Therapeutic Development

Adeno-Associated Virus (AAV) Gene Therapy
Applications and Solutions Guide

Visit bio-rad.com/GeneTherapy for more information.

AAV Gene Therapy
Key Application Map: Discovery to Release

Discovery
Viral Titer

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and services that advance scientific discovery and improve healthcare.
The journey of every therapeutic is unique. We offer a suite ofcustomizable products and services that ensure efficiency,
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and our experienced global support team is available to provide
technical and regulatory audit support.

Characterization
PotencyPreclinical and Clinical Research
Viral Titer
Characterization
Potency
Immunogenicity
Biodistribution and Viral Shedding

Process Development
Viral Titer
Characterization
Potency
Method Development
Residual Contaminants and Purity

Manufacturing and QC
Viral Titer
Characterization
Potency
Residual Contaminants and Purity

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Rapid Results — Real-Time PCR
In the early development of AAV gene therapies, sensitivity, dynamic range, and time
to results are often criteria in developing initial titer assays.

AAV VIRAL TITER
Accurately measuring viral titer is a key metric throughout genetherapy bioprocess development, whether you are in the early stage,
scaling production, or running assays for quality control (QC) release.
However, the stages of development may require different levels ofanalytical rigor or throughput.
Additional challenges arise due to AAV inverted terminal repeat(ITR) secondary structure and the difficulty of achieving acceptable
amplification efficiency for use with traditional quantification methods.
Regardless of your challenge or development stage, we havecomprehensive PCR technology solutions to address your needs.

“ddPCR is the gold standard method
to titer [AAV vector genomes].”(Alliance for Regenerative Medicine, 2021)

When screening production cell lines or in process development, a large number ofsamples are generated requiring quick results to increase productivity and advance tothe next stage.
Real-Time PCR (qPCR) has been established to provide the speed, throughput, andsensitivity required when these needs are most relevant.

CFX Opus Real-Time PCR System

QX ONE™ Droplet Digital™ PCR (ddPCR™) System

Increasing Precision — Droplet Digital PCR
As AAV therapies progress through development, the need for validated and highly
reproducible assays increases. Also, achieving acceptable amplification efficiency using
real-time PCR can be difficult due to the AAV genome structure, potentially leading toimprecise viral genome measurements. The endpoint PCR amplification used by ddPCR
technology enables high accuracy and precision without the requirement for perfectamplification efficiency.
Bio-Rad’s family of qPCR and ddPCR instruments and validated assays provides acomplete and scalable method to implement viral titer determination and characterizationfrom discovery to QC.

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Capsid Integrity and Serotype Identity

VIRAL CHARACTERIZATION
Across the gene therapy development process, understanding
the consistency of iterations of your AAV gene therapy is essential.
Start with reliable tools to measure the physical properties ofyour viral vector, including capsid integrity, serotype identity,
and genome integrity.

Stain-Free western blotting and SDS-PAGE
are powerful tools to confirm serotype
identity and capsid protein ratios, while
Droplet Digital PCR enables high accuracy
in assessing the AAV genome integrity.

Routine characterization of AAV vectors includes analyzing
their vector capsid protein ratios and validating theserotype identity. As more recombinant AAV vectors target
different tissue types, it is important to validate thesecharacteristics through development and into production.
Our Stain-Free technology offers the fastest method forchecking capsid protein ratios to verify that the AAV viralparticles are intact, and ChemiDoc™ MP Imaging Systemprovides best-in-class sensitivity and flexibility across afull set of imaging methods.

ChemiDoc MP Imaging System

Genome Integrity
One common challenge is assessing the completenessof incorporated DNA packaging in vectors. By targeting
multiple distinct regions of the viral genome withmultiplex ddPCR technology, AAV vector genome
integrity can be evaluated with high accuracy andprecision. Custom ddPCR assays can be created withthe ddPCR assay design tool, allowing rapid evaluationof novel AAV vectors.

QX600™ Droplet Digital PCR System

This in-depth characterization can be used to optimize
manufacturing and maximize potency.

