Preparation And Quality Evaluation Of Pig Spleen Transfer Factor Against Porcine Reproductive And Respiratory Syndrome By Tangential Flow Filtration

As the development of large-scale pig industry in China,the low immune function and poor disease-resistance of pigs were caused by combination of high pig breeding density,immunosuppressive disease,mycotoxin in feed and many other reasons.The pig industry in China were faced with a great risk from the ineffective protection and even failed immunization after vaccination.Among them,serious economic losses were caused by one of important immunosuppressive diseases called porcine reproductive and respiratory syndrome(PRRS).Some protection effect showed mainly by vaccine immunization used to prevent and control PRRS.However,great pressure still remains to be faced in the prevention and control of PRRS in China.In order to improve the immune efficacy of PRRS vaccine,the research and development of adjuvant for PRRS vaccine was regarded as an important work.Transfer factor(TF)was an enhanced immune adjuvant,which played an important role in the prevention of infectious diseases and immunodeficiency diseases while ineffective measures by antibodies or antibiotics.But at present,the production of TF for veterinary use in China was entirely nonspecific TF(NTF).In this case,the manufacture process and quality evaluation of pig spleen specific TF against PRRS(PRRS-STF)would aim the TF-based research,development and production.Based on this,in order to provide a reliable reference for the large-scale production of PRRS-STF,in this study we established a step by step tangential flow filtration technology to produce PRRS-STF,optimized the technical conditions in the preparation process and evaluated the quality of PRRS-STF prepared by the process.There are six parts in the following.1.Study on the preparation and inspection of PRRS-STFIn this experiment,the spleens without virus contamination of bovine viral diarrhea virus(BVDV),porcine parvovirus virus(PPV),classical swine fever virus(CSFV),porcine circovirus type 2(PCV-2),PRRS virus(PRRSV),foot and mouth disease virus(FMDV)and pseudorabies virus(PRV)from PRRS antibody positive immunized pigs were selected after inspection.The large-scale manufacture process and specific potency test method(leukocyte adhesion inhibition method)of PRRS-STF were established,and 3 batchs of trial-producing isosmotic PRRS-STF in the laboratory were inspected.The results showed that the polypeptide contents,ribose contents,nonspecific potencies by doffed E receptor method,specific potencies by leukocyte adhesion inhibition method were at the same level for 3 batchs of PRRS-STF,and results of the above mentioned tests were respectively all above 2.5 mg/mL,55 μg/mL,12%and 30%,In identification test,the OD260nm/OD280nm ratios of PRRS-STF were entirely greater than 1.9 and the maximum absorption peaks were all at 250 nm～260 nm.None of the contents of endotoxin were more than 10 EU/mL.The results of protein qualitative test,aseptic test,mycoplasma test,extraneous virus test(BVDV,PPV,CSFV,PCV-2,PRRSV,FMDV,PRV,cytopathic effect tests and erythrocyte adsorption tests for extraneous virus test)were all negative.These results indicated that the 3 batchs of PRRS-STF prepared had a good specificity against PRRS,and its polypeptide content,ribose content and nonspecific potency by doffed E receptor method and other indicators were far higher than standard for TF compared to both the national standard for human medicine(《Transfer Factor Injection national drug standard》)and the highest quality standard for veterinary use(《Quality standard for pig spleen Transfer Factor Injection(the third new veterinary new medicine)》),and PRRS-STF was suitable for combined immunization with PRRS attenuated vaccine because of the technological effect that neutral isosmotic PRRS-STF without residual inactivator.2.Study on the tangential flow filtration process of PRRS-STFIn biopharmaceutical,the tangential flow filtration technology was widely applied from the purification,clarification,enrichment to the separation of target molecule.In addition,the separation process of TF was the key technology for the preparation of TF.Based on this,in this experiment the effects of transmembrane