Optimisation of the Microbial Cell Concentration Procedure in Plague Vaccine Production

To date, the technology of live plague vaccine production uses a combined method of concentrating microbial cells which consists of three operations with a total duration of 18 hours. The procedure of obtaining concentrate, which is used in the current vaccine production technology, has a number of disadvantages, namely: multiple operations, high energy consumption, long duration, and, as a consequence, low yield of concentrated suspension (0.04 l from 1 l of native culture). The aim of the study was to optimise the procedure of Yersinia pestis ЕV microbial cell concentration using the system for tangential flow microfiltration with the ASF-020 filter support unit. Materials and methods: the vaccine strain used in the study was Yersinia pestis ЕV derived from NIIEG cell line (the strain of the Research Institute of Epidemiology and Hygiene). Submerged cultivation of the native culture was performed using the BIOR-0.25 reactor. The content of live microbial cells was determined by cytorefractometry. Oxidative metabolism was assessed using the chronoamperometric method. Physico-chemical and immunobiological properties of the dry live plague vaccine were assessed according to the monograph FS.3.3.1.0022.15 of the State Pharmacopoeia of the Russian Federation, 14 edition. Results: the equipment’s design features made it possible to carry out membrane filtration of the microbial suspension using the BIOR-0.25 reactor as an intermediate storage unit, thereby excluding three technological stages. The total concentration of microbes in the suspension obtained by the routine and the optimised methods was not less than 120 billion microbial cells/ml. A comparative study of the effect of various hydrodynamic regimes in the working cavities of ASF-009 and ASF020 filter units did not significantly affect the morphometric and physiological properties of microbial cultures. Experimental data helped to determine the process mass balance of membrane filtration. The optimised technology gave 0.17 l yield of the concentrate from 1 l of native culture, and the process duration was reduced to 4 hours. Conclusions: the process of concentrating Y. pestis EV microbial cells during production of plague vaccines was optimised. A comparative study of morphometric and physiological properties of plague microbe cultures that was carried out during their concentration using the optimised technology did not reveal any significant differences as compared to the routine one.

1. Mikshis NI, Kudryavtseva OM, Kutyrev VV. Contemporary tendencies in constructing recombinant vaccines for specific prophylaxis of plague. Zhurnal mikrobiologii, ehpidemiologii i immunologii = Journal of Microbiology, Epidemiology and Immunology. 2015;(3):116–26 (In Russ.)

2. Biryukov VV. Fundamentals of Industrial Biotechnology. M.: KolosS Khimiya; 2004 (In Russ.)

3. Ezhov AV, Sadovoy IN, Biryukov VV, Mokhov DA, Bagin SV, Chernyadyev AV, et al. The possibility of using the microfiltration method in the plague live dry vaccine technology. Proceedings of the AllRussian scientific conference devoted to the 80th anniversary of the founding of the Federal State Research Institute of the 48th Central Research Institute of the Ministry of Defense of Russia. Kirov: FSI «48 Central Research Institute of the Ministry of Defense of Russia», 2008. Issue 1. P. 313–6 (In Russ.)

4. Teterin VV, Ezhov AV, Biryukov VV, Mokhov DA, Bagin SV, Khonin AZ, Logviov SV. Method of obtaining preparation based on vaccine strain of plague microbe. Patent of the Russian Federation No. 2510825; 2014 (In Russ.)

5. Zagoskina NV, Nazarenko LV, Kalashnikova EA, Zhivukhina EA. Biotechnology: theory and practice. Moscow: Onyx; 2009 (In Russ.)

6. Martynov NV, Chebotarev YeV, Lebedinskiy VA, Chicherin YuV, Dodonov NP, Nesterenko AA. Polarographic method in assessing the cultural properties of the vaccine strain EB. Zhurnal mikrobiologii, ehpidemiologii i immunologii = Journal of Microbiology, Epidemiology and Immunology. 1990;(12):18–21 (In Russ.)

7. Krasnyuk II, Mikhaylova GV., Practice on the technology of medicinal forms. M.: Academy; 2007 (In Russ.)

8. Leshchenko AA, Teterin VV, Lazykin AG, Ezhov AV, Mokhov DA, Biryukov VV, et al. Experimental justification of the possibility of obtaining microbial Yersinia pestis EV strain concentrate by microfiltration. BIOpreparaty. Profilaktika, diagnostika, lecheniye = BIOpreparations. Prevention, Diagnosis, Treatment. 2014;(1):31–5 (In Russ.)