Studies On The Application Of Near-infrared Spectroscopy In Heparin Sodium Production Process

Heparin is a linear polysaccharide with a repeating disaccharide unit of1,4-linked uronic acid (D-glucuronic (GlcA) or L-iduronic (IdoA) acid) and D-glucosamine (GlcN) residues. It is a kind of macromolecule glycosaminoglycans (GAGs) drugs which was first developed and used in clinical application. It will participate, affect and regulate metabolism and physiological function. It is mainly used as an anticoagulant in clinical, meanwhile it can regulate the blood fat and can be used as anti-inflammatory, antiallergic drugs and immunomodulator. Heparin, one of the most important biological raw material drug, takes up as high as87%of the world share of trade. The production process of heparin refers to the whole process from evaluation of heparin API to production storage. Oversulfated chondroitin sulfate (OSCS), a contaminant in heparin. can cause serious adverse reactions so thant it should be avoided doped. The biological activity of heparin is based on the integrated structure so that structure destruction should be avoided in the production. In addition, the protein combined with heparin is prone to allergic reactions and it must be removed as much as possible in the production process. As for one of the key unit operations affecting the quality and yield of heparin, the process of alcohol precipitation should be strictly controlled. Therefore, it is great important to find a precise method to ensure the quality of heparin. And a vaild process analytical technology can give a deep understanding the process to precise control of the production process. Near-infrared spectroscopy (NIRS) analysis technology used as a fast and effective method has been widely used in chemical industry for process monitoring, but there is yet no research paper about near-infrared spectroscopy reported in the application of heparin manufacture process.Under these consideration, this thesis aimed at reseaech on the discriminant of OSCS in heparin active pharmaceutical ingredient (API), ion exchange process of crude heparin, and the procrss of ethanol precipitation using near-infrared spectroscopy and chemometric. And the relationship between potency of heparin and near-infrared spectra of heparin was also studied. These studies will lay a theoretical foundation for application NIRS on the production process control of heparin.The results and conclusion of the research about heparin process based on NIRS are listed belows:1. Research on a rapid detection of OSCS in heparin API by NIRSIn this study, two pattern recognition methods, Partial Least Squares Beta Classification (PLSBC) and K-nearest Neighbor (KNN) were used to qualitative discriminant analysis of93heparin API samples (47heparin samples not containing OSCS,46heparin samples containing OSCS) based on near-infrared spectroscopy.34heparin samples not containing OSCS and34heparin samples containing OSCS were used to establish qualitative analysis model, and the model was validated using the remaining samples. The results of PLSBC indicate that NIRS can detect the OSCS effectively when the content greater than0.7%in heparin. Then a method KNN combined with PCA was investigated in detecting OSCS. The17validation samples were predicted correctly, the accuracy rate was100%. In order to further validate the relationship betwent the heparin samples not containing OSCS and heparin samples containing OSCS, we treated heparin samples not containing OSCS as A level, and divided heparin samples containing OSCS into five different content levels such as B level, C level, D level, E level and F level according to OSCS content. Then the result showed that there were two samples which were predicted wrongly in different OSCS content levels, the accuracy rate in OSCS was83.33%.2. Application of NIRS in monitoring of crude heparin purification processIon-exchange chromatography is a widely used purification technology in the heparin manufacturing process. To improve the efficiency and understand the process directly, a rapid and equally precise method needs to be developed to measure heparin concentration in chromatography process. Here, two robust partial least squares (PLS) models were established for quantification of heparin based on NIRS with80samples of adsorption process and76samples of elution process. Several variables selection algorithms, including correlation coefficient method, successive projection algorithm (SPA) and interval partial least squares (iPLS), were performed to remove non-informative variables. The results showed that the correlation coefficient of validation (Rp) and the residual predictive deviation (RPD) corresponded to0.957and3.4472for adsorption process,0.968and3.9849for elution process, respectively. The approach was found considerable potential for real-time monitoring the heparin concentration of chromatography process.3. Application of NIRS in determination of heparin content in ethanol precipitation processEthanol precipitation process is a key unit operation in the heparin production, and fluctuations of process parameters will lead to an unstable product quality and differences among batches. First, score control charts were established to identify the characteristic trajectory of all batches by Principal Component Analysis (PCA). And the all five batches had a similar trajectory. In addition, the plot showed that the first principal component reflected the content change trend of ethanol, while the second principal component reflected the stability of ethanol precipitation internal system. Then, a PLS model to predict the heparin content in the precipitation process. Five batches of heparin mother liquor were collected under the same ethanol precipitation conditions, and the transmission spectra were associated with the heparin content which was determined by sulfuric acid method. A PLS model was established to predict the content in mother liquor. And the R2was0.974, ang the values of RMSEP and RMSECV was1.1054g/1and1.4089g/1. Throughout the study, NIRS was proved to be an effective tool for determination of heparin content in ethanol precipitation process.4. Association studies on heparin potency and NIRSIn this study,47heparin samples with pontency among154IU/mg and162IU/mg were investigated to study the association between the potency and NIRS. The PLS model was established to predict the potency and meanwhile the variables effect was investigated to find the ralationship between the absorbance of heparin, potency and the structure of the heparin. Several variables selection algorithms, including correlation coefficient method, SPA and iPLS, were performed to remove non-informative variables. The results showed that the correlation coefficient method having the best model parameters, and the value of Rp was0.8868, while the values of RMSEP and RMSECV was0.7925IU/mg and1.2786IU/mg. A wide distribution of variables was selected by correlation coefficient method, which covered the first overtone, second overtone and some combination overtone of C-H. In addition, the strong absorption of1800-2100nm of was due to the combination of OH bond. And these selected had great correlation with the structure of heparin, so the variables selected by the correlation coefficient method was able to produce the priority prediction results.Major innovative achievements:1. Established a method to identify OSCS in heparin API using NIRS combined with PLSBC and KNN.2. Established a method to monitor the heparin content in the ion-exchange process of crude heparin using NIRS combined with multivariate analysis. 3. Established a method to qualitative and quantitative analysis of heparin content in ethanol precipitation process4. Studied the association between the heparin potency and NIRS, and optimized NIRS quantitative analysis model of heparin potency.5. Revealed the associated regularity between NIRS and heparin in the different systems, and studied the different characteristics among substances.