Determination of indicators for tests of polysept (polyhexamethylene guanidine hydrochloride) for flocculation properties

In vaccine production, it is particularly important to purify the virus-containing suspension in order to remove ballast proteins and fats, which, when present in high concentrations, are responsible for depression or allergic reactions in animals. Polyguanidine and its derivatives have long been used for such purposes. At present, the market offers polyhexamethylene guanidine hydrochloride, a cationic polyelectrolyte with aunique combination ofphysico-chemical and biocidal properties allowing for it to be used in nearly all spheres of economy. Flocculation properties of polysept (polyhexamethylene guanidine hydrochloride) vary from batch to batch, and this has necessitated the development of a test system for determination of the incoming material quality, which has a significant impacton virus antigen concentration during vaccine production. Seven batches of polyhexamethylene guanidine were tested for flocculation properties, changes in FMDV immunogenic component concentration in the virus-containing suspension, osmolality of solutions at different percentage concentrations. Indicators of incoming material suitability for FMD vaccine production were determined. The batches of polysept should be tested for flocculation properties at different concentrations of thepolymer (0.007, 0.0105 and 0.01575%) in dynamics during 24 hours. After this period, the turbidity of solutions should not exceed 30 FNU (formazin turbidity) at concentrations of 0.0105 and 0.01575%. It is also necessary to determine the osmolality of polysept solutions at different percentage concentrations (6, 8, 10, 12, 14%). Osmolality values should be within the following ranges: 260 ± 20 mOsm fora 6% solution; 330 ± 25 mOsm foran 8% solution; 400 ± 25 mOsm fora 10% solution; 460 ± 30 mOsm fora 12% solution; 520 ± 20 mOsm fora 14% solution

CHANGES IN AMINO ACID COMPOSITION OF BLOOD PROTEIN HYDROLYSATE DEPENDING ON THE RAW MATERIAL PREPARATION MATERIAL

The raw material for blood protein hydrolysate preparation is whole animal blood, its clots and other serum production wastes. The dependence of amino acid composition of blood protein hydrolysate on the season of the raw material preparation was studied. The research lasted three years. It was demonstrated that the amino acid composition changed depending on the season. The peak, as a rule, was during summer months when their amount increased by 1.2–2.3 times and during autumn and winter it went down by 1.2–1.4 times (the difference is considerable, р < 0,05). The peak of glutamic and asparagine acid growth was in November when their amount was 1.4 times higher then during the previous months (р < 0,01). The increase of alanine, asparagine, valine, lysine, methionine, histidine, proline, tyrosine, threonine, and phenylalanine by 1.3–1.8 times was observed in March (the difference is considerable, р < 0,05). The amount of histidine, glycine, leucine, serine, and tryptophane in the beginning of spring was at the same level and the amount of arginine, asparagine, isoleucine in March decreased by 1.2–1.6 times (the difference is considerable, р < 0,01). So, it was determined that the dynamics of BPH amino acid composition was directly associated with the seasonal dynamics of physiological and biochemical cattle blood values. It was noted that in case of considerable change in absolute amino acid parameters their relative amount, in general, remained constant

TESTING OF EMULSION VACCINES BASED ON WHITE OILS OBTAINED AS A RESULT OF HYDROCATALYTIC PROCESSING OF OILSTOCK

The paper demonstrates the possibility to use white mineral oils resulting from hydrocatalytic processing of unconverted vacuum gas oil as a basis for an oil adjuvant used in the production of FMD vaccines intended for livestock. Samples of the developed white oils were tested for their reactogenicity and pyrogenicity, and emulsion vaccines were tested for their reactogenicity, potency, and emulsion stability. Tests results were compared with the ones for a foreign analogue. Blood samples were taken from animals before vaccination and 14 days after it to obtain sera which were further tested for FMD antibodies using VNT. Assessment of the white oil sample reactogenicity demonstrated that sample BM 1.4 obtained during hydroisomerization was the least reactogenic. Sample V-BM 1.4 demonstrated the best result in comparison with other samples within the group as well as in comparison with the control sample during the emulsion stability test. According to the potency test results, the emulsion vaccine based of the white oil sample BM 1.4 demonstrated the greatest difference in antibody titers in 14 days - 4.15 log2, while the one based on the control sample - 3.50 log2. This means that it the use of this vaccine results in 17.7-fold increase in FMD antibodies, while the use of control emulsion vaccine in 11.3-fold increase. Vaccines based on BM 1.3 and BM 2.2 oil samples demonstrated lower performance in comparison with the control vaccine in terms of these parameters

Studies of biological properties of continuous suspension ВНК-21/SUSP/ARRIAH cell line

