Recently, technologies for dispersed cultures of M. bovis BCG in synthetic media in small-scale bioreactors were developed.13 These cultures allow recording and adjusting of culture parameters and give rise to single bacilli with a high degree of
live bacteria. In mouse studies, bioreactor-grown M. bovis BCG exhibited slightly stronger replication and persistence than
the vaccine produced under the classical conditions. The protective efficacy in the mouse against challenge with M. tuberculosis was identical for both vaccine preparations. However, the novel manufacturing technique is unlikely to be implemented in
commercial production of the BCG vaccine against tuberculosis since such a change would require a clinical trial that may
take over a decade.
The production of the S. typhi Ty21a vaccine is based on a master and working seed lot system. During the production process
of the Ty21a vaccine, bacteria derived from working seed lot ampoules are inoculated in shake flask cultures, followed by
growth in medium-and large-scale bioreactors. Bacteria were harvested by centrifugation (Figure 1). For downstream-processing,
the bacteria are mixed with a stabilizer containing sucrose, ascorbic acid, and amino acids, and then lyophilized. The lyophilized
bacteria are subsequently mixed with lactose and magnesium stearate as excipients and filled into gelatine capsules that are
coated with an organic solution to render them resistant to dissolution in stomach acid. The enteric, coated capsules are
then packaged into blister packs for distribution. Each capsule contains 2–10 x 109 (2–6.8 x 109 in the US) lyophilized live bacteria and they are administered orally.14
Alternatively, a double chambered sachet formulation has been developed, with one sachet containing the lyophilized vaccine
and the other containing a bicarbonate buffer to neutralize stomach acidity. The contents of the two sachets are dissolved
in 100-mL water and ingested by the vaccinee. The production process is similar for CVD 103-HgR. However, for this vaccine,
only the double-chambered sachet formulation was developed, with 2–10 X 108 live bacteria per sachet for travellers from nonendemic regions and 2–10 X 109 for residents of endemic regions.
Figure 1. Ty21a vaccine production: During the production process of the Ty21a vaccine, bacteria derived from working seed
lot ampoules are inoculated in shake-flask cultures, followed by growth in medium-and large-scale bioreactors. The bacteria
are harvested by centrifugation. For downstream-processing, the bacteria are mixed with a stabilizer and lyophilized.
The release of commercial batches of both vaccines is based on microbiological and biochemical tests. The potency assay for
both vaccines relies on determining the live bacteria, and therefore, special attention has to be given to this assay. Another
critical test is determining the vaccine purity. Contaminating microorganisms may not be easily detected in a vaccine dose
containing more than one billion live vaccine bacteria, and hence, special purity assays needed to be developed. Finally,
the attenuated phenotype of the bacteria has to be demonstrated for each vaccine batch.
The manufacturing, quality-control, and release testing of all vaccines have to follow the guidelines issued by regulatory
authorities covering cGMP requirements for pharmaceuticals, biologicals, and vaccines. For example, Annex 2 of the PIC-guide
to good manufacturing practice for medicinal products, clearly specifies that dedicated facilities should be used for producing
the BCG vaccine.15 There are additional challenges posed by LBVs in comparison to other vaccines. When working with lyophilized live bacteria
in large quantities, special cleanroom design and monitoring procedures are required to maintain appropriate cleanroom conditions.
Also, relevant biosafety guidelines have to be followed for large-scale manufacturing of live bacteria.
Pharmacopoeia monographs are in place for the release testing of Ty21a and BCG.16,17 However, after internal testing and release by the vaccine manufacturers, each vaccine batch must undergo additional quality-control
testing by regulatory authorities before it can be commercialized.