Streamlining AAV Production for Gene Therapy
eBook
Published: October 4, 2024
Credit: Thermo Fisher Scientific
With over 30 therapies approved globally, the gene therapy field is unlocking new possibilities for treating previously untreatable conditions.
However, ensuring the purity, potency and safety of viral vectors requires robust purification and analytical methods to effectively eliminate impurities.
Unfortunately, existing methods are often difficult to scale, time-consuming and costly.
This eBook explores the complexities of AAV purification and highlights innovative solutions aimed at enhancing the safety, potency and consistency of gene therapy treatments.
Download this eBook to discover:
- Insights into the latest advancements in analytical methods and their impact on AAV production
- Solutions that support safer and more effective AAV therapies
- Efficient, accurate and scalable methods for AAV purification
Optimizing Gene
Therapy
A comprehensive guide
to AAV PurificationForeword
We are at the forefront of a gene therapy revolution, with
over 30 groundbreaking treatments for a wide range of
conditions already approved by the FDA. Central to these
advancements are adeno-associated virus (AAV) vectors,
which have proven to be highly effective and versatile in
therapeutic applications.
However, as the demand for AAV-based therapies grows, so
does the need for scalable production methods. Meeting this
demand requires a profound optimization of manufacturing
processes to ensure efficiency and scalability, while still
maintaining high quality, purity and potency.
Through a comprehensive exploration of current
methodologies, technological advancements and industry
best practices, this eBook aims to equip readers, whether
clinician, researcher or industry professional, with a
deeper understanding of the obstacles and opportunities
in AAV production.
1 thermofisher.com/aav-purificationThe importance of AAV purification and analytics 3
Viral vectors in gene therapy:
Innovations and simplified solutions 6
AAV purification trends and
techniques: Ask the experts 7
Harnessing CDMOs for optimized AAV
production: ask the expert 13
Top 5 benefits of affinity chromatography
for AAV purification 16
Viral clearance in a downstream AAV process: Case
study using a model virual panel and noninfectious
surrogate 18
Productivity optimization and process calculations
for AAV affinity chromatography 25
Optimizing AAV purification for gene therapy
manufacturing 34
Resources & digital tools 35
Contents
23 thermofisher.com/aav-purification
Introduction
The rise of gene therapies marks a radical shift from
traditional treatment approaches by addressing the underlying
genetic causes of a disease rather than merely treating the
symptoms. Initially conceived in the 1970s, gene therapy has
rapidly evolved with the advent of more precise and safer
delivery methods, leading to several breakthroughs in clinical
applications. With the approval of over 30 gene therapies by
regulatory agencies worldwide, the field has transitioned from
experimental to therapeutic, offering hope for conditions once
deemed untreatable.1
A key component of gene therapy is the efficient delivery
of therapeutic genes into host cells. Viral vectors are often
chosen due to their natural capability to efficiently infect human
cells. These vectors are derived from viruses like adenovirus,
lentivirus and adeno-associated virus (AAV) that have been
engineered to be safe and effective. AAVs are one of the most
promising vectors in gene therapy due to their ability to provide
long-lasting gene expression, target specific cell types and
exhibit relatively low immunogenicity.2
Large-scale production of AAV-based treatments relies on
robust purification and analytical methods to ensure the
consistency, potency and safety of the final product. A primary
concern is the removal of impurities, such as residual host cell
proteins, DNA and empty capsids, which can compromise
the efficacy and safety of gene therapies. However, current
The Importance of AAV
Purification and Analytics
methods are often difficult to scale, time-consuming and limited
to specific AAV serotypes.
As the number of approved AAV-based therapies and clinical trials
continues to grow, there is an increasing demand for rigorous
quality control measures and improved purification techniques.3
Continued development and refinement of purification and
analytical technologies are therefore pivotal in advancing gene
therapy, ensuring that these innovative treatments can safely and
effectively reach the patients who need them.
This article explores the challenges associated with large-scale
AAV purification and the latest advancements in purification
techniques and analytical methods.
Challenges of AAV purification
The production of AAV vectors follows a complex workflow,
beginning with vector design and cloning, where the therapeutic
gene of interest is inserted into an AAV plasmid vector. This
vector is then transfected into host cells, typically HEK293 cells.4
Following transfection, the cells are cultured under specific
conditions to produce the AAV particles. After an incubation
period, the cells are harvested and lysed to release the AAV
particles. The crude lysate then undergoes several purification
steps to isolate and concentrate the AAV particles, followed by
formulation into a final product suitable for clinical use (Figure 1).
Purifying AAVs is a critical step in the production process,
ensuring that the final product is effective and safe for
Cell line development Cell culture Purification Formulation
Expression vector
development
Bioreactors
Harvest/cell
removal
Harvest media
Transfection
Clone selection
Cell banks
Virus clearance
Concentration
Column chromatography
Bulk drug
substance
Finished batch
of active substance
Figure 1. AAV manufacturing process for gene therapy products.thermofisher.com/aav-purification 4
therapeutic use. The primary goal of purification is to
remove impurities such as host cell proteins, DNA and other
contaminants while maximizing the yield and potency of
the AAV vectors. A range of techniques can be used for
purification, including centrifugation and chromatography.
Downstream purification of AAV vectors presents several key
challenges that significantly impact the yield and efficacy of the
final product. One major issue is the increased impurity burden
due to cell lysis, which releases a substantial amount of host
cell proteins, adventitious viruses and other debris into the
crude lysate. However, complex purification processes aimed
at removing these impurities often lead to substantial loss of
viral particles. Thus, improved purification techniques are key to
balance efficient purification with a good recovery yield.
The large variety of AAV serotypes also complicates the purification
process. AAV vectors come in many different serotypes, each
with distinct capsid proteins and surface properties.5 This diversity
creates challenges in developing a standardized purification
process, as different serotypes may require specific conditions or
methods for optimal purification. Tailoring purification protocols to
accommodate the variety of AAV serotypes can add complexity
and cost to the production process.
Perhaps the most difficult challenge is the enrichment of full
capsids. The production process often results in a mix of full,
empty and partially filled capsids. Full capsids are crucial
for delivering the therapeutic gene, while partial and empty
capsids can dilute potency and increase the total viral load
needed for effective treatment. Moreover, empty capsids
can trigger immune responses, compromising transduction
efficiency and limiting the potential for repeat dosing due to
the development of neutralizing antibodies.6 Hence, advanced
separation techniques are needed to enrich the full capsids
effectively.
Analytical techniques in AAV purification
Analytical techniques are vital for accurately assessing critical
quality attributes (CQAs), such as the purity, potency and safety
of AAV products. Several CQAs are essential for characterizing
AAV products, including (i) virus titer, (ii) capsid aggregation and
(iii) the ratio of full to empty viral particles (Figure 2).7 Several
analytical methods are employed to assess these attributes,
each offering unique insights into the quality of AAV products.
For example, analytical ultracentrifugation (AUC) is widely
regarded as the gold standard for determining the ratios of full,
partial and empty capsids, in addition to providing detailed
aggregation profiles. Viral titer and content ratios can be
measured using a combination of quantitative PCR (qPCR) and
enzyme-linked immunosorbent assay (ELISA), where qPCR
measures the viral genome and ELISA quantifies capsid protein
content. Additionally, transmission electron microscopy (TEM)
can provide detailed visualization of AAV particles, allowing for
the evaluation of capsid integrity, aggregation and morphology.
To date, manufacturing purification methods for AAV vectors
typically rely on a sequence of chromatography techniques
due to their ease of scalability. The process generally begins
with affinity chromatography which purifies the AAV particles
from crude mixtures. In this stage, specific ligands within
the affinity column are engineered to selectively bind to AAV
capsid proteins. As the mixture flows through the column,
AAV particles are captured by these ligands, while impurities
are allowed to pass through, resulting in a significantly
purified AAV product. This product can then be further refined
through additional chromatography techniques, such as
anion-exchange high-performance liquid chromatography
(HPLC). Anion-exchange chromatography (AEC) is particularly
effective in enriching full capsids based on their distinct charge
properties, thus enhancing the overall quality and consistency
of the final AAV preparation.
However, these techniques come with various challenges.7,8
Many of them face issues related to scalability and throughput,
which are crucial for large-scale production. For instance,
TEM and AUC are labor-intensive and have long turnaround
times, making them less suitable for high-throughput analysis.
Techniques like qPCR and ELISA, while highly sensitive, can
Figure 2. Several techniques can be used to measure the potency,
purity and safety of AAV therapies including optical density (OD), size
exclusion chromatography (SEC) and static/dynamic light scattering
(SLS/DLS).
Product
and Process
Understanding
Potency,
Purity &
Safety
rAAV
CQAs
Virus titer Content ratio Aggregation
• qPCR
• ELISA
• OD
• etc.
• AUC
• TEM
• AEC
• etc.
• SEC
• AUC
• SLS / DLS
• etc.5 thermofisher.com/aav-purification
only quantify limited serotypes and often require multiple steps,
increasing the complexity and time required for analysis. Anionexchange HPLC, although effective in separating AAV particles,
can be hampered by resins with small pores, limited binding
capacities and long turnaround times, leading to suboptimal
performance in large-scale operations.
Hence, developing more robust and streamlined analytical
workflows is essential to support the growing demand for
AAV-based therapies. By addressing these limitations, the
gene therapy field can achieve more efficient production and
quality control, ultimately leading to safer and more effective
treatments for patients.
Trends and innovations
As the field of AAV-mediated gene therapy progresses, there
is an increasing need for highly scalable methods for AAV
purification. As a result, one-step affinity chromatography
protocols have become attractive for accelerating the
purification of viral vectors while meeting good manufacturing
practice (GMP) requirements.9
HPLC-based affinity chromatography is quick and efficient,
enabling the determination of both capsid titer and content
ratio. With a quick run time and no need for manual sample
handling or pretreatment steps, the process is ideal for rapid,
high-throughput analysis in both research and production
environments.10 However, the specificity that makes affinity
chromatography effective also presents a notable drawback.
Most ligands are designed to capture only one or a few AAV
serotypes, meaning that any change in the virus particle
often necessitates a new capture ligand. This can be timeconsuming and costly, hindering the flexibility needed for
platform production processes.
As a result, affinity columns based on AAV-specific camelid
antibodies have started to dominate the field. These resins can
target a broad range of natural and synthetic AAV serotypes,
significantly simplifying the purification process across various
vector types.11,12 Additionally, they have very high binding
capacities, demonstrate robust viral clearance and are stable
against harsh clean-in-place and regeneration methods, making
them suitable for repeated use.12,13 Similar advances in AEC are
also contributing to more effective polishing steps, resulting in
the enrichment of full capsids by 90% in some cases.14
These trends and innovations are pivotal in meeting the
growing demand for AAV-based therapies, facilitating the
production of safe and effective treatments on a larger scale.
As the field continues to evolve, these advancements will likely
play an integral role in the future of gene therapy, driving further
improvements in both efficiency and product quality.
Future directions
As the number of gene therapy programs advancing to the
clinical phase and commercialization continues to rise, the
optimization of large-scale AAV production is essential for the
future of the field. Achieving this requires analytical tools and
methods capable of providing rapid and accurate assessments
of sample purity at increasing scales.
Innovations in purification techniques, particularly in
chromatography, are paving the way for more efficient and
effective viral vector purification. Future developments will
likely focus on optimizing these methods, integrating advanced
analytical tools and leveraging automation to enhance
consistency and scalability. Overall, continued advancements
in viral vector purification will be essential to meet the growing
needs of the gene therapy field and bring innovative treatments
to patients worldwide.
References
1. Approved cellular and gene Therapy products. US Food and Drug Administration. https://
www.fda.gov/vaccines-blood-biologics/cellular-gene-therapy-products/approved-cellularand-gene-therapy-products. Published online February 8, 2024. Accessed September 10,
2024.
2. Li C, Samulski RJ. Engineering adeno-associated virus vectors for gene therapy. Nat Rev
Genet. 2020;21(4):255–272. doi: 10.1038/s41576-019-0205-4
3. Kuzmin DA, Shutova MV, Johnston NR, et al. The clinical landscape for AAV gene therapies.
Nat Rev Drug Discov. 2021;20(3):173–174. doi: 10.1038/d41573-021-00017-7
4. Naso MF, Tomkowicz B, Perry WL, Strohl WR. Adeno-associated virus (AAV) as a vector for
gene therapy. BioDrugs. 2017;31(4):317–334. doi: 10.1007/s40259-017-0234-5
5. Issa SS, Shaimardanova AA, Solovyeva VV, Rizvanov AA. Various AAV serotypes and
their applications in gene therapy: An overview. Cells. 2023;12(5):785. doi: 10.3390/
cells12050785
6. Schnödt M, Büning H. Improving the quality of adeno-associated viral vector preparations:
The challenge of product-related Impurities. Hum Gene Ther Methods. 2017;28(3):101–
108. doi: 10.1089/hgtb.2016.188
7. Gimpel AL, Katsikis G, Sha S, et al. Analytical methods for process and product
characterization of recombinant adeno-associated virus-based gene therapies. Mol Ther
Methods Clin Dev. 2021;20:740–754. doi: 10.1016/j.omtm.2021.02.010
8. Werle AK, Powers TW, Zobel JF, et al. Comparison of analytical techniques to quantitate
the capsid content of adeno-associated viral vectors. Mol Ther Methods Clin Dev.
2021;23:254–262. doi: 10.1016/j.omtm.2021.08.009
9. Smith RH, Levy JR, Kotin RM. A simplified Baculovirus-AAV expression vector system
coupled with one-step affinity purification yields high-titer rAAV Stocks from insect cells.
Mol Ther. 2009;17(11):1888–1896. doi: 10.1038/mt.2009.128
10. Heckel J, Martinez A, Elger C, et al. Fast HPLC-based affinity method to determine capsid
titer and full/empty ratio of adeno-associated viral vectors. Mol Ther Methods Clin Dev.
2023;31:101148. doi: 10.1016/j.omtm.2023.101148
11. Mietzsch M, Smith JK, Yu JC, et al. Characterization of AAV-specific Affinity ligands:
Consequences for vector purification and development Strategies. Mol Ther Methods Clin
Dev. 2020;19:362–373. doi: 10.1016/j.omtm.2020.10.001
12. Florea M, Nicolaou F, Pacouret S, et al. High-efficiency purification of divergent AAV
serotypes using AAVX affinity chromatography. Mol Ther Methods Clin Dev. 2023;28:146–
159. doi: 10.1016/j.omtm.2022.12.009
13. Viral Safety in AAV production: How Affinity Chromatography effectively contributes.
Genetic Engineering & Biotechnology News. https://assets.thermofisher.com/TFS-Assets/
BPD/Reference-Materials/viral-safety-in-aav-production-affinity-article.pdf. Accessed
September 10, 2024.
