In response to the market demand for therapeutic antibodies, the upstream cell culture scale and expression titer of antibodies have been significantly improved, while the production efficiency of downstream purification process is relatively fall behind, and the downstream processing capacity has become a bottleneck limiting antibody production throughput. Using monoclonal antibody mab-X as experimental material, we optimized the caprylic acid (CA) precipitation process conditions of cell culture fluid and low pH virus inactivation pool, and studied two applications of using CA treatment to remove aggregates and to inactivate virus. Based on the lab scale study, we carried out a 500 L scale-up study, where CA was added to the low pH virus inactivation pool for precipitation, and the product quality and yield before and after precipitation were detected and compared. We found that CA precipitation significantly reduced HCP residuals and aggregates both before and after protein A affinity chromatography. In the aggregate spike study, CA precipitation removed about 15% of the aggregates. A virus reduction study showed complete clearance of a model retrovirus during CA precipitation of protein A purified antibody. In the scale-up study, the depth filtration harvesting, affinity chromatography, low pH virus inactivation, CA precipitation and depth filtration, and cation exchange chromatography successively carried out. The mixing time and stirring speed in the CA precipitation process significantly affected the CA precipitation effect. After CA precipitation, the HCP residue in the low pH virus inactivation solution decreased 895 times. After precipitation, the product purity and HCP residual meet the quality criteria of monoclonal antibodies. CA precipitation can reduce the chromatography step in the conventional purification process. In conclusion, CA precipitation in the downstream process can simplify the conventional purification process, fully meet the purification quality criterion of mab-X, and improve production efficiency and reduce production costs. The results of this study may promote the application of CA precipitation in the purification of monoclonal antibodies, and provide a reference for solving the bottleneck of the current purification process.
Cricetinae ; Animals ; Antibodies, Monoclonal/metabolism* ; Caprylates/chemistry* ; Cell Culture Techniques ; Chromatography, Affinity ; CHO Cells ; Cricetulus ; Chemical Precipitation

Microbial induced calcium carbonate precipitation (MICP) refers to the natural biological process of calcium carbonate precipitation induced by microbial metabolism in its surrounding environment. Based on the principles of MICP, microbial cement has been developed and has received widespread attention in the field of biology, civil engineering, and environment owing to the merits of environmental friendliness and economic competence. Urease and carbonic anhydrase are the key enzymes closely related to microbial cement. This review summarizes the genes, protein structures, regulatory mechanisms, engineering strains and mutual synergistic relationship of these two enzymes. The application of bioinformatics and synthetic biology is expected to develop biocement with a wide range of environmental adaptability and high performance, and will bring the MICP research to a new height.
Calcium Carbonate/metabolism* ; Chemical Precipitation ; Urease/metabolism*

Taking Pudilan Xiaoyan Oral Liquid as a demonstration, the effective delivery of quality markers in alcohol precipitation of Chinese medicine oral liquid preparations was studied. With the transfer rates of adenosine, corynoline, cichoric acid, baicalin, and wogonin as evaluation indexes, the effect of the density of concentrate before alcoholic precipitation, volume fraction of ethanol, stirring speed, temperature of concentrated solution, stirring time, alcohol concentration, alcohol precipitation time, alcoholic precipitation temperature, alcohol addition rate, and the pH of concentrate on the alcohol precipitation process was investigated by Plackett-Burman trial design, thus obtaining the key factors that influenced the alcohol precipitation process. The key factors were further optimized by Box-Behnken design to determine the optimal alcohol precipitation conditions. When the density of concentrate before alcoholic precipitation was 1.12 g·mL~(-1), the pH of concentrate was 6.86, and the alcohol concentration was 50.00%, the transfer rates of baicalin and wogonin were 91.86% and 87.78%, respectively. When the density of concentrate before alcoholic precipitation was 1.13 g·mL~(-1), the concentration of alcohol was 74.50%, and the alcoholic precipitation temperature was 17.0 ℃, the transfer rates of adenosine, corynoline, and cichoric acid were 85.95%, 71.62% and 83.19%, respectively. The method of optimizing alcohol precipitation techniques and determining the parameters of Pudilan Xiaoyan Oral Liquid by response surface methodology is reasonable and feasible, which provides guidance and experience for the effective delivery of quality markers in Chinese medicine oral liquid preparations.
Drugs, Chinese Herbal ; Ethanol ; Adenosine ; Chemical Precipitation

