Phosphatase Activity in Chemical Cholera Vaccine and its Components

Presented are the data on detection and characterization of phosphomonoesterase and phosphodiesterase activities in detoxicated cultural fluid of production Vibrio cholerae strains 569B and M41, and in choleragen-anatoxin and O-antigenic fraction, the vaccine components. These enzymes were demonstrated to be present in the cholera vaccine tablet, thus its biochemical properties were characterized more completely

Antigenic Components of Chemical Bivalent Cholera Vaccine, Methods of their Isolation and Control

The paper presents a review of the data on the methods of isolation and control of Vibrio cholerae antigens – cholerogen-anatoxin and O-antigens of Inaba and Ogawa – components of the oral bivalent chemical cholera vaccine produced by the RusRAPI “Microbe”, the only prophylactic drug against cholera registered in the territory of the Russian Federation. Currently, the vaccine is produced using the method of segregated manufacturing of cholerogenanatoxin and O-antigens Inaba and Ogawa with step-by-step control of their main properties, which ensures the production of a high-quality finished product. Ultrafiltration is an effective method for concentrating a semi-finished product, which helps to reduce losses and increases the yield of the final product. It remains promising to develop a method for gentle steril ization of O-antigens to maximize the preservation of specific activity. To control the specific activity of the antigenic components and the finished vaccine preparation, a complex of in vivo and in vitro methods is applied. However, the multi-stage process and duration, the use of several types of laboratory animals, as well as modern WHO requirements determine the need for the introduction of alternative in vitro control methods. The use of cell cultures as a replacement for the biological method appears prospective, and demonstrates a positive correlation with animal tests. To assess the activity of antigens, the use of an immunochemical method – dot-immunoassay with gold nanoparticles – is put forward, which will make it possible to harmonize the control method at all stages of the production process, as well as to determine the serovar specificity of Vibrio cholerae O-antigens. The development of molecular-genetic, microbiological, immunochemical methods is relevant for a more complete and comprehensive control of the main immunogens of industrial strains of cholera vibrio. The introduction of promising methods for obtaining antigens and monitoring their properties will allow for a more complete characterization of the component composition of the finished dosage form of the chemical cholera vaccine

Experimental Substantiation of the Possibility to Use Finite Cell Line CHO-K1 for Determination of Specific Activity of Components of Chemical Cholera Vaccine

Objective was to experimentally substantiate the possibility to use the finite cell line CHO-K1 for measuring specific activity of cholera toxin and component of the vaccine choleragen-anatoxin in the process of chemical cholera vaccine manufacturing. Materials and methods. The studies involved the finite cell line CHO-K. The registration of results of bio-indication method was performed visually with the help of inverted microscope and photometrically - in colorimetric test for the assessment of metabolic activity of the cells at the wave length of 595 nm. Results and discussion. The proposed method allows for determining the toxin-production activity of Vibrio cholerae 569B strain during submerged cultivation in bioreactor and specific activity of choleragen-anatoxin by anatoxin binding measuring using cell cultures. The results correlate with the data obtained using intradermal Craig’s technique, GM1-ELISA and radial passive immune hemolysis (RPIH). Introduction of cell culture method into practice will provide for significant decrease in the volumes of usage of animals at the stages of manufacturing of chemical bivalent cholera vaccine

Enhancement of Manufacturing Technology for Finished Dosage Form of Bivalent Chemical Tableted Cholera Vaccine

Objective of the study is to experimentally substantiate the possibility to improve manufacturing efficiency by means of mass reduction of a vaccine tablet from 300 to 100 mg. Materials and methods. Inaba O-antigen lyophilizate serves as the specific immunogenic component of the vaccine. Results and conclusions. It is identified that it is expedient to produce tablets of 6 mm in diameter. Justified is the quantitative content of additive substances (lactose monohydrate, micro-crystal cellulose, and polyvinylpyrolidone). Moreover, the studies have specified target values for technological parameters of such processes as fluid bed granulation of the formula with overfeed of the binder, tablet compression and enteric-coating (Acryl-eze) application to finished dosage form. Using Inaba O-antigen lyophilizate manufactured has been model experimental series of the vaccine. Investigated have been its characteristics. Verified vaccine quality indicators testify to the compliance of the product with the requirements of manufacturer’s pharmacopoeial monograph. The studies exercised showed the possibility in principle to enhance manufacturing efficacy through the decrement of additives amounts, and thus the mass of a vaccine tablet from 300 up to 100 mg