pressure(TMP),inlet flow rate,concentration multiple,dialysis multiple and other key parameters on the membrane flux(Flux)were studied by 0.1 m2 0.22μm tangential flow microfiltration and 0.1 m2 5 KD tangential flow ultrafiltration,and the above key parameters of tangential flow filtration were determined.5 batchs of PRRS-STF were prepared with the magnified parameters linearly on 2 m2 microfiltration and 2.5 m2 ultrafiltration system,and compared with 5 batchs of PRRS-STF prepared by the traditional Lawrence method on the process and test results(character,identification test,protein qualitative test,polypeptide content,ribose content,nonspecific potency by doffed E receptor method and specific potency by leukocyte adhesion inhibition method).The results showed that the optimal conditions for tangential flow filtration:the inlet flow rate,TMP,concentration multiple,dialysis multiple and average Flux were 9.3 L/min/m2,2 psi,3 times,0.5 times and 11LMH respectively at 0.22μm microfiltration,and 5 L/min/m2,19.5 psi,6 times,1 times and 51 LMH respectively at 5 KD ultrafiltration.The Flux curves after linear amplification were close to that of 0.1 m2 microfiltration and 0.1 m2 ultrafiltration.The polypeptide content,ribose content,nonspecific potency and specific potency of PRRS-STF prepared by the tangential flow filtration method were higher than that of the traditional Lawrence method,and decreased by about 24 h than the traditional Lawrence method.These findings indicated that the tangential flow filtration method had good linear amplification and high production efficiency.The active ingredient content and activity of PRRS-STF prepared by the tangential flow filtration method were high.3.Study on the virus removal process of PRRS-STFIn this experiment,different:final concentrations of β-propiolactone(0.1%,0.02%,0.01%,0.005%)were used to inactivate PPV in the centrifugal supernatant(12 h,24 h,36 h,48 h)with 105 TCIDso/mL final virus titer.The samples were hydrolyzed 2 h at 37℃ after inactivation,and then passaged three generations continuously on swine testis(ST)cells.Cytopathic effect was observed,and the inactivation effect of the third generation cell culture was detected by indirect immunofluorescence antibody method.The results showed that three concentrations of 0.1%,0.02%and 0.01%β-propiolactone were all inactivated for PPV test negative from 12 h to 48 h,and 0.005%β-propiolactone was PPV positive in 48 h.To ensure the safety of product,it was suggested that the final concentration of 0.01%β-propiolactone could be better inactivated 24 h at 2-8 C and then hydrolyze 2 h at 37℃.The 5 KD ultrafiltration was used to filtrate PCV-2,PRRSV and PRV respectively in the outcome of microfiltration with 106 TCIDso/mL final virus titer.The PCV-2 group’s outcome of ultrafiltration was passaged three generations continuously on porcine kidney-15(PK-15)cells.The third generation cell culture was detected by indirect immunofluorescence antibody method.PRRSV group’s and PRV group’s outcome of ultrafiltration were detected by PCR respectively.The results showed that the tests of PCV-2,PRRSV and PRV were negative.These findings indicated that PRRS-STF was free from extraneous virus contamination ensured by β-propiolactone treatment,5 KD ultrafiltration and other virus removal method process.4.Pharmacological study of PRRS-STFIn this experiment,the pharmacological activity of 3 batchs of PRRS-STF was studied.The results showed that the 3 batchs of PRRS-STF were all qualified in pyrogen test on rabbit,toxicity test on mice and anaphylaxis test on guinea pig.In the test of E rosette rate on rabbit the percentage of E rosette in the experiment groups(23.3%,23.6%and 26.2%)were significantly higher than that in the control group(13.2%)(P<0.05).In the delayed allergy test on mice the swelling degree of the experiment groups(14.3,14.5 and 15.8 mg)were significantly higher than that of the control group(9.8 mg)and blank group(0.5 mg)(P<0.05).In the peritoneal macrophage phagocytosis test on mice the phagocytic percentage(30.50%,30.83%,31.17%)and phagocytic index(0.68,0.67,0.71)of the experiment groups were significantly higher than those of the control group(21.33%and 0.45)(P<0.05).In the test of carbon clearance on mice the K