Potential effects of alginate-pectin biocomposite on the release of folic acid and their physicochemical characteristics.

Potential effects of folates on the treatment of several human diseases like cognitive function, neural tube defects, coronary heart disease and certain kinds of cancers have been discovered. However, the stability of folic acid against adverse conditions is a great concern. The present study investigates various alginate (A)-pectin (P) gastrointestinal-resistant hydrogel to immobilize folic acid. This involves evaluating different compositions of alginate-pectin to achieve higher encapsulation efficiency and stability during simulated gastric (SG) and simulated intestinal (SI) conditions. Coated alginate hydrogels with pectin resulted significant (p < 0.05) better protection of folic acid compared to non-coated alginate hydrogel when exposed to SG condition and when exposed to SI condition, sustained release behavior obtained with the ratio of A70-P30. The structural and physicochemical properties of blended A-P hydrogel were characterized using scanning electron microscopy, Fourier transform infrared spectroscopy and X-ray diffractometer, indicating the presence of folic acid into the matrix and signified no covalent reaction between components. Therefore, this adequate composition of alginate-pectin showed to be a potential carrier for folic acid stability.

Application of recurrent neural network for online prediction of cell density of recombinant Pichia pastoris producing HBsAg.

Artificial neural networking (ANN) seems to be a promising soft sensor for implementing current approaches of quality by design (QbD) and process analytical technologies (PAT) in the biopharmaceutical industry. In this study, we aimed to implement best-fitted ANN architecture for online prediction of the biomass amount of recombinant Pichia pastoris (P. pastoris) - expressing intracellular hepatitis B surface antigen (HBsAg) - during the fed-batch fermentation process using methanol as a sole carbon source. For this purpose, at the induction phase of methanol fed-batch fermentation, carbon evolution rate (CER), dissolved oxygen (DO), and methanol feed rate were selected as input vectors and total wet cell weight (WCW) was considered as output vector for the ANN. The obtained results indicated that after training recurrent ANN with data sets of four fed-batch runs, this toolbox could predict the WCW of the next fed-batch fermentation process at each specified time point with high accuracy. The R-squared and root-mean-square error between actual and predicted values were found to be 0.9985 and 13.73, respectively. This verified toolbox could have major importance in the biopharmaceutical industry since recombinant P. pastoris is widely used for the large-scale production of HBsAg.

Exploration of Recombinant Streptokinase Degradation Products Under Various pH Reduction Conditions in Downstream Processing.

BACKGROUND: Methods of producing streptokinase, which can be used in the treatment of myocardial infarction, by hemolytic streptococci and recombinant E. coli have been described in patents since 1955. Degradation products in active pharmaceutical ingredients (APIs) and finished pharmaceutical products are considered as impurities and it is required that these degradation impurities are minimized or rather avoided throughout manufacturing process. OBJECTIVE: The aim of this study was to explore the occurrence of rSK degradation during acidification step in downstream processing. METHODS: The polyclonal antibody was produced by immunization of New Zealand white (NZW) rabbit with pure rSK (purity>98%). The solubilized inclusion bodies with various pH values (4.2, 5.0 and 6.0) were analyzed by Western blotting using rSK polyclonal antibody. RESULTS: Western blot analysis demonstrated the generation of rSK degradation products (with the molecular weight of about 27, 20 and 17 kDa) when the pH value of the solubilized inclusion bodies was reduced to 5.0 and 4.2, while no degradation of rSK observed at pH 6.0. CONCLUSION: This study demonstrates that the level of pH reduction in the solubilized inclusion bodies during downstream processing plays an important role in generating rSK degradation products, and substantial post-solubilization degradation of rSK occurs at pH lower than 6.0. Development of these degradation impurities, which cannot be eliminated by subsequent chromatographic purifications, can be exclusively avoided during acidification procedure by appropriate pH adjustment approach in downstream processing.

Effect of post-solubilization conditions on the yield and efficiency of recombinant streptokinase purification at large-scale.

Streptokinase, a plasminogen activator which converts plasminogen to plasmin and consequently promotes fibrinolysis, is the leading drug for treating acute myocardial infarction in developing countries and its production is industrially demanded. In this work, the substantial influence of inclusion body (IB) post-solubilization condition on the performance of a sequential chromatography method for large-scale purification of recombinant streptokinase was demonstrated. In the preliminary experiments, various post-solubilization pH conditions were studied, and it was shown that the pH value of solubilized inclusion bodies (i.e., in 4M urea) had a marked impact on the purity of streptokinase obtained at the end of post-solubilization process. When the pH value of the solution containing solubilized IBs was decreased from 7.5 to 6.5 and 6.0, the greatest increases (10% and 27%, respectively) in streptokinase purity occurred. The influence of different post-solubilization pH conditions on the efficiency and yield of large-scale chromatographic purification methods was next investigated. When the solubilized IBs solution with pH adjusted to 6.0 was utilized for subsequent sequential chromatography process, the complete elution peak with high overall yield (91.3%) and purity (98%) was achieved. In comparison to this, while the sequential chromatography procedure was instigated by using the solubilized IBs solution with pH 4.2, four elution fractions (EF1 to EF4) with disparate target protein purities (i.e., 57%, 77.3%, 91.4% and 86.7%, respectively) were attained, the process was incompletely effective, and the highest recovery and purity figures (81.8% and 91.4%, respectively, belonging to EF3) were much lower than those for the earlier process.