Generic manufacturing process of Foot & Mouth ... - EMD Millipore

Application Note

Generic manufacturing process of Foot & Mouth Disease Vaccines Introduction Foot and mouth disease (FMD) is a highly contagious, and sometimes fatal, viral disease that affects cloven-hoofed animals, including domestic and wild bovids. Humans are very rarely affected by FMD virus (FMDV). FMD is a severe issue for animal farming, since it is highly infectious. Containment of FMD demands considerable efforts in vaccination, strict monitoring, trade restrictions and quarantines, and occasionally the elimination of millions of animals. The virus causes a high fever for two or three days, followed by blisters inside the mouth and on the feet that may rupture and cause lameness. FMDV can be transmitted in a number of ways, including close contact animal-to-animal spread, long-distance aerosol spread, and inanimate objects, typically fodder and motor vehicles. Other infectious sources include the clothes and skin of animal handlers, standing water, uncooked food scraps and feed supplements containing infected animal products, and even the semen of infected bulls. FMD occurs throughout the world. Currently, the World Organization for Animal Health classifies countries to be in one of three disease states with regards to FMD: • FMD present with or without vaccination • FMD-free with vaccination • FMD-free without vaccination

The virus responsible for the disease belongs to the family of Picornaviridae in the genus Aphthovirus. FMDVs are small (25-30 nm), non-enveloped icosahedral viruses that contain single-stranded RNA. FMDV is extremely labile in vitro, as whole particles dissociate into monomers at temperatures above 56 °C and pH below 7. The virus is highly variable, which limits the effectiveness of vaccination. Like other viruses, FMDV continually evolves and mutates, making vaccination difficult. There are seven FMD serotypes: O, A, C, SAT-1, SAT-2, SAT-3, and Asia-1, Serotype O being most common. There is no cross-protection between serotypes, since two strains within a given serotype may have nucleotide sequences that differ by as much as 30% for a given gene. As a result, FMD vaccines must be highly specific to each strain. Vaccination only provides temporary immunity that lasts from months to years. Many early vaccines used dead samples of FMDV to inoculate animals, but those early vaccines sometimes caused real outbreaks. Typical FMD vaccines, first genetically engineered in the US in 1981, are killed virus preparations grown in mammalian cell culture. This document outlines the basic manufacturing process of FMD vaccine.

Countries designated FMD-free without vaccination have the greatest access to export markets for animalderived components, and therefore many developed nations, including Canada, the United States, and the UK, work hard to maintain their current status.

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FMD vaccine process flow

Roller Bottle Media Prep

Cell Lysis

Figure 1.

Generic FMD Vaccine Process

Inoculums Propagation

Secondary Clarification

Suspension Culture

FMD vaccine manufacturing follows a simple multi-step process. Though each manufacturer follows a different process for manufacturing, a general outline of the process is provided in Figure 1. The GMP requirements for this process, and all veterinary vaccine processes, are very similar to GMP required for human vaccine manufacturing. Manufacturing is performed in a biosecure environment. When the starting material is the inactivated FMD antigen, the time to release FMD vaccine to market is about 5 days subject to special regulatory provisions. The process for manufacturing FMD vaccines from raw materials averages 10 weeks, including all required regulatory testing prior to market

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Centrigfugation

release. Manufacturing cost of FMD vaccine in the EU is less than around $0.50-$0.60 (US$) per dose. Vaccine production costs are15 times lower for Indian or Latin American manufacturers, resulting in a typical selling cost of FMD vaccine in India of $0.07-$0.20 per dose.

UF/DF

Sterile Filtration

Formulation & Blend with Adjuvant

Finish Fill

Final Vaccine

Antigen Bank

Virus Inactivation Chromatography UF/DF

Sterile Filtration

Antigen Bank

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1.Media Preparation

spinner flasks, roller bottles, or small bioreactors. Later cell culture expansion is done in large bioreactors in suspension culture, followed by virus propagation. A typical pooled roller bottle batch volume is 500-700L and suspension culture is 1000-2000L. Cell cultures are grown in low oxygen tension in the presence of ~5% CO2. The main culture is seeded at an initial density of 0.2-0.5 x 106 cells/ml, which is expanded to 2-3 x 106 cells/ml before being infected with virus.