The present study was designed to determine the relationships between the performance of ethanol precipitation and seven process parameters in the ethanol precipitation process of Re Du Ning Injections, including concentrate density, concentrate temperature, ethanol content, flow rate and stir rate in the addition of ethanol, precipitation time, and precipitation temperature. Under the experimental and simulated production conditions, a series of precipitated resultants were prepared by changing these variables one by one, and then examined by HPLC fingerprint analyses. Different from the traditional evaluation model based on single or a few constituents, the fingerprint data of every parameter fluctuation test was processed with Principal Component Analysis (PCA) to comprehensively assess the performance of ethanol precipitation. Our results showed that concentrate density, ethanol content, and precipitation time were the most important parameters that influence the recovery of active compounds in precipitation resultants. The present study would provide some reference for pharmaceutical scientists engaged in research on pharmaceutical process optimization and help pharmaceutical enterprises adapt a scientific and reasonable cost-effective approach to ensure the batch-to-batch quality consistency of the final products.
Chemical Precipitation ; Chromatography, High Pressure Liquid ; methods ; Drugs, Chinese Herbal ; analysis ; chemistry ; Ethanol ; Injections ; Principal Component Analysis

Design space approach is applied in this study to enhance the robustness of first ethanol precipitation process of Codonopsis Radix (Dangshen) by optimizing parameters. Total flavonoid recovery, dry matter removal, and pigment removal were defined as the process critical quality attributes (CQAs). Plackett-Burman designed experiments were carried out to find the critical process parameters (CPPs). Dry matter content of concentrated extract (DMCE), mass ratio of ethanol to concentrated extract (E/C ratio) and concentration of ethanol (CEA) were identified as the CPPs. Box-Behnken designed experiments were performed to establish the quantitative models between CPPs and CQAs. Probability based design space was obtained and verified using Monte-Carlo simulation method. According to the verification results, the robustness of first ethanol precipitation process of Dangshen can be guaranteed by operating within the design space parameters. Recommended normal operation space are as follows: dry matter content of concentrated extract of 45.0% - 48.0%, E/C ratio of 2.48-2.80 g x g(-1), and the concentration of ethanol of 92.0% - 92.7%.
Chemical Precipitation ; Chemistry, Pharmaceutical ; methods ; Codonopsis ; chemistry ; Drugs, Chinese Herbal ; chemistry ; isolation & purification

The theoretical basis of the alcohol precipitation process control was provided, the alcohol precipitation was optimized and the relationship equation was got. The monod glycoside, loganin and paeoniflorin were used as the evaluation indexes to determine the impact factors of alcohol precipitation techniques of Liuwei Dihuang decoction by the Plackett-Burman experimental design and the levels of non-significant factors were identified. Then, Box-Behnken response surface methodology was used to research and discuss the critical process parameters influence the effect of alcohol precipitation and draw interaction between key process parameters and the correlation equation with index components. Through the establishment and solving the quadratic regression model of composite score, the optimum preparation conditions of alcohol precipitation techniques of liuwei were as follows: stirring speed was 580 r x min(-1), standing time was 17 hours, alcohol concentration was 34%, the density of Liuwei Dihuang decoction was 1.13. The response surface methodology for optimized alcohol precipitation techniques of Liuwei Dihuang decoction is. reasonable and feasible.
Chemical Precipitation ; Chemistry, Pharmaceutical ; methods ; Drugs, Chinese Herbal ; chemistry ; Ethanol ; chemistry