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Target Persistence and Stability

POTENCY
Potency tests establish the quality, stability, and strength of the
product. Although regulatory agencies have not determined apreferred set of assays, multiple techniques are typically needed
to provide complementary and accurate data.
This unique and product-dependent combination of assays is often
developed from initial work performed in early-stage discovery andrefined in preclinical and clinical research with the purpose of being
used as lot-release testing upon approval.

“This one-step RT-ddPCR method simplifies
the workflow, allows for duplexing reactions,and enables absolute quantification oftranscripts without standard materials.”(Clarner et al. 2021)

Verifying that an AAV vector delivers sufficient and stable
expression of the target protein is a common goal when
protein replacement is the mechanism of action. Western
blotting is a fast and flexible tool to measure protein
expression across cell lines, tissue types, and time points.
This can be challenging, as any one reference protein may
exhibit varying signal levels across these samples. Using a
total protein signal to serve as a reference enables reliable
quantification and normalization in these experiments.
The Stain-Free Western Blotting Workflow easily facilitates
total protein normalization (TPN) to allow a flexible
reference signal with minimal steps.

Infectivity
Infectivity and transduction efficiency of AAV
vectors are often measured using one-step reverse
transcription Droplet Digital PCR (RT-ddPCR)
methods when precision and accuracy are critical,
as Droplet Digital PCR offers a reduced coefficientof variance (CV) regardless of testing site or operator.
Conversely, reverse transcription quantitative PCR(RT-qPCR) is used if sample throughput or faster time
to results are desired.
Regardless of your challenge, Bio-Rad offers thequality and reliable solutions you have come tocount on to address your needs.

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Stain-Free Western Blotting

PCR Instruments Portfolio

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Multiplex Immunoassays

IMMUNOGENICITY
While validating the effects of your AAV vector, it is important toassess the immune response both toward the vector and any processrelated impurities such as host cell protein or DNA/RNA. Potential
immunogenicity is a common safety concern; therefore, avoiding severe
immune reactions at the time of therapeutic infusion is a top priority.
By monitoring the immune response in preclinical and clinical stages,
it allows characterization of the therapeutic effect and observation ofboth target and off target activation, i.e., immunotoxicity.

Immunogenicity and cellular toxicity
are critical considerations whenassessing the potential efficacy of
AAV therapeutic approaches.

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When analyzing the immunogenicity andimmunotoxicity of your AAV gene therapy,
Bio-Plex™ Pro Cytokine and Chemokine Panels
provide high sensitivity across the physiologically
relevant ranges, ensuring detection of theseimportant immunoproteins. These multiplex
assays also provide a high-throughput workflow,
allowing characterization of the immune profile
with low sample input. Choose from fixed
multiplex panels or customize assays for yourexperimental requirements.

Bio-Plex Pro Cytokine Panel

Phenotypic Assays
Monitoring immune responses across individuals
or time points often involves analyzing numerous
samples. These can be varied in terms of complexity,
throughput, and cell type used. Assays thatinvestigate more complex cellular responses suchas those involved in immunogenicity require highnumbers of cells and ten or more parameters.
The ZE5 Cell Analyzer stands out with advanced
multiparameter capabilities, efficiently processing
high sample volumes with walk-away automation.
This facilitates rapid evaluation of cellular immune
profiles and accurate comparison of responses
across samples.

ZE5 Cell Analyzer

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Established and Reliable — Real-Time PCR
Real-time PCR is often considered ideal for biodistribution and viral shedding assays
because of its wide dynamic range, high throughput, and speed (Ma et al. 2020). A
well-designed assay in combination with a thorough sample prep method can meetthe regulatory LLOD guideline of 50 copies/g of human genomic DNA.

BIODISTRIBUTION & VIRAL SHEDDING
To accurately measure biodistribution, detection of both the vector
and the transgene protein is recommended. This allows collectionof correlative evidence showing the presence, persistence, activity,and effect of the AAV vector. In addition, viral shedding is monitored
to assess the clearance of the AAV gene therapy product. AAV
vectors may be present at vanishingly low levels in some tissues,
necessitating highly sensitive detection techniques.

