FMD Vaccine Manufacture: Controls and Constraints TDCD FMD Vaccine Manufacture: Controls and Constraints Tim & Claudia Doel Global Foot and Mouth Disease Research Alliance Hanoi, Vietnam 20-22 October, 2015 Hanoi, October 2015

Compliance Most vaccine manufacturers must comply with national or international standards of manufacture (GMP) and submit a product dossier to Regulators before it can be sold. Additionally, there may be mandatory requirements in pharmacopoeias of which the European Pharmacopoeia Monograph on FMD Vaccines is particularly important. TDCD GMP Certificate For Premises Located at 1 Penrose Way, Glasgow, Scotland. Dated 05 October. 2015 FMD Vaccine Dossier Hanoi, October 2015

Compliance Costs!!! Under full GMP, every employee will commit some time to GMP and QC/QA staff may represent 20% or more of the total headcount. Perversely, Compliance requirements can inhibit potential product improvements because of the time and costs required to conduct and gain approval for a revised product dossier. TDCD GMP Certificate For Premises Located at 1 Penrose Way, Glasgow, Scotland. Dated 05 October. 2015 FMD Vaccine Dossier Hanoi, October 2015

New Product Registration Changes to media, inactivation method, downstream processing, adjuvant/delivery systems etc require major work including safety and efficacy of dose, overdose and repeated dose in the target species, including specific claims relating to pregnant animals, juveniles etc. A few Regulators have argued that even a change of virus strain should prompt such studies with particular reference to efficacy! TDCD Hanoi, October 2015

Media Most production media will be based on MEM, GMEM, DMEM etc and supplemented with a protein hydrolysate, antibiotics, some miscellaneous additives and bovine serum. Because of TSEs in particular, media components of animal origin attract special attention from Regulators. TDCD Hanoi, October 2015

Media Bovine serum is expensive. In Europe, it must be sourced from a minimal risk TSE country (notably NZ) using the supplier(s) registered in the dossier. Each batch must be tested for freedom from general and specified adventitious agents before being gamma irradiated. It is necessary to prove the serum is of bovine origin – Adulteration in transit of high value NZ serum with human plasma (ex-blood banks) was reported many years ago in the UK. TDCD Hanoi, October 2015

Cells Cell production requires a 2-3 day growth cycle and the conditions used are also optimal for bacterial contaminants. In Europe, strong antibiotic cover using Penicillin and Streptomycin is not permitted. After 3 days, cell numbers in the range 2 - 6 million per ml can be achieved. Many years ago, titres of up to 9 million cells per ml were achieved but there seemed to be no additional virus productivity above 3 million cells per ml. TDCD Hanoi, October 2015

Cells While all BHK cells originated from Glasgow and, later, Pirbright in the case of the suspension line, virus growth in different lineages varies significantly (panel A). In the 1970s, Clarke derived a range of cell lines of which the AC9 lineage is reputed to give high virus yields (panel B). TDCD Hanoi, October 2015

TDCD CF Titre Panel A Panel B Hanoi, October 2015 Clarke and Spier, 1979

Virus Growth of established, ‘classical’ strains is generally straightforward. New vaccine strains are not always so easy. To increase the chances of obtaining a high yielding vaccine strain with good stability and able to confer relevant protection, a comprehensive selection of field isolates will be the minimum requirement. Regardless, a new vaccine virus will need full, lengthy and costly testing to ensure freedom from adventitious agents! TDCD Hanoi, October 2015

Virus In the case of an emergency situation, a position paper by the European Medicines Evaluation Agency does allow ‘fast-tracking’ using risk assessment to rule out at least a substantial number of potentially contaminating viruses. Fortunately, FMDV is resistant to organic solvents such as ether and chloroform whereas many potential contaminants are not and can be excluded from the testing process. TDCD Hanoi, October 2015

