PROCESS DEVELOPMENT SCHEME FOR A CELL-CULTURE BASED VACCINE
Current technologies for vaccine manufacturing can help ensure quality and reduce time. Some of these technologies include
analytical methods such as surface plasmon resonance, upstream technologies such as microcarrier beads for adherent cell lines,
high-throughput screening methods, and downstream technologies such as membrane chromatography and cross-flow filtration.
Figure 1 outlines the schematic flow for complete vaccine-process development.
Figure 1: Process-development scheme for vaccine manufacturing.
Upstream process development
The productivity of large-scale cell culture can be increased either by scaling up to larger volumes with cell densities of
2–3 x 106 /mL, or by intensifying the process in smaller volumes but with higher cell densities (up to 2 x 108 cells/mL). When intensifying cell densities, more frequent media changes are needed and perfusion is eventually applied
Many alternative technologies are also available. Cross-linked dextran beads (i.e., microcarriers), for instance, provide
an extended surface and a stable environment for optimal cell growth. Microcarrier culture of anchorage-dependent or entrapped
cells reduces volume and thus belongs to the latter of the options cited above (8). The technique, in general, has many advantages
for the commercial manufacturer. It can be operated in batch or perfusion modes during cell culture and it is well-suited
to efficient process development and smooth scale-up. Washing and changing culture media just before viral infection is easier
because there is no virus inside and one does not have to follow additional precautions as required while handling a virus.
The reactors can also be modified to grow other organisms.
Downstream process development
As evident from Figure 1, the downstream processing and purification steps occupy a major portion of the vaccine-manufacturing
process. As a result, the use of robust and economical steps to develop and optimize parameters for purification is beneficial.
For example, in general, downstream steps are more expensive to carry out than upstream steps because of the use of chromatography
columns and membranes. The onus for generating a product that is absolutely free of pathogens and other contaminants such
as host cell protein (HCP), host cell DNA, or endotoxin, relies on downstream steps. If this cost escalates, the manufacturer
could run into business risks.
The current industry trend for purification of biological therapeutics, including vaccines, involves the use of membrane chromatography
to purify viruses as well as for polishing applications. Membrane chromatography offers many benefits compared with density
gradient ultracentrifugation, including the removal of HCPs, contaminating DNA and endotoxins. Membrane chromatography compared
with centrifugation offers many advantages (e.g., processing time is faster, virus yield is higher, cleaning validation is
not needed, the carbon footprint is smaller) (see Table I). This method can therefore bring greater effectiveness to the
key stages of bioprocessing.
Table I: Comparison between the use of membrane chromatography and density gradient ultracentrifugation for virus purification.
The implementation of membrane chromatography for virus purification, in place of density gradient ultracentrifugation, is
gaining prominence in many areas, even though the latter remains the gold standard among traditional vaccine manufacturers.
Membrane chromatography has been successfully used in multiple applications for virus purification. In a recent study, human
and equine influenza A virus in cell-culture supernatant (i.e., serum-free and serum-containing cultivation) was directly
adsorbed onto Sartobind Q 75 anion-exchanger, and eluted out by displacement with sodium chloride (up to 1.5 M, pH 7.0), which
resulted in average yields of 86% based on hemagglutinin activity (9). In another instance a virulent wild type NIA3 strain
of Pseudorabies, grown in porcine kidney epithelial (PK15) cell monolayers was purified using Sartobind S cation exchanger membranes (10).
In another study, Sartobind D membrane adsorber was used in a larger scale downstream process, for the purification of rotavirus
VLPs to a clinical grade at 46% global recovery yield, and with nearly 100% removal of host bulk DNA together with approximately
98% of HCPs (11). Sartobind D was also used for purification of recombinant baculoviruses that are widely used as vectors
for the production of recombinant proteins in insect cells (12).
Another robust technology is cross-flow filtration (using tangential-flow filters), which is best suited for higher solid
contents, more viscous feed solutions, or in cases where concentration, recovery, and purification of cells or target species
is desired. This technology is largely used for concentrating and washing feed streams before chromatography (3).
For vaccine manufacturing, particularly virus purification, fully scalable macrovoid-free hollow-fiber technology applied
to ultrafiltration and microfiltration offers great advantages in the virus-purification process due to its open porous structure.
The open-flow path design of hollow fibers gently processes cell suspensions and other particulate feed streams and reduces
shear forces, thereby maintaining the integrity of the virus. Hence, recovery rates of the target virus and overall process
economics are improved (13).
Figure 2: A generic process for egg-based vaccine manufacturing using platform technologies. SPF is specific pathogen free.
UF is ultrafiltration. DF is diafiltration.