20
Downstream
thirty seven
Viral vectors and viral vaccines play an
important role in current medical approaches.
Viral vectors like adenoviruses, adeno-
associated viruses, or retro-viruses are the
vehicles being developed for delivering genetic
material to the target cell in gene therapy. Viral
vaccines, such as attenuated or inactivated
rabies virus, influenza virus, or hepatitis virus
are powerful tools used to limit the number of
serious viral infections and pandemics.
Purifying human influenza virus
vaccine towards a generic strategy
H
igher safety demands by the
Food and Drug Administration
(FDA) and the European
Agency for Evaluation of Medicinal
Products (EMEA) to reduce side effects
are forcing improvements to the
production and purification processes for
viral vectors and vaccines. For example,
producers of influenza viral vaccines are
beginning to switch from egg cultivation
to mammalian cell culture systems.
Within the purification procedure, there
is a clear move away from traditional
purification methods like sucrose
gradient centrifugation towards
ultrafiltration and liquid chromatography
techniques. Here we describe a generic
strategy for the purification of viruses
and viral vaccines, exemplified by human influenza virus
vaccine, using filtration and chromatography techniques.
Purification strategy for
influenza virus vaccine
Human influenza virus particles,
like all viruses, are much larger in
dimension than other molecules
such as proteins, peptides,
glycoproteins, sugars, DNA and
RNA. This feature can easily be
used to separate viruses out of
fermentation broth. In principle
there are two methods to separate
molecules by size via membrane
separation techniques such as
ultrafiltration, and via chromato-
graphic separation techniques such
as size exclusion (gel filtration) in
group-separation mode. In the case
of influenza virus vaccine, both techniques have been
combined to develop a complete downstream process.
Cell culture supernatant with human influenza virus
Ultrafiltration
MidGeeTM cross flow filter cartridge, 750 NMWC
Ultra-/Diafiltration
MidGeeTM cross flow filter cartridge, 750 NMWC
Group Separation
SepharoseTM 6 Fast Flow
"Negative" AEX Chromatography
Q SepharoseTM 6 Fast Flow
Purified human influenza virus
Surface view illustration of influenza virus. The red spikes represent Haemagglutinin
and are arranged all over the surface, roughly equally spaced. The yellow box like
surface features represent Neuramindase. Each 4-fold assembly is a single tetramer
of Neuraminidase. The Neuraminidase tetramers are not distributed evenly over
the virus but cluster in groups. The green surface peeping through between the
spikes represents the viral envelope, which was derived from the host cell during
budding. Copyright Russell Kightley Media, www.rkm.com.au