TDCD Source: Position Paper on FMD Vaccines, 2004 EMEA Committee for Medicinal Products for Veterinary Use. Hanoi, October 2015

Virus – Matching the Vaccine Strain with the Field Strain TDCD Virus – Matching the Vaccine Strain with the Field Strain How closely should the vaccine strain match with the prevalent field strain? Should the serological relationship between the viruses be the deciding factor? What about characteristics such as stability and immunodominance (whatever that means!)? Hanoi, October 2015

TDCD 1 Hanoi, October 2015 Adapted from Pay, 1994

Virus – Matching the Vaccine Strain with the Field Strain While the relationship between the two viruses is clearly important, it is worthwhile reflecting that many of the popular and effective current vaccine strains have been used successfully for decades (eg A24 Cruzeiro, O Campos, Asia1 Shamir, O Manisa, A22 Iraq). Whereas, there has been a tendency for new strains to come and go quite quickly (Iran 96/99/2001). TDCD Hanoi, October 2015

146S Analysis A critical test which will be used for yield analysis as well as vaccine payload decisions TDCD Our experience is that it is quite common to find deficiencies in the method including the calibration of the spectrophotometer. This doesn’t really matter as long as the deficiencies are constant over a period of years. Payload and yield trends will still be (internally) meaningful. Naturally, it matters greatly if the method is variable from day to day. Extreme caution is needed if a customer decides to compare the payloads claimed by different manufacturers. Hanoi, October 2015

TDCD Hanoi, October 2015 Doel and Mowat, 1985

A critical stage of FMD Vaccine Manufacture. Inactivation A critical stage of FMD Vaccine Manufacture. TDCD Many manufacturers favour a two dose, two tank BEI process to ensure full inactivation. In fact, the European Pharmacopoeia only requires that the inactivation kinetics are linear and there must be less than 1 infectious particle per 10,000 litres bulk virus at the end* of the inactivation process . Log10 Virus Titre Hours of Inactivation For the first dose of BEI, complete inactivation is generally required by 67% of the inactivation period. Eg 16 hours assuming the first cycle is 24 hours. Hanoi, October 2015

A critical stage of FMD Vaccine Manufacture. Inactivation A critical stage of FMD Vaccine Manufacture. TDCD Failure to get a statistically acceptable regression line means one thing for the antigen batch!!! Hanoi, October 2015

Instability of 146S During Inactivation with AEI Virus Before AEI* After AEI* % Loss 146S A24 Cruzeiro 1.7 1.8 A27 3.5 3 14.3 2.3 2.2 4.4 O Campos 3.2 (216) 1.3 (190) 59.4 0.8 0.1 87.5 O 9489 0.2 88.9 1.9 0.3 84.2 TDCD *Production samples. Same viruses grown and inactivated at Pirbright did not show significant breakdown. High instability of the Colombian O preparations was never explained, but was not due to pH. Hanoi, October 2015 Gomez and Doel, 1984

TDCD Downstream Processing Many manufacturers concentrate and partially purify their bulk antigens. Historically, one of the aims was to reduce allergic responses of animals to non-viral proteins. Semi-purified concentrates also opened the door for antigen banks as well as giving significant commercial and manufacturing flexibility for some companies. Increasingly, however, there is a major perceived benefit in removing NS proteins in order to minimise NS antibody responses. TDCD Hanoi, October 2015

Downstream Processing How is it done? Three basic methods used individually or in combination. 1. Ultrafiltration. Primarily a method of concentration but will remove molecules up to 1 million daltons depending on the filtration cartridge. 2. Gel Permeation Chromatography. Used only by one manufacturer to our knowledge. 3. PEG Precipitation. An old but very widely used and proven method. By cycles of different PEG concentrations, it is possible to get very high levels of purification. TDCD Hanoi, October 2015

