PharmaceuticalOutsourcingQ42020 - 42

ANALYTICAL TESTING

acceptable precision for recombinant and endogenous material.
To assess the parallelism of the recombinant and endogenous
material, endogenous QCs are prepared from samples from healthy
individuals and are analyzed at multiple dilutions to determine
which dilutions return consistent (between dilution) results. When
endogenous protein levels are low in comparison to the calibration
range, calibrator protein can be added to endogenous QCs to assess
the accuracy of the full quantitation range of the calibration curve
in matrix. Alternatively, QC samples can be prepared by adding
recombinant protein to the assay buffer to assess calibrator curve
accuracy. An additional endogenous QC sample can be added in
this case to represent actual study samples.
In addition to determining the presence of protein, the function
of the protein also must be assessed in the case of gene therapy
applications. The gold standard for determining drug efficacy is
clinical outcome, but a comprehensive bioanalytical package also
should inform on the mechanism of action of the therapeutic. In the
case of many gene therapies, this is accomplished by using pseudoPK/PD (pharmacodynamics) functional assays, e.g., enzyme activity
assays or bioassays. Functional assays are most informative when
paired with protein quantification assays because functional assays
may have greater assay variability and reduced dynamic ranges.
These factors can reduce the granularity of the data produced
and may reduce the ability to detect subtle differences between
treatment groups. Additionally, enzymatic activity is often more
prone to instability in patient samples, as compared to protein
levels. Enzymatic assays can be validated with a similar approach to
the endogenous QC strategy described previously and should use a
mixture of recombinant enzyme and/or endogenous QCs to monitor
potential shifts of the assay performance from day to day due to
environmental conditions.10
In the case of cell therapies like CAR-T, options for quantitating the
number of therapeutic cells in the body include flow cytometry and
quantitative polymerase chain reaction (qPCR). Recently, digital PCR
(dPCR),11 including droplet digital PCR (ddPCR),12 has been used
for patient samples to quantitatively assess CAR-T levels with high
sensitivity and precision. These methodologies allow for assessment
of persistence of CAR-T therapeutics over time. Although dPCR
and ddPCR report absolute values and do not rely on calibrators or
standards for quantitation, the lack of a standard CAR-T reference
material has complicated analysis for cell-based therapies.13
Assays using qPCR- and ddPCR-based technologies have been
successfully used to monitor both biodistribution and viral shedding
in CGT therapies.14 These assays allow for the detection and
quantification of viral insertion and integration within the tissues of
the target organism or patient, resulting in a vector copy number
(VCN), which is the average number of integrated copies of the target
gene within each diploid cell. These integration events risk aberrant
inactivation of necessary genes or expression of unwanted genes.
To safely monitor the integration of virus within a study, integration
site analysis (ISA) is typically performed using an unbiased approach
through next-generation sequencing (NGS).15 ISA both confirms
the presence of the target gene within the cell or tissue of interest
and identifies the location of the integration event. Together, these
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strategies can monitor the quantity and location of a therapeutic
driven by lentiviral and AAV vectors.

Additional Assessments:
Safety and Immunogenicity
Detection of introduced or newly expressed proteins or cells is
only one component of the overall bioanalytical package for CGTs.
Depending on the specific product and its applications, safety and
immunogenicity also will need to be assessed as part of the clinical
bioanalytical package.
For CAR-T cell therapies, a biomarker-based approach is important
to monitor safety risks. For example, patients receiving CAR-T cells
are at risk of CRS (cytokine release syndrome), an overreaction of the
immune system. The use of assays to detect C-reactive protein, as
well as inflammatory cytokines, can monitor for CRS development.
These assays also can determine the risk of CRS development in
CAR-T patients.16
Immunogenicity assessments also are essential to CGT programs. In
addition to the standard anti-drug antibody (ADA) immunogenicity
screening, pre-existing antibodies to the vector and expressed
protein also must be characterized before treatment initiation and
throughout the study. The viral vectors commonly used for CGT are
adeno-associated viruses (AAV), adenoviruses (AdV) and lentiviruses
(LV). Due to the widespread natural occurrence of these viruses,
patient immunogenicity to the viral vector must be assessed prior
to patient enrollment to ensure the therapeutic can be delivered
to the target site and to assure a therapeutic benefit.17,18 Many CGT
studies include viral vector immunogenicity screening as part of
their inclusion screening process, so patients with high levels of
pre-existing antibodies may be excluded from the study or have
their dose adjusted or supplemented with empty capsid to ensure
target delivery. For example, cell-based assays are regularly used for
assessing the presence of neutralizing antibodies to AAV delivery
vectors. Viral immunogenicity also must be monitored during the
conduct of the study to determine if adverse events or low efficacy is
due to emergent immunogenicity.
Viral immunogenicity assays, monitoring B-cell mediated immunogenicity, usually have a format similar to ADA immunogenicity assays;
they generally are bridging or sandwich immunoassay formats. The
challenges of designing a viral immunogenicity assay include reagent
acquisition and viral strain cross-reactivity. Reagents usually can be
commercially obtained, but nonstandard viral strains and a positive control antibody with sufficient sensitivity and specificity may
need to be custom produced since they may be difficult to obtain.
Additionally, viral vectors with a reporter gene insert (i.e., luciferase)
will be required for Nab assays. It should be noted that poor transfection efficiency resulting in a high degree of empty capsids and subsequent poor reporter gene expression can have severe negative effects
on the precision and sensitivity of AAV-based Nab assays. Sponsors
should investigate the availability of reagents early in the drug development process and begin contracting or producing high-quality
reagents early on. The presence of pre-existing AAV antibodies in the
population caused by environmental exposure to related viruses can
| October/November/December 2020

PharmaceuticalOutsourcingQ42020

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