14. Joshi PRH, Bernier A, Moço PD, Schrag J, Chahal PS, Kamen A. Development of a scalable
and robust AEX method for enriched rAAV preparations in genome-containing VCs of
serotypes 5, 6, 8, and 9. Mol Ther Methods Clin Dev. 2021;21:341–356. doi: 10.1016/j.
omtm.2021.03.016Viral Vectors in
Gene Therapy:
Innovations
and Simplified
Solutions
Gene therapy involves the introduction of specific genetic
material into a patient to alter and improve cell function. Recent
advancements have resulted in over 30 approved cell and
gene therapies worldwide, addressing a variety of conditions
ranging from congenital disorders to solid cancers.1, 2
These breakthroughs have been possible thanks to the
development of sophisticated delivery systems and the
refinement of gene-editing technologies. Innovations such as
viral vectors, including adeno-associated viruses (AAVs), have
enabled precise delivery of genetic material to the target cells,
enhancing the efficacy and safety of treatments.
This infographic explores current trends, delivery mechanisms
and manufacturing challenges in gene therapy.
JAPAN
MHLW - Ministry
of Health, Labour
and Welfare
SOUTH KOREA
MFDS - Ministry
of Food and Drug
Safety
EUROPE
EMA - European
Medicines
Agency
CHINA
NMPA - National
Medical Product
Administration
CANADA
Health Canada
USA
FDA - Food
and Drug
Administration
AUSTRALIA
TGA - Therapeutic
Goods Administration
PHILIPPINES
FDA - Food
& Drug
Administration
UK
MHRA -
Medicines and
Healthcare
products
Regulatory
Agency
7 6 5 4 3 2 1 0 2004
2005
2006
2007
2008
2009
2010
2011
2012
2013
2014
2015
2016
2017
2018
2019
2020
2021
2022
2023
2024
Number of gene therapies approved worldwide3
The two paths to gene therapy
There are two main ways to deliver gene therapy: ex vivo and in vivo. Each method offers its own set of benefits and considerations.
Enabling efficient gene delivery
The plasma membrane acts as a barrier to large molecules, meaning that specialized methods are needed to ensure the genetic
material can enter the cells effectively.3
The vast majority of gene therapies use viral vectors, as they are remarkably efficient at gene delivery.4 There are different types of
viral vectors available; however, AAV vectors are often chosen due to their efficiency, low risk of insertional mutagenesis and longterm gene expression.
The future is in our genes
Gene therapies are transforming the future of medicine. As we advance our understanding and technology, these therapies are
expected to become more precise, effective and accessible. Thermo Fisher is at the forefront of this evolution, providing innovative
tools and solutions that help streamline the development and production of gene therapies. By enhancing purification techniques,
Thermo Fisher is working to pave the way for safer, more effective treatments that can improve patient outcomes and redefine the
future of personalized medicine.
AAV purification is a critical yet challenging step
The first stage in AAV production involves the expansion of viral producer cells in culture, prior to their transfection with one or more
AAV-encoding plasmids. Following transfection, the cells are broken down and the lysate is harvested for AAV purification.
However, AAV purification is associated with several challenges:
Enhanced AAV purification with targeted solutions
Thermo Scientific™ POROS™ CaptureSelect™ resins are engineered to meet the diverse needs of AAV purification across multiple
serotypes. Additionally, pre-packed chromatography columns can help ensure rapid evaluation, simplified scalability and deliver
high yields with increased purity in a single step. With these efficient manufacturing solutions, biomanufacturers can streamline AAV
production, accelerating the development of gene therapies without compromising quality.
In vivo methods are often used when treating a singular
gene or when targeting an internal organ, like the heart,
brain or lungs.
Prepacked
Chromatography
Columns
CaptureSelect and
POROS Chromatography
Resins
AAV-MAX
Transfection Kit
Analytical Testing
Solutions
Ex vivo gene therapies are most commonly used to
treat blood disorders.
For Research Use Only. Not for use in diagnostic procedures. © 2024 Thermo Fisher Scientific Inc. All rights reserved. All
trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified.
Simplify your AAV Purification Process
Adenovirus
Herpes simplex
virus-1
Retrovirus
Lentivirus
Adeno associated
virus (AAV)
Non-pathogenic
Can target dividing
and non-dividing cells
Long-term gene
expression
Can target a broad
range of cell types
Genetic material
is delivered either
intravenously
or locally to the
patient
Viral
vector
Modified
DNA
Modified DNA
Cells are edited to create
modified genes
Patient’s
cells are
collected
Engineered cells
get re-infused
in patient’s
system to target
diseased cells
Specialized resins can only
capture certain AAV serotypes
Difficult to clear host
cell proteins (HCPs) and
adventitious viruses
Designed to capture a wide
range of AAV serotypes
Ready-to-run columns for
quick and easy evaluation
Simplified scalability
Disadvantages of traditional affinity chromatography resins
References
1. Approved Cellular and Gene Therapy Products. U.S. Food and Drug Administration. https://www.fda.gov/vaccines-blood-biologics/cellular-gene-therapy-products/approved-cellularand-gene-therapy-products . Published February 8, 2024. Accessed August 13, 2024.
2. Gene, Cell & RNA Therapy Landscape Report. American Society of Gene & Cell Therapy. https://www.asgct.org/publications/landscape-report. Accessed August 13, 2024.
3. 2024 Archives. American Society of Gene and Cell Therapy. https://www.asgct.org/global/documents/asgct-citeline-q2-2024-report.aspx. Accessed September 11, 2024.
4. Giacca M, Zacchigna S. Virus-mediated gene delivery for human gene therapy. J Cont Release. 2012;161(2):377-388. doi:10.1016/j.jconrel.2012.04.008
5. Bulcha JT, Wang Y, Ma H, Tai PWL, Gao G. Viral vector platforms within the gene therapy landscape. Sig Transduct Target Ther. 2021;6(1). doi:10.1038/s41392-021-00487-6
Key government agencies overseeing therapy approvals
Multiple processing steps
result in yield loss and
increased costs
Challenges in scaling up
production
Excellent separations of
HCPs and adventitious
viruses from AAVs
Delivers high yields and
increased purity in one step
Adventitious
virus
Adventitious
virus
AAVs
AAVs
Host cell
protein
Cell
debris
Cell
debris
Host cell
protein7 thermofisher.com/aav-purification
Experts
Alejandro Becerra
Principal Scientist and Global Technical
Lead for Purification Products, Thermo
Fisher Scientific
Dr. Alejandro Becerra is a Principal
Applications Scientist and Global
Purification Technical Lead. Alejandro has over 15 years of
experience in downstream processing and customer support
having worked as Purification Team Manager and other
bioprocess engineering roles prior to joining Thermo Fisher
Scientific in 2018. Dr. Becerra is a subject matter expert in
preparative chromatography with expertise in the development,
optimization and scale-up of antibodies, recombinant proteins,
viral vectors, and nucleic acid purification processes. Alejandro
holds a Ph.D. in Chemical Engineering from Cornell University.
Chantelle Gaskin
Field Applications Scientist, Purification
Business Unit, Thermo Fisher Scientific
Chantelle is a Field Applications Scientist,
specializing in protein and viral vector
purification and downstream process development. She held
leadership positions at Applied Genetic Technology Corporation
and Brammer Bio, prior to joining the Thermo Fisher Scientific
Bioproduction Division in 2020. With over 10 years of experience
in gene therapy, Chantelle has accumulated comprehensive
knowledge of standard industry practices and regulatory
standards, applying this knowledge to advance the development
of therapies for a variety of indications including ocular, CNS
and systemic disease. Chantelle holds a Master’s degree in
Chemistry from the University of Florida and a Bachelor’s in
Chemistry from Smith College.
AAV Purification Trends and Techniques:
Ask the Experts
Adeno-associated viral (AAV) vectors are an increasingly
popular choice for gene therapies; however, a major bottleneck
in the production of AAVs is their efficient purification. A broad
range of serotypes, residual contaminants and a disparity
in genetic filling increases the complexity of purification
processes. These factors can affect the consistency and
quality of the final product, making it challenging to achieve the
high purity levels required for safe and effective therapies.
We asked two in-house specialists at Thermo Fisher Scientific
for their advice on overcoming AAV purification challenges and
implementing innovative strategies to enhance the efficiency
and reliability of AAV purification.
Q: What are the current trends in AAV purification?
What challenges do process development scientists
typically face in this process?
Alejandro Becerra (AB): The field of AAV purification has been
advancing rapidly, particularly in the seven years since the approval
of Luxturna®. Today, most of the industry is adopting a similar
approach to purification with two chromatography steps after cell
lysis and clarification. The primary challenge now lies in further
optimizing this standardized process. Unlike other purification
processes, AAV purification can be limited by difficulties in obtaining
sufficient material for proper process development.
Another challenge closely related to purification is the quality of
analytics. The effectiveness of any purification process depends
heavily on the robustness of the associated analytical techniques,
in addition to the sample type and the stage of the process.
These limitations impact the ability to detect and quantify various
elements accurately and develop robust unit operations.
Current efforts in clinical and commercial manufacturing are
focused on eliminating product-related impurities, as these are
more difficult to address. In contrast, process-related impurities
are largely removed during the pre-capture and capture stages.
These product-related impurities include empty particles, partially
filled capsids and, occasionally, over-packaged and aggregated
AAVs. The primary focus is on achieving high product purity,
particularly a high percentage of full particles. As previously
mentioned, the field has adopted a common downstream
approach for AAV particle purification, with affinity and anion
exchange chromatography serving as key unit operations.thermofisher.com/aav-purification 8
Chantelle Gaskin (CG): There are three main trends in AAV
purification that I’d like to highlight. Firstly, there’s an increasing
focus on engineered capsids. Many companies generate novel
capsids, driven by their R&D teams, for various reasons. This
includes developing viral vectors with intellectual property
protection and capsids with specific tropism to target particular
tissues. Additionally, there’s a strong emphasis on safety and
reducing immunogenic responses.
This trend towards novel and engineered capsids has
escalated, and many customers are using the POROS™
CaptureSelect™ AAVX resin for this purpose. However, purifying
engineered capsids requires downstream purification steps
to be further optimized. Our role as field application scientists
(FAS) is to support customers in optimizing their entire
downstream process for these novel capsids.
Another significant trend is the enrichment of full capsids,
which is generally achieved using non-affinity polish resins
combined with different buffer compositions. Moreover, there is
a growing focus on characterizing partially filled and overfilled
capsids. Historically overlooked, this aspect is now gaining
attention as companies are exploring purification processes
and upstream strategies to address these issues.
Lastly, the importance of analytics in the purification process
cannot be overstated; effective purification is contingent upon
robust analytical methods. We’re seeing ongoing trends in
developing analytical assays for titer determination of various
capsid species and methods for quicker titer readouts. These
advancements are critical for accurately characterizing the
purification process and are increasingly prevalent in the field.
Q: How does affinity chromatography contribute to
achieving high purity and yield in AAV purification?
Can you explain the underlying principles and
mechanisms involved?
CG: Affinity chromatography has become a staple in biologics
purification due to its efficiency and specificity. The technique
relies on highly specific binding sites on the chromatography
media, allowing only the target molecule to bind to the column
while other impurities in the starting material flow through.
This results in highly purified material in just one step,
unlike the two or three steps often required with non-affinity
chromatography.
Our CaptureSelect portfolio, for example, uses camelid
antibody fragments to achieve this level of purification.
These antibody fragments have high specificity for the target
molecule, helping to ensure that only the desired AAV particles
are retained on the column. This approach not only shortens
the purification process but also increases its efficiency,
delivering high purity and yield with fewer steps.
AB: Affinity chromatography offers numerous advantages
for AAV purification. For example, it eliminates the need to
adjust the sample before loading it onto the chromatography
column. Thus, high purity can be achieved in a single
affinity chromatography step. For instance, the initial load
sample may contain less than 1% of the target product,
with the rest being process-related impurities. After affinity
chromatography, purity levels can exceed 90–95%, effectively
removing these impurities. Additionally, this step concentrates
the sample by several hundred-fold, depending on the
specific process.
Affinity chromatography is highly specific, eliminating the need
for additional unit operations, and thereby reducing the number
of steps in the process. Each additional step can lead to
product losses, so minimizing these steps enhances the overall
AAV recovery.
Q: What are the recent advancements in affinity
chromatography for AAV purification?
AB: One recent development in AAV purification using
affinity chromatography involves optimization of the overall
downstream process to reduce product losses. Traditionally,
many processes include a concentration step using tangential
flow filtration (TFF) before the affinity chromatography step.
This concentration step helps to increase the amount of AAV
in the sample, thereby reducing the time needed for affinity
chromatography. However, it also introduces an additional unit
operation, which may lead to potential product losses.
However, an innovative approach to eliminate the TFF step can
be achieved by using chromatography resins with high capacity
and high permeability, such as POROS CaptureSelect AAVX
or AAV9. By operating at higher flow rates and using shorter
bed heights, these resins can process larger volumes quickly
without the need for prior concentration.
This method not only simplifies the process but also helps
to minimize product losses associated with additional steps.
This approach has been increasingly adopted in the field, with
companies like Bristol Myers Squibb presenting related work at
the American Chemical Society (ACS) meeting last year.
CG: Recent advancements in affinity chromatography
for AAV purification have primarily focused on optimizing
the surrounding downstream processes to enhance the
efficiency of the affinity purification step. One approach
involves implementing DNA removal protocols prior to affinity
purification. This step helps increase the purification efficiency
of the affinity chromatography process.9 thermofisher.com/aav-purification
Additionally, there has been a focus on optimizing the
cleaning and reuse of AAV resins to extend their lifecycle.
This is particularly important in process development and
GMP manufacturing, where the ability to reuse columns can
significantly reduce costs. Historically, single-use affinity
columns were preferred in GMP settings due to concerns
around handling viral vector product. However, to support
the development of larger-scale AAV processes, we have
demonstrated that POROS CaptureSelect AAVX resin can be
used for multiple cycles, enabling more sustainable and costeffective manufacturing.
These advancements, while not the most glamorous aspects
of AAV purification, are crucial for improving the efficiency and
cost-effectiveness of both process development and largescale production. We have been actively supporting customers
in implementing these strategies to achieve better results in
their purification workflows.
Q: The POROS CaptureSelect AAVX can capture a
wide range of AAV serotypes. How does it do that?
