| Human Growth Hormone(hGH)is one of the peptide hormones,which main role is to promote the synthesis of proteins in human body.But this natural source of growth hormone from the pituitary extract is shortage and expensive,which limited the clinical research.Recombinant Human Growth Hormone(rhGH)is one of bio-recombinant products expressed in Escherichia coli.During the whole purification process,residual host cell protein(HCP)of Escherichia coli in recombinant products is the key issues to measure the purity.Fine purification can effectively remove HCP of recombinant protein products.This paper is mainly study on setting up the detection method of residual HCP and recombinant protein content of rhGH.The two methods are jointly together to applly to the purification of rhGH in the production and in order to control the quality in purifying process.Through compared with linear regression fitting and cubic spline interpolation analysis method,compared whether used closed liquid,compared four species diluted liquid detection,using Orthogonal designer to select the best condition of experiment by multi-factors,and using method validation of the new detection method of residual HCP;used Pharmacopoeia method to detect protein content of rhGH,and method validation;According to two methods of detecting residual HCP and protein content of rhGH in original every step of the purification process,the results as the guide to optimize purification processes as a standard to meet the Pharmacopoeia in new purification processes.Due to antigen-antibody reaction existing plateau,cubic spline curve fitting is better.Comparing with several blocking solution in common,the final results are similar to unclosed one.Dilution test shows that citrate buffer is better than the others.Contrast to different pH,the pH 6.0 of citrate buffer is the best one.Then,through recoveries,sensitivity and precision tests,the paper rebuild the new method for detecting residual HCP of rhGH.Validation method of protein content verified the relevant provisions;Optimized purification process enables HCP residue from 13~16ng/mg down to 2ng/mg before the optimization and meet Pharmacopoeia requirements(<10ng/mg).This paper successfully establishes the determination of protein content and the detection method of residual HCP of rhGH,and the two detection methods for validation method.It has a vital significant control that in every step of the purifying process of rhGH.At the same time,there is also the foundation studies of rhGH. |
