PharmaceuticalOutsourcingQ42020 - 41


significant improvements over tissue-derived cells. For cancer therapies, scientists are trying to turn autologous chimeric antigen receptor T cell (CAR-T) approaches into allogeneic treatments by silencing proteins that mediate host-vs-graft immune responses. Some
examples of these adaptations are using CAR-T cells with silenced T
cell receptors3 or switching from T cells to less immunogenic natural
killer (NK) cells.4
Owing to its targeted and personalized nature, ex vivo gene therapy
generally has an improved safety profile. However, cell retrieval and in
vitro manipulation can be a costly and intricate process. In vivo gene
therapy is potentially more straightforward; but faces challenges such
as toxicity or the induction of immune responses. Clinically the most
frequently observed toxicities for in vivo gene therapies are hepatic
toxicity and cytokine release syndrome (CRS).5,6 In addition, genomeediting technology is being explored as a strategy to advance the
scientific engineering of the CAR. Compared with conventional
CAR-T cells, CRISPR/Cas9-edited CAR-T cells have shown an enhanced
potency as well as delayed differentiation and exhaustion.7,8

Conceptualizing a Bioanalytical Package
for CGT
Due to the unique delivery and therapeutic mechanisms employed
in cell and gene therapeutic products, nontraditional and
comprehensive bioanalytical testing strategies must be developed to
demonstrate the CGT's safety and efficacy. The bioanalytical package
for a CGT must be able to determine that the therapeutic protein is
present and functional at the site of action, sometimes systemically.
Because many gene therapies are delivered by a vector, usually a
virus, the existence and emergence of anti-vector antibodies also
should be monitored. Special consideration also should be given
to pre-existing and emergent immunogenicity to the therapeutic
protein, particularly in heavily pretreated disease populations. For
example, patients with enzymatic deficiencies that have been treated
with enzyme replacement therapy for long periods of time may have
pre-existing antibodies to the therapeutic enzyme9 Additionally, the
host immune system may interpret the newly expressed protein as
foreign in deficient patients and mount an immune response.

Approaches for Measuring Therapeutic
Protein Levels
Creating a pharmacokinetic (PK) profile of a CGT is a complicated
undertaking. The bioanalytical strategy for therapeutic protein
pharmacokinetics or exposure does not follow the normal
pharmacokinetic model that is used for traditional small and large
molecule drugs. For example, the translated products of transgenes
only may be expressed at the site of action, so the bioanalytical strategy
must be able to detect the expressed protein at the site of action and/
or systemically, depending on the specific therapeutic. Additionally,
since the protein is constitutively expressed, exposure does not
follow the typical elimination phase. Instead, exposure is monitored
for persistent expression of the functional replacement protein or
enzyme. Adsorption, distribution and metabolism also have unique
profiles from conventional biologics and small molecules. There are |


additional considerations for the delivery vehicle in many cases, e.g.,
for viral delivery vectors the vector copy number, biodistribution and
functional gene insertion must be monitored.
Determining the ideal matrix to use to identify changes in the
level of the therapeutic protein is an important component of the
bioanalytical program. This may require expressed protein monitoring
in multiple matrices (e.g., serum and cerebrospinal fluid) and may
even necessitate monitoring of protein levels in human tissues from
biopsy of accessible sites such as skin or muscle. With the advent of
precision medicine, these procedures also must be minimally invasive
for the patient in question, particularly if a repeat biopsy is required.
For gene therapies that result in expression of enzymes, the instability
of proteins in collected matrices may require special considerations
for sample handling, including rapid collection at the clinical site,
reduction of freeze-thaws, addition of stabilizing excipients to
collection tubes, minimization of thaw time and thawing on ice.
Therefore, investigation of the impact of upstream processing steps
prior to arrival of samples at the bioanalytical lab may increase in
importance as compared to more standard pharmacokinetic testing
of antibody therapeutics in patient serum samples.
In the case of gene therapies targeted to rare disease applications,
the patients at enrollment may have little to no protein expression
and after treatment the protein may be many orders of magnitude
higher. This necessitates using a method that is highly sensitive,
sometimes to the pg/mL range, and has a wide dynamic range
to fully characterize the patient response to treatment. The
preferred methodology for protein PK assays is the immunoassay
format. Immunoassays are simple to design, have readily available
reagents and can be easily adapted to be automated to reduce
operational changes over time and to enable high-throughput
analysis. Immunoassay design will need to consider whether
the antibody pairs can properly discriminate between truncated
or other nonfunctional proteins that may be present, for which
options such as enzyme-linked immunosorbent assay (ELISA), meso
scale discovery electrochemiluminescence (MSD-ECL) and Luminex
assays are all available. Ultra-sensitive immunoassay platforms can
be evaluated when designing a protein PK method to maximize
sensitivity and dynamic range.
Many gene therapies involve dosing of the components to express
proteins with similar endogenous counterparts, which adds
complexity to protein PK immunoassay design. The presence
of endogenous material can complicate assay design: the
calibrator must be prepared in matrix that has been stripped of
the endogenous protein, a laborious and variable process, or the
calibrator must be prepared in buffer/assay diluent. One common
tactic is to utilize a standard curve made in buffer/assay diluent with
a corresponding recombinant protein, produced either in-house or
obtained from a commercial source. To confirm the appropriateness
of the recombinant protein as a calibration material, specific assay
validation experiments are recommended. The assay design strategy
also should ensure that the capture and detection antibodies used
are able to suitably detect both the recombinant and endogenous
proteins. An assessment of endogenous quality control sample
(QC) precision should be performed to ensure that the assay has
| October/November/December 2020