CG: The AAVX ligand was developed through an extensive
screening process of multiple ligand candidates to find the
one with the highest specificity across a broad range of AAV
serotypes. The key to its success lies in its ability to bind to
specific sequences on the AAV capsid. These sequences are
conserved across different viral vector serotypes, allowing
AAVX to effectively capture a wide variety of AAV serotypes.
This binding mechanism is documented in a white paper that
highlights that these conserved sequences are crucial for the
ligand’s broad specificity.1 However, when customers engineer
capsids and alter these proteins, the binding efficiency of AAVX
can decrease. To assist with this, we provide access to the
published epitopes so that customers can avoid modifying
these critical binding sites during their engineering processes.
Q: What are the key factors to consider when
selecting an affinity chromatography method for
AAV purification? Are there any specific ligands
or matrices that have demonstrated superior
performance?
AB: When discussing AAV purification, it’s crucial to recognize the
wide range of serotypes used and the field’s efforts to engineer
these particles for various applications. Therefore, the first factor to
consider is the specificity of the affinity chromatography resin, i.e.,
ensuring the affinity resin can target the specific serotypes used
by an organization. The POROS CaptureSelect AAVX resin has
demonstrated broad specificity, effectively binding to all natural
serotypes as well as engineered capsids.
The second consideration is scalability. Chromatography has a
long history and is easily scalable. However, given the typically
low concentration of AAV and the relatively long processing
times, it’s essential to consider the binding capacity of the
affinity resin, particularly at shorter residence times and higher
flow rates. In this regard, chromatography resins like POROS
CaptureSelect AAVX are particularly advantageous as they
offer high binding capacity at high flow rates. This capability
reduces overall processing time and allows smaller columns to
be used, thereby lowering the overall costs.
CG: There are several key factors to consider when selecting
an affinity chromatography method for AAV purification. First,
you need to look at binding capacity, as this will impact the
efficiency and yield of your purification process. Next, consider
the material of construction, which affects flow pressure and
flow characteristics. These factors can be critical when scaling
up your process.
Another important aspect is the ability to clean your resins.
Efficient cleaning protocols are essential for maintaining resin
performance and longevity, especially in large-scale operations.
Additionally, the ability to pack resins effectively is crucial. While
many AAV purification processes use pre-packed columns,
those who pack their own columns need a resin that is easy to
pack consistently.
Q: Can you provide examples where affinity
chromatography has successfully enabled one-step
capture of AAV with high purity and yield?
AB: It’s important to note the difference between producing
recombinant AAVs for research or preclinical purposes versus
clinical studies in humans. Several research groups and
industry specialists have used one-step purification with affinity
resins and demonstrated their effectiveness in in vitro or earlystage in vivo models.
A nice example is recent work by scientists at a biotechnology
company showing that using just affinity resins can be effective
for producing AAV for early stages.2 Some of their work
focused on the removal of endotoxins, which are undesirable
in the context of in vivo studies. They were able to effectively
remove those endotoxins using an intermediate wash with a
detergent after binding AAV particles to POROS CaptureSelect
resins. Most researchers working in research settings primarily
use just one-step purification with affinity resins which provide
sufficient purity for their work.
CG: One example comes from a recent study, in which they
used AAVX to purify 15 divergent AAV serotypes, including
AAV2, AAV9 and even the ancestral AAV serotype Anc80,
known for its excellent tropism but difficult purification.3 Thethermofisher.com/aav-purification 10
results demonstrated high levels of purification in a single step.
They compared this approach to ultracentrifugation with an
iodixanol gradient, a common method in early-stage research
that, while effective, is difficult to scale. This comparison
highlighted the advantages of AAVX, particularly in scalability,
making it suitable for larger-scale applications like preparing
materials for extensive animal studies.
A case study published earlier this year utilized an AAVX affinity
column for analytical purposes.4 This method is particularly
beneficial for titer determination of crude samples.
Q: Are there some serotypes that prove to be more
challenging when developing an AAV affinity capture
step? Have these challenges been addressed?
AB: While many AAV serotypes are quite similar, which is why
we can use a single affinity resin to capture all of them, they
also have some key differences. One notable difference is the
stability of the AAV particle itself. For example, serotypes like
AAV2 are more prone to aggregation, especially under low
conductivity conditions where there’s not enough salt.
In affinity chromatography, we bind the particles at neutral
pH and elute them at low pH. Generally, low conductivity is
beneficial for good recovery. However, for serotypes prone to
aggregation under these conditions, we need to address the
challenge of balancing recovery and stability. We do this by
including excipients like arginine to prevent aggregation while
still achieving good recoveries. Additionally, after elution, we
can add different salts to the neutralization buffer to prevent
aggregation.
Another example is AAV5, which binds very strongly to the
AAVX ligand. This means we need slightly more stringent
conditions for elution, such as a lower pH – maybe half to one
pH unit below what we’d use for other serotypes. We can also
use excipients or modifiers to facilitate elution and maintain
good recoveries.
For engineered capsids, the situation can be different.
Sometimes, the binding to the resin isn’t sufficient. In such
cases, we can adjust the binding conditions or explore
alternative custom ligands or resins to achieve the desired
capture efficiency.
CG: AAV9 and AAV9-like serotypes tend to resist binding, making
purification difficult. This serotype crosses the blood-brain barrier,
making it particularly useful for neurological applications and
diseases involving the central nervous system (CNS).
Some companies are making small modifications to the AAV9
capsid to improve its suitability as a viral vector. Despite these
challenges, the AAVX resin is capable of purifying AAV9
capsids. We recommend certain considerations to optimize the
purification process for AAV9, but overall, AAVX shows great
binding capacity for this serotype. Additionally, we have also
developed the POROS CaptureSelect AAV9 resin, which is
made specifically to bind this species.
Q: In your experience, what are the main benefits of
affinity chromatography compared to alternative AAV
purification methods?
AB: One of the main benefits is the ability to take the
sample from the previous step without needing to adjust
pH or conductivity. For example, if you use cation exchange
chromatography for capture, you need to lower the pH and
adjust the conductivity. Some impurities may precipitate after
these adjustments. This adds extra steps that require further
optimization and can lead to product losses.
The second benefit is achieving very high purity levels in a single
step. Affinity chromatography is scalable, and in that same
step, it also concentrates the load sample. Depending on the
initial concentration and specific conditions, you can achieve a
concentration increase of 100- to several hundred-fold.
Compared to ultracentrifugation, the scalability of
chromatography resins is also clear. Ultracentrifugation faces
scalability issues, especially as the field moves toward larger
doses for larger patient populations, making it challenging to
produce the required amount of vector.
CG: Affinity chromatography offers significant benefits by
effectively reducing the number of purification steps needed. With
just one affinity chromatography step, you can achieve the same
level of purification that might otherwise require two or three ion
exchange steps. This translates to greater process efficiency, as
you’re eliminating additional chromatography steps, along with
their associated costs for resins, buffers and manpower.
Q: How does affinity chromatography fit into the
overall process of AAV production? Are there any
considerations regarding scalability and costeffectiveness?
AB: Affinity chromatography plays a crucial role in both
research and larger-scale AAV production. Typically, it fits
into the process after several initial steps and before any final
processing steps. Chromatography resins have been used
for decades, so the underlying physical principles remain the
same, with a range of column diameters and bed heights
providing flexibility compared to other adsorptive methods.
In terms of cost-effectiveness, affinity chromatography offers
significant benefits by potentially eliminating the need for11 thermofisher.com/aav-purification
additional steps. While the cost of affinity chromatography
resins is a factor, it should be compared to other expensive
raw materials, such as nucleases and plasmids. Importantly,
these resins can be reused in both research and GMP settings.
They can be cleaned and utilized multiple times, which helps
reduce the overall cost of the process. We have demonstrated
that these resins can maintain comparable performance over
35 cycles. Similarly, research by Florea et al. has shown good
reproducibility over six cycles.3
The ability to reuse chromatography resins significantly lowers
the cost per cycle, and this exponential reduction in cost with
reuse makes affinity chromatography a cost-effective choice.
However, it’s crucial to validate the resin reuse using a qualified
scale-down model and ensure the necessary analytics are in
place to support this approach.
CG: Affinity chromatography streamlines the purification
process. For instance, compared to ultracentrifugation – which
is labor-intensive and has significant scalability issues, such
as the need for precise manual band extraction from gradient
tubes – affinity chromatography offers a more efficient and
consistent approach.
The manual aspect of ultracentrifugation, often described
as tedious or even an art form, can vary greatly between
operators, further complicating scalability. In contrast,
affinity chromatography using POROS AAVX resins provides
excellent scalability. The resin’s robust material construction
supports large-scale applications and enables multiple
reuse cycles, leading to significant cost savings. This allows
affinity chromatography to be not only more cost-effective
but also more scalable compared to traditional methods.
Overall, the efficiency, consistency and reusability of affinity
chromatography contribute to its advantages in AAV
production.
Q: Are there any limitations associated with affinity
chromatography in AAV purification? How can these
be addressed or optimized?
AB: One key limitation is specificity. While the AAVX ligand has
been effective for many engineered capsids, there have been
instances where the resin, or even the AAV9 resin, hasn’t bound
to certain capsids, particularly with AAV particles similar to AAV9.
Future engineered serotypes might also face similar issues.
When these challenges arise, there are a couple of options.
One is to explore non-affinity approaches, such as cation
exchange chromatography. However, this method involves
an additional step before chromatography and requires
optimization for each specific case.
The second option is to develop a custom ligand. At Thermo
Fisher, we offer the capability to create tailored affinity ligands
and resins for various biomolecules, including AAVs. We’ve
successfully developed custom solutions in the past, and this
could be a viable route when dealing with new engineered
capsids that don’t bind well with standard resins.
CG: There are some limitations and challenges with affinity
chromatography in AAV purification, particularly when dealing
with novel capsids. Novel capsids can present unique issues,
as they may not bind as effectively or predictably to the affinity
resin. This challenge extends to upstream processes, where
suboptimal production conditions for the novel capsid can lead
to lower viral titers, complicating downstream purification.
Our AAV-MAX system is designed to enhance upstream AAV
production by optimizing culture media, additives and cell
lines. Despite these advancements, issues with novel capsids
can still arise, and overcoming them often requires careful
troubleshooting and optimization.
FAS and purification specialists work closely with customers to
navigate these difficulties, developing workarounds and refining
processes to support effective purification even with novel capsids.
Q: What are the current methods to remove any
additional impurities that remain after an optimized
AAV capture chromatography step?
AB: The main methods used are anion exchange
chromatography and ultracentrifugation. Anion exchange
chromatography, especially with specific resins, is commonly
employed. Each has its advantages and disadvantages
depending on whether you’re working in research or scaling up
for GMP production.
These are the primary approaches because removing productrelated impurities – similar in size and charge to the target
product – is quite challenging. Fine separation is required,
which is something that ion exchange chromatography and
ultracentrifugation currently handle most effectively.
CG: After capturing AAV through affinity chromatography,
the next step typically involves using ion exchange
chromatography, with anion exchange being the most common
choice. Anion exchange chromatography effectively addresses
the remaining impurities, such as empty, partially filled and
overfilled AAV capsids, as well as trace amounts of host cell
DNA and proteins. These impurities usually account for about
3–5% of the purified material.
The focus of the anion exchange step is often on enriching
the full capsid population. This step is crucial for removing
empty capsids, which could potentially trigger an immunogenicthermofisher.com/aav-purification 12
response in patients. Since most of the AAVs produced
upstream are empty, it is essential to effectively separate these
from the full capsids.
POROS HQ and POROS XQ are strong anion exchangers that
are particularly effective in this process. Recent publications
have explored advanced techniques, such as using dual salt
buffer systems to create step gradients rather than linear
gradients. Step gradients are more suitable for large-scale
purification, enabling better separation of different capsid
species and improving scalability.
Q: What particular resins are used and how does
a process scientist evaluate and choose the best
candidate for the process?
AB: To select the best resin, a process scientist needs to start
by defining the goals of the step. This involves understanding
the target enrichment of the full particles required for the
process and determining the acceptable levels of other
impurities, like residual ligands or DNA.
Once those targets are set, it’s crucial to leverage existing
knowledge and resources. For instance, since the approval
of AAV therapies in the U.S. about six or seven years ago,
the field has accumulated significant insights, particularly
in anion exchange chromatography. Scientists should use
this knowledge to guide their initial conditions and step
development.
Scalability is another key factor. Anion exchange resins
offer more size options compared to other adsorbents, like
membranes or convective materials. Typically, this polishing
step is conducted at an alkaline pH (between 8 and 9.5)
because AAV particles exhibit poor binding at lower pH
levels. The separation is also performed at low conductivity.
Additionally, different counter ions or salts, like magnesium,
have been found to positively impact the separation. While the
exact mechanism might not be fully understood, it’s generally
considered as an additive during the process evaluation.
CG: In downstream purification, POROS HQ and POROS XQ
are commonly used strong anion exchange resins. These are
preferred because anion exchange chromatography effectively
handles the diverse characteristics of AAV and its impurities,
such as isoelectric points and binding strengths. While anion
exchange is the predominant choice, there are instances where
cation exchange might be used, for which POROS HS and
POROS XS are available.
When selecting the best resin, process scientists evaluate
several factors, including the specific binding properties and
the nature of the impurities. Downstream scientists have many
options here, but POROS XQ and POROS HQ are highly
recommended due to their robust performance and extensive
published data supporting their efficacy.
Q: Do you have any case studies that showcase the
successful polishing of AAVs?
AB: Fortunately, the field is starting to share more insights and
case studies on this topic. One example involves a thorough
evaluation and scale-up of a polishing step using POROS HQ.5
It’s a great example of how to approach developing this step for
a specific serotype, but the underlying principles are applicable
to other serotypes as well.
While I’ve focused mainly on POROS HQ, we’ve found through
recent customer interactions that POROS XQ might actually
perform better in many cases. There aren’t as many examples
yet, but scientists have investigated these interactions and
used POROS XQ as well.6 These case studies highlight how our
understanding and approaches are evolving.
CG: We have some notable case studies, with one of the
most recent being a paper published a couple of months ago.7
This study focused on AAV9 and demonstrated a successful
full capsid enrichment step using POROS HQ. Their results
were impressive, achieving over 60% full capsids, surpassing
their initial target of 50%. This is just one example; there are
numerous other cases, both published and unpublished, where
our POROS HQ resin has been used effectively to achieve high
levels of full capsid enrichment.
References:
1. Drulyte I, Raaijmakers H, Hermans P, Adams H, Radjainia M. Cryo-EM structure of
AAV8 and epitope mapping of CaptureSelect AAVX. Thermo Fisher Scientific. https://
assets.thermofisher.com/TFS-Assets/MSD/Reference-Materials/pharma-biotechaav-space-support-wp0030.pdf. Published online in 2022. Accessed September 10,
2024.
