APR Sept/Oct 2023 - 69

CGT CIRCUIT
the viral vector which is dependent on both
infectivity of the particle and expression/
function of the transgene.
To determine
potency, infectious titer assays and cellbased
assay are employed that examine the
ability of the viral vector product to infect
cultured cells, express their transgene, and
show functionality.
utilized. There are multiple methods for the
detection
of adventitious
virus,
including
The ultimate readout
from these assays is dependent on the
transgene and can be assessed through a
variety of techniques including qPCR, ELISAs,
or cell reporter systems.
Quality Testing involves evaluating the
physicochemical
properties
of
the
viral
vector and determining the level of process
and product related impurities.
The
physicochemical properties evaluated usually
include appearance, pH, and osmolality.
Process related impurities includes residual
host cell protein that is usually determined by
ELISA and residual host cell DNA and plasmid
DNA that are analyzed using qPCR methods.
Other process-related impurities are residual
compounds that come from the host cell
growth media (i.e. antibiotics, raw materials)
or the purification procedure (i.e. detergents,
column leachables) and are evaluated using a
variety of techniques including ELISAs, liquid
chromatography, and mass spectrometry.
Assessment of key product-related impurities
includes determining the ratio of viral vector
particles that contain nucleic acid (full
capsids) to those that do not contain nucleic
acid (empty capsids) or that contain the
incorrect nucleic acid (illegitimate capsids).
For determining the ratio of full to empty viral
particles a variety of methods can be utilized
including liquid chromatography, analytical
ultracentrifugation,
electrophoresis,
and electron microscopy.
qPCR,
Illegitimate capsid
levels can be determined using qPCR or
sequencing methods.
Safety Testing
involves
the presence of any
determining
harmful microbial
contaminants including bacteria, molds,
fungi, mycoplasma, and adventitious viruses.
Furthermore, the viral vector product is
evaluated for the presence of bacterial
endotoxins as well as viral vector particles that
are replication competent.
any bacteria, mold, and fungi is determined
by a sterility test while for mycoplasma either
a culture method or qPCR method can be
PCR methods, ELISA methods, and electron
microscopy that detect specific viruses,
however, non-specific methods that look for
cytopathic effects in a cell-based assay may
also be employed. To detect for the presence
of replication competent viral vectors, a
permissive cell line is treated with the viral
vector and the presence of the sequences
unique to the viral vector detected using qPCR
or Southern blotting.
Containing Closure Integrity
Testing
(CCIT) and Extractable and Leachables
Testing. In addition to the testing described
above, viral vector drug products are also
evaluated for the integrity of the container
closure system and in later stage clinical
development any potential compounds
that could migrate from the container
closure system. There are many methods for
evaluating CCIT including dye ingress, high
voltage leak detection, oxygen headspace
and helium leak detection. Extractable and
leachable components are determined using
liquid and/or gas chromatography with mass
spectrometry detection.
Stability Testing is a critical component of
any drug development program and involves
analysis of the potency and quality attributes
of the product over time at the storage
temperature, accelerated temperatures, and
at stress temperatures. The goals of a stability
study are to determine the shelf life of the
product but also refine the attributes of the
product that are predictive of stability and
potency.
For viral vectors, stability studies
The presence of
often examine characteristics such as the
appearance, pH, content, structural features,
degree of aggregation, sterility, and potency.
Overall, viral vectors are complex biologics
that requires sophisticated testing throughout
the manufacturing process to ensure that
they are safe and effective for use in patients.
With many biopharma companies in the
race to release gene therapies to market, it is
important to choose a CRO with specialized
viral vector and other gene therapy expertise,
deep regulatory knowledge, ample capacity
and agility, and consistent, transparent
communication to support your viral-vector
product from development to market.
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APR Sept/Oct 2023

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