RU2307166C2 - Method for proximate determination of non-sterility of broth for microorganism, animal cell and virus cultivation in bioreactors - Google Patents

Abstract

FIELD: biotechnology, in particular biopreparation production.

SUBSTANCE: claimed method includes feeding of sterilized broth into presterilized inoculator or bioreactor equipped with means for redox-potential (eH) controlling, including eH electrode and microprocessor unit for controlling and adjustment of eH and pH measurement of redox potential value for 1 h under stirring and comparison of steady-state eH values with steady-state values. When redox-potential value deviates from steady-state value of broth redox-potential by 10 % said broth is recognized as non-sterile one.

EFFECT: process of decreased cost.

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Description

The invention relates to biotechnology and can be used in scientific research and in the production of biological products (vaccines, diagnosticums and other biologically active substances) by the method of deep cultivation.

Known microbiological methods for determining the sterility of culture media for the cultivation of microorganisms, animal cells and viruses by seeding samples on meat-peptone broth (MPB), meat-peptone agar (MPA) or Saburo agar, then leave a few control bottles with the medium at first at 37 ° C for 3-4 days, and then at room temperature for 10-12 days (Handbook "Veterinary preparations" under the editorship of DF Ozidze. - M: Kolos, 1981 - S. 47.) - prototype.

However, this method is long and does not allow promptly (1 hour) to give a qualitative assessment of the sterility of the nutrient medium used in the preparation of seed (inoculation) and for cultivation in bioreactors.

It is known that the metabolism reactions of microorganisms and animal cells are redox reactions that are characterized by electron transfer from the reducing agent to the oxidizing agent. Each nutrient medium for the deep cultivation of microorganisms and animal cells, depending on its composition, has a certain initial constant value of the redox potential (eH) in the range from -1200 to +1200 mV, which can be measured using a redox electrode. The electrode in its design is similar to an electrode for measuring the concentration of hydrogen ions (pH), except that its sensitive element is made of metal (platinum, gold or silver) or glass with electronic conductivity. The reference electrode, as in the measurement of pH, is a silver chloride electrode.

The aim of the invention is to develop a high-quality method for the rapid determination of medium sterility for deep cultivation of microorganisms, animal cells and viruses in the manufacture or transfer of culture media to the inoculator and bioreactor for 1 hour before inoculation.

This goal is achieved by the fact that the determination of the non-sterility of the nutrient medium is carried out by changing the value of the redox potential (eN) from the constant value of ЕН established for 1 hour for the used nutrient medium before the inoculation and cultivation of microorganisms, animal cells and viruses, which leads to a decrease the cost of production of biological products due to the exclusion of non-sterile operations of deep cultivation processes.

The method is as follows. Inoculators and bioreactors for the deep cultivation of microorganisms, animal cells and viruses should be equipped with technical means for monitoring the redox potential (eH), including an eH electrode and a microprocessor-based analytical unit for controlling and regulating pH and eH. The sterilized growth medium (thermal or filter sterilization) is transferred to a previously sterilized inoculator or bioreactor and the eH value of this medium (in mv) is measured with stirring.

If within 1 hour there is no change in the eH value from the steady-state value, then it is possible to say with probability P = 0.9 that the nutrient medium is sterile, and a deviation of the eH value from the steady-state value within more than 10% indicates contamination of the nutrient medium by extraneous microflora, which is subject to re-sterilization, in order to avoid non-sterility in the preparation of the inoculum and cultivation.

As a control, take at least 8 vials or tubes (statistically significant sample) with sterilized medium from an inoculator and a bioreactor, which are seeded on meat-peptone agar and left for 3-4 days in an incubator at 37 ± 0.5 ° C and then carried out visual or microscopic analysis of the growth of a contaminating culture, which should confirm the express analysis of the presence of non-sterility by changing eH from the initial initial value for a given culture medium

Example 1. For the cultivation of the vaccine strain Brucella abortus 19 in bioreactors in the production of anti-brucellosis vaccine, a nutrient medium based on the Hottinger pass is used.

The finished nutrient medium is sterilized in a bioreactor at a temperature of 127 ± 1 ° C and a pressure of 0.08-0.09 MPa for 40 min and then cooled to a cultivation temperature (37 ± 0.5 ° C). For biological control, samples of the finished nutrient medium are plated on MPA or MPB and incubated at 37-38 ° C for 3-4 days.

