Facing the Challenges in Vaccine Upstream Bioprocessing - 11

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

not readily distinguished from completely empty
capsids using density centrifugation or electron
microscopy.

dual-labeled hybridizable probe for detection
and quantification of amplified DNA junction
sequence.

Helper virus-dependent replication-competent
AAV (rcAAV), also referred to as "wild-type" or
"pseudo-wild-type" AAV, is an AAV capsid particle
containing AAV rep and cap flanked by ITR. This
type of AAV (rcAAV) is able to replicate in the presence of a helper virus.

rcAAV DNA sequence titer is calculated by direct
comparison to the fluorescent signal generated
from known plasmid dilution bearing the same
DNA sequence. A positive signal indicates an intact
left IRT-rep gene junction has been detected and
amplified, representing the maximum possible
rcAAV contamination level present in the rAAV
vector sample being analyzed. It does not indicate whether the DNA sequence is infectious or
capable of helper-virus assisted replication.

Though wild-type AAV is unable to replicate
autonomously and requires co-infection with
helper viruses, such as adenovirus, the expression of AAV rep or cap from rcAAV present in an
AAV vector increases the risk of immunotoxicity in
vector-transduced tissues. Replication competent
rcAAV is a rare (<10-8) and yet deleterious event.
To assess rcAAV generation, target DNA sequence
spans left AAV2 ITR D-Sequence and AAV2 rep
sequence. An intact left AAV ITR-rep gene junction
is a requisite feature for AAV replication to occur
in vivo in the presence of a helper virus. The assay
employs sequence-specific PCR primers and a
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Potency
High potency of AAV vectors is achieved by carefully selecting and isolating full capsids. Physical
and functional titers can be measured to assess
the actual potency of AAV production. Physical
titer measures the encapsidated AAV vector
genome, a key mediator and indicator of therapeutic effect. Measurement of vector genomes by
quantitative real-time PCR is the closest physical

indicator of rAAV vectors. Functional titer is
established by measuring transgene protein
expression in a dose-dependent manner,
following transduction into appropriate cell lines.

Safety
Safety concerns comprise infectious agents
used to generate AAV vectors. Mechanisms
to inactivate infectious viruses include heat
inactivation of adenoviruses and detergent
inactivation of enveloped viruses. A complete
list of product release tests can include adventitious virus tests of porcine, canine, and bovine
viruses (Table).

Payload Increase
Developing viral vector comes with key features
scientists strive for, including large payload
capacity. With rAAV, the limited packaging
capacity precludes the design of vectors for
the treatment of diseases associated with larger
genes; AAV has a packaging capacity of up to
4.5 kb for packaging foreign 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

Contents
Facing the Challenges in Vaccine Upstream Bioprocessing - 1
Facing the Challenges in Vaccine Upstream Bioprocessing - 2
Facing the Challenges in Vaccine Upstream Bioprocessing - 3
Facing the Challenges in Vaccine Upstream Bioprocessing - Contents
Facing the Challenges in Vaccine Upstream Bioprocessing - 5
Facing the Challenges in Vaccine Upstream Bioprocessing - 6
Facing the Challenges in Vaccine Upstream Bioprocessing - 7
Facing the Challenges in Vaccine Upstream Bioprocessing - 8
Facing the Challenges in Vaccine Upstream Bioprocessing - 9
Facing the Challenges in Vaccine Upstream Bioprocessing - 10
Facing the Challenges in Vaccine Upstream Bioprocessing - 11
Facing the Challenges in Vaccine Upstream Bioprocessing - 12
Facing the Challenges in Vaccine Upstream Bioprocessing - 13
Facing the Challenges in Vaccine Upstream Bioprocessing - 14
Facing the Challenges in Vaccine Upstream Bioprocessing - 15
Facing the Challenges in Vaccine Upstream Bioprocessing - 16
Facing the Challenges in Vaccine Upstream Bioprocessing - 17
Facing the Challenges in Vaccine Upstream Bioprocessing - 18
Facing the Challenges in Vaccine Upstream Bioprocessing - 19
Facing the Challenges in Vaccine Upstream Bioprocessing - 20
Facing the Challenges in Vaccine Upstream Bioprocessing - 21
Facing the Challenges in Vaccine Upstream Bioprocessing - 22
Facing the Challenges in Vaccine Upstream Bioprocessing - 23
Facing the Challenges in Vaccine Upstream Bioprocessing - 24
Facing the Challenges in Vaccine Upstream Bioprocessing - 25
Facing the Challenges in Vaccine Upstream Bioprocessing - 26
Facing the Challenges in Vaccine Upstream Bioprocessing - 27
Facing the Challenges in Vaccine Upstream Bioprocessing - 28
Facing the Challenges in Vaccine Upstream Bioprocessing - 29
Facing the Challenges in Vaccine Upstream Bioprocessing - 30
Facing the Challenges in Vaccine Upstream Bioprocessing - 31
Facing the Challenges in Vaccine Upstream Bioprocessing - 32
Facing the Challenges in Vaccine Upstream Bioprocessing - 33
Facing the Challenges in Vaccine Upstream Bioprocessing - 34
Facing the Challenges in Vaccine Upstream Bioprocessing - 35
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