Facing the Challenges in Vaccine Upstream Bioprocessing - 10

Facing the Challenges in Vaccine Upstream Bioprocessing * Scalable Production of AAV Vectors

However, achieving preclinical efficacy testing,
especially in large animal models and toxicology
studies, requires vector quantities that simply
cannot be produced in a laboratory setting or in
most research-grade vector core facilities. Current
methods for transfection require use of adherent
HEK 293 cell cultures, expanded by preparing
multiple culture plates. Ideally, a single large-scale
suspension culture would be a replacement for
multiple culture plates.
In this tutorial, we examine some of the currently
available schemes used in generating rAAV from
suspension cultures, and describe what it takes to
achieve scalable rAAV production.

Scalable Production
Two basic systems for growing cells in culture
exist: monolayers on an artificial substrate
(i.e., adherent culture) and free-floating in
culture medium (i.e., suspension culture).
rAAV vector production uses a triple transfection
method performed in adherent HEK 293 cells,
which is the most common and reliable method
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(Lock et al., 2010), albeit resource intensive.
Due to its scalability and cost, rAAV cell suspensions are more desirable.
To simplify scalability and dramatically decrease
operational costs and capital investments, use
of bioreactors provides process simplification,
from pre-culture to final product. Two examples
are the iCELLis from Pall Life Sciences, designed
for adherent cell culture applications, and the
WAVE Bioreactor from GE Healthcare Life
Sciences, ready for batch culture, fed-batch
culture, perfusion culture, and cultivation of
adherent cells.
Both are designed for convenient handling
and control of cell cultures up to 25 L. Both
enable rapid and scalable rAAV production.
Recently, Grieger et al. (2016) showed suspension
HEK293 cell lines generated greater than 1×105
vector genome-containing particles (vg)/cell
or greater than 1×1014 vg/L of cell culture when
harvested 48 hours post-transfection, a protocol
developed and used to successfully manufacture
GMP Phase I clinical AAV vectors.

Large-scale productions require consistent and
reproducible. AAV produced for clinical uses
must be thoroughly analyzed to identify the
main purity, potency, safety, and stability factors
described below.

Purity
Empty capsids typically take 50-95% of the total
AAV particles generated in cell culture, depending
on specific serotypes and protocols used. Empty
capsids may solicit deleterious immune response
against AAV (Zaiss and Muruve, 2005). It is desirable they be minimized during production and
removed during purification.
AAV empty capsids are composed of an AAV
capsid shell identical to that of the desired
product, but lacking a nucleic acid molecule
packaged within. Gradient ultracentrifugation
using iodixanol is effective in separating empty
capsids. Assessment and measurement can be
done by either electron microscopy or A260/A280
spectrometry. It may be difficult to distinguish
AAV capsids containing small fragments of DNA

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Facing the Challenges in Vaccine Upstream Bioprocessing

Table of Contents for the Digital Edition of Facing the Challenges in Vaccine Upstream Bioprocessing