Following the cell amplification phase, a viral production phase is initiated. In a

first cell culture stream, cells are infected for viral production with a working virus

seed stock, derived from the master virus bank according to pre-established process

parameters. A second uninfected cell culture stream is processed under similar

operating conditions and monitored in parallel to the cell culture viral production

stream as a control to demonstrate the absence of non-adventitious agents in the

whole cell-culture production process. Depending on the infection/replication ki-

netics, the virus bulk is harvested after days to weeks.

The virus bulk harvest is purified following pre-established downstream process

steps optimized and validated during the early process development phase and

involve multiple filtration/ultra-filtration or chromatographic steps highly de-

pending on the virus type, its structure, and stability. It is not uncommon to use

large-scale ultracentrifugation in the vaccine manufacturing process. For example,

hundreds of millions of influenza vaccine doses are produced using large-scale

ultracentrifugation.

The upstream, downstream, and analytical protocols are detailed in dedicated

core chapters in this textbook (Chapters 5 to 8) and examples are extensively

presented and discussed in the case study reports of cell-culture produced vaccines

and associated processes (Chapters 9 to 12). A special emphasis is placed on the

development and execution of different sets of assays for vaccine lot release. As an

example, single-radial immunodiffusion assay (SRID) (which is the only validated

assay approved by regulatory agencies for the release of influenza vaccine lots)

would require the preparation of a standardized antigen and specific polyclonal

antibody. The preparation of the antibody might take weeks to months, and it is

produced through immunization of animals, such as mice, rabbits, or sheep, col-

lecting the serum from animals after immunization with the specific antigen. The

overall process for preparation of these reagents is very time-consuming, taking

weeks to months and might delay the approval of a vaccine for eventual eva-

luation in humans. A report from the WHO [21] identified the timelines required

for the deployment of the SRID assay as one of the many reasons that made the

response to the 2009 H1N1 pandemic and the timely availability of vaccine in-

adequate. Although the vaccine was manufactured, delays due to unavailability of

standardized SRID reagents in a timely fashion were observed, raising significant

concerns on readiness to respond to pandemic situations. The search for alter-

native assays to SRID assays fueled significant research in this area underlining

the critical role of assays in vaccine development and manufacturing. Ideally,

when the mechanism of action of the vaccine is known, a potency assay that is

predictive of the clinical response should be available, serving as a “clinical

correlate of protection.” The correlation of protection is the minimum immune

response that has been demonstrated to provide protection against the infectious

disease. It is estimated by experts in the vaccine field that 70% of the time re-

quired for vaccine manufacturing is dedicated to quality control and represents

several hundreds of analytical tests.

Specific to the vaccine field regulation, virus bulk batches require formal release by

agents of the territory competent regulatory agency such as Health Canada, FDA, or

12

Bioprocessing of Viral Vaccines