Table of Contents for the Digital Edition of PharmaceuticalOutsourcingQ42020

Editor's Message
Editorial Advisory Board
CN Perspectives
Social Media Connections
Insider Insight - Price
Insider Insight - Ventura
Contract Manufacturing
Supply Chain
Contract Manufacturing
Interview with Yourway
Supply Chain
Clinical Trials
Supply Chain
Analytical Testing
Supply Chain
Clinical Trials
Analytical Testing
Horizon Lines
Industry News
Advertiser's Index
PharmaceuticalOutsourcingQ42020 - Cover1
PharmaceuticalOutsourcingQ42020 - Cover2
PharmaceuticalOutsourcingQ42020 - 1
PharmaceuticalOutsourcingQ42020 - Editor's Message
PharmaceuticalOutsourcingQ42020 - 3
PharmaceuticalOutsourcingQ42020 - 4
PharmaceuticalOutsourcingQ42020 - 5
PharmaceuticalOutsourcingQ42020 - Editorial Advisory Board
PharmaceuticalOutsourcingQ42020 - 7
PharmaceuticalOutsourcingQ42020 - CN Perspectives
PharmaceuticalOutsourcingQ42020 - Social Media Connections
PharmaceuticalOutsourcingQ42020 - Insider Insight - Price
PharmaceuticalOutsourcingQ42020 - 11
PharmaceuticalOutsourcingQ42020 - Insider Insight - Ventura
PharmaceuticalOutsourcingQ42020 - 13
PharmaceuticalOutsourcingQ42020 - Contract Manufacturing
PharmaceuticalOutsourcingQ42020 - 15
PharmaceuticalOutsourcingQ42020 - 16
PharmaceuticalOutsourcingQ42020 - 17
PharmaceuticalOutsourcingQ42020 - Supply Chain
PharmaceuticalOutsourcingQ42020 - 19
PharmaceuticalOutsourcingQ42020 - Contract Manufacturing
PharmaceuticalOutsourcingQ42020 - 21
PharmaceuticalOutsourcingQ42020 - Interview with Yourway
PharmaceuticalOutsourcingQ42020 - 23
PharmaceuticalOutsourcingQ42020 - Supply Chain
PharmaceuticalOutsourcingQ42020 - 25
PharmaceuticalOutsourcingQ42020 - 26
PharmaceuticalOutsourcingQ42020 - 27
PharmaceuticalOutsourcingQ42020 - 28
PharmaceuticalOutsourcingQ42020 - 29
PharmaceuticalOutsourcingQ42020 - Clinical Trials
PharmaceuticalOutsourcingQ42020 - 31
PharmaceuticalOutsourcingQ42020 - 32
PharmaceuticalOutsourcingQ42020 - Roundtable
PharmaceuticalOutsourcingQ42020 - 34
PharmaceuticalOutsourcingQ42020 - 35
PharmaceuticalOutsourcingQ42020 - Supply Chain
PharmaceuticalOutsourcingQ42020 - 37
PharmaceuticalOutsourcingQ42020 - 38
PharmaceuticalOutsourcingQ42020 - 39
PharmaceuticalOutsourcingQ42020 - Analytical Testing
PharmaceuticalOutsourcingQ42020 - 41
PharmaceuticalOutsourcingQ42020 - 42
PharmaceuticalOutsourcingQ42020 - 43
PharmaceuticalOutsourcingQ42020 - Supply Chain
PharmaceuticalOutsourcingQ42020 - 45
PharmaceuticalOutsourcingQ42020 - 46
PharmaceuticalOutsourcingQ42020 - 47
PharmaceuticalOutsourcingQ42020 - Clinical Trials
PharmaceuticalOutsourcingQ42020 - 49
PharmaceuticalOutsourcingQ42020 - 50
PharmaceuticalOutsourcingQ42020 - Analytical Testing
PharmaceuticalOutsourcingQ42020 - 52
PharmaceuticalOutsourcingQ42020 - 53
PharmaceuticalOutsourcingQ42020 - Horizon Lines
PharmaceuticalOutsourcingQ42020 - 55
PharmaceuticalOutsourcingQ42020 - 56
PharmaceuticalOutsourcingQ42020 - 57
PharmaceuticalOutsourcingQ42020 - Industry News
PharmaceuticalOutsourcingQ42020 - 59
PharmaceuticalOutsourcingQ42020 - 60
PharmaceuticalOutsourcingQ42020 - 61
PharmaceuticalOutsourcingQ42020 - 62
PharmaceuticalOutsourcingQ42020 - 63
PharmaceuticalOutsourcingQ42020 - Advertiser's Index
PharmaceuticalOutsourcingQ42020 - Cover3
PharmaceuticalOutsourcingQ42020 - Cover4