The steady state values of the redox potential of this nutrient medium should be in the range from +300 to +400 mV with an accuracy of ± 10%. In the presence of contamination by extraneous microflora within 1 hour, the deviation from the steady-state value is at least ± 50 + 60 mV. In control samples in the case of non-sterility by deviation of the eH value, cultures of Escerichia coli and Pseudomonas aeuruginosa were detected and microscopically infected in an amount of 50 / CFU per 1 ml. The results of determining the non-sterility of culture media for the cultivation of Brucella abortus 19 in the production of cattle brucellosis vaccine are shown in table 1.

Table 1 The results of the rapid determination of the sterility of the nutrient medium for the deep cultivation of Brucella abortus 19 pcs in the bioreactor No. n / n Type and capacity of bioreactor Culture medium Culture Sterilization The initial value of the eH medium at 37 ° C (mv) EN value after 1 hour (mv) The presence of extraneous microflora in the control after 4 days one Bior-0.1 (100 L), Kirishi, Russian Federation Based on Hottinger Pass with additives In the bioreactor at (127 ± 1 ° С) for 10 min + 350 ± 10% + 280 ± 10% Escerichia coliu Pseudomonas aeuruginosa 50 CFU per 1 ml 2 Bioflo-6000 (180 L) "NBS" USA "-" In a bioreactor at 127 ± 1 ° С for 40 min + 400 ± 10% + 396 ± 10% Absent

Table 1 shows that insufficient sterilization of the nutrient medium in the BIOR-01 bioreactor led to the emergence of non-sterility, as evidenced by a change in the initial value of the pH of the nutrient medium in the bioreactor without aeration with stirring for one hour from +350 to +280 mV. This was confirmed by 8 control samples on day 4 (culture medium contamination with Escerichia coli and Pseudomonas aeuruginosa cultures). Sterility in the Bioflo-6000 bioreactor was confirmed by the fact that the value of eH of the nutrient medium did not change for an hour within the accuracy of measuring eH (± 10%). The same was confirmed by control seeding on MPA or MPB.

Example 2. A serious problem is the purification of the air supplied to the bioreactors for aeration, from suspended aerosol solid and liquid particles and, especially, from microorganisms. In 1 m ^{3 of} air contains up to 10 ⁴ microorganisms. From 1 to 10 volumes of air per volume of medium per hour (for animal cell cultures during surface aeration) up to 120 rpm · h ^-1 (for microorganisms during bubble aeration in stirrer bioreactors) are supplied to bioreactors for aeration of the culture medium. The cultivation cycle lasts from 4 to 200 hours (bacteria and fungi) and up to 360 hours (cells of animals and higher plants). The air entering the bioreactor and inoculator must be sterile. The required degree of purification of the air entering the aeration after the fine filter (individual filter) should be 99.9999999%. The results of determining the sterility of culture media for the cultivation of Pasteurella multocida pcs. A-1231 in the manufacture of a vaccine against pig pasteurellosis, with insufficient cleaning and sterilization of the air supplied for aeration in the bioreactor, are shown in table 2.

Nutrient medium was prepared on the basis of meat hydrolyzate. The resulting nutrient medium was sterilized in a bioreactor at a temperature of 127 ± 1 ° C and a pressure of 0.08-0.09 MPa for 40 minutes. Table 2 shows that insufficient purification of the air entering the aeration into the AK-210 bioreactor, caused by the absence of an individual air filter, when exposed to the nutrient medium for 30 minutes, leads to its contamination by extraneous microflora, as evidenced by a change in eH +240 to + 160 ± 10% mv for an hour with vigorous stirring, which was confirmed by control samples on the 4th day of exposure.