2. Zhao H, Meisen WH, Wang S, Lee KJ. Process development of recombinant adenoassociated virus production platform results in high production yield and purity. Hum
Gene Ther. 2023;34(1–2):56–67. doi:10.1089/hum.2022.153
3. Florea M, Nicolaou F, Pacouret S, et al. High-efficiency purification of divergent
AAV serotypes using AAVX affinity chromatography. Mol Ther Methods Clin Dev.
2023;28:146–159. doi:10.1016/j.omtm.2022.12.009
4. Heckel J, Martinez A, Elger C, et al. Fast HPLC-based affinity method to determine
capsid titer and full/empty ratio of adeno-associated viral vectors. Mol Ther Methods
Clin Dev. 2023;31:101148. doi:10.1016/j.omtm.2023.101148
5. Lavoie RA, Zugates JT, Cheeseman AT, et al. Enrichment of adeno-associated virus
serotype 5 full capsids by anion exchange chromatography with dual salt elution
gradients. Biotechnol Bioeng. 2023;120(10):2953–2968. doi:10.1002/bit.28453
6. Khanal O, Kumar V, Jin M. Adeno-associated viral capsid stability on anion exchange
chromatography column and its impact on empty and full capsid separation. Mol
Ther Methods Clin Dev. 2023;31:101112. doi:10.1016/j.omtm.2023.101112
7. Kish WS, Lightholder J, Zeković T, et al. Removal of empty capsids from high-dose
adeno-associated virus 9 gene therapies. Biotechnol Bioeng. 2024;121(8):2500–
2523. doi:10.1002/bit.2873713 thermofisher.com/aav-purification
Expert
Pouria Motevalian
Director, Viral Vector Process and
Analytical Development, Thermo Fisher
Scientific
As Director of Process Development,
Pouria Motevalian oversees the
development, scale-up and analytical characterization of novel
and compliant manufacturing processes for gene therapy. He
also holds a key position as a member of the Senior Leadership
Team for Thermo Fisher Scientific’s Plainville, MA site, our
largest viral vector development and manufacturing site in North
America. In this role, Pouria provides strategic guidance, shaping
scientific and operational plans for implementing bioprocess
technologies to meet the needs of viral vector clients. Pouria
received his PhD in chemical engineering with a minor in
computational science from Pennsylvania State University.
Harnessing CDMOs for Optimized AAV
Production: Ask the Expert
Adeno-associated viruses (AAVs) are a versatile tool in gene
therapy, promising to treat a range of previously incurable
genetic disorders by delivering therapeutic genes directly
into patients’ cells. Despite their potential, the large-scale
production and purification of AAVs still faces significant
challenges. Contract development and manufacturing
organizations (CDMOs) play a crucial role in addressing these
complexities, using their expertise to streamline and enhance
production.
To gain deeper insights into how CDMOs can tackle these
purification challenges and optimize AAV production, we
spoke with Pouria Motevalian, Director of Viral Vector Process
Development at Thermo Fisher Scientific.
Q: What have been the key advances in AAV
manufacturing technology over the last few years?
A: One of the most notable advancements has been the
development of scalable production platforms, particularly in
the areas of triple transfection-based systems, baculovirus
expression systems, and producer cell lines. These platforms
have evolved significantly, enabling the establishment of
scalable production processes for each approach.
Another major advancement is in capsid design and
engineering, which enables the development of vectors with
improved specificity, stability, and a reduced immune response.
Equally notable are improvements in downstream processing,
particularly in chromatography and filtration. Enhanced affinity
chromatography, with improved resins for the affinity capture
step, has resulted in more robust and efficient processes.
Depth filtration technologies have also been significantly
improved, increasing throughput and reducing process and
product impurities. These advancements in recent years have
propelled the field forward.
Q: What is the main bottleneck for manufacturing of
viral vectors?
A: Scaling up the production process while maintaining purity,
consistency and potency is a major challenge. Despite all the
advancements we’ve seen in recent years, inconsistencies can
still arise during scale-up, particularly with titer levels, impurity
removal and overall product quality. These inconsistencies canthermofisher.com/aav-purification 14
ultimately affect the final product’s potency, making this a key
bottleneck in viral vector manufacturing today.
Q: Are there any unique considerations or challenges
that arise when purifying viral vectors compared to
other types of biologics?
A: The goal is always to reduce process- and product-related
impurities. Removing upstream impurities, such as host cell
DNA or proteins, is standard for any biologic. However, for
viral vectors, particularly AAVs, the downstream process must
also remove empty and partial capsids. This is especially
challenging because empty, full, and partial capsids are similar
in size and have minimal differences in isoelectric points—often
just 0.2 to 0.6 units—making their separation extremely difficult.
To address this challenge, we rely on high-throughput resin
and mobile phase screening for optimization of anion exchange
and affinity chromatography steps. Anion exchange is
particularly crucial for empty/full capsid separation, and utilizing
high-throughput technologies for screening is essential for
developing a well-optimized process. This approach allows for
substantial removal of empty and partial capsids, significantly
enhancing the effectiveness of the purification process.
Q: How do CDMOs ensure the scalability and
reproducibility of viral vector purification processes,
particularly when dealing with large-scale production
for gene therapies?
A: There are two key aspects to consider. First, it’s crucial
to rely on a scalable, well-developed scale-down model for
each unit operation. Ensuring that the scale-down model
used during process development accurately reflects largerscale operations is essential. If the scale-down model doesn’t
faithfully represent large-scale production, the development
process loses value as the results won’t be transferable.
Second, it’s important to keep the end goal of large-scale
production in mind throughout the course of process
development. The focus should always be on ensuring that
the process designed in the lab can be successfully scaled
for clinical and commercial manufacturing. This means that
when developing processes and determining normal operating
ranges, we must work to ensure that these parameters are
feasible for large-scale production.
Q: How do gene therapy developers ensure
compliance with regulatory guidelines and standards
when purifying viral vectors?
A: The answer is straightforward: embrace Quality by Design
(QbD) throughout the entire development process and ensure
strict compliance with GMP best practices and guidelines
during clinical and commercial manufacturing. By adhering to
these principles—integrating QbD from the outset and following
GMP guidelines—developers can ensure they meet regulatory
requirements and align with industry standards.
Q: What further innovations would you like to see in
viral vector manufacturing in the future?
A: First, plasmid design and optimization should be prioritized
early on, because the optimized design of the plasmid
(especially ITR regions) has been shown to significantly
boost productivity, especially for AAV viral vectors. Second,
we would like to see advancements in resins that allow for
enhanced separation – not just when it comes to separating
empty and full capsids, but also in removing impurities. Lastly,
implementing process analytical technology (PAT) tools such
as Raman, FTIR and NIR for real-time measurement of critical
quality attributes is highly desired. This would help reduce
bottlenecks in QC testing without compromising product
quality, enabling real-time measurement and release.
Q: What are the advantages of working with a CDMO
to solve purification challenges early on in the
process?
A: The first major advantage is the expertise and experience
that CDMOs bring. Specializing in specific areas, CDMOs
possess the technical knowledge and deep understanding
necessary to develop and scale processes efficiently for clinical
and commercial manufacturing. Their experience working with
a variety of vectors and clients, each with unique requirements,
gives them a broad perspective on industry challenges. This
accumulated expertise allows them to provide tailored solutions
to the specific challenges each client faces.
Another key advantage is the ability to accelerate development
timelines. CDMOs, equipped with advanced tools and
technologies, can offer accelerated development. This is partly
due to economies of scale—they handle multiple projects
and pipelines simultaneously, enabling a more standardized
and efficient approach. As a result, they can speed up the
development process, which is particularly beneficial when
time is critical.
Q: Are there any particular purification challenges
for which your company has interesting and valuable
solutions for AAV manufacturers?
A: One of the major challenges in AAV purification is the
separation of empty and full capsids, as well as the removal
of residual host cell impurities like DNA and proteins. Now,15 thermofisher.com/aav-purification
the tricky part is that each client’s process is unique, so the
challenges they face are unique too. Because of that, the
solutions we offer need to be tailored to each client’s specific
needs.
Instead of providing a one-size-fits-all purification solution, we
take a more versatile approach to downstream processing,
development and optimization. We rely on high-throughput
technologies and techniques, which have consistently proven
effective in tackling these major downstream challenges across
multiple clients.
Q: Are there any specific recommendations for AAV
therapeutic downstream scientists when developing a
process planned to be transferred to a CDMO?
A: First, ensuring process robustness is essential, though
it’s a broad concept. To clarify, when transferring process
parameters for a specific unit operation, it’s important to
provide a range of acceptable parameters rather than just
a single target. This flexibility allows for a better facility fit,
particularly in a GMP setting for clinical and commercial
manufacturing.
Another key aspect of process robustness is the
manufacturability of mobile phases used in critical
chromatography steps. At times, the passing criteria for
parameters like pH and conductivity are so stringent that it
becomes challenging to prepare and release these buffers
in a GMP environment. When developing mobile phase
formulations, it’s important to consider manufacturability,
ensuring that the release criteria are broad enough to be
practical without compromising quality.
The second point is establishing appropriate hold times
for intermediates. Undefined hold times can force critical
operations to be performed during less optimal shifts,
increasing operational risk. Planning for these operations to
occur during shifts with full manpower and expertise reduces
this risk.
Lastly, it’s crucial to ensure that unit operations are scalable
throughout the process. For example, ultracentrifugation is
often used for viral vector purification but becomes difficult
to scale beyond a certain point, leading to the need to scale
out rather than up, which poses operational challenges and
increases capital and space requirements. Designing scalable
unit operations from the start is critical for a smooth and
successful tech transfer to a CDMO.
Pharmaceutical Grade Reagent. For Manufacturing and Laboratory Use Only.16 thermofisher.com/aav-purification
Introduction
Affinity chromatography is an advanced and highly effective
technique widely employed in downstream purification
processes, especially for adeno-associated viruses (AAVs).
By selectively binding and isolating target molecules, affinity
chromatography can greatly enhance both the efficiency and
yield of AAV purification. Here, we will explore the five main
benefits of affinity chromatography for AAV purification and
discuss various techniques and strategies to optimize your
downstream purification of viral vectors.
1. High specificity and selectivity
Affinity chromatography relies on the specific interaction
between a target molecule, such as a specific protein of the
AAV capsid, and an immobilized ligand on the chromatography
resin. This targeted binding enables the selective isolation
of AAV particles from complex mixtures, yielding a highly
pure sample. The high specificity and selectivity of affinity
chromatography help to ensure minimal contamination and
maximize the recovery of AAV particles.
Top 5 Benefits of Affinity Chromatography
for AAV Purification
2. Increased purity and yield
By specifically targeting AAVs from a complex feedstock,
affinity chromatography effectively recovers AAV particles
and allows unrelated impurities to pass through unretained.
When optimized, this technique achieves both high purity and
yield in a single step, streamlining the purification process and
maximizing the recovery of AAV particles.
3. Versatility and adaptability
Affinity chromatography can employ a wide range of ligands
that can be tailored to specific AAV purification needs. A key
advancement in this area involves the use of VHH antibodies –
camelid-derived single-domain antibodies – that can be finely
tuned to bind with high specificity to a vast array of biomolecules.
Through the use of advanced ligand generation platforms,
researchers can design VHH antibodies that target specific
properties of AAV, enabling either serotype-specific targeting or
broad, pantropic AAV binding. This adaptability ensures that the
purification process can be customized to address various needs
and improve the effectiveness of AAV isolation.17 thermofisher.com/aav-purification
4. Gentle purification conditions
A key advantage of affinity chromatography is its ability to
operate under mild purification conditions. This is especially
important for the purification of delicate viral vectors like AAV,
as harsh purification methods may compromise their structural
integrity and functionality. This ensures that the quality and
functionality of the AAV particles are maintained throughout the
process.
5. Scalability and automation
Affinity chromatography is highly scalable, making it wellsuited for large-scale AAV production. The technique can be
easily scaled up to meet increasing production demands while
maintaining efficiency. Additionally, the purification process
can be automated, enabling high-throughput purification
and significantly reducing the time and labor involved. This
combination of scalability and automation makes affinity
chromatography a practical and efficient choice for industrialscale AAV production.
Conclusion
Affinity chromatography presents substantial advantages
for the downstream purification of AAV vectors, making it a
cornerstone technique for process development scientists
in this field. Its exceptional specificity, ability to achieve high
purity and yield, versatility, gentle purification conditions and
scalability collectively contribute to its effectiveness in AAV
purification. By exploring and optimizing various techniques
and strategies within affinity chromatography, scientists can
further enhance the efficiency and yield of AAV purification
processes, thereby advancing the development of viral vectorbased therapies.
Learn more about optimizing your
purification process today. Visit our
webpage to find out more.18 thermofisher.com/aav-purification
38 BioProcess International 19(4) April 2021
B I O P R O C E S S TECHNICAL
Viral clearance in a
downstream AAV process
Case study using a model virus panel
and a noninfectious surrogate
Michael Winkler, Mikhail Goldfarb, Shaojie Weng, Jeff Smith, Susan Wexelblat, John Li,
Alejandro Becerra, Sandra Bezemer, Kevin Sleijpen, Aleš Štrancar, Sara Primec, Romina Zabar,
April Schubert, Akunna Iheanacho, and David Cetlin
Product Focus: Viruses
Process Focus: Downstream
processing
Audience: Process engineers,
analytical, manufacturing
Keywords: Monoliths,
affinity chromatography,
adenoassociated virus, TCID50,
Immuno-qPCR, SPR analysis
Level: Intermediate
O ver the past decade, adenoassociated virus (AAV) vectors have become established as leading genedelivery vehicles. In 2017, the pipeline
for gene therapies included 351 drugs in
clinical trials and 316 in preclinical
development (1–4). As those candidates
advance, significant efforts are being
made in process development and
manufacturing for viral vectors, with
the overall goal of reducing process
impurities while maintaining the
highest possible process yield.
To address that goal, industry
suppliers have developed innovative
AAV-specific separation technologies.
Thermo Fisher Scientific’s POROS
CaptureSelect AAVX affinity resin
provides a capture method for a number
of natural and synthetic AAV serotypes
irrespective of the expression system
used to produce them. By leveraging a
proprietary recombinant camelid
antibody technology immobilized onto
the highly permeable POROS backbone,
the resin achieves a fine-tuned
specificity for AAV recognition with an
increased surface area and capacity for
AAV binding. The significant impurity
reduction benefits and rapid scalability
of this affinity resin have led to its
incorporation into several noted AAV
downstream process designs.