Downstream Processing TDCD In some countries, manufacturers are required to purify antigens such that NS antibodies are not induced. Certainly, it will reduce the possibility of adverse reactions in livestock. Concentrates can be stored almost indefinitely in freezers providing commercial and manufacturing flexibility and strategy. Most probably, only a modest level of purification is necessary. Garland showed many years ago that even crude vaccines did not induce anti-3D (VIAA) except after three doses. High levels of purification can significantly reduce yields. This can mean only 50 or 60% of the original culture yield is recovered. It is absolutely vital to quickly purify and store the antigens if proteolysis of the VP1 is to be avoided/minimised. Hanoi, October 2015

Proteolytic Cleavage of the VP1 of O and SAT2 Reduces Neutralising Antibody Titres by over 90% TDCD Antibody Titres of Vaccinated Guinea Pigs Vaccine Virus Control Trypsin Treatment Cells Mice OBFS 2.4 6.4 1.5 1.1 A24 2.6 6.5 1.8 6.1 C 2 5.9 1.7 6 Asia1 4.9 2.1 4.4 SAT1 3.4 SAT2 0.9 SAT3 3.9 3.6 Hanoi, October 2015 Doel and Collen, 1982

TDCD ‘Innocuity’ Testing Another critical test is the demonstration that the inactivated antigen contains no infectious virus. The European Pharmacopoeia requires that it is necessary to inoculate and passage not less than 200 dose equivalents of antigen in a sensitive cell line and demonstrate absence of CPE in all of the passages. This test is far more sensitive than animal testing. Hanoi, October 2015

Vaccine Design A fundamental complication is that FMD vaccines are expected to cover all scenarios. This invites the question of whether formulations should/could be developed to suit the ages and species of animals as well as livestock systems, with particular reference to pigs. 1. With short-lived animals, the vaccine needs to induce rapid immunity, often in the face of maternal antibodies, and protective immunity is required for a relatively short time. Duration of immunity is less important and immune memory probably irrelevant. 2. With longer-lived animals, immune memory and duration of immunity are essential. TDCD Hanoi, October 2015

TDCD Vaccine Design Possible Approaches: Higher payload primary dose vaccines designed to establish a good primary immune response and, possibly, overwhelm the effect of maternal antibodies. Secondary dose vaccines with lower payloads to ensure boosting and development of immune memory. All of this would require extensive animal studies + a new product dossier + full cooperation between the vaccine supplier and customer to ensure product was used correctly in the appropriate animals. TDCD Hanoi, October 2015

Vaccine Design TDCD Manufacturers will use their 146S assays to routinely determine the payloads of the antigen to be used in the vaccine. A perception that very high payloads are desirable for general vaccine efficacy is not supported either by experimental work (Rweyemamu) or from a fundamental immunological perspective. Hanoi, October 2015

TDCD Effect of Antigen Payload on Antibody Titre 7 14 21 28 60 90 120 0.5 1 1.5 2 2.5 3 3.5 Days Post Vaccination Log Neutralising Antibody Titre 330 ug 55 ug 9.2 ug 1.5 ug 0.25 ug 0.04 ug 0.007 ug ug 146S TDCD Hanoi, October 2015 Rweyemamu et al, 1984

Don’t throw the baby out with the bathwater Vaccine Design With some serotypes, there are perceived benefits in using a mixture of strains: When a new field strain emerges and is recommended for use, it is prudent to use a mixture of the current and new vaccine strains for at least a year until it is clear that the current strain is no longer as relevant and the new strain remains appropriate. In fact, it is not uncommon to use a mixture of related vaccine strains to broaden the serological response and cover more effectively the population of field viruses. TDCD Don’t throw the baby out with the bathwater Hanoi, October 2015

Repeated Vaccination Doesn’t Fundamentally Change the Heterologous r1 Relationship Between a Vaccine Strain and Other Related Strains. TDCD VN Titre Number of Vaccinations Hanoi, October 2015 Dubourget et al, 1987

TDCD Thank You for Your Attention Hanoi, October 2015