“The endpoint assay in ddPCR minimizes
the matrix effect, facilitating preparationof a single surrogate calibration curve in
combination with external control geneand DNA recovery normalization, which
is superior to the qPCR-based method interms of animal ethics and cost.”(Nakayama et al. 2023)
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The CFX Opus Real-Time PCR System offers reliability, sensitivity, and multiplexing
capability, along with user-intuitive software with regulatory compliance–enabling
capabilities. From flexibility of platform choice to automation incorporation, the
CFX Opus platform can assist your biodistribution studies.

QX600 Droplet Digital PCR System

CFX Opus Real-Time PCR System

Expanding Sensitivity — Digital PCR
Droplet Digital PCR has been shown to be more reproducible and sensitive for raretargets in limited samples (Nazir 2023). It also has a higher tolerance for inhibitors, as
it does not depend on amplification efficiency for quantification.
The use of digital PCR is on the rise in the pharmacology phases of therapeutic
development, especially in cases where assay sensitivity requirements are not being
met with conventional methods.

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Process Optimization

METHOD DEVELOPMENT
While characterization and analytical assays are critical to AAV lot
release, downstream method development and process optimization
for the capture and polishing steps are essential to obtain the yield,
purity, and process economics necessary. Bio-Rad has a dynamic
portfolio of chromatography instrumentation, prepacked and empty
columns, media, and resins that can be utilized for AAV development
and manufacturing.

Our team of scientists is here to assist
you with purification method developmentfrom bench to pilot scale through processtransfer to manufacturing.
Contact our scientists today to accelerate
your process development.

From initial resin screening to processdevelopment through production andmanufacturing scale, Bio-Rad has a wide
range of tools in multiple formats to assist in
downstream purification. Our chromatography
resins enable straightforward processoptimization to deliver both high purity andyield. From plate-based resin screening togood manufacturing practice (GMP) readyprepacked columns, we have solutions
for your purification needs. Contact us forsamples today.
Bioprocess Chromatography Products

Method Optimization
Our NGC Chromatography System is the perfectcompanion for lab-scale purification and method
development. The customizable design allows
the system to be upgraded to provide higher
flow rates, sophisticated detection capabilities,
pH monitoring, column or resin scouting, andmultidimensional chromatography.

NGC Chromatography System

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Host Cell Proteins

RESIDUAL CONTAMINANTS & PURITY
Impurities and contaminants in AAV products can cause adverse
responses upon administration. Therefore, purity of the viral vector
must be confirmed prior to administration in both preclinical andclinical trials, as well as throughout the manufacturing QC process.
To ensure patient safety, purity analysis requires a multitude of tests
using techniques that measure host cell proteins (HCP), host cell DNA(HC DNA), and common contaminants such as Mycoplasma.

When you are part of a team working in the earlystages of viral vector development, SDS-PAGE analysis
is a quick and relatively inexpensive way to check forthe presence of nonviral proteins in your prep. Whileconventional fluorescent protein stains, such as SYPRORuby, are highly sensitive, they can slow you down with
their overnight protocols.
Bio-Rad’s Stain-Free imaging enables detection ofcontaminating proteins in minutes rather than overnight,
for rapid assessment of protein purity when your priority
is speed.

Stain-Free Imaging

Mycoplasma

The probe-based chemistry, Vericheck ddPCR
Mycoplasma Detection Kit brings confidence to
Mycoplasma contamination testing. The specificity
and reproducibility of this kit allows for a precise
measurement of up to 112 Mycoplasma species
using Droplet Digital PCR.

Vericheck ddPCR
Mycoplasma Detection Kit

Host Cell DNA
Vericheck ddPCR HEK 293
The standard methods used for host cell DNA
Residual DNA Quantification Kit
quantification can face challenges due to lack of
specificity and reproducibility. Vericheck ddPCR
HEK293 Kits feature novel ddPCR assays that havebeen designed to quantify residual HEK293 DNA andits size with high specificity and reproducibility without a
Vericheck ddPCR HEK 293
reference curve.
Residual DNA Size Kit
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Support
With more than 50 years of dependable service supporting the scientific community,
we are here when you need us.
Ensure coverage for routine maintenance and emergency repairs at no
additional cost with a Bio-Rad service contract plan. Plans include:
Service■■■

SUPPORT, SOFTWARE, & COMPLIANCE
From hardware and instruments to consumables and software withtools to assist with compliance, we are ready to partner with you toaddress your needs in the AAV gene therapy space.