BIA Separations (now part of
Sartorius AG) has developed and
commercialized CIMmultus QA
monoliths, which have been cited in
several AAV downstream processes for
their ability to separate empty and full
virus particles effectively. Monolithic
supports represent a unique type of
stationary phase for liquid
chromatography, bioconversion, and
solid-phase synthesis. Aside from
increased processing speed, monolithic
flow-through pores (channels) also
provide easy access for large molecules,
which supports both purification and
depletion of nanoparticles such as
plasmid DNA (pDNA) molecules and AAV
particles.
One elusive aspect of AAV process
development is viral clearance (VC). As
outlined in the ICH Q5A guidelines, VC
validation is a key regulatory
requirement governing all recombinant
biopharmaceuticals (5). According to
these guidelines, the risks of viral
contamination should be assuaged by a
three-pronged approach: prevent, test,
and remove. Over the past few decades,
Similarity in structures of adenoassociated
virus serotype 8 (top) and minute virus
of mice (bottom). PROTEIN DATA BANK
(HTTPS://WWW.RCSB.ORG)
38 BioProcess International 19(4) April 2021
B I O P R O C E S S TECHNICAL
Viral Clearance in a
Downstream AAV Process
Case Study Using a Model Virus Panel
and a Noninfectious Surrogate
Michael Winkler, Mikhail Goldfarb, Shaojie Weng, Jeff Smith, Susan Wexelblat, John Li,
Alejandro Becerra, Sandra Bezemer, Kevin Sleijpen, Aleš Štrancar, Sara Primec, Romina Zabar,
April Schubert, Akunna Iheanacho, and David Cetlin
Product Focus: Viruses
Process Focus: Downstream
processing
Audience: Process engineers,
analytical, manufacturing
Keywords: Monoliths,
affinity chromatography,
adenoassociated virus, TCID50,
Immuno-qPCR, SPR analysis
Level: Intermediate
O ver the past decade, adenoassociated virus (AAV) vectors have become established as leading genedelivery vehicles. In 2017, the pipeline
for gene therapies included 351 drugs in
clinical trials and 316 in preclinical
development (1–4). As those candidates
advance, significant efforts are being
made in process development and
manufacturing for viral vectors, with
the overall goal of reducing process
impurities while maintaining the
highest possible process yield.
To address that goal, industry
suppliers have developed innovative
AAV-specific separation technologies.
Thermo Fisher Scientific’s POROS
CaptureSelect AAVX affinity resin
provides a capture method for a number
of natural and synthetic AAV serotypes
irrespective of the expression system
used to produce them. By leveraging a
proprietary recombinant camelid
antibody technology immobilized onto
the highly permeable POROS backbone,
the resin achieves a fine-tuned
specificity for AAV recognition with an
increased surface area and capacity for
AAV binding. The significant impurity
reduction benefits and rapid scalability
of this affinity resin have led to its
incorporation into several noted AAV
downstream process designs.
BIA Separations (now part of
Sartorius AG) has developed and
commercialized CIMmultus QA
monoliths, which have been cited in
several AAV downstream processes for
their ability to separate empty and full
virus particles effectively. Monolithic
supports represent a unique type of
stationary phase for liquid
chromatography, bioconversion, and
solid-phase synthesis. Aside from
increased processing speed, monolithic
flow-through pores (channels) also
provide easy access for large molecules,
which supports both purification and
depletion of nanoparticles such as
plasmid DNA (pDNA) molecules and AAV
particles.
One elusive aspect of AAV process
development is viral clearance (VC). As
outlined in the ICH Q5A guidelines, VC
validation is a key regulatory
requirement governing all recombinant
biopharmaceuticals (5). According to
these guidelines, the risks of viral
contamination should be assuaged by a
three-pronged approach: prevent, test,
and remove. Over the past few decades,
Similarity in structures of adenoassociated
virus serotype 8 (top) and minute virus
of mice (bottom). PROTEIN DATA BANK
(HTTPS://WWW.RCSB.ORG)thermofisher.com/aav-purification 19
April 2021 19(4) BioProcess International 39
certain VC strategies for monoclonal
antibodies (MAbs), such as low-pH
inactivation and nanofiltration, have
become standard for most downstream
processes. However, because AAVs are
viruses themselves (within the family
Parvoviridae), it may not be possible to
apply the same purification strategies to
them that have served so well in MAb
processes. As a result, the gene-therapy
industry may depend increasingly on
chromatographic modes of separation to
demonstrate sufficient viral clearance.
In the study reported herein, we
addressed viral removal by performing
scale-down–model spiking studies and
measuring VC using a clinically relevant
AAV8 downstream purification process.
The two-step chromatography process
begins with affinity capture using
POROS CaptureSelect AAVX affinity
resin followed by anion-exchange
polishing using a CIMmultus QA
monolith. We selected as spiking agents
a wide range of viruses with different
sizes, molecular makeups, and
physiochemical properties. For DNA
viruses, we used enveloped
pseudorabies virus (PRV) and
nonenveloped minute virus of mice
(MVM). For RNA viruses, we used
enveloped xenotropic murine leukemia
virus-related virus (XMuLV) and
nonenveloped reovirus type 3 (Reo-3).
We also included human contagions
hepatitis A (HAV) and herpes simplex
virus 1 (HSV-1) based on a risk
assessment of possible adventitious
virus contaminations with a HEK293
human-derived producer cell line used
for upstream production. For the benefit
of future process developers who may
wish to perform similar VC testing on
their own purification process steps but
lack the ability to conduct such
resource-intensive studies, we assessed
the MockV MVM kit from Cygnus
Technologies, LLC, as an economical
and rapid means to generate predictive
MVM clearance data (6). We used the kit
in parallel with MVM spiking
experiments throughout our study.
Materials
All AAVs used for these viral clearance
studies were produced at representative
scale by a platform process at
REGENXBIO Inc.
Production of Virus and Mock Virus
Particles (MVPs): Viruses were
propagated and purified by Texcell NA
of Frederick, MD, according to standard
protocols. Noninfectious MVM-MVPs
were assembled after expression of the
major MVM capsid protein (VP2) in a
baculovirus expression-vector system
(BEVS) using Spodoptera frugiperda 9
(Sf9) cells at Cygnus Technologies.
Particles were purified with affinity and
ion-exchange chromatography (IEC).
Transmission electron microscopy
(TEM) was used to verify MVP
morphology, size, and concentration.
Chromatography Products: Thermo
Fisher Scientific supplied prepacked,
5-mL POROS CaptureSelect AAVX
affinity resin columns; BIA Separations
provided scale-down CIMmultus QA
monolith devices in 4-mL and 8-mL
diameters. Control POROS resins were
custom made by Thermo Fisher
Scientific with identical base beads to
Table 1: Adenoassociated virus (AAV) process spiking runs for xenotropic murine leukemia virus (XMuLV), hepatitis A (HAV), reovirus
type 3 (Reo-3), pseudorabies virus (PRV), herpes simplex virus 1 (HSV-1), minute virus of mice (MVM), and the MVM mock virus particle
(MVM-MVP)
Spiking Agent
POROS CaptureSelect AAVX CIMmultus QA
Center Point
Run 1 Run 2 POROS Alternative Ligand POROS Base Matrix AAV Null Load Worst Case Center Point Worst Case
XMuLV ✓ ✓ ✓ ✓ ✓ ✓ ✓
HAV ✓ ✓ ✓ ✓
Reo-3 ✓ ✓ ✓ ✓
PSV ✓ ✓ ✓ ✓
HSV-1 ✓ ✓ ✓ ✓
MVM ✓ ✓ ✓ ✓ ✓ ✓ ✓
MVM-MVP ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓
Figure 1: (a) Immuno-quantitative real-time polymerase chain reaction (Immuno-qPCR)
assay; (b) Immuno–qPCR standard curve
Mock
virus
particle
(MVP)
Anti-MVP
antibody
conjugated
to target DNA
Anti-MVP
capture
antibody
Cycle Threshold (Ct) Value
MVM-MVP/mL
40
30
20
10
0
104 106 108 1010
y = –0.915ln(x) + 43.542
R² = 0.9815
A B
Figure 2: Viral clearance using affinity resin; see Table 1 for full virus names
Log Reduction Value
XMuLV MVM Reo-3 HAV PRV HSV-1
76543210
Manufacturing Center-Point Process Worst-Case Process
6.4
4.6
4.4
3.6
2.7
2.5
4.9 5.0
4.0
3.8
3.1
3.620 thermofisher.com/aav-purification
40 BioProcess International 19(4) April 2021
the affinity resin used but incorporating
either an alternative V
HH ligand
specificity (nonbinding for AAVs) or no
V
HH functionalized ligand.
Methods
Study Design: To assess the robustness of
the affinity resin and monolith polishing
step within REGENXBIO’s downstream
process (7), we selected “center-point”
and “worst-case” processing parameters
for our viral clearance spiking
experiments (Table 1). For each run, we
spiked in-process AAV material with
model viruses or MVM-MVPs to a target
of 10.0 log10 MVM-MVP/mL and
processed accordingly.
For affinity resin center-point and
alternative-ligand runs, we loaded
150 mL of spiked material according to
standard manufacturing load ratios and
residence time. For worst-case
conditions, we loaded 200 mL of spiked
material (133% of the target) and
decreased the flow rate to lengthen
residence time to 170% of the centerpoint target. For the monolith polishing
study, we applied center-point load
volumes of 90 mL to 8-mL monoliths
and 65 mL to 4-mL monoliths for worst
case — except for the MVP- and XMuLVspiked runs. For those, we loaded 45 mL
and 65 mL onto 4-mL columns,
respectively, for the center-point and
worst-case conditions.
Samples were collected from each
run during each step phase (flowthrough, wash, and so on). We analyzed
the virus samples immediately with a
50% tissue culture infectious dose
(TCID50) assay or quantitative real-time
polymerase chain reaction (qPCR).
MVM-MVP samples were stored at
–80 °C before Immuno-qPCR analysis
(described below). From those results,
we determined log reduction values
(LRVs) using a standard calculation (5).
During this study, we performed
affinity-capture experiments to probe
potential nonspecific binding
interactions (Table 1). Both AAV-null
(produced by pooling the flow-through
fractions of previous AAVX runs) and
AAV8-containing load materials were
spiked with model virus, then affinity
purified using center-point conditions
and compared for viral clearance.
Additionally, we evaluated interactions
between viruses and base beads by
performing AAV8 center-point runs
using the POROS base matrix without a
functionalized V
HH ligand; we evaluated
virus–V
HH ligand interactions using a
POROS resin with an alternative V
HH
ligand specificity to the Fc portion of
MAbs that cannot bind AAVs.
We applied an orthogonal test
method — surface plasmon resonance
(SPR) — to confirm the specificity of the
AAVX ligand for AAV. For that, a
biotinylated AAVX VHH ligand was
immobilized onto a detection surface so
that binding sensograms could be
generated by injection of free MVM-MVP
or AAV.
Analytical Assays and LRV
Determinations: Texcell scientists
quantified infectious titer of XMuLV using
a validated plaque-forming infectivity
assay. They quantified HAV, Reo-3, HSV-1,
and MVM using validated TCID50
infectivity assays. PRV was quantified
with a validated qPCR assay. From those
titer determinations, we calculated LRVs
by a standard method (5).
To analyze the concentration of
noninfectious MVM-MVP within each
sample, Cygnus Technologies scientists
performed an Immuno-qPCR assay
(Figure 1a) as described elsewhere (8).
In brief, samples were added to
microwells coated with an anti–MVMMVP capture MAb. After incubation and
washing, a DNA-conjugated anti–MVMMVP detector MAb was added. Following
another incubation and washing step, a
dissociation buffer was added to each
Figure 4: Characterization of nonspecific
interactions based on log reduction
values (LRVs) for xenomorphic murine
leukemia virus (XMuLV) and minute virus
of mice (MVM)
XMuLV MVM
LRV
76543210
Control
Alternative
VHH Ligand
AAV-null
POROS
Base Matrix
Figure 3: Log reduction value (LRV)
determinations for affinity resin runs
spiked with minute virus of mice (MVM)
and noninfectious MVM mock virus
particles (MVM-MVPs)
LRV
5 4 3 2 1 0
4.35
5.035
3.58
4.07
3.79
4.28
MVM MVM-MVP
Center
Point
Alternate
Ligand
Worst
Case
Table 2: Affinity resin data from experiments with minute virus of mice (MVM) and
noninfectious MVM mock virus particles (MVM-MVPs)
Run Type Phase
Total Particles (log10) Percentage of Particles
MVM
MVM-MVP
MVM
MVM-MVP
Run 1 Run 2 Run 1 Run 2
Center Point Load 8.1 12.3 12.2 NA NA NA
FT 7.9 12.0 12.0 66.1% 52.6% 67.0%
Wash 1 6.1 10.0 9.7 1.0% 0.5% 0.3%
Wash 2 5.4 11.3 11.2 0.2% 10.4% 10.4%
Wash 3 4.7 8.7 8.7 0.0% 0.0% 0.0%
Elution 3.8 7.4 7.0 0.0% 0.0% 0.0%
CIP 5.0 6.9 6.7 0.1% 0.0% 0.0%
Worst Case Load 7.9 11.9 NT NA NA NA
FT 7.6 11.8 55.0% 79.1%
Wash 1 NT 9.9 NA 1.1%
Wash 2 NT 11.0 NA 14.3%
Wash 3 NT 9.0 NA 0.1%
Elution 4.3 7.8 0.0% 0.0%
CIP NT 6.8 NA 0.0%
NT = not tested; NA = not applicable; FT = flow-through fraction; CIP = clean-in-place solutionthermofisher.com/aav-purification 21
April 2021 19(4) BioProcess International 41
well for five minutes. Then 5 µL of
sample was transferred from each well
to a qPCR plate containing TaqMan
primers/probes (Thermo Fisher
Scientific) directed against the
conjugated DNA. To determine the
quantity of particles in unknown
samples, threshold cycle (Ct) values
were interpolated into a standard curve
generated by including a 10-fold dilution
series of a known MVM-MVP standard
(Figure 1b). From those concentration
values, we could calculate MVM-MVP
LRVs for each experiment.
Results and Discussion
Viral clearance strategies for AAV
downstream processes are limited by
difficulty in performing viral
inactivation and filtration steps without
also inactivating or removing the AAV
product. Therefore, chromatographic
modes of separation are paramount to
achieving the desired levels of removal
for viruses of concern.
To complicate matters further, MVM
(a small, nonenveloped virus used
internationally as a model spiking
agent) is a member of the same
parvovirus family as AAV. Morphology
and physicochemical properties (size,
surface charge, and surface
hydrophobicity) are similar in the two
virus species. Because of those
physicochemical similarities, a step
optimized to bind and elute AAV
through affinity or ion interactions also
might bind MVM. That would result in
poor removal of such viral contaminants
— or conversely, a step optimized to
remove MVM could compromise AAV
yield as the product is washed or eluted
away along with MVM.
