Studies On PEGylation And Preliminary Properties Of Recombinant Protein HCH-2

The conjugation of polyethylene glycol (PEG), PEGylation, is an important part of second-generation biopharmaceuticals. It has received increasing attention as a well-established technique that has the capacity to overcome several problems of native proteins for therapeutic uses, and demonstrated reduced immunogenicity, extended circulating half-life, and improved proteolysis stability for these therapeutic agents. PEGylated protein-drugs are gradually becoming new high efficiency and low toxicity therapeutic drugs and extensively applied on clinical trials. In this study, we have accomplished mostly preclinical basic researches of PEGylated hCH-2 such as the selection of eligible PEGylation reagents, the optimization and verification of technologies of PEGylation and purification by amplification experiment, the establishment of methods and requirements for quality control of mono-PEG-hCH-2 and the investigation of preliminary characteristics of mono-PEG-hCH-2, and gained the target product, mono-PEG-hCH-2. These studies have laid the solid foundation for developing the second generation prolonged-release PEGylated drugs of hCH-2. The detailed contents and conclusions are shown as follows:(1) A rapid and reliable reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed and validated for the determination of PEG-hCH-2 and hCH-2 in the reaction mixture. PEG-hCH-2 and hCH-2 were separated by HPLC on a C4 column on the basis of the hydrophobicity difference and quantified by ultraviolet detection at 280nm. The chromatography analysis was performed on C8 (250mm×4.6mm i.d., 300?, with a particle size of 5μm) column. The elution strategy was the combination of the linear gradient and isocratic elution with the changes of the mobile phase A and B. The compositions of the mobile phase A and B were 999:1(v/v) water/TFA and 100% acetonitrile at a flow rate of 1.0ml/min. Detection was done by spectrophotometry at 280nm and the column temperature was 30±1°C.For PEG-hCH-2 and hCH-2, the standard curves were linear in the range of 0.049-0.456mg/ml and 0.112-1.796mg/ml, the regression equation were y=2.11×106x -3.30×10~4 (r~2=0.9994, n=5) and y=1.08×10~6x-8.45×10 (r~2=1.0000, n=5), the lower limit of quantitative analysis were established at 0.044mg/ml and 0.056mg/ml, the relative standard deviation (RSD) values of intra-day precision were <1.34% and <2.18% (n=6), the RSD values of inter-day precision were <1.49% and <2.21% (n=6), the RSD values of recovery experiment were <2.62% and <2.32% (n=3), the average recovery rates...