Manufacturing of Conjugate Vaccines

The steps in the production of conjugate vaccines can be summarized as follows:

  • Protein production:  fermentation of host organism,
    collect, wash, homogenize, recover and purify protein
  • Polysaccharide production: fermentation of donor organism, disrupt cells, purify polysaccharide, trim by hydrolysis (acid, periodate)
  • Derivativization: make both components reactive
  • Conjugation: React components together
  • Purification: Remove unwanted compounds
  • Finish and fill: Concentrate to final strength, dialyse into appropriate solute, sterile fill as liquid or lyophilize

Each of these simple activities does require a number of very precise intermediate steps.

A large number of reagents are used to derivatize and conjugate these vaccines. While preparation and purification of the protein is a relatively straightforward procedure, preparing the saccharide components, adding, and then removing reagents is less so.

Purified bulk saccharides, when trimmed, have to be sorted into size classes to meet the specifications of the product. This can be done by size exclusion chromatography, but tends to result in larger volumes of dilute material, which needs to be concentrated by ultrafiltration. Many vaccines are therefore produced by ultrafiltration membrane sizing; the saccharide mixture is filtered through two or more different ultrafiltration membranes. For example, a 50 kiloDalton (KD) membrane may be used to exclude large contaminants, polysaccharide aggregates and larger polysaccharide molecules.

The filtrate is then passed over a 10KD membrane, which removes small molecules, salts and solvents that have been used in the process, leaving a range of oligosaccharides with a fairly tight size range “trapped” between these two membranes.

Upon derivativization, the unreacted species can also be flushed through the 10KD membrane and the treated oligosaccharides diafiltered into a solution appropriate for the subsequent conjugation reaction.

A similar process can be used to remove unwanted reagent from the protein. Once conjugation is completed, the conjugate can once again be passed over a small porosity ultrafiltration membrane to remove the unreacted conjugation chemicals, and once again concentrate or diafilter the conjugate into the final formulation strength and carrier fluid, before formulation with any adjuvants and preservatives that may be required.

For some vaccines, the product is multivalent – that is, it is comprised of mutltiple saccharide serotypes. In this case, each serotype is prepared and conjugated separately, and then the serotypes are mixed together in final formulation. Prevnar (Wyeth) is an example of such a vaccine, with seven different polysaccharides – serotypes 4, 6B, 9V, 14, 18C, 19F and 23F, representing the most frequently encountered. New vaccines are in development that address even more serotypes. This further complicates manufacturing, as each must be produced and controlled separately. This complexity is one barrier to transfer in a cost effective way of this type of vaccine to developing areas of the world that could use such vaccines, and who may not be able, without intervention of organizations such as WHO and the World Bank, to purchase such vaccines.

There is some simple equipment available, however, which can at least go some way to mitigating this cost and is, in fact, used in several vaccine manufacturers in the USA, India and Asia. For the ultrafiltration stages, flat sheet membrane cassettes are typically used with dedicated holders and systems for each stage of usage. For multivalent vaccines, and vaccines with many UF processing steps for even a single serotype, the cost of such multiple systems can be high. Such costs are more easily justified when producing large quantities of a given vaccine on a continuous basis. Disposable technologies allow the preparation of many of the reagents, sterile filtration, and compounding with little need for capital equipment. However, larger scale UF systems are not currently available as disposables. What is available are systems with plastic liners, so that only a single set of stainless steel tanks (which do not contact the liquids) is required, and numerous sets of liners used, each one dedicated to a single part of the process or even used as a disposable item.