With that in mind, in our study we
wanted to elucidate whether viruses
(including MVM) could be resolved from
AAV through the combination of a
POROS CaptureSelect AAV column and a
CIMmultus QA monolith, thereby
providing effective VC.
Affinity Capture: Figure 2 summarizes
the results from all VC spiking
experiments using POROS CaptureSelect
AAVX affinity resin. At manufacturing
center-point process conditions, effective
viral clearance of ≥4 LRV was
demonstrated for XMuLV, MVM, HAV,
and PRV. The AAVX resin also
contributed to clearing ≥2.5 LRV for
Reo-3 and HSV-1. During worst-case–
conditions testing, similar levels of
clearance were observed for all model
viruses tested. Taken together, this
demonstration of robust VC using POROS
CaptureSelect AAVX affinity
chromatography is consistent with the
highly specific nature of the affinity
interaction between AAVX resin and AAV
vectors. Its high degree of specificity and
capacity is mediated by the camelid VHH
antibody ligands functionalized to the
custom-designed base beads, which in
combination provide high-affinity
binding to AAV vectors while minimizing
nonspecific interactions.
Figure 5: Binding selectivity of camelid VHH antibody-fragment affinity ligand analyzed
by surface plasmon resonance (SPR)
MVM-MVP
AAV1
Binding Response Units (RU)
2,500
2,000
1,500
1,000
500
0
Time (seconds)
0 200 400 600 800 1,000
Association Dissociation Regeneration
Table 3: Monolith results for xenotropic murine leukemia virus
(XMuLV)
Phase
Total XMuLV (log10) Percentage of XMuLV
Center Point Worst Case Center Point Worst Case
Load 6.7 6.9 NA NA
FT ≤5.0 ≤5.1 ≤2.0% ≤1.8%
Pre-peak 1 ≤4.8 NT ≤1.2% NA
Pre-peak 2 ≤4.7 NT ≤0.9% NA
Pre-peak 3 ≤3.8 NT ≤0.1% NA
Elution ≤1.6 ≤1.6 ≤0.0% ≤0.0%
Post-peak 1 ≤0.8 NT ≤0.0% NA
Post-peak 2 ≤1.3 NT ≤0.0% NA
Strip 4.1 4.5 0.3% 0.4%
NT = not tested; NA = not applicable; FT = flow-through fraction
Table 4: Monolith results for hepatitis A virus (HAV)
Phase
Total HAV (log10) Percentage of HAV
Center Point Worst Case Center Point Worst Case
Load 7.5 7.4 NA NA
FT ≤3.3 ≤3.1 ≤0.0% ≤0.0%
Pre-peak 1 ≤3.0 NT ≤0.0% NA
Pre-peak 2 ≤3.0 NT ≤0.0% NA
Pre-peak 3 ≤2.3 NT ≤0.0% NA
Elution ≤2.9 ≤2.6 ≤0.0% ≤0.0%
Post-peak 1 ≤2.0 NT ≤0.0% NA
Post-peak 2 ≤2.5 NT ≤0.0% NA
Strip 7.3 7.2 58.6% 70.3%
NT = not tested; NA = not applicable; FT = flow-through fraction
MVM (a small,
nonenveloped virus used
as a model spiking agent)
is a member of the same
parvovirus family as AAV.
Morphology and
physicochemical
properties (size, surface
charge, and surface
hydrophobicity) are
SIMILAR in the two
virus species.22 thermofisher.com/aav-purification
42 BioProcess International 19(4) April 2021
Among the panel of six model
viruses tested, MVM is potentially
problematic to remove based on its
similarity in size to AAV and high
resistance to inactivation. Being a
nonenveloped DNA virus that is a
member of the same Parvoviridae
family as AAV, MVM has a similar
capsid structure and therefore
potentially similar viral morphology
and physicochemical properties (size,
surface charge, surface
hydrophobicity).
Table 2 details our results for MVM
and MVM-MVP (the noninfectious MVM
surrogate created by Cygnus
Technologies as a biosafety-level 1 safe
analytical tool). Most MVM initially
loaded onto the column was contained
within the flow-through fractions for
both center-point and worst-case runs
(66.1% and 55.0%, respectively).
Similarly, most MVM-MVPs also were
found within the flow-through fraction
(52.6–67.0% and 79.1% for center-point
and worst-case runs, respectively).
Affinity wash steps did little to strip the
column further of MVM (0.2% for center
point); moderate quantities of MVMMVPs were removed (10.4% and 14.3%
for center-point and worst-case runs,
respectively). Small quantities of both
MVM and MVM-MVPs were found in the
elution fractions collected.
From MVM center-point and worstcase runs, 3.8 and 4.3 log10 total
particles were detected in the elution,
respectively, leading to LRV calculations
of 4.35 ± 0.38 and 3.58 ± 0.46 (Figure
Table 5: Monolith results for herpes simplex virus 1 (HSV-1)
Phase
Total HSV-1 (log10) Percentage of HSV-1
Center Point Worst Case Center Point Worst Case
Load 7.2 7.1 NA NA
FT ≤3.9 ≤3.7 ≤0.0% ≤0.0%
Pre-peak 1 ≤3.7 NT ≤0.0% NA
Pre-peak 2 ≤3.6 NT ≤0.0% NA
Pre-peak 3 ≤2.8 NT ≤0.0% NA
Elution ≤3.5 ≤3.2 ≤0.0% ≤0.0%
Post-peak 1 ≤2.0 NT ≤0.0% NA
Post-peak 2 ≤2.5 NT ≤0.0% NA
Strip 6.1 5.2 7.0% 1.3%
NT = not tested; NA = not applicable; FT = flow-through fraction
Table 6: Monolith results for pseudorabies virus (PRV)
Phase
Total PRV (log10) Percentage of PRV
Center Point Worst Case Center Point Worst Case
Load 9.9 9.7 NA NA
FT ≤5.1 ≤4.8 ≤0.0% ≤0.0%
Pre-peak 1 ≤4.8 NT ≤0.0% NA
Pre-peak 2 ≤4.8 NT ≤0.0% NA
Pre-peak 3 ≤3.9 NT ≤0.0% NA
Elution ≤4.7 ≤4.3 ≤0.0% ≤0.0%
Post-peak 1 ≤3.9 NT ≤0.0% NA
Post-peak 2 ≤4.2 NT ≤0.0% NA
Strip 8.9 8.7 10.4% 9.6%
NT = not tested; NA = not applicable; FT = flow-through fraction
Table 7: Monolith results for reovirus 3 (Reo-3)
Phase
Total Reo-3 (log10) Percentage of Reo-3
Center Point Worst Case Center Point Worst Case
Load 8.8 8.7 NA NA
FT ≤3.6 ≤3.4 ≤0.0% ≤0.0%
Pre-peak 1 ≤3.4 NT ≤0.0% NA
Pre-peak 2 ≤3.3 NT ≤0.0% NA
Pre-peak 3 ≤2.4 NT ≤0.0% NA
Elution ≤3.2 ≤2.9 ≤0.0% ≤0.0%
Post-peak 1 ≤2.0 NT ≤0.0% NA
Post-peak 2 ≤2.5 NT ≤0.0% NA
Strip 6.4 6.3 0.4% 0.4%
NT = not tested; NA = not applicable; FT = flow-through fraction
Table 9: Monolith results for noninfectious minute virus of mice
mock virus particles (MVM-MVPs)
Phase
Total MVM-MVP (log10) % of MVM-MVP
Center Point Worst Case Center Point Worst Case
Load 11.3 11.9 NA NA
FT ≤7.1 ≤7.3 ≤0.0% ≤0.0%
Pre-peak 1 ≤6.5 NT ≤0.0% NA
Pre-peak 2 ≤7.0 NT ≤0.0% NA
Pre-peak 3 ≤6.1 NT ≤0.0% NA
Elution ≤7.4 ≤6.8 ≤0.0% ≤0.0%
Post-peak 1 ≤6.6 NT ≤0.0% NA
Post-peak 2 ≤7.1 NT ≤0.0% NA
Strip 11.0 11.5 43.1% 45.1%
NT = not tested; NA = not applicable; FT = flow-through fraction
Table 8: Monolith results for minute virus of mice (MVM)
Phase
Total MVM (log10) Percentage of MVM
Center Point Worst Case Center Point Worst Case
Load 6.5 6.4 NA NA
FT ≤3.6 ≤3.4 ≤0.1% ≤0.1%
Pre-peak 1 ≤3.0 NT ≤0.0% NA
Pre-peak 2 ≤3.3 NT ≤0.1% NA
Pre-peak 3 ≤2.1 NT ≤0.0% NA
Elution ≤1.2* ≤0.9* ≤0.0% ≤0.0%
Post-peak 1 ≤2.3 NT ≤0.0% NA
Post-peak 2 ≤2.8 NT ≤0.0% NA
Strip 7.5 7.6 941.7% 1,635.7%
NT = not tested; NA = not applicable; FT = flow-through fraction
* large-volume sampling to increase sensitivity
SIMILAR clearance results were
achieved for the two particle types at each
condition, and the trend in reduced clearance
seen for MVM could be monitored through
the use of MVM-MVPs. Our data demonstrate
the utility of MVM-MVPs as a spiking/analysis
tool for process development and
characterization.thermofisher.com/aav-purification 23
3). That difference in LRV probably can
be attributed to the influence of process
parameters (load ratio and residence
time) on virus–ligand interactions. As
predicted, a higher load ratio and
residence time yielded nearly a 1.0 log10
decrease in MVM clearance. For MVMMVP center-point and worst-case runs,
7.0–7.4 and 7.8 log10 total particles were
determined, respectively, giving LRV
calculations of 4.91–5.16 and 4.07. Thus,
similar clearance results were achieved
for both particle types at each condition,
and the trend in reduced clearance seen
for MVM could be monitored through the
use of MVM-MVP. Figure 3 also shows
LRV results for a center-point run using
an alternative base matrix.
Overall, those results demonstrate
the high selectivity of the POROS
CaptureSelect AAVX affinity resin,
which can differentiate between the
surface epitopes of AAV and the
evolutionarily similar virus MVM. Such
high specificity enables the resin to
partition those two particle types from a
heterogenous mixture containing both
of them. Our data also reveal
comparable results between MVM and
MVM-MVP, demonstrating the utility of
MVM-MVP as a spiking/analysis tool for
process development and
characterization.
To probe nonspecific binding, a more
detailed interaction study was
performed using MVM and XMuLV,
which are the two most commonly used
model viruses for VC spiking studies (9).
To probe virus–AAV interactions, we
performed an AAV-null run wherein the
spiked virus load was devoid of AAV8
product. As Figure 4 shows, the null run
demonstrated similar performance to
the manufacturing control run for both
MVM and XMuLV, indicating that the
presence of AAV8 had minimal effect on
clearance of model viruses.
Next, to probe interactions among
viruses and V
HH ligands or POROS base
beads, we used two control resins
designed by Thermo Fisher Scientific.
The first control was an AAVX-like resin
with an identical base bead but a
functionalized V
HH ligand with an
alternate specificity that cannot bind
AAV. The second control was POROS
CaptureSelect AAVX resin without a
functionalized V
HH ligand. Using these
control resins, we observed similar VC
levels to those of the center-point run
using unmodified POROS CaptureSelect
AAVX affinity resin, which indicates
that minimal interactions occurred
between viruses and the ligand or base
beads. These results strongly indicate
that model viruses show no nonspecific
binding to either the VHH ligand or to
the POROS base bead — and that
interactions between those viruses and
the AAV product are minimal.
SPR results demonstrated that the
AAVX V
HH ligand bound to the injected
AAV1, but not to MVM-MVP (Figure 5).
For an experimental control, the binding
signal was recovered when AAV was
spiked back into a 0.1-µg/mL MVM-MVP
background. These data suggest that the
presence of virus particles (infectious or
otherwise) does not interfere with the
ability of AAV to bind to the specific
AAVX V
HH ligand.
Monolith Polishing: Tables 3–9 show
complete VC results (including MVMMVP) from our CIMmultus QA spiking
experiments. During each experiment,
we captured flow-through, three preAAV elution peaks, two postproduct
peaks, and a strip fraction along with
the load and main AAV elution pool. As
the data show, no virus (or MVM-MVP)
was detected in any fraction other than
the strip for either center-point or worstcase conditions. That indicates complete
clearance for all virus types in this
downstream AAV process step. Viruses
also were undetectable in the flowthrough, pre– and post–main-peak
collections.
Virus titers within the strip fractions
were significant but differed by virus.
The amount of MVM detected in the
strip fraction was greater than the
overall challenge, which could indicate
interference with the assay. In some
cases, the mass-balance of total virus
detected within the collected fractions
did not equate to the amount of virus
challenged. That may be attributable to
(partial) degradation of the virus by the
stripping agent and/or to using the
stripping agent for too short a time to
elute all the virus.
Figure 6 shows LRVs from each
experiment. The monolith offered
effective removal for a wide range of
physicochemically distinct viruses. In
addition, the LRV data demonstrate
comparability between MVM and MVMMVP clearance at both center-point and
worst-case conditions.
Final Results: Table 10 lists overall
process LRVs achieved after using both
Figure 6: Monolith log reduction values (LRVs); see Table 1 for full virus names
Log Reduction Value
7 6 5 4 3 2 1 0
XMuLV HAV HSV-1 PRV Reo-3 MVM MVP
Center Point Worst Case
>5.1 >5.3
>4.6>4.8
>3.8 >3.9
>5.1
>5.4 >5.6 >5.7
>3.8
>5.1
>5.4 >5.5
Table 10: Step-by-step and overall process log reduction values (LRVs) achieved at
center-point operation
XMuLV HAV HSV-1 PRV Reo-3 MVM MVM-MVP
POROS CaptureSelect
AAVX resin
≥6.4 ≥4.9 3.1 4.0 2.7 4.4 5.0
CIMmultus QA column ≥5.1 ≥4.6 ≥3.8 ≥5.1 ≥5.6 ≥5.4 ≥3.9
Overall ≥11.5 ≥9.5 ≥6.9 ≥9.1 ≥8.2 ≥9.8 ≥8.924 thermofisher.com/aav-purification
44 BioProcess International 19(4) April 2021
POROS CaptureSelect AAVX affinity
resin and CIMmultus QA monolith
polishing steps operated at center-point
manufacturing conditions.