Experimental Evaluation of Application of Cross-Flow Ultrafiltarion Method for O Antigen Concentrating in Cholera Chemical Bivalent Vaccine Production

Demonstrated is possibility to apply cross-flow ultrafiltration method for O antigen of Vibrio cholerae M-41 Ogawa concentrating from germ-free centrifugate. Technological process of concentrating was optimized. Worked out were the regimes of conservation and cleaning of the ultrafiltration device. The prospects of cross-flow ultrafiltration method introduction in technology of cholera chemical bivalent vaccine production were determined

Experimental Substantiation of Feasibility of Using Enzymatic Fibrin Hydrolyzate-Based Medium to Obtain Components of Chemical Cholera Vaccine

The aim of the study was to experimentally substantiate the possibility of using a nutrient medium based on enzymatic fibrin hydrolyzate in order to obtain specific components of chemical cholera vaccine: cholerogen-anatoxin and O-antigen. Materials and methods. We used production strains of Vibrio cholerae 569B and V. cholerae M-41. Submerged low-volume cultivation was carried out in a laboratory fermenter for 8 hours, with automatic maintenance of cultivation parameters and feeding with glucose on the nutrient medium based on enzymatic fibrin hydrolyzate, containing (1.0±0.1) g/l of amine nitrogen, pH being (8.0±0.1). Cholerogen-anatoxin and O-antigens were obtained from detoxified formalin-treated centrifugates of culture liquids. The specific activity of V. cholerae antigens at the stages of cultivation and isolation was determined applying immunochemical methods. The preparation of the finished dosage form of the cholera vaccine and the coating of the tablets with an enteric coating was carried out in accordance with the regulatory documentation. Results and discussion. It has been shown that cultivation on the medium based on enzymatic fibrin hydrolyzate provides a stable growth of the biomass of V. cholerae production strains with a high level of specific activity of antigens. Comparative analysis of the main properties of the finished dosage form of laboratory batches with a commercial batch of chemical cholera vaccine has demonstrated compliance with the requirements of regulatory documentation. The results obtained has led us to conclusion that it is feasible to use the nutrient medium based on enzymatic fibrin hydrolyzate for cultivating production strains and obtaining specific components of the cholera vaccine

Usage of nutrient Medium Based on Dry Hydrolysate of Casein in Manufacturing Bivalent Chemical Cholera Vaccine

Objective of the study was to select the standardized substrate containing dry hydrolysate of casein for preparation of nutrient medium utilized for manufacturing bivalent chemical cholera vaccine under submerged cultivation of cholera vibrio strains in fermenters. Materials and methods. We used Vibrio cholerae O1 strains of classical biovar: strain 569B Inaba and strain M-41 Ogawa. Examined were two dry substrates of the medium: enzymatic hydrolysate of casein, Type I Himedia (India) and pancreatic hydrolysate of casein, produced by the State Scientific Center of Applied Microbiology and Biotechnology (Russian Federation). Produced under laboratory conditions at the premises of the RusRAPI “Microbe” medium was used as a control. Submerged cultivation was conducted in bioreactors during (9±1) h with aeration and automatic feeding of glucose and ammonia. Production of protective antigens was measured applying immunochemical and biological methods. Results and discussion. It is demonstrated that submerged cultivation of cholera vibrio production strains on nutrient media under study provides for synthesis of protective antigens the parameters of which comply with the requirements of normative documentation. More standardized and higher indicator values of the target product are ensured by cultivation of producer strains on nutrient medium with a substrate from dry enzymatic hydrolysate of casein, containing (1.5±0.1) g/l of amino nitrogen for the strain V. cholerae M-41 and (2±0.1) g/l – for V. cholerae 569 B. Transition to the use of standardized dry protein components of cultivation media does not lower the quality of the chemical cholera vaccine, but allows for the reduction of cost price and duration of technological process

Assessment of Stability of Chemical Cholera Vaccine in a New Primary Packaging

The bivalent chemical cholera vaccine is the only drug for the prevention of cholera registered in the Russian Federation. The vaccine has been produced in glass bottles containing 210 tablets. At the same time, modern trends dictate the need to produce the drug in varying dispensing and more practical packaging for the convenience of the consumer.The aim of the work was to study the stability of the properties of the immunobiological medicinal product “Bivalent chemical cholera vaccine” with modified filling and in new primary packaging.Materials and methods. When studying the quality of bivalent chemical cholera vaccine batches, physicochemical parameters, formaldehyde content, specific activity and safety, abnormal toxicity, immunogenicity, and microbiological purity were assessed. Stability in terms of “specific activity” was evaluated using dot immunoassay.Results and discussion. As a result of this work, the use of several dispensing options and new primary packaging of cholera vaccine has been experimentally substantiated. The stability of the finished vaccine preparation has been established in the “accelerated aging” test and during long-term storage. The possibility of using dot immunoassay with a conjugate based on staphylococcal protein A, labeled with colloidal gold, to monitor the stability of cholera vaccine has been experimentally demonstrated

Ways to Reduce the Level of Contamination at the Stages of Tableted Chemical Cholera Vaccine Production

Objective of the study was an assessment of the degree of contamination of cholera chemical vaccine at the stages of preparation and determination of the ways to reduce it.Materials and methods. Liquid and lyophilized components of the cholera chemical vaccine used in the study: cholerogen-anatoxin and O-antigens of Vibrio cholerae 569B and V. cholerae M-41 strains, as well as auxiliary substances (sucrose, talc, calcium stearate, starch). Granulation was carried out in a device that works on a fluidized bed principle, GPCG 2 (GLATT, Germany). Subsequent tabletizing of the mixture was performed using MiniTabT compression machine (LUXNER, Germany). Studies were conducted on the evaluation of “microbiological purity” at the stages of manufacturing of the cholera chemical vaccine, tablets coated with an enteric coating. Positive or negative growth of microorganisms on Petri dishes with nutrient media was determined on visual inspection.Results and conclusions. The dynamics of changes in microbial contamination at certain technological stages of vaccine production has been revealed. It is shown that the solutions of antigens in the process of separation are subject to microbial contamination which is associated with the use of ammonium sulfate during precipitation and non-sterile water at the stage of dialysis. Sterility of semi-finished products has been achieved through twophase filtration of choleragen-anatoxin and sterilization of O-antigens of V. cholerae 569B and V. cholerae M-41 strains with flowing steam at (100±1) °C for 30 minutes. In order to decrease microbial contamination at the stage of granulation additional fine filters were installed in the air-supply system. Further on comparative assessment of microbial purity of vaccine batches obtained using both, direct compression and preliminary granulation, was carried out. It has been experimentally demonstrated that granulation of the components of a tablet mixture of cholera vaccine leads to a decrease in the level of bacterial contamination and improves the microbiological purity of the finished dosage form

Toxicity of Carbon Based Nanomaterials against Escherichia coli Depends on Dispersion Efficacy of Their Water Suspensions

The relationships between surface wettability, dispersion efficacy in water suspensions and biotoxicity of nine carbon_based nanomaterials (CBN) samples represented by nanotubes, nanofibres and fullerenes are established. It is shown that presence of polar groups on the surface of similar in structure CBNs increases their hydrophilicity, reduces the particles size in water suspensions, and manifests itself by growth of the toxicity level in the luminescent screening assay based on Escherichia coli with the cloned luxCDABEgenes Photobacterium leiognathi. The artificial increase in individual CBN dispersion efficacy by primary suspension in the aprotic solvent dimethyl sulfoxide with the following transfer into an aqueous environment, also has led to growth of the registered biological activity. At the same time, the dispersion efficacy of morphologically diverse CBN is not the main cause of distinctions in their biotoxicity