Improving Rev.1 Vaccine Stability Produced in Iran بسم الله الرحمن الرحیم Improving Rev.1 Vaccine Stability Produced in Iran Dr. Ramin Bagheri Nejad Head of Brucellosis Department Razi Vaccine & Serum Research Institute

Rev.1 Vaccine Importance B. melitensis as the main cause of human brucellosis Primary cause of Small ruminants brucellosis Sheep & goats: Preferred natural hosts Rev.1 vaccine the most effective and the only approved vaccine by the OIE

Role of Animal Vaccination Necessary measure for the control of animal brucellosis but not all required Effective on decreasing human disease incidence proved by several studies around the globe (Greece, Mongolia and…) Vaccination efficacy depends on: Vaccination coverage Prevalence of infection Animal production practices Sanitary measures Control of animal movements

Quality of Rev.1 Vaccine Quality: fitness for the intended use Safety & potency Stability: no specified duration Determined based on standard stability studies Done on 3 successive batches The product should retain its potency during shelf life Important factors: safety, identity, purity & potency

Standards for Rev.1 Vaccine Production & Quality Control International standards: OIE Manual of diagnostic test & vaccines (2012) European Pharmacopeia (2015) WHO: Requirements for Rev.1 vaccine production (1970)

Standard Quality Control Tests Identity: Biochemical and microbiological tests Purity: free from contaminating agents Safety in target animal species Potency: Smoothness Enumeration of live bacteria per dose: EP:0.5- 4×109, OIE: 0.5-2×109 Immunogenicity in mouse model No requirement for testing on target animal species

Standard Quality Control Tests

Standard Quality Control Tests

Rev.1 vaccine in Iran Rev.1 production since 1341 Collaboration with the WHO Field trials among first ones in the world First supplied as liquid Then lyophilized Shelf life of 4 months

Rev.1 Vaccine Quality Control in Razi Institute Production of each batch Sampling by QC department and sending production documents QC tests Report of QC tests to Release Office Final decision of release or fail by Release Office

Materials & Methods Focus on the stabilizer composition Stabilizer: The most important factor to assure vaccine strain viability Protects the vaccine strain during lyophilization Protects final products against environmental stresses during storage Conventional stabilizer composition as recommended by the WHO and OIE: Pancreatic digest of casein Saccharose Sodium L-glutamate Different studies by adding cryoprotectants and lyophilization exepients

Materials & Methods Evaluation of stability by accelerated testing Production of a lab-scale batch for extended studies1 Real-time real-temperature stability study Safety evaluation Determination of immunogenicity in mice model as recommended by the OIE and European Pharmacopeia

Number of live bacteria (cfu/dose) Results Identification of 3 more substances Accelerated Stability study: Day post-incubation Number of live bacteria (cfu/dose) 1.66 × 109 1 1.96 × 109 3 1.28 × 109 4 1.33 × 109 5 1.16 × 109 7 1.02 × 109 8 1.11 × 109

Results Real-time stability study:

Results Safety: According to European pharmacopeia (2012) Injection to 2 sheep Observation of animals for 21 days No local and systemic adverse effects

Results Immunogenicity in mice OIE (2012) & EP protocol 3 mice groups Challenge with B. abortus strain 544 one month after vaccination Euthanizing all mice 2 weeks post-challenge Enumeration on challenge strain in spleens

Log of challenge strain in spleen Results 8 months after production Mouse Group Log of challenge strain in spleen (M±SD) Y=log(X/logX) Original seed 2.55±0.95 a 2.1±0.7 a New Rev.1 3.04±0.57 a 2.5±0.4 a Non-vaccinated Control 5.32± 0.34 b 4.6± 0.3 b

Future Plan Production of 3 successive industrial batches Performing stability studies on all 3 batches to show consistency of production Identity Purity Enumeration of live bacteria Safety in target animal species Immunogenicity in mice Application to Razi Quality Assurance Deputy and IVO to acquire permission for introduction of changes in industrial production

Thanks for your Attention