mean(0.0455,0.0487,0.0492)and a mean(6.97,6.95,7.39)of the macrophage in the experimental groups were significantly higher than those in the control group(0.0263 and 5.97)(P<0.05).These findings indicated that PRRS-STF could significantly increase the activity of T lymphocyte and macrophage,promote the proliferation of T lymphocyte into sensitized lymphocyte and improve the immune function.5.Safety study of PRRS-STFThe safety of injection was studied after 3 batchs of trial-producing PRRS-STF and PRRS attenuated vaccine injected jointly or singly.The 1 day old piglets,4-6 weeks old nursery pigs,about 30 days of pregnant sows,about 30 days before the birth pregnant sows and the stock boars were intramuscularly injected with a single dose,repeated single dose and an overdose PRRS-STF in the laboratory.In addition,a single dose and an overdose PRRS-STF were combined immunization with PRRS attenuated vaccine to intramuscularly inject the 3-4 weeks old pigs,about 1 weeks before the mating sows and the stock boars.The results showed that there was no adverse reaction in piglets,nursery pigs,sows and stock boars after PRRS-STF and PRRS attenuated vaccine injected jointly or singly.The offsprings by the sows injected with and without PRRS-STF were no difference.The average daily gains of the piglets and the nursery pigs were higher than that of the groups without PRRS-STF injected,but the difference was not significant(P>0.05).These findings indicated that PRRS-STF was safe for inoculating piglet,nursery pig,sow and stock boar.6.The effectiveness of PRRS-STFIn this experiment in order to evaluate the effectiveness of trial-producing PRRS-STF,the influence of PRRS-STF on that lymphocyte transformation rate by MTT method and E rosette rate in Pigs,PRRSV antibody level by Elisa,IFN-γ by liquid chip platform technology Luminex X-MAP method,the number and ratio of lymphocyte(Lym)cells,T lymphocytes,CD4+CD8+ double positive T(DP T)lymphocytes and CD4-CD8-double negative T(DN T)lymphocytes in the peripheral bloods of pigs immunized with PRRS attenuated vaccine and challenging protection of PRRS attenuated vaccine was studied.The results showed that single injection of PRRS-STF could increase lymphocyte transformation rate,E rosette rate and enhance the immune function of pigs.PRRS-STF could increase PRRSV antibody level and IFN-γ content of pigs combined immunization with PRRS attenuated vaccine,and enhance the immune effect of PRRS attenuated vaccine.PRRS-STF could improve the challenging protection of PRRS attenuated vaccine(CH-1R strain),and challenging protection attacked by highly pathogenic PRRSV(HP-PRRSV)TJ-F5 strain was increased from 40%in single PRRS attenuated vaccine immunized group to 80%in PRRS attenuated vaccine combined immunization with PRRS-STF.From 3 day post vaccination(dpv)to 21 dpv,the number of DP T cells and the ratio of DP T/T in the experimental group were always higher than those in the other two groups.And from 7 dpv to 21 dpv,the number of DN T cells and the ratio of DN T/T were always lower than those in the other two groups.On 3 day post challenge(dpc)by HP-PRRSV TJ-F5,the number of Lym,T,DP T and DN T cells in each groups decreased significantly(P<0.01),and meanwhile,the number of Lym,T,DP T,DN T cells and the ratio of DP T/T,DN T/T in each groups were 35%,38%,27%,51%and 72%,134%before challenge respectively.On 28 dpc,the number of Lym,T,DP T,DN T cells and the ratio of DP T/T,DN T/T in survival pigs of each groups were 72%,60%,122%,36%and 197%,59.8%before challenge respectively.These findings indicated that the increase of DP T cells and the decrease of DN T cells played an important role in anti-infection immunity against PRRS.PRRS-STF could play immunopotentiation effect by regulating the number of DP T,DN T,T and Lym cells in pigs immunized with PRRS attenuated vaccine.These results indicated that the preparation of PRRS-STF by the tangential flow filtration process was easy to control the key point of production and make the linear amplification,which was suitable for large-scale production.The active ingredients content,nonspecific potency and specific potency of the prepared PRRS-STF were high.PRRS-STF could be used to improve the immune function of pigs and the immune effect of PRRS attenuated vaccine.