An Accurate and Economic
Prediction Model
We sought to determine whether effective
viral clearance could be achieved through
chromatographic methods in an AAV
purification process. Through spiking
studies using a broad and inclusive panel
of viruses, we determined that
chromatographic modes of separation
indeed can provide an effective VC
strategy. Both POROS CaptureSelect AAVX
affinity resin and CIMmultus QA anionexchange monoliths demonstrated superb
ability to reduce viral levels and
contribute to high overall process LRVs.
During this study, we also sought to
determine whether a biosafety-level 1
compliant, noninfectious mock MVM
particle could serve as an accurate
surrogate for predicting MVM removal.
High correlation between the MVM and
MVM-MVP results obtained throughout
this study suggest that such an
approach could provide an accurate and
economic model for predicting the VC
efficacy of other AAV chromatographic
separation techniques.
References
1 Rininger J, Fennell A, SchoukrounBarnes LR. AAV Vector Manufacturing
Platform Selection and Product Development.
BioProcess Int. December 2019 sponsored
insert; https://bioprocessintl.com/sponsoredcontent/aav-vector-manufacturing-platformselection-and-product-development.
2 Scull L. Gene Therapy Pipeline
Overview. ATHN Data Summit: Chicago, IL,
25–26 October 2018; https://alliancerm.org/
wp-content/uploads/2018/10/Gene-TherapyPipeline-Overview_FINAL-2.pdf.
3 Ginn SL, et al. Gene Therapy Clinical
Trials Worldwide to 2017: An Update. J. Gene
Med. 20(5) 2018: e3015; https://doi.
org/10.1002/jgm.3015.
4 Lloyd I, et al. Pharma R&D Annual
Review. Pharmaprojects: London, UK,
February 2017; https://pharmaintelligence.
informa.com/~/media/Informa-ShopWindow/Pharma/Files/PDFs/whitepapers/
RD-Review-2017.pdf.
5 ICH Q5A: Viral Safety Evaluation of
Biotechnology Products Derived from Cell
Lines of Human or Animal Origin. US Fed.
Reg. 63(185) 1998: 51074; http://www.fda.
gov/downloads/Drugs/GuidanceCompliance
RegulatoryInformation/Guidances/
ucm073454.pdf.
6 Orchard JD, et al. Using a
Noninfectious MVM Surrogate for Assessing
Viral Clearance During Downstream Process
Development. Biotechnol. Prog. 36(1) 2020:
e292; https://doi.org/10.1002/btpr.2921.
7 Goldfarb M, et. al. Downstream
Purification of Adeno-Associated Virus for
Large-Scale Manufacturing of Gene
Therapies. Cell Gene Ther. Ins. 6(7) 2020:
955–963.
8 Cetlin D, et al. Use of a Noninfectious
Surrogate to Predict Minute Virus of Mice
Removal During Nanofiltration. Biotechnol.
Prog. 34(5) 2018: 1213–1220; https://doi.
org/10.1002/btpr.2694.
9 Herbig K, et al. Modeling Virus
Clearance: Use of a Noninfectious Surrogate
of Mouse Minute Virus As a Tool for
Evaluating an Anion-Exchange
Chromatography Method. BioProcess Int.
17(5) 2019: 34–40; https://bioprocessintl.
com/downstream-processing/viral-clearance/
modeling-virus-clearance-use-of-anoninfectious-surrogate-of-mouse-minutevirus-as-a-tool-for-evaluating-an-anionexchange-chromatography-method. c
Corresponding author Michael Winkler (mwinkler@
regenxbio.com) is director of downstream process
development and validation, Mikhail Goldfarb*
was a principal scientist in process development,
Shaojie Weng is a scientist II, Jeff Smith is a senior
associate scientist, and Susan Wexelblat is a CMC
technical writer at REGENXBIO Inc., 9600 Blackwell
Road, Suite 210, Rockville, MD 20850. John Li is a
staff scientist, Alejandro Becerra is a principal
applications scientist, and Sandra Bezemer and
Kevin Sleijpen are scientists at Thermo Fisher
Scientific, 35 Wiggins Avenue, Bedford, MA 01730.
Aleš Štrancar is chief executive officer, Sara
Primec is sales and product manager, and Romina
Zabar is head of quality assurance at BIA
Separations, Mirce 21, SI-5270 Ajdovščina, Slovenia.
April Schubert is director of business development
for North America, and Akunna Iheanacho is
scientific director at Texcell, 4991 New Design Road,
Frederick, MD 21703. David Cetlin is senior director
of R&D at Cygnus Technologies LLC, 4332 SouthportSupply Road SE, Southport, NC 28461.
*Goldfarb is now director of protein purification at
Arcellx, Inc.
To share this in PDF or professionally printed
format, contact Jill Kaletha: jkaletha@
mossbergco.com, 1-574-347-4211.
Originally cited and published by Bioprocess International.25 thermofisher.com/aav-purification
Bioprocessing
Learn more at thermofisher.com/purification-contact
Productivity optimization and process calculations for
AAV affinity chromatography
INTRODUCTION
The use of recombinant adeno-associated virus (rAAV) as a delivery method for gene
therapies continues to be successful with hundreds of ongoing clinical trials and some
recent approvals. The diversity of applications for rAAV ranges from rare diseases
affecting small patient populations to more prevalent inherited ailments such as
hemophilia. The doses required vary widely from ~4E11 vg/eye for subretinal
administration to 3.5E14 vg for intrathecal applications [1]. From a manufacturing
perspective the field has moved to common approaches for production and
purification of rAAV. Upstream approaches typically use transfection of HEK293 cells
and titers are routinely in the 1-2E10 vg/mL although higher titers of up to 6E11 vg/mL
at a 2000 L scale were recently reported [2]. These high titers will be needed for
large dose and/or patient populations to meet the demand of these therapies and
reduce costs. For rAAV purification the majority of the field has moved to scalable
processes employing an affinity capture chromatography step [3] and commonly
utilizing POROS™ CaptureSelect™ AAVX resin. In this work, dynamic binding capacity
(DBC) data for multiple AAV serotypes were leveraged to estimate an optimal
productivity of rAAV using the AAVX resin. An analysis of process conditions and
column geometries that would fit maximum processing times and resin utilization was
conducted for two case scenarios representing current titers for clinical manufacturing
and high titers for commercial manufacturing scales.
Alejandro Becerra-Arteaga, Ph.D. and Jett Appel, Thermo Fisher Scientific, Bedford, MA, USA
Dynamic Binding Capacity
✓ Limited DBC data are available due to high capacity of AAVX resin, relatively low titers,
and sample availability.
✓ DBC for AAV2 is relatively high (~1E15 capsids/mL resin) even at 30 sec residence time.
✓ Data fit to equation (I) approximates the dependence of DBC to residence time.
TRADEMARKS/LICENSING
© 2023 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless
otherwise specified. This information is not intended to encourage use of these products in any manner that might infringe the intellectual property
rights of others. Intended use: For research use only. Not for use in diagnostic procedures
CONCLUSIONS
• The relatively high binding capacity of POROS CaptureSelect AAVX resin was
confirmed to be >1E15 capsids/mL resin at residence times >= 0.5 min for AAV2.
• Productivity is maximized at load residence times <= 0.5 min depending on titer
but hardware and/or system considerations limit operation closer to 1 min.
• For clinical manufacturing the high DBC allows for a range of process conditions
and requires small column volumes.
• For large bioreactor volumes and high titers the model suggests columns 20-30
cm diameter to meet typical processing limits while maximizing resin utilization.
Scale-up and process considerations
Productivity
✓ Productivity maximum is achieved at residence times below 0.5 min.
✓ Productivity increases by ~3.5x with an increase in titer of ~12x.
✓ Increased titer shifts productivity maximum from ~7 to ~24 seconds RT for loading.
METHODOLOGY
Dynamic binding capacity:
AAV2 breakthrough curves were generated using HEK293 clarified lysate to
determine DBC at 10% breakthrough. AAV8 and rh10 DBC data were obtained from
references 4 and 5, respectively.
Equation I was fitted to the DBC data using a linear regression numerical method.
Productivity:
Productivity curves were generated using equations I and II.
Column volumes and residence time for the non-loading steps were 25 CV and 2 min.
Column volumes and residence time for CIP steps were 10 CV and 3 min.
Scale-up and process considerations:
GMP pre-packed column pressure limitations were based on literature from multiple
vendors.
Pressure drop at 3 bar was based on pressure-flow curves for POROS CaptureSelect
AAVX resin (internal pressure-flow data).
Case scenarios
Processing time and resin utilization calculations were performed using Microsoft ®
Excel ® assuming 20% full capsids and the results were further analyzed and plotted
using MODDE® software.
Scenario 1. Clinical manufacturing, 200 L, Co=2.5E11 capsids/mL
1.0E+12
1.0E+13
1.0E+14
1.0E+15
1.0E+16
0 1 2 3
Dynamic Binding Capacity @10% breakthrough
(capsids/mL resin)
Residence time (min)
AAV2
AAV8
rh10
Fit
Qd= Dynamic Binding Capacity @10% breakthrough
Qdmax = DBC at long residence times
RT = Load residence time
q = Residence time constant
𝑃𝑃 =
𝐴𝐴𝑚𝑚𝑜𝑜𝑢𝑢𝑛𝑛𝑡𝑡 𝑜𝑜𝑓𝑓𝐴𝐴𝐴𝐴𝑉𝑉 𝑐𝑐𝑎𝑎𝑝𝑝𝑠𝑠𝑖𝑖𝑑𝑑𝑠𝑠 𝑝𝑝𝑢𝑢𝑟𝑟𝑖𝑖𝑓𝑓𝑖𝑖𝑒𝑒𝑑𝑑
𝑈𝑈𝑛𝑛𝑖𝑖𝑡𝑡 𝑟𝑟𝑒𝑒𝑠𝑠𝑖𝑖𝑛𝑛 𝑣𝑣𝑜𝑜𝑙𝑙𝑢𝑢𝑚𝑚𝑒𝑒 × 𝑈𝑈𝑛𝑛𝑖𝑖𝑡𝑡 𝑡𝑡𝑖𝑖𝑚𝑚𝑒𝑒
P = Productivity
h = Loading safety factor (% DBC)
C0 = Load sample concentration
CV
non-load= Column volumes for non-loading steps
RT
non-load = Residence time for non-loading steps
CVCIP= Column volumes for non-loading steps
RT
CIP = Residence time for non-loading steps
No breakthrough
0
500
1000
1500
2000
2500
3000
3500
5 10 15 20
Linear velocity (cm/h)
Bed height (cm)
RT=0.2 min
RT=0.5 min
RT=1 min
RT=2 min
RT=3 min
DP = 3 bar
Window of operation
outside GMP pre-packed
column (4 bar) and/or
system limitations
REFERENCES
1. Burdett and Nuseibeh, Gene Therapy (2023)
2. Van Lieshout, et al. Molecular Therapy-Methods in Clinical Development (2023)
3. Adams et al., Biotechnology and Bioengineering (2020)
4. Ravault and Laroudie, Cell & Gene Therapy Insights (2022)
5. Hurwit and Morrison, ASGCT Meeting (2018)
✓ Pre-packed columns are widely used in GMP manufacturing (4 bar limit).
✓ Owing to hardware limitations the optimal productivity can only be achieved with
a 5 cm bed height and 30 sec residence time only with 10 cm bed heights.
✓ For larger columns (e.g. >25 cm i.d.) commonly used chromatography systems
may limit operation to residence times >0.5 min.
(I)
(II)
POROS™ base bead technology (polystyrene divinylbenzene, left) and CaptureSelect™ ligand
technology (single-domain antibody, right) are combined in the manufacturing of AAVX resin
✓ Residence times <0.8 min meet acceptance criteria for processing time and capacity utilization.
✓ Capacity utilization is low but CV are <0.4 L resin, i.e. low contribution to overall process cost.
✓ All column configurations in acceptable space require only 1 process cycle (data not shown).
✓ Broader window of operation bed heights of 10 and 15 cm max res. time at 1.1-1.2 min.
✓ Capacity utilizations of 60-80% for column configurations meeting acceptable criteria.
✓ Only some configurations require 1 process cycle (data not shown). Considering potential
system pump limitations optimal configurations are 20 cmD x 15cmL or 25 cmD x 10cmL.
Scenario 2. Commercial manufacturing, 2000 L, Co=3.2E12 capsids/mL
Analysis criteria:
Processing Time Maximum = 12 hours
Minimum Resin Capacity Utilization = 10%
Analysis criteria:
Processing Time Maximum = 12 hours
Minimum Resin Capacity Utilization = 50%
𝑄𝑄𝑑𝑑 =
𝑄𝑄𝑑𝑑𝑚𝑚𝑎𝑎𝑥𝑥 𝑅𝑅𝑇𝑇
𝜃𝜃 + 𝑅𝑅𝑇𝑇
𝑃𝑃 =
h 𝑄𝑄𝑑𝑑
𝑄𝑄𝑑𝑑
𝐶𝐶𝑜𝑜
𝑅𝑅𝑇𝑇 + 𝐶𝐶𝑉𝑉
𝑛𝑛𝑜𝑜𝑛𝑛−𝑙𝑙𝑜𝑜𝑎𝑎𝑑𝑑 𝑅𝑅𝑇𝑇𝑛𝑛𝑜𝑜𝑛𝑛−𝑙𝑙𝑜𝑜𝑎𝑎𝑑𝑑 + 𝐶𝐶𝑉𝑉𝐶𝐶𝐼𝐼𝑃𝑃 𝑅𝑅𝑇𝑇𝐶𝐶𝐼𝐼𝑃𝑃
0.0E+00
2.0E+13
4.0E+13
6.0E+13
8.0E+13
1.0E+14
1.2E+14
1.4E+14
0.0 0.5 1.0 1.5 2.0
Productivity (capsids/L resin/hr)
Residence time (min)
Co=2.5E11 capsids/mL
Co=3.2E12 capsids/mL26 thermofisher.com/aav-purification
Integrating Advanced Purification and
Analytical Tools into the Workflow
AAV Downstream Process
and Product Characterization
S P E C I A L R E P O R T27 thermofisher.com/aav-purification
2 BioProcess International 20(1–2)si January–February 2022 Sponsored
A denoassociated virus (AAV) vectors are a leading platform for gene delivery in the treatment of many human diseases. Efficient production of high-yield, high-quality AAV
vectors is essential for continued advancement of the
gene-therapy field, which can deliver profound and
curative outcomes for patients. AAV vector mediated
gene delivery has been approved for treating
inherited blindness and spinal muscular atrophy,
and long-term therapeutic effects have been achieved
in patients with other rare diseases, including
hemophilia.