table 2 The results of rapid determination of the sterility of the nutrient medium for deep cultivation of Pasteurella multocida pcs. A-1231 No. n / n Type and capacity of bioreactor Culture medium Culture Sterilization Air sterilization for aeration Initial eN value of the medium at 37 ° С (mv) EH value after 1 hour The presence of extraneous microflora in the control after 4 days one Bior-0.2 (250 L), Kirishi of the Russian Federation Based on meat hydrolyzate with additives In the bioreactor at 127 ° C for 40 min Individual membrane titanium filter + 200 ± 10% + 195 ± 10% Absent 2 AK-210 (10 l) Institute of Biological Instrument Engineering, Pushchino, Oka "-" In a bioreactor and in an autoclave at 127 ° С for 40 min Nutrient aeration for 30 min with individual filter removed + 240 ± 10% + 160 ± 10% Eserichia coli you. megaterium you. stearother mophilis. 70 CFU in ml

Example 3. For deep cultivation in suspension of transplantable kidney cells of a Syrian hamster (BHK-21) in the production of FMD vaccine, a standard synthetic minimal Eagle medium was used. The membrane sterilizing filtration of the synthetic nutrient medium when fed into the bioreactor was carried out at the UPB filtration unit using Millipor or Vladipor membranes with pore sizes from 0.2 to 0.3 μm. The results of determining the sterility of the synthetic nutrient medium for the cultivation of animal cells are shown in table 3.

From table 3 it follows that the puncture or rupture of the sterilizing and filtering membranes leads to contamination of the nutrient medium, as evidenced by control microbiological samples.

Table 3 The results of rapid determination of the sterility of the synthetic medium for the cultivation of BHK-21 cells in the production of FMD vaccine No. n / n Type and capacity of bioreactor Culture medium Culture Sterilization The initial value of the eH medium at 37 ° C (mv) EH value after 1 hour The presence of extraneous microflora in the control one Celly-Gen (5 L) "NBS" USA Minimal needle environment Sterilizing filtration in a filtration unit with membrane filters "Millipor" or "Vladipor" -50 ± 10% -49 ± 10% Absent 2 BB (3.5 L) "Belico Biotechnology" USA "-" Membrane puncture -50 ± 10% + 15 ± 10% Eserichia coli you. subtilis 50 CFU per ml

Example 4. For deep cultivation of primary cells of quail embryos on microcarriers "Cytodex-3" (pseudosuspension) in the production of virus vaccines against Marek's disease, synthetic medium 199 was used, which was sterilized through Millipor membrane filters, and the microcarriers were sterilized in an autoclave with a bioreactor, filled with distilled water. In the case when the microcarriers were only rinsed with distilled water, but not sterilized and placed in a nutrient medium, a change in eH value was observed for 1 hour from +135 to + 160 ± 10% mV, which indicates a contamination of the nutrient medium. The same was confirmed in microbiological control (see table 4).

Table 4 The results of rapid determination of the sterility of a synthetic nutrient medium for the cultivation of primary cells of quail embryos on microcarriers in the production of virus vaccines against Marek’s disease of chickens No. n / n Type and capacity of bioreactor Nutrient medium Micro media Culture Sterilization Air sterilization for aeration The initial value of the eH medium at 37 ° C (mv) EH value after 1 hour The presence of extraneous microflora in the control after 4 days one Bioflo 111 (3.3 L) "NBS" USA Wednesday 199 Cytodex-3 Sterilizing Filtration Using Millipor Membranes Together with a bioreactor filled with distilled water in an autoclave at Т = 127 ° С ± 1 for 30 min + 120 ± 10% + 125 ± 10% Absent 2 "-" "-" "-" "-" Microcarriers were not sterilized + 135 ± 10% + 160 ± 10% Eserich ia coli you. megaterium 150 CFU per ml

Thus, by controlling the change in the initial value of the redox potential, the possibility of express determination of the sterility of nutrient media used for the cultivation of microorganisms, animal cells, and viruses in all cases of their possible contamination by extraneous microflora was confirmed.

Claims (1)

A method for expressly determining the sterility of nutrient media for the deep cultivation of microorganisms, animal cells and viruses in bioreactors, comprising supplying a sterilized nutrient medium to a pre-sterilized inoculator or bioreactor equipped with technical means for monitoring the redox potential (eH), including an eH electrode and a microprocessor analytical unit control and regulation of eH and pH, measuring the values of the redox potential of pit medium with stirring for 1 hour, and a comparison of the values of eH with the level of steady-state values, and the conclusion about the non-sterility of the nutrient medium is made when the value of the redox potential deviates from the steady-state value of the redox potential of the nutrient medium by more than 10%.