Astragalus polysaccharide has been widely used in food and medicinal industry owing to its health-promoting properties. In order to characterize better the relationship among molecular weight, structure-activity and activities, a simple method was used different concentration of ethanol including 30% (PW30), 50% (PW50), 70% (PW70), 75% (PW75), 80% (PW80) and 90% (PW90) to precipitate Astragalus polysaccharides into different molecular weight. As a result, PW90 showed smooth surface and the strongest antioxidant activity among these six fractions (P < 0.05). In conclusion, graded ethanol precipitation was a simple method to separate Astragalus polysaccharides into different molecular weight with different antioxidant activity fractions.
Antioxidants ; pharmacology ; Astragalus Plant ; chemistry ; Chemical Precipitation ; Ethanol ; chemistry ; Polysaccharides ; chemistry ; pharmacology

Near infrared (NIR) spectroscopy as a kind of rapid process analysis technology has been successfully applied in Chinese medicine pharmaceutical process. In this research, the technology was adopted to establish the rapid quantitative analysis models of main indicators from the Lonicera japonica and Artemisia annua alcohol precipitation process of Reduning injection. On-line NIR spectra of 142 samples from alcohol precipitation process were collected and the content of main indicators for each sample were detected through off-line HPLC. With eliminating outliers, determination of spectra pretreatment method and selecting optimal band, the NIR quantitative calibration model for each indicator was established using partial least squares (PLS). These models were used to predict the unknown samples from precipitation process of Reduning injection to achieve the goal of rapid detection. The results showed that the models were ideal. The correlation coefficients of models for neochlorogenic acid, chlorogenic acid, 4-O-caffeoylquinic acid and secoxyloganin were 0.973 872, 0.985 449, 0.975 509 and 0.979 790, respectively and their relative standard errors of prediction (RSEP) were 2.922 49%, 2.341 37%, 2.930 40% and 2.184 60%, respectively. This study indicated that the NIR quantitative calibration model showed good stability and precision, and it can be used in rapid quantitative detection of main indicators of efficacy in order to on-line monitor the alcohol precipitation process of Reduning injection.
Chemical Precipitation ; Chromatography, High Pressure Liquid ; Drugs, Chinese Herbal ; chemistry ; Ethanol ; chemistry ; Lonicera ; chemistry ; Quality Control ; Spectroscopy, Near-Infrared ; methods

A set of central composite design experiments were designed by using four factors which were ethanol amount, ethanol concentration, refrigeration temperature and refrigeration time. The relation between these factors with the target variables of the retention rate of schizandrol A, the soluble solids content, the removal rate of fructose and the removal rate of glucose were analyzed with Bayesian networks, and ethanol amount and ethanol concentration were found as the critical process parameters. Then a network model was built with 2 inputs and 4 outputs using back propagation artificial neural networks which was optimized by genetic algorithms. The R2 and MSE from the training set were 0.983 8 and 0.001 1. The R2 and MSE from the test set were 0.975 9 and 0.001 8. The results showed that network analysis method could be used for modeling of Schisandrae Chinensis Fructus ethanol precipitation process and identify critical operating parameters.
Bayes Theorem ; Chemical Precipitation ; Cold Temperature ; Cyclooctanes ; chemistry ; Ethanol ; chemistry ; Fructose ; analysis ; Fruit ; chemistry ; Glucose ; analysis ; Lignans ; chemistry ; Neural Networks (Computer) ; Polycyclic Compounds ; chemistry ; Reproducibility of Results ; Schisandra ; chemistry ; Time Factors

The purpose of this research was to prepare total salvianolic acids-phytosome-HA coprecipitate to improve drug dissolution and its micromeritic properties. Firstly, the coprecipitate was prepared by solvent method and in vitro dissolution of tripterine was performed with the salvianolic acid B and danshensu as criteria. At the same time, the micromeritic properties was characterizated, the structure of samples was characterized by TEM, DSC, XRD and FTIR. Results showed that when the ratio of drug to HA was 1:2, it had a better dissolution, the accumulative drug-release percent in vitro at 60 min was over 90%. At the same time, it has good liquidity and low moisture absorption. Its micromeritic properties have improved. It is proved that the drug still existed amorphously by microstructure analysis. The preparation process is simple and feasible, it has practical value.
Alkenes ; chemistry ; Chemical Precipitation ; Chemistry, Pharmaceutical ; methods ; Durapatite ; chemistry ; Phospholipids ; chemistry ; Polyphenols ; chemistry ; Time Factors