Different complex cell culture media can be used for BHK21 cell propagation. Most propagation media contain serum, as high as 10%, but during the virus infection stage the serum is typically depleted to1-2%. Fetal calf serum used in media is typically certified to be free from bovine spongiform encephalopathy (BSE) and transmissible spongiform encephalopathy (TSE) agents and mycoplasma. Alternatively, newer processes may utilize serum-free media. Typical media is formulated with high peptone content (25-40g/L of media). The media can be filter sterilized using pre-filters (Polysep™ II 1.0/0.5µm) followed by sterile filtration using Millipore Express® filters. Millipore Express® SHC filter offers a capacity of ~1500-2000 L/m2 for pre-filtered cell culture media containing 5-10% serum. During the growth phase, most of the cell culture undergoes media exchange or addition of fresh medium, either in continuous or batch mode.

If serum is used during the cell expansion phase, media exchange is done to a serum-free virus propagation medium, usually via transfer of bioreactor cell suspension into a new tank, sedimentation of cells during 18h at 4°C, and resuspension of cells in fresh media. Cellvento™ BHK200 (BHK21 serum-free medium) can be used to grow cells for FMD vaccine production. Cellvento™ BHK200 is a serum-free medium, formulated without any animal derived component, optimized for suspension culture of BHK21 cells at high-density and viability in serum-free conditions. Cells grown in Cellvento™ BHK200 medium are first adapted via serial passaging in the medium supplemented with decreasing concentration of serum. Adapted BHK21 cells can then be grown as suspension cultures in T-flasks, shaker flasks, spinner bottles or stirred tank bioreactors with a higher growth in comparison to GMEM/TPB/serum cultures (Figure 2).

2. Cell Culture/ Virus Propagation Cell lines used to grow FMDV are usually BHK21 suspension cells derived from the BHK21 C13-2P clone, available at the American or European cell culture collections. This cell line is banked to grow in Glasgow Minimum Essential Medium (GMEM) supplemented with 5% tryptose phosphate broth (TPB) and 10% serum in

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Culture Time (days) Cell Counts (GMEM/TPB/serum) Cell Counts (Cellvento™ BHK200)

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Viability (GMEM/TPB/serum) Viability (Cellvento™ BHK200)

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Viability (%)

Viable density (x106 cells/ml)

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Figure 2. Comparison of the growth of BHK21 C13-2P Cells in GMEM/TPB/ Serum or in Cellvento™ BHK200 Medium (after cell line adaptation). Represented are the viable cell density and the viability over an 11 days batch experiment done in 250ml shaker flasks. Cells are incubated at 37°C, 5% CO2 , 80% humidity with a rotating speed of 110 rpm (5 replicates, Mean ± SD)

The serum-free medium Cellvento™ BHK200 has also been shown to be an effective media formulation for the growth and maintenance of BHK21 suspension cell lines used in FMDV processes. In this serum-free medium, the cells can reach higher viable cell densities (e.g 6.105 cells/ml) leading to higher viral antigen production. When using serum-free media for cell growth, the media exchange is not necessary, allowing for a simpler process, reduced processing space and shorter process time. Virus inoculation is done aseptically to cells after 2-3 days in cell culture. However, the duration of infection depends on the viral strains (O1 9-15h, C3 11-16h, A2 15-22h). Virus harvesting is performed 20-40hr after virus inoculation, and cells are lysed to increase virus release. The virus load in the harvest generally ranges from 106-108 pfu/ml, corresponding to typical mass values of 2-3 g/ml. Optimum temperature for cell-infected virus growth is 37°C, and is reduced to 4-6 °C during other stages of manufacture. Virus is maintained at approximately pH 7.3-7.6 and should never be below pH 7.0. Typical bioreactor size for high-volume FMD vaccine manufacture ranges from 4000-5000L, while the typical bioreactor size for small volume manufacturer is 500L1000L. Generally one liter of cell culture produces 35-50 doses of finished FMD vaccine, depending on the type of strain, antigen payload and formulation (monovalent or trivalent). A manufacturer having 200-240 batches of 4000L bioreactor can produce 240-250 million doses of FMD type O vaccine per year.

