pharmacological Properties of Certolizumab Pegol , 2014, BioDrugs.

[31]  R. Suryanarayanan,et al.  Investigation of PEG crystallization in frozen and freeze-dried PEGylated recombinant human growth hormone-sucrose systems: implications on storage stability. , 2011, Journal of pharmaceutical sciences.

[32]  W. Jiskoot,et al.  Plain and mono-pegylated recombinant human insulin exhibit similar stress-induced aggregation profiles. , 2011, Journal of pharmaceutical sciences.

[33]  Robert W. Payne,et al.  Product development issues for PEGylated proteins , 2011, Pharmaceutical development and technology.

[34]  Theodore W Randolph,et al.  The effect of protein PEGylation on physical stability in liquid formulation. , 2014, Journal of pharmaceutical sciences.

[35]  G. Pasut,et al.  Chemical and Enzymatic Site Specific PEGylation of hGH: The Stability and in vivo Activity of PEG-N-Terminal-hGH and PEG-Gln141-hGH Conjugates. , 2016, Macromolecular bioscience.

[36]  R. Houghten,et al.  Human somatotropin. Reaction with hydrogen peroxide. , 1977, Archives of biochemistry and biophysics.