The results of the studies of cytomorphological, karyological, cultural properties of continuous suspension ВНК-21/SUSP/ARRIAH subline of newborn Syrian hamster kidney cells intended for foot-and-mouth disease, rabies, bovine parainfluenza-3, Aujeszky’s disease virus reproduction, as well as for production of diagnostic veterinary biologicals are presented. When cultured in suspension, BHK-21/SUSP/ARRIAH cell subline undergoes selection towards hypoploidy: modal class is represented by cells with 41 chromosomes (32–40% of cells); the share of cells containing 40–42 chromosomes is 78–80%; the share of polyploids averages around 1%; the range of variation in the number of chromosomes is from 36 to 54. BHK-21/SUSP/ARRIAH cell subline cultured in suspension with cell seeding concentration of 0.6–0.8 million cells/cm3 demonstrates growth rate of 6.67–11.00 and 96–99% cell viability. After 48 hours, G1-phase (diploid-2n) cells prevail in the cell population of the new subline (30.0–75.0% of cells); cells that undergo preparation for mitosis (S-phase) and mitosis (G2+M-phase) account for 3.0 to 20.0% of the entire population; the number of meganucleated and multinucleated cells (>4n) at the beginning and at the end of the logarithmic phase increases to 2%. BHK-21/SUSP/ARRIAH cells recover rapidly after cryopreservation and demonstrate 95–99% viability and growth rate of 3.36–5.88 at passages 1 to 3 and 6.85–10.95 at passages 4 to 12. Continuous suspension BHK-21/SUSP/ARRIAH cell line ensures virus accumulation at the following titres: FMD virus – 7.30– 8.00 lg TCID50/cm3, rabies virus – 7.25–8.00 lg CCID50/cm3, bovine parainflunza-3 virus – at least 6.00 lg TCID50/cm3, Aujeszky’s disease virus – 7.50–7.80 lg TCID50/cm3

PURITY TESTING OF ARRIAH FMD VACCINES

The paper demonstrates results of FMD vaccine testing for their inability to induce antibodies to FMD virus non-structural proteins (FMDV NSP antibodies) in cattle at different dates post vaccination. Subtype A, O, Asia-1 and SAT-2 cultural FMD virus replicated in baby hamster kidney suspension culture (ВНК-21/2-17) was used for vaccine production. FMDV antigen was inactivated with aminoethyl ethylenimine solution, and the suspension was subsequently purified. Non NSP-purified preparation was made to simulate presence of NSPs in the vaccine. ELISA test-kits were used for testing cattle sera collected before and after immunization for presence of 3ABC polyprotein-specific antibodies. Inoculation of cattle with non-purified preparation induced antibodies specific to FMDV NSPs in 87.5% animals on day 30, in 75% animals on day 44 and in 62.5% animals on day 132 post third immunization. All tested vaccines manufactured by the FGBI “ARRIAH” were demonstrated to induce no FMDV NSP antibodies suggesting their complete removal from the virus-containing suspension

The use of specialised Sheff-Vax ACF supplements for ВНК-21/SUSP/ARRIAH cell cultivation and FMDV reproduction

Compliance with the existing purity and safety requirements for immunobiologicals can be effectively achieved by the use of serum-free nutrient media and specialised supplements of non-animal origin. The paper shows the possibility of using Sheff-Vax ACF® supplements (Kerry, Inc., Ireland) for ВНК-21/SUSP/ARRIAH cell cultivation and FMDV reproduction. By passage 7, cell concentration and growth rate with Sheff-Vax Plus PF ACF were found to be 40–60% higher than with Sheff- Vax PF ACF and Sheff-Vax Plus ACF. No differences were observed as regards changes in pH. During FMDV reproduction in the cells, it was found that the number of 146+75S components in the test samples containing 1 million cells was 2.3–2.4 higher compared to the controls. Cells cultured with the use of Sheff-Vax Plus PF ACF supplement had normal morphology and multiple dynamic protrusions. In the presence of this supplement, growth rate and suspension concentration in the test and control samples became equal by passage 7. The number of immunogenic components of FMDV reproduced in the cells grown using Sheff-Vax Plus PF ACF was 20–30% higher than in the cells grown using other supplements. ВНК-21/SUSP/ARRIAH cell concentration and growth rate in the presence of specialised supplements were found to be lower than those in the control samples with serum and blood protein hydrolysate added to the nutrient medium. The virus yield from 1 million cells was higher in the culture grown using Sheff-Vax ACF supplements. Sheff-Vax Plus PF ACF was found to be the most suitable for ВНК-21/SUSP/ARRIAH cell cultivation and FMDV reproduction in the said cells out of the three tested supplements

Optimization of nutrient medium composition for BHK-21/2-17 suspension culture

The paper demonstrates results of the research aimed at optimization of amino acid composition of the nutrient media used for BHK-21-2/17 suspension cultivation by change of amino acid amount in such media. Growth level in the control and tested media amounted to 5.6; cell growth dynamics was also similar. In-media utilization of different amino acids was different. The cells mostly consumed glutamin, serine, tyrosine, methionin (up to 90%, 55%, 40% and 60%, respectively). Growth of alanine level was reported (from 10% in the control sample and up to 60% in the test sample). Amount of tryptophan and glutamic acid remained stable. Glycine, threonine, isoleucine were actively utilized within the first 24 hours (7-13%, 10-18%, 34-41%, respectively in the control experiment). Hereafter, the concentration slightly increased (by 7-45%, by 16-21%, by 20-50%, respectively in the control experiment). During the FMDV reproduction in the cells grown in the test and control media no significant differences in the yield of immunogenic virus components were reported