3. Clarification The objective of clarification is to remove cell debris and recover virus. Zonal centrifugation is commonly used for primary clarification. Some manufacturers also use a body feed sparkler assembly. Solid content in viral vaccine harvest is low. Hence, normal flow filters typically work well for primary clarification (Polygard® CR (5 µm), Milligard®, Polysep™ II). The Polysep™ II with Durapore® 0.65 µm is commonly used as secondary clarification filter. Some manufacturers also use cellulosic pad filters for this application. Durapore® 0.45 µm is commonly used as bioburden reduction filter. Clarification is done at 4 °C.

4. Virus inactivation Formaldehyde inactivation was previously popular for FMDV inactivation; however, it has become increasingly apparent that its use carries a slight risk of residual contamination of inactivated vaccines with live virus. Due to the effectiveness and reliability of the process at the industrial scale, use of 3% binary ethyleneimine (BEI) for FMDV inactivation is now most common. Using BEI in a two-tank system according to GMP, it is possible to achieve the European Pharmacopoeia requirement of less than 1 infectious particle per 10,000 liters of FMD antigen preparation. Inactivation by a combination of binary ethyleneimine (BEI) and formaldehyde (FA) can inactivate FMD very rapidly (>2 log10 ID50 per hour). Inactivation is usually performed at 26°C in a tank. Due to the fact that the BEI-FA may not enter some parts of the tank, such as dip-tubes, the virus might not be inactivated completely and might, at the end of the process, contaminate the inactivated virus antigen. Therefore, after approximately 4 hours the whole inactivation mixture is transferred into a second inactivation tank (with BEI only after 24 hours). Samples are taken every 20 minutes to monitor the inactivation and verify the correct shape of the regression line. In the present method, in batches of 150 liters, the extrapolated inactivation plot must reach below −6.3 log10 ID50 at the end of the inactivation (at 24 h). The inactivation is stopped/ quenched by the addition of 2% sodium thiosulphate, which neutralizes BEI. Preparation BEI-FA mixture require binary ethyleneimine, formaldehyde, µ-naphtol violet solution and NaOH. Both BEI and formaldehyde are toxic substances and must be handled with extreme care and prepared fresh in a fume hood. BEI remains must be neutralized by the addition 1/10 vol. of a 20% Na-thiosulfate solution before it is discarded.

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5. Ultrafiltration Tangential flow filtration (TFF) is commonly used to remove inactivating agent by diafiltration and concentration of virus. Typically 10-15X concentration and 5-8N diafiltration is performed. Sodium phosphate buffer [137018 and 137036] (pH 7.6) or glycol buffer (pH 8.7) is the typical diafiltration buffer. Pellicon® 2, 100kD or 300kDa Biomax®, C screen works well for this application. TMP of 10 psi, cross flow of 7 L /min/m2 can result in average flux of 85 LMH. At this step, concentrated antigens can be stored as an antigen bank in low temperature until formulation. The FMD virus antigens in the banks are stored at ultra-low temperatures (usually -130 °C) to guarantee a shelf life of at least five years compared to a shelf life of 1-2 years for vaccines stored at +4 °C. When concentrated, a 50L volume of antigen can contain up to 15 million cattle doses as per the standard potency specifications in the OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. Earliest concentration methods used in industrial scale production of FMD vaccine were precipitation by polyethylene glycol or polyethylene oxide. Some have replaced ultrafiltration with a PEG process. Adsorption and elution from polyethylene oxide or PEG at an industrial scale helps to achieve concentration factors of 1000-fold and substantial removal of the bulk of the extraneous proteins (95%). Decivac FMD Monovalent vaccine by MSD is purified and concentrated by precipitation with polyethylene glycol. Typically FMD virus is concentrated by 8% PEG 6000.

6. Chromatography (optional) Some manufacturers produce an FMD antigen bank or FMD marker vaccine to differentiate the immune response of a vaccinated animal from a naturally infected animal. Such marker vaccine purification requires complete removal of low molecular weight non-structural proteins (NSP). Traditional manufacturing methods remove NSPs but do not guarantee 100% removal. Some manufacturers use industrial scale chromatography to purify the antigens previously concentrated by ultrafiltration to further remove extraneous proteins, including the NSPs, and produce highly purified, highly concentrated, inactivated antigens. Antigen is usually stored in the gaseous phase of liquid nitrogen until it is required.