As the gene-therapy field rapidly expands,
regulatory guidance is evolving to help ensure the
safety of such complex therapies and driving the
need for efficient and effective methods of process
and product characterization. To ensure proper
characterization and meet regulatory expectations
for product quality and safety, vector production
workflows must integrate advanced purification and
analytical tools. As Figure 1 shows, vector quality
and purity should be monitored throughout the
entire workflow using a number of methods.
Regulatory guidance recommends monitoring
mycoplasma and other contaminants in upstream
processes, which span a set of unit operations from
plasmid development through viral vector
production. For downstream processes, guidance
increasingly focuses on removal of empty or
incomplete capsids and clearance of adventitious
viruses in addition to residual host-cell proteins
and host-cell/plasmid DNA. Lot-release test
expectations are comprehensive, including
demonstrated clearance of process-related
impurities to ensure patient safety and product
quality (see box above). The final release step is
governed by specifications for residual host-cell
DNA and plasmid removal.
Here, we describe advanced purification strategies
for AAV capture and polishing steps along with
analytical tools that can be integrated seamlessly
into vector production processes for simplified
upstream and downstream workflows.
Mycoplasma Testing
A known contaminant of mammalian cell cultures,
mycoplasma can affect the safety, quality, and
efficacy of biotherapeutic products. Given the risks
associated with this contaminant and the need to
comply with regulatory requirements, it is critical to
AAV downstream process
and product characterization
Integrating advanced purification and
analytical tools into the workflow
Chantelle Gaskin, Ilaria Scarfone, and Julia Beck
Lot-Release Test Expectations
Identity
• Capsid/serotype and transgene
Strength
• Viral genome titer
• Total viral particles
Potency
• Infectious titer
• Functional analysis
Purity
• Host-cell protein (HCP) and DNA clearance
• Residual bovine serum albumin (BSA), endonucleases,
ligands, plasmids, transfection reagents, and detergents
• Genome integrity
• Protein purity
• Aggregation status
• Ratio of full to empty capsids
Compendial Assays
• Appearance
• pH
• Osmolarity
Safety
• Absence of adventitious/replication-competent viruses
• Sterility (mycoplasma, endotoxin, bioburden)thermofisher.com/aav-purification 28
Sponsored January–February 2022 20(1–2)si BioProcess International 3
ensure that upstream cell cultures are free of
mycoplasma before feeding bulk harvest material
into a downstream process.
Historically, the only test method for
mycoplasma accepted by regulatory agencies was
based on a 28-day cell culture. Such a long testing
cycle could delay lot disposition, so the industry
has moved toward using nucleic-acid amplification
techniques (NATs) as a faster alternative. One
example with an extensive regulatory acceptance
track record is the Applied Biosystems® MycoSEQ™
mycoplasma detection system, which incorporates a
highly sensitive and specific real-time polymerase
chain reaction (PCR) assay that delivers
mycoplasma contamination results in under five
hours.
Because the MycoSEQ system was designed to
fulfill regulatory requirements, more than 40
marketed biologic manufacturing processes have
received acceptance from regulatory agencies
worldwide to use this assay for testing applications
across multiple therapeutic modalities, including
gene-therapy applications (following validation,
regulatory filing, and review). More than 30
customers are now in the process of validation and
regulatory submission using the MycoSEQ assay as a
lot-release test.
The MycoSEQ system can detect more than 90
different species of mycoplasma and related species.
Sensitivity has been demonstrated in both internal
and external validations showing that it can detect
fewer than 10 mycoplasma genome copies or colonyforming unit (CFU) equivalents. That is the required
sensitivity for mycoplasma NAT detection methods
according to regulatory guidelines. For a sample to
be considered positive for mycoplasma, it must meet
three objective analysis parameters established
during validation (Figure 2):
Figure 1: A typical adenoassociated virus (AAV) vector production workflow showing key points in the process at
which analytical methods are integrated
Residual DNA
quantitation
Plasmid
development
and production
Cell
expansion
Plasmid
transfection
Viral vector
production
Capture
purification
Fill and
finish
UPSTREAM DOWNSTREAM
Mycoplasma
testing
Capsid
titer
determination
Polish
purification
Viral
clearance
Empty/full
analysis
Figure 2: Analytical parameters
Ct
(Cycle threshold)
Tm
(Melting temperature)
DV
(Derivative Value)
Amplification Plot Dissociation Curve
Threshold
Ct
Tm
DV29 thermofisher.com/aav-purification
4 BioProcess International 20(1–2)si January–February 2022 Sponsored
• cycle threshold (Ct), a measure of the target DNA
level at the start of the PCR reaction
• derivative value, a measure of specific amplicon
quantity generated during the PCR reaction
• melting temperature (Tm), a measure of
amplicon size and base composition that is known
for mycoplasma using this assay.
A unique discriminatory positive control (DPC)
significantly reduces the possibility of false-positive
and false-negative results. It is used to confirm
extraction and PCR performance without risk of
false positives from accidental cross-contamination.
The DPC facilitates amplification of a modified PCR
product with a Tm well outside the range of real
mycoplasma amplicons. With post-PCR melting
analysis, users can determine whether samples
contain mycoplasma or a positive control. Because a
DPC maintains the same extraction behavior as
genuine mycoplasma DNA, it can be used as a
sample-extraction positive control for spiking test
samples without risk of unrecognized crosscontamination. In addition, the DPC can be used as
a surrogate for mycoplasma DNA during method
optimization and early qualification, mitigating the
requirement to use live mycoplasma and thus
serving as a precaution against introduced
mycoplasma contamination at early stages of
method adoption. For validation, mycoplasma
genomic DNA provides a suitable alternative to live
mycoplasma.
The MycoSEQ system’s AccuSEQ™ real-time
detection software generates automated presence or
absence results during data analysis. Automated
calls are made based on the Ct, T
m, and derivative
values of a test sample and inhibition control, as
previously described. The software’s security, audit,
and electronic-signature capabilities are designed to
enable 21 CFR part 11 compliance required in a good
manufacturing practice (GMP) environment.
Capture Purification
The most common first purification step in an AAV
downstream process is capture of capsids from cell
lysate using affinity chromatography. POROS®
CaptureSelect® affinity resins for AAV purification
offer broad selectivity, high capacity and elution
recovery (>90% recovery and 90% purity from a
single step), and excellent scalability (Figure 3). The
affinity ligands immobilized onto POROS beads are
VHH camelid antibody fragments recombinantly
expressed in yeast. Because their production process
is free from animal-derived components, the resins
are compatible with commercial processes. Two such
ligands are serotype specific (AAV8 and AAV9), and
a third ligand acts as a universal capsid affinity
resin (AAVX). The AAVX resin serves as a platform
solution for manufacturers developing therapies that
include a range of serotypes.
The POROS backbone is a rigid, polystyrenedivinylbenzene–based solid support that allows for
robust chemical stability and a linear pressure flow
curve, independent of column diameter. The large
pore structure reduces mass transfer resistance and
results in an increased surface area, which in turn
raises the binding capacity. The 50-µm bead size
Figure 3: Purification of a synthetic AAV serotype
using the AAVX resin; recovery (blue bar) and yield are
>90%.
Clarified FT Eluate
100
90
80
70
60
50
40
30
20
10
0
Recovery (%)
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
Total VG (1013)
Figure 4: Viral clearance capabilities of AAVX affinity resin; LRV = log reduction value
76543210
XMuLV MVM Reo-3 HAV PRV HSV-1
Log Clearance
Worst Case Manufacturing Centerpoint Process
Clearance Contributing
(1–3 LRV)
Eective
(≥4 LRV)
Contributing
(1–3 LRV)
Eective
(≥4 LRV)
Eective
(≥4 LRV)
Eective
(≥4 LRV)thermofisher.com/aav-purification 30
Sponsored January–February 2022 20(1–2)si BioProcess International 5
gives increased resolution without compromising
process efficiency.
Viral Clearance
In addition to host-cell and process-related impurity
clearance, the AAVX resin has been demonstrated to
provide effective viral clearance. Figure 4 shows
robust clearance of model viruses achieved using
AAVX affinity chromatography. The process was
tested with a panel of six RNA- and DNA-based
enveloped and nonenveloped viruses of different
sizes. The AAVX resin achieved >4 log reduction of
four of those and 1–3 log reduction of the remaining
two viruses on the panel.
Capsid Titer Determination
CaptureSelect ligands also come in free conjugated
forms. Biotinylated and horseradish-peroxidase
(HRP)–labeled AAVX ligands can be used to develop
enzyme-linked immunosorbent assays (ELISAs) for
determination of total capsid titer. The method can
be applied to both in-process and purified samples
and used for monitoring the mass balance in harvest
and capture unit operations.
Figure 5 illustrates use of AAVX-conjugated
ligands in a highly sensitive total–capsid-titer ELISA
for multiple AAV serotypes. Streptavidin-coated plates
bind the biotinylated capture ligand, and the HRPlabeled ligand is used for detection. Standard curves
of each serotype were prepared separately for AAV1,
AAV2, AAV5, AAV6, and AAV9, then aliquoted onto
the coated wells. Following a one-hour incubation,
the wells were treated with a diluted preparation of
the anti-AAVX HRP detection ligand and incubated
for 10 minutes with a 3,3′5,5′-tetramethylbenzidine
(TMB) substrate. The reaction was stopped by
addition of acid. This method is intended as a
starting point, and method optimization is always
recommended. Under most conditions, it yields a
valid assay covering the ranges of 1 × 108 and 1 × 1011
capsids/mL for most serotypes.
Empty/Full Capsid Analysis
Another critical step in vector-production workflows
is analyzing the ratio of empty to full capsids, which
can be determined using a ProPac™ SAX-10 highperformance liquid chromatography (HPLC) column.
Robust separation of full and empty particles enables
users to determine the ratio following both affinity
purification and polishing steps. The results can be
used to confirm successful removal of empty capsids
and provide a baseline for further downstream
purification.
ProPac SAX-10 columns are packed with polymer
resin coated with a hydrophilic layer that prevents
unwanted hydrophobic interactions, and the grafted
polymer chains carry strong anion-exchange
functional groups. Either salt or pH gradient elution
can be used. With a salt gradient, protein samples
bind to the stationary phase through charge
interaction and elute with an increase in the salt
concentration. With a pH gradient, negatively
charged AAV particles become neutral as pH
Figure 5: (top) Total AAV capsid enzyme-linked
immunosorbent assay (ELISA) and (bottom) results;
HRP = horseradish peroxidase
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5×107 5×108 5×109 5×1010
Absorbance (450 nm)
AAV Empty Capsids (capsids/mL)
AAV1
AAV2
AAV5
AAV6
AAV9
Anti-AAV biotin
ligand (capture)
Streptavidincoated ELISA
plate
AAV particle Anti-AAV HRP
ligand (detection)
Table 1: Chromatography settings and mobile phases
for analysis of empty and full capsids
Column Format 4 × 50 mm or 2 × 50 mm
Detection UV: Full (260 nm) and empty (280 nm)
capsids
Fluorescence: higher sensitivity, more
accurate quantitative data, full capsids only
Temperature 30 °C (temperature and pressure can affect
capsid structure stability)
Mobile Phase:
Salt Gradient
A: 20 mM Bis-Tris propane at pH 9.5 or 8.5
(depending on serotype)
B: 20 mM Bis-Tris propane at pH 9.5 or 8.5,
1 M tetramethylammonium chloride or
tetraethylammonium chloride (for better
resolution with a higher background signal)
Mobile Phase:
pH Gradient
A: 20 mM ammonium bicarbonate and
15 mM ammonium hydroxide at pH 9.2
B: 30 mM acetic acid and 15 mM formic acid
at pH 2.831 thermofisher.com/aav-purification
6 BioProcess International 20(1–2)si January–February 2022 Sponsored
decreases, so they elute from the column. Small
differences between the isoelectric points (pI) of full
and empty particles allow for such separations.
Either UV or fluorescence can be used for
detection. With UV detection, information on full and
empty capsids is provided by the intensity of signals
at 260-nm and 280-nm wavelengths, respectively.
The sensitivity of UV detection is lower than that of
fluorescence detection, which also provides better
quantitative data because it monitors the AAV capsid
signal, derived from the tryptophan residue of the
capsid protein.
Table 1 summarizes conditions and mobile phases
recommended for salt and pH gradients. Baseline
separation of empty and full capsids from different
serotypes was achieved using those conditions with
tetraethylammonium chloride as the salt (Figure 6,
top row). The pH gradient also provided good
baseline separation for both AAV8 and AAV9
serotypes (Figure 6, bottom row).
Polishing Purification
A range of weak and strong POROS anion-exchange
resins can be used for removal of empty capsids in
polishing chromatographic operations. The 50-µm
POROS base bead is made of polystyrene-divinyl
benzene, which provides rigidity for a stable column
bed and enhanced chemical stability. As described
above, these resins provide high binding capacity
and linear pressure-flow curves without
compromising on resolution during scale-up.
Qu et al. described empty and full capsid
separation using POROS HQ resin (3). Capsids
purified by cesium chloride gradient were applied to
a POROS HQ column and then eluted using a linear
sodium-acetate gradient. Empty capsids,
characterized by a higher absorbing A280 peak,
eluted sooner than the full capsids, characterized by
a higher absorbing A260 peak. When the empty and
full capsids were combined at a ratio of 16:1 and
applied to the same column with the same gradient
conditions, the empty capsid peak presented first
followed by the full capsid peak with baseline
separation, demonstrating the resin’s resolving
power and providing a feasible and scalable process
for AAV empty capsid removal.
Residual DNA Quantitation
Purification workflows for AAV processing must
remove residual host-cell DNA and plasmid DNA
impurities effectively. The World Health Organization
(WHO) requires documented residual DNA per
therapeutic dose to be <10 ng; the US Food and Drug
Administration (FDA) requests that host-cell DNA
should be as low as possible and that a highly
sensitive method be used to determine DNA levels.
The agency also encourages companies to conduct
vigorous clearance studies throughout their
Figure 6: Salt (top row) and pH (bottom row) gradient analysis of full and empty particles
Time (minutes)
0 10
0
2.4 × 107
Full
Empty
0 12
0
2.2 × 107
Partial?
Full
Empty
Time (minutes)
AAV 6 AAV 8
Absorbance (mAU)
0 12
5 0
Full
Empty
Time (minutes)
Time (minutes)
Fluorescence (counts)
0 16
2.5 × 107
Empty
AAV 8 Full AAV 9
Fluorescence (counts)
0
.
Fluorescence (counts)thermofisher.com/aav-purification 32
Sponsored January–February 2022 20(1–2)si BioProcess International 7
downstream processes both to demonstrate removal
of the vast majority of DNA from product streams
and to monitor for process deