Development of the Experimental Technology for Protective Antigens of &lt;i&gt;Vibrio cholerae&lt;/i&gt; 569B Inaba Concentration by Means of Tangential Ultrafiltration

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.

Section: материалы и методыunclassified

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

Вольников

Durakova

Salikhov

et al. 2023

“…Был апробирован процесс обессоливания диафильтрацией в периодическом режиме. Техно логические режимы проведения процесса диафильтрации были выбраны исходя из результатов собственных исследований по концентрированию О-АГ и ХА методом тангенциальной ультрафильтрации [2,3]: давление на входе и выходе фильтрационной установки -(2,5±0,1) и (0,5±0,1) кгс/см 2 соответственно. Для обессоливания О-АГ были апробированы ультрафильтрационные модули с НОММ 300 кДа с площадью фильтрации 0,02 м 2 , ХА -30 кДа с площадью фильтрации 0,1 м 2 .…”

Section: результаты и обсуждениеunclassified

Hardware-Controlled Method of Desalting Antigen Components of Cholera Chemical Vaccine

Комиссаров¹,

Никифоров²,

Перепелица³

et al. 2014

Self Cite

Experimentally substantiated is the possibility to apply tangential ultrafiltration for desalting antigen components of the tableted bivalent chemical cholera vaccine. Specified are the technological parameters of the process. It is demonstrated that the properties of choleragen-anatoxin (produced by Vibrio cholerae strain 569B Inaba) and O-antigens (produced from V. cholerae 569B Inaba and M-41 Ogawa strains) obtained using the designed methodology are as efficient as the ones manufactured using certified procedure and satisfy regulatory requirements. Experimentally substantiated technology for the desalting of antigen components of chemical cholera vaccine provides for the reduction of the time elapsed up to 5-6 hours from the original 3 to 4 days. It also allows for the manufacturing under controlled conditions. This hardware controlled method of desalting has been implemented into the vaccine production practice.

“…совершенствование технологической стадии концентрирования микробной культуры при производстве вакцинных препаратов является одним из направлений развития биотехнологии и требует свое временного аппаратурного переоснащения, использования высокоэффективных технологических методов и современных конструкционных материалов [4,5].…”

unclassified

“…получение концентрата в рамках данной технологической стадии производства вакцины характеризуется многооперационностью и продолжительностью до 21 ч. следствием комбинированного способа является малый выход концентрированной суспензии (0,04 л с 1 л нативной культуры). современный уровень развития биотехнологического оборудования, в том числе для мембранного разделения биологических продуктов, позволяет обеспечить увеличение выхода продукта с единицы объема полуфабриката при минимизации энергетических и временных затрат [4,5].…”

unclassified

Improvement of Microbial Cell Concentration Technology in the Production of Live Plague Vaccine in the Form of Orodispersible Tablets

Шаров¹,

Лещенко²,

Багин³

et al. 2021

The aim of the study was to improve the procedure for the Yersinia pestis EV strain cell concentration using the system for tangential flow microfiltration with the ASF-020 filter support unit.Materials and methods. The study used the vaccine Y. pestis EV strain derived from NIIEG cell line. Submerged cultivation of the native culture was performed using BIOR-0.25 reactor with automated control system. Microbial suspension concentrate was produced through microfiltration applying (Adaptive filtration system) AFS-009 and AFS-020 installations. The content of live microbial cells was determined by cytorefractometry. Assessment of the resistance of Y. pestis EV strain cells to technological factors was performed by photometric registration of changes in the optical density of bacterial suspension during the lytic response of cells to sodium dodecyl sulfate. Physical-chemical and immunobiological properties of the dry live plague vaccine were determined in accordance with the pharmacopoeial item.3.3.1.0021.15 of the State Pharmacopoeia of the Russian Federation, 14th edition.Results and discussion. The design features of the equipment introduced made it possible to carry out membrane filtration of microbial suspension, using BIOR-0.25 reactor as an intermediate storage unit, thereby excluding three technological stages. The total concentration of microbes in the suspension obtained by routine and improved methods was more than 150 billion microbial cells per ml. A comparative study of the effect of various hydrodynamic regimes in the working cavities of AFS-009 and AFS-020 filter units did not significantly affect the morphometric properties and resistance of microbial cultures to extreme (technological) factors. Based on the experimental data, the mass balance of the membrane filtration process has been determined. The optimized technology gave 0.13 liter yield of concentrate from 1 liter of native culture, and the process duration was reduced to 5 hours, the yield of the finished product in one production cycle was increased by 3 times. Thus, the process of concentrating Y. pestis EV strain cells during the production of the tablet form of live plague vaccine has been enhanced. A comparative study of the morphometric properties and resistance of plague microbe cultures to technological factors in the process of their concentration using optimized technology did not reveal any significant differences as compared to the routine one. Technological stage of concentrating has been reduced to 5 h term with a three-fold increase in the yield of finished product.