Shenzhen Sciprogen Bio-Pharmaceutical Co., Ltd.

To establish a useful PEGylation reaction method of recombinant human erythropoietin (EPO) with high yield of mono-substitution product (mono-PEG-EPO), a consecutive PEGylation reaction method had been developed.At first, the reaction mixture of PEG-EPO after being stored under 4°C for 24 mo had been analyzed and it showed significant long-term stability.Meanwhile, the CD test results of EPO in various buffer solutions suggested that the character of secondary structure of EPO was slightly different.And then the direct second-time PEGylation of EPO under the same buffer solution, and the consecutive second-times PEGylation of EPO in different dual buffer solution systems were conducted.After that, consecutive three-times PEGylation of EPO was carried out successfully in a dual buffer solution system.The resulting purified mono-PEG-EPO products had been analyzed by different methods, including HPLC test, FL spectrum test, electrophoresis test, content of sialic acid test, in vivo activity test and PEGylation position test.From these research results, it could be concluded that the reaction mixture of PEGylation of EPO could not conduct the second-set PEGylation directly.However, applying with dual buffer solution system, consecutive PEGylation of EPO could be carried out with yield of mono-PEG-EPO above 54% without obvious change of PEGylation position.And the purified monoPEG-EPO products maintained their biol. activity and protein structure integrity.These findings provided a basis for the development of a new type of PEG-EPO manufacturing process with high yield of mono-PEG-EPO product to reduce the drug production cost.

To establish a simple and correct detection method of Nadroparin calcium(NC) in its practical production process, a new UV spectrophotometry based on the system of 1, 10-phenanthroline monohydrate and zinc sulfate(phen-ZnSO₄) is developed.The results showed that NC had a significant UV absorption peak(λ_max=302 nm) in the system of phen-ZnSO₄ with 1 mol/L NaAc as buffer, and there is a linear proportional relationship between the absorbance and the concentration of NC solutionIn order to optimize the detection method, effects of the amount of NaAc solution, the amount of ZnSO₄ solution and the color time have been studied.The further research results indicated that the resulting method had a linear range of 5.38*10^-3∼3.87*10^-2 mol/L and a detection limit of 2.06*10^-3 mol/L.It has been applied in the practical production process of NC, and the anal. results are satisfied and useful in process monitoring.Furthermore, the detection mechanism of this method has been explored by the test of the detection of unfractionated heparin(UFH) and the study of the effect of addnl. NaNO₂ solutionThe results suggest that the hydroxyl groups on the pentasaccharidic antithrombin-binding sequence of the NC structure take part in the formation of the color system.These findings provide a basis for the establishment of a new type of process control technol. in the pharmaceutical production process of NC.