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Hao C.-Y.,Chengdu Rongsheng Pharmaceutical Co. | Ye X.-S.,Chengdu Rongsheng Pharmaceutical Co. | Yuan P.,Chengdu Rongsheng Pharmaceutical Co. | Zeng L.-X.,Chengdu Rongsheng Pharmaceutical Co. | And 4 more authors.
Chinese Journal of Biologicals | Year: 2017

Objective: To optimize the two-wavelength microplate Reitman-Frankel assay for ALT testing. Methods: The blank reagent (PBS or purified water) and concentration of sodium hydroxide for two-wavelength microplate Reitman-Frankel assay were optimized by orthogonal test, and the optimized method was evaluated for stability, linearity, precision and accuracy. Results: The optimized method showed good stability and linearity when purified water was used as bland reagent and 1. 0 mol/L sodium hydroxide as stop solution (R2 = 0. 998 3 > 0. 990 0). Both the CVs of A492/630 values in intra-assay on 38 and 57 Karmen units of standard pyroracemic acid were less than 10%, and all the determination results of the samples were within the acceptable range. Conclusion: The optimized two-wavelength microplate Reitman-Frankel assay showed good stability, linearity, precision and accuracy and high flux, which was suitable for large-scale determination of ALT.

Nie Y.-T.,Chengdu Rongsheng Pharmaceutical Co. | He T.-P.,Chengdu Rongsheng Pharmaceutical Co. | Lei T.,Chengdu Rongsheng Pharmaceutical Co. | Ge Y.-H.,Chengdu Rongsheng Pharmaceutical Co. | And 6 more authors.
Chinese Journal of Biologicals | Year: 2010

Objective: To construct an immune antibody library and screen sub-nM affinity human antibody. Methods: The genes encoding variable region were amplified from the high titer human B lymphocytes immunized with tetanus toxoid by PCR to construct an immune antibody library, from which high affinity human antibody was screened. The obtained antibody gene fragments were transferred into whole molecules which were expressed and purified then compared with human polyclonal antibody against TT. Results: An immune phage antibody library against human tetanus, with a capacity of 4. 5 × 106, was constructed, of which the diversity met the requirements for design of primers. The human antibody with a relative affinity of 10-10 M was obtained, of which the neutralizing activity was higher than that of human polyclonal antibody. Conclusions: Sub-nM affinity human antibody was obtained by the construction of immune antibody library with small capacity by designing primers according to the frequency of antibody gene. The strategy may be used for the development of human antibody against infectious diseases.

Mu L.,Chengdu Rongsheng Pharmaceutical Co. | Lu T.,Chengdu Rongsheng Pharmaceutical Co. | Wang Q.-C.,Chengdu Rongsheng Pharmaceutical Co. | Li W.,Chengdu Rongsheng Pharmaceutical Co. | Yu W.,Chengdu Rongsheng Pharmaceutical Co.
Chinese Journal of Biologicals | Year: 2015

Objective To develop a method for preparation of human coagulation factor VIII (FVIII) by ion exchange chromatography as well as the formula of stabilizer. Methods The plasma-derived cryoprecipitate was purified by PEG precipitation plus ion exchange chromatography, in which the virus was inactivated by S/D and dry heating at 80°C for 72 h. The inactivation effect was verified, and the optimal formula of stabilizer was developed. Results The purity of FVIII increased by about 110 folds after PEG precipitation, while by more than 30 folds after ion exchange chromatography. Both S/D method and dry heating at 80°C for 72 h showed satisfactory inactivation effects. The optimal formula of stabilizer consisted of 0.01 mol/L sodium citrate, 0.001 mol/L calcium chloride, 8 mg/ml albumin and 40 mg/ml arginine hydrochloride. Conclusion The developed method may be used for preparation of FVIII with high purity, stability and safety. The screened formula of stabilizer was resistant to inactivation by dry heating.

Lin T.,Chengdu Rong Sheng Pharmaceutical Co. | Liu X.,Chengdu Rong Sheng Pharmaceutical Co. | Tan L.-J.,Chengdu Rong Sheng Pharmaceutical Co. | Pi Y.-K.,Chengdu Rong Sheng Pharmaceutical Co. | And 3 more authors.
Chinese Journal of Biologicals | Year: 2016

Objective: To develop a chromogenic method for determination of biological activity of human coagulation factor VIII (FVIII) based on the principle of biometric verification method in European Pharmacopoeia 8. 0. Methods: The procedure of chromogenic method was designed by randomized block in slope ratio model, while the data were analyzed statistically, and the result was calculated. The method was determined for linear range by using international standard, bulk and final product of FVIII, and verified for reproducibility, intermediate precision and specificity. The determination result by the developed method was compared with that by one phase method in Chinese Pharmacopoeia (Volume III, 2010 edition). Results: Both the linear correlation coefficients (r values) of the developed method for determination of standard and samples at FVIII titers of 0 ∼ 17. 85 IU/L were more than 0. 99, while the relative standard deviation (RSD) in reproducibility test was 2. 1%. The determination results by two staff were analyzed by t test and showed no significant difference (P > 0. 05). The confidence limit of the method was 80% ∼ 120%. The determination results by the developed method showed no significant difference with that by one phase method in Chinese Pharmacopoeia (Volume III, 2010 edition) (P > 0. 05). Conclusion: The developed method was simple, rapid, stable and accuracy, which met the requirements in European Pharmacopoeia 8. 0 and might be used for the determination of biological activity of FVIII.

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