Research On The Testing Method Of The Biological Activity Of Mouse Nerve Growth Factor For Injection

Nerve growth factor is a kind of nerve trophic factor with functions to guarantee the survival of neurons and promote the directional regeneration of axons and the generation of myelin sheaths. Currently, the NGF activity testing methods that are internationally accepted include the dorsal root ganglia of chicken embryo culture and differentiation of cell strain of rat pheochromocytoma of adrenal medulla. Traditional dorsal root ganglia of chicken embryo culture method is typically complicated with testing results vulnerable to the subjective judgments of the operation staffs. It can only be invoked for qualitative and semi-quantitative assessment. The PC12cell culture method is based on the induction of NGF towards the PC12cell differentiation. NGF receptors exist on the PC12cytomembrane which is subjected to the induction by NGF. PC12cell stops its division and produces the nervous process. It is differentiated into a cell with superior cervical ganglia features. The differentiated PC12cell number has certain linear relationship with the NGF activity, which can be used to assess the NGF activity.This research is conducted to fulfill the objective to establish and optimize the testing methods on NGF activity by PC12cell, which mainly includes:1. the optimization of PC12cell culture methods like the dilution ratio of NGF, inoculum density of cells, number of cleaning the cell suspensions, time needed for cell culture, and dyeing time for the staining fluids. The result of the experiment suggested that:under the serum-free culture condition, multiple proportion dilution and the optimal inoculum density of NGF was (3-4)*104/well. The optimal number of cleaning was2times while the optimal time needed for culture was48hours. The optimal dyeing time, according to the experiment, was4hours. The coefficients of variance were all smaller than15%if applying the optimized culture method to test the biological activity of NGF from two different production batches.2. Methodology Validation. Apply the methodology validation to the optimized PC12cell culture method, including testing its accuracy, precision, linear range of test, anti-interference capability and others. The test on the anti-interference capability was mainly for measuring the influence of auxiliary materials the mannitols and human blood albumins on the NGF activity. The result revealed that the optimized PC12cell culture method for analysis on the NGF activity contributed by this research had the strong anti-jamming capability. The auxiliary materials did not have the conspicuous influence on NGF activity testing. The accuracy and precision were positive. The response rate was ranging from80%to120%with coefficient of variance less than10%. The linear range of test was about1-100AU/ml. The linear regression equation was y=-0.873/[1+(x/17.977)2092]+1.350. The result of the testing on the activity of NGF from different batches produced by Wuhan Hite suggested that the established method performed the positive feasibility.