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This material is particularly suited to the needs of antigen banks as well as facilitating vaccine formulation when more than one strain is required in a vaccine. Ion-exchange chromatography can be employed as well as gel filtration chromatography. It has been estimated that 20-25L gel filtration resin is sufficient to process virus harvested from 4000 L bioreactor.

7. Sterile Filtration Final FMD virus vaccine bulk is very much water-like and easily passes through 0.22 µm sterilizing grade filtration filters. Typical final sterilizing grade filter is 0.22 µm Durapore®.

8. Formulation FMD vaccine is typically formulated as quadrivalent inactivated antigens of O, A, C & Asia1 strains, adjuvanted with oil or saponin and aluminium hydroxide gel increase the immune response. FMD vaccine can be formulated alone or with other vaccines, such as the haemorrhagic septicaemia and black quarter vaccines. Combination vaccines are mixed with light mineral oil and homogenized to form a homogenous oil emulsion. Single vaccine (FMDV alone) is typically adsorbed on aluminum hydroxide gel. Other components of the final blend include antifoam, phenol red dye (if permitted by the country requiring vaccine), lactalbumin hydrolysate, tryptose phosphate broth, antibiotics, amino acids, vitamins and buffer salts. One single FMD vaccine dose contains 1-5 μg of 146S antigen. Emergency FMD vaccines are normally formulated to a higher potency than its conventional counterpart and there are banks who stipulate a requirement of at least 6 PD50 (50% protective dose) per dose for cattle, in contrast to the minimal statutory requirement of 3PD50. The antigenicity of the vaccine deteriorates if the temperature is allowed to rise above 8 o C, hence cold chain of 2-8 °C is maintained for distribution of vaccine.

9. FMD Vaccine Analytics The efficacy of inactivated vaccines is critically dependent on the integrity of FMD virus particles. The recommended method to quantify the active ingredient of vaccines is the 146S quantitative sucrose density gradient analysis. Some manufacturers quantify FMDV particles during the vaccine manufacturing process based on separation of components by size-exclusion chromatography and measurement of virus by absorption at 254 nm.

Testing is performed as follows: Inactivation. A portion of each batch of bulk inactivated antigen representing at least 200 doses is tested for freedom from infectious virus by inoculation into sensitive cell cultures. Antigenicity. The antigen content of each batch of bulk inactivated antigen is determined by an in vitro method (for example, 146S-particle measurement by sucrose density gradient centrifugation and ultraviolet spectophotometry at 259 nm). Safety. A representative sample of the bulk inactivated antigen may be diluted and blended with the adjuvants for safety testing in cattle. Sterility. The bulk inactivated antigen, the adjuvants and all dilution buffers comply with the test for sterility prescribed in the monograph on Vaccines for veterinary use (0062). Potency. A representative sample of the bulk inactivated antigen may be diluted and blended with the adjuvants for potency testing in cattle.

Chemicals used in FMD vaccine process Chemical Name

Catalogue No.

Cellvento™ BHK200

1.02376

Formaldehyde

8.17043

GMEM

1.00575

Sodium thiosulfate pentahydrate cryst., suitable for use as excipient EMPROVE® exp Ph Eur,BP,USP

1.06514

β-Naphthol violet

1.01306

Sodium hydroxide pellets suitable for the biopharmaceutical production EMPROVE® bio Ph Eur,BP,JP,NF,ACS

1.37020

Sodium dihydrogen phosphate dihydrate suitable for the biopharmaceutical production EMPROVE® bio Ph Eur,BP,USP,JPE

1.37018

di-Sodium hydrogen phosphate dihydrate suitable for the biopharmaceutical production EMPROVE® bio Ph Eur,BP,USP

1.37036

Polyethylene glycol 6000 Ph Eur

8.17008

Polyethylene glycol 20000 Ph Eur

8.17018

Antifoam (Poloxamer 188 suitable for biopharmaceutical production Ph.Eur.,NF)

1.37065

Phenol Red

1.37038

Table 1. Chemicals used in FMD vaccine process which are supplied by EMD Millipore

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