■

For regulated environments, equipment qualification services are available.
Services include:
Validation

■

■

■

Bio-Rad offers software editions enabling
traceability features that facilitate
compliance with regulations such as
U.S. FDA 21 CFR Part 11.

Service and repair coverage
Replacement parts, travel, and labor
Prioritized response time
Preventative maintenance■Installation Qualification (IQ)
Operational Qualification (OQ)
Preventative Maintenance (PM)Thermal validation

Software

QX Manager Software
Supports QX200™ and QX600
Droplet Digital PCR Systems

QX ONE Software
Supports QX ONE Droplet Digital
PCR System

Image Lab Software
Supports GelDoc™ Go andChemiDoc Imagers, GS-900
Densitometer
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CFX Maestro Software
Supports CFX Real-Time PCR Systems

Everest Software
Supports ZE5 Cell Analyzer

ChromLab Software
Supports NGC Chromatography
Systems

Bio-Plex Manager Software
Supports Bio-Plex 200 System

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Applications
Residual
MethodContaminants
Development & Purity

Technique

Key Products

Viral
Viral Titer Characterization Potency Immunogenicity Biodistribution

Digital PCR

QX200 System

X

X

X

X

X

X

QX600 System

X

X

X

X

X

X

QX ONE System

X

X

X

X

X

X

Assays

X

X

X

X

X

X

ChemiDoc System

X

X

X

X

ChemiDoc MP System

X

X

X

X

Gels and blots

X

X

X

X

Trans-Blot™ Turbo System

X

X

X

X

Imaging

Real-time PCR

Chromatography

CFX Opus 96 System

X

X

X

X

X

CFX Opus 384 System

X

X

X

X

X

Assays

X

X

X

X

X

Reagents and consumables

X

X

X

X

X

NGC Chromatography System

X

Screening tools

X

Prepacked columns

X

Process resins

X

Analytical columns
Flow cytometryX

ZE5 Cell Analyzer

X

PanelsX

Antibodies

X

Multiplex immunoassay Bio-Plex 200 System

X

Bio-Plex 3D System

X

Bio-Plex Pro Wash Station

X

AssaysX

Raw beads and coupling
reagents

X

References
Alliance for Regenerative Medicine (ARM) and the National Institute for Innovation in Manufacturing Biopharmaceuticals (NIIMBL) (2021). Project A-Gene: A case study-based
approach to integrating QbD principles in gene therapy CMC programs. https://alliancerm.org/wp-content/uploads/2021/09/ALL-PROJECT-A-GENE-V10.pdf, accessed
November 27, 2023.
Clarner P et al. (2021). Development of a one-step RT-ddPCR method to determine the expression and potency of AAV vectors. Mol Ther Methods Clin Dev 23, 68–77.
Dobnik D et al. (2019). Accurate quantification and characterization of adeno-associated viral vectors. Front Microbiol 10, 1570.
Ma H et al. (2020). qPCR and qRT-PCR analysis: Regulatory points to consider when conducting biodistribution and vector shedding studies. Mol Ther Methods Clin Dev 20,
152–168.
Nakayama M et al. (2023). Novel cell quantification method using a single surrogate calibration curve across various biological samples. AAPS J 25, 26.
Nazir S. (2023). Medical diagnostic value of digital PCR (dPCR): A systematic review. Biomedical Engineering Advances, 6, 100092.

Visit bio-rad.com/GeneTherapy for more information.
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Purchase of Digital PCR and/or Single-Cell NGS Sample Preparation products (the “Products”) from Bio-Rad Laboratories is subject to Bio-Rad Laboratories, Inc. Standard Terms and Conditions ofSale, which can be accessed at https://www.bio-rad.com/en-us/ terms-conditions. Unless we expressly state otherwise in additional Terms and Conditions, no rights are granted for you to distribute orresell the Products. Unless we expressly state otherwise in additional Terms and Conditions, no rights are granted for the development or commercialization of diagnostic assays for use with the Products
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Image Lab Touch software is based in part on the work of the CImg project (http://cimg.eu/). See license for details at www.cecill.info/licences/Licence_CeCILL-C_V1-en.html.
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