Human Gene Therapy - April 2023 - 269

SHORT COURSE IN AAV MANUFACTURING
269
amount of diafiltration buffer fed relative to the product
retentate in the system-and the extent of buffer exchange
determined. The data gathered are then discussed in the
context of scaling up to manufacturing.
Analytical methods section
The analytical portion of the courses comprises 3.5 h of
lectures, 2.75 h of hands-on laboratories, and 1.25 h of data
analysis discussion and workshop, as detailed (Table 1).
This format provides course participants with a theoretical
framework for understanding gene therapy analytical
methods and with hands-on experience generating and
analyzing data from real AAV samples.
The initial lecture covers where, in a biopharmaceutical
process, analytical testing is taking place and what aspects
of AAV are to be analyzed to evaluate product identity,
purity, safety, and potency. This lecture also covers the
International Conference on Harmonization (ICH) guidelines
for assay validation. The next two lectures focus on
the specifics of assays for AAV titer and SDS-PAGE for
AAV purity, which participants conduct during the handson
portion of the course. The lecture on AAV titer gives
particular attention to qPCR because it is an important
dosing assay that includes technical aspects of the assay
and considerations for sample preparation for AAV viral
genome quantification. There is also a discussion of
droplet digital PCR (ddPCR) in this lecture, and a comparison
between ddPCR and qPCR. The final lecture focuses
on assays for safety, purity, and potency.
This lecture addresses many of the complicated
product-related impurities that are unique to gene therapy
and pose significant analytical challenges, including
empty capsids, encapsidated host or plasmid DNA, and
noninfectious particles. The goal of this portion of the
course is to ensure that participants have a foundation in
analytical method development, unique analytical aspects
of AAV, and an overview of the assays used to characterize
and assess the quality of AAV. To date, students do
not perform potency assays or other advanced characterization
techniques (transmission electron microscopy,
analytical ultracentrifugation, etc.); however, BTEC offers
a 3-day short course entitled Analytical Methods for
Gene Therapy Vector Characterization and Testing in
which these methods are covered in detail.
To prepare course participants not only for some of the
unique assays in gene therapy but also more broadly for
analytical testing of biopharmaceuticals, the hands-on
portion of the analytical section seeks to highlight method
validation principles using gene therapy assays. The participants
receive hands-on training in two assays: qPCR
and SDS-PAGE. These methods were chosen because they
cover different aspects of AAV analysis (quantity and
purity), and they can be completed in sufficient time to
allow for data analysis. While these methods are currently
used, as newer methods are adopted, such as ddPCR and
capillary electrophoresis, the newer methods will be incorporated
into the course.
In the qPCR laboratory, participant groups prepare
AAV-containing samples to evaluate DNase-treatment
effectiveness, linearity, intermediate precision, and matrix
effects. These aspects were chosen because they teach
important lessons in sample preparation, assay development
and optimization, and assay validation. A similar
strategy is utilized during the SDS-PAGE laboratory,
where students prepare samples and run SDS-PAGE to
evaluate validation concepts such as linearity, precision,
and accuracy.
The samples and qPCR plate prepared by the students in
the laboratory are run on an ABI 7500 Fast qPCR instrument
while students attend the lecture. The data are retrieved
and quickly analyzed by the instructor for a data
analysis laboratory in the afternoon. In this laboratory, the
students begin by evaluating the aspects of system suitability
in qPCR. This includes assessing the standard curve
to determine amplification efficiency, linearity, and contamination
in the no-template control. To prepare the
qPCR plate, all samples are loaded in triplicate, and the
relative standard deviation of the triplicate is calculated.
This allows for a discussion of the importance of the pipetting
technique on assay precision. The topics discussed
in the data analysis for each laboratory group are presented
(Table 3). The SDS-PAGE data are also discussed, with
particular attention to the pharmacopeial considerations in
assessing SDS-PAGE.
PARTICIPANTS AND PARTICIPANT PROFILE
Since the first Hands-On cGMP Biomanufacturing of
Vectors for Gene Therapy course in March 2019, seven
offerings have been held with nearly 80 participants.
Table 3. Description of quantitative polymerase chain
reaction laboratory groups and topics covered in data
analysis laboratory
Sample
Laboratory
Group
1
2
3
or Validation
Aspect Evaluated
Matrix effects
DNase I treatment
conditions
Data Analysis Topics
Precision of triplicate samples
Impact of PCR inhibitors in matrices
Calculation of sample recovery from spike-in
Precision of triplicate samples
Impact of DNase I conditions
Calculation of DNase I efficiency
Linearity, LOD, LOQ Precision of triplicate samples
Differences between plasmid standard
curve and AAV
Appropriate operating range and use
of standard curves for quantitation
4
Intermediate
precision
Precision of the triplicate samples
Intermediate precision in comparing operators
Discussion of accuracy
LOD, limit of detection; LOQ, limit of quantitation.
Human Gene Therapy - April 2023

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