Fermentation

FERMENTATION

The production of a bacterial vaccine batch begins with the recovery of the bacterial seed contained in an ampoule of the seed lot stored at −70°C or below, or freeze-dried. The resuscitated bacteria are first cultivated through one or more passages in preproduction media. Then, when the bacteria have multiplied sufficiently, they are used to inoculate a batch of production medium. Again, all media used must be from sources certified free of TSEs. Wherever possible, medium components of animal origin, especially human and ruminant, should be avoided.

The production medium is usually contained in a large fermenter, the contents of which are continuously stirred. Usually the pH and the oxidation/reduction potential of the medium are monitored and adjusted throughout the growth period to provide conditions that will ensure the greatest bacterial yield. In the case of rapidly growing bacteria the maximum yield is obtained after about a day but in the case of bacteria that grow slowly, e.g. M. bovis BCG, the maximum yield may not be reached before 2 weeks. At the end of the growth period the contents of the fermenter, which are known as the harvest, are ready for the next stage in the production of the vaccine

i)  Processing of bacterial harvests

The harvest is a very complex mixture of bacterial cells, metabolic products and exhausted medium. In the case of a live attenuated vaccine it should be innocuous, and all that is necessary is for the bacteria to be separated and resuspended under aseptic conditions in an appropriate diluent, possibly for freeze-drying. In a vaccine made from a virulent strain of pathogen the harvest may be intensely dangerous and great care is necessary in the subsequent processing. Adequate containment will be required and for class 3 pathogens such as S. enterica serovar Typhi or Yersinia pestis or bulk production of bacterial toxins, dedicated facilities that will provide complete protection for the operators and the environment are essential.

• Killing. This is the process by which heat and disinfectants are used to render the live bacteria in the culture non-viable and harmless. Heat and/or formalin or thiomersal are used to kill the cells of Bordetella pertussis used to make whole-cell pertussis vaccines, whereas phenol was used to kill the V. cholerae and the S. enterica serovar Typhi cells used in the now obsolete whole-cell cholera and typhoid vaccines.

• Separation. The process by which the bacterial cells are separated from the culture fluid and soluble products. Centrifugation using either a batch or continuous flow process, or ultrafiltration, is commonly used. Precipitation of the cells by reducing the pH has been used as an alternative. In the case of vaccines prepared from cells, the supernatant fluid is discarded and the cells are resuspended in a saline diluent; where vaccines are made from a constituent of the supernatant fluid, the cells are discarded.

• Fractionation. This is the process by which components are extracted from bacterial cells or from the medium in which the bacteria are grown and obtained in a purified form. The polysaccharide antigens of N.meningitidis are usually separated from the bacterial cells by treatment with hexadecyltrimethylammonium bromide followed by extraction with calcium chloride and selective precipitation with ethanol. Those of Streptococcus pneumoniae are usually extracted with sodium deoxycholate, deproteinized and then fractionally precipitated with ethanol. The purity of an extracted material may be improved by resolubilization in a suitable solvent and reprecipitation. These procedures are often supplemented with filtration through membranes or ultrafilters with specific molecular size cut-off points. After purification, a component may be freeze-dried, stored indefinitely at low temperature and, as required, incorporated into a vaccine in precisely weighed amount at the blending stage.

• Detoxification. The process by which bacterial toxins are converted to harmless toxoids. Formaldehyde is used to detoxify the toxins of Corynebacterium diphtheriae, Clostridium botulinum and Cl. tetani. The detoxification may be performed either on the whole culture in the fermenter or on the purified toxin after fractionation. Traditionally the former approach has been adopted, as it is much safer for the operator. However, the latter gives a purer product. The pertussis toxin used in acellular vaccines may be detoxified with formaldehyde, glutaraldehyde, or both, hydrogen peroxide or tetranitromethane. In the case of genetically detoxified pertussis toxin, a treatment with a low concentration of formaldehyde is still performed to stabilize the protein.

• Further processing. This may include physical or chemical treatments to modify the product. For example polysaccharides may be further fractionated to produce material of a narrow molecular size specification. They may then be activated and conjugated to carrier proteins to produce glycoconjugate vaccines. Further purification may be required to eliminate unwanted reactants and by-products. These processes must be done under conditions that minimize extraneous microbial contamination. If sterility is not achievable then strict bioburden limits are imposed.

• Adsorption. This describes the adsorption of the components of a vaccine on to a mineral adjuvant or carrier (aluminium hydroxide or aluminium phosphate; rarely calcium phosphate). Their effect is to increase the immunogenicity and decrease the toxicity, local and systemic, of a vaccine. Diphtheria vaccine, tetanus vaccine, diphtheria/tetanus vaccine and diphtheria/tetanus/pertussis (whole-cell or acellular) vaccine, are generally prepared as adsorbed vaccines.

• Conjugation. The linking of a vaccine component that induces an inadequate immune response with a vaccine component that induces a good immune response. For example, the immunogenicity for infants of the capsular polysaccharide of H. influenzae type b is greatly enhanced by the conjugation of the polysaccharide with diphtheria or tetanus toxoid, or with the outer-membrane protein of N. meningitidis. More recently, in attempts to improve efficacy, protein carriers that themselves induce a protective immune response against the pathogen have been conjugated to the capsular polysaccharide e.g. Panton-Valentine leucocidin conjugated to Staph. aureus capsular polysaccharide.