Design, development and evaluation of PEGylated rhGH with preserving its bioactivity at highest level after modification.

Modification of recombinant proteins with polyethylene-glycol (PEG) can improve their pharmacokinetic properties, although their bioactivity may be reduced after PEGylation due to structural changes. In this study, simultaneous optimization of PEGylation efficiency and preserved bioactivity of recombinant human growth hormone (rhGH) was investigated. In this regard, experiments were designed by the response surface methodology (RSM)-central composite design (CCD) utilizing design expert software. Under the obtained optimum conditions of 6.73 molar ratio of PEG to protein and pH 7.71 as the main factors affect the process, 54% PEGylation efficiency and 63% preserved bioactivity can be achieved. Based on the ANOVA table, model F-values equal to 31.16 and 20.8 for PEGylation efficiency and preserved bioactivity, respectively, demonstrated the validity and importance of the models. High performance liquid chromatography (HPLC) and gel electrophorese analyses verified the purity of the PEGylated form of rhGH. Findings showed that the modified protein would be stable for six months at 4 °C. In vitro cell growth assessments revealed Nb2-11 cell proliferation during 48 h, although proliferation rate decrease with the increase of PEGylated rhGH concentration. Half-life prolongation in serum observed for PEGylated form in comparison with the non-modified one on in vivo. In overall, the results are promising for the utilization of the PEGylated form of rhGH for the treatment of human growth deficiency after further investigations.