Research On Near Infrared Spectroscopy Quantitative Analysis In Production Processes Of Human Coagulation Factor â…§

Coagulation factor Ⅷ is an indispensable medicine for the treatment of Hemophilia A and the bleeding caused by acquired deficiency of FⅧ, but China’s current total blood products industry can’tmeet the need for a long period of state, so how to increase the activity yield and specific activity of the product is very important. DomesticFⅧproduction process use more of ion exchange chromatography, which isolated and purified FⅧ from human plasma cryoprecipitate, including acid precipitation, A50 adsorption, S/D inactivation, chromatography, ultrafiltration, freeze-dried, and other major steps. Due to the lack of a good process control technology, resulting in low production yield and low specific activity. Therefore, it is necessary to introduce the modern process analysis and control technology into the key steps of the FⅧ production process, in order to increase the understanding of the production process, and effectively control of the process, finally enhance the yield and specific activity of the product. Near infrared spectroscopy, the most important one of Process Analytical Technology with the advantages of fast, accurate and non-destructive, has been used widely inthe pharmaceutical industry, and also in blood product.This thesis selected 3 processes--acid precipitation, elution process in chromatography and freeze-drying as research object, and used NIRS combined with different Chemometrics methods, finally established quantitative analysis models in connection with FⅧ specific activity, potency and moisture, thus laid the foundation for its online application.The details are listed below:1. The acid precipitation process analysis and the establish of the quantitative analysis model.This study investigated the rationality of the endpoint control method of FⅧ acid precipitation process, and then build a partial least squares quantitative model for FⅧ specific activity by NIRS. The research selected 108 samples which have been determined the reference data, and randomly classified six trials including 64 samples as calibration set, four trials including 44 samples as validation set. Through the analysis of the distribution of the calibration set and validation set samples content as well as the principal component score plot, we found that the classification results are ideal and meet the requirements of the sample collection division. Through statistical analysis of calibration set samples, judged the samples with high leverage value and studentized residual value, and excluded abnormal samples. The spectrummeasurement process will inevitably introduce noise and generate baseline drift, so it is necessary for proper pretreatment of the spectrum before the establishment of the calibration model. Derivative methods can effectively eliminate the baseline drift and amplify the signal difference, Smooth combined with Derivative can effectively improve the signal to noise ratio, Autoscale can give the same right variable weight to spectrum at all wavelengths and highlight the differences between the sample spectra, so eventually we selected these 3 methods to pretreatment the spectrum. Then build the PLS calibration model, and the parameters of the model are as follows:Rc=0.9649, Rp=0.9229, RMSEC=0.0576, RMSEP=0.0493. We executed the predicting precision investigation of the model, and the relative standard deviation of the predicted value was about 5%, proving that the analysis method has a good reproducibility and can be used to predict unknown samples.2. Research of NIRS quantitative analysis for the FⅧ potency in the elution process of chromatography.This study established a quantitative model for the determination of FⅧ potency in the chromatography elute process. We got 68 samples from the elute process, which had a uniform potency distribution to meet the sample set requirement. Compared different PLS modeling results with kinds of methods, we knew that Meancenter and Genetic Algorithm were the best methods combination with the result as follows: R2=0.9010, RMSEP=0.4090, which has a high linear and a low prediction error. However, the BP-ANN modeling method with the transfer function sigmoid, has a better result than PLS and other methods, which has parameters as follows:R2=0.942, RMSEC=0.3754, RMSEP=0.4272. The FⅧ potency is the activity information of the substance, which is very complex and the concentration of the substance is not a linear relationship between the quantitative, so the nonlinear regression modeling results can be better. By using NIRS and this quantitative model, we can monitor the F Ⅷ potency specifically in the chromatographic elution process, and control the start and end points more accurately.3. Research of NIRS quantitative analysis for the product moisture in the FⅧ freeze-dried process.Utilized NIRS and Chemometrics, this study established a quantitative model for the determination of moisture in the product during the freeze-dried process. After investigated different pretreatment and spectral region selection methods, we eventually chose OSC combined with second derivative plus SG smooth as pretreatment methods and GA as spectrum region selection method. The model parameters are as follows:R2=0.918, RMSEC=0.2141, RMSECV=0.4102, RMSEP=0.3848. Through validation, the model linear, resolution, accuracy and precision are adhere to the requirements, which proved the practicality and effectiveness of the model. The establishment of the model is the foundation for on-line monitoring FⅧ moisture content changes in lyophilized preparation process, which can optimize drying time and avoid the activity loss caused by too long dying time. Multi-point monitoring using fiber can ensure that the moisture content of bulk products reach the standard.The innovation points are listed below:This research applied NIRS in the FⅧ production process for the first time, respectively established PLS quantitative model for the FⅧ specific activity analysis in the acid precipitation process, quantitative model for the FⅧ potency analysis in the chromatography elution process and PLS quantitative model for the moisture analysis in the freeze-dried process.