Facing the Challenges in Vaccine Upstream Bioprocessing - 13

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

Table(con't). Method of removal and measurement of AAV vector production impurities
AAV Vector Production
Impurity

Method of Removal

Method of Measurement

qPCR using amplicons to specific sequences
of concern (e.g., E1A)
Encapsidated helper
component DNA
(nuclease-resistant)
replication-competent AAV
noninfectious AAV particles

qPCR using amplicons for helper backbone
sequences

Other, including aggregated,
degraded, and oxidized AAV
vectors

Ad-dependent amplification; Ad-dependent
infectivity in susceptible cells; various, including
size-exclusion chromatography, dynamic light
scattering, electrophoresis

Utilizing the split vector system, which exploits
head-to-tail concatamerization formation, has
been developed to circumvent AAV small packaging capacity. Two main approaches include
trans-splicing and homologous recombination
methods; both depend upon recombination
between two vector genomes, with each genome
encoding approximately half the transgene,
within the same cell to achieve gene expression.
ViGene has developed a trans-splicing system
that can expand the payload to 8 kb.

13

| GENengnews.com

We setup a screen for a more efficient transsplicing system. Here, the GFP reporter was split
and cloned into two AAV vectors. Based on GFP
expression, after co-transfection in HEK-293 cells,
we scored expression efficiency of the combination of different trans-splicing elements and
included the selection of a splicing donor and
acceptor sequence, and the annealing sequence in
the intron. Our best vector generated about 70%
expression compared to single vector (Figure 1).

http://www.GENengnews.com

Facing the Challenges in Vaccine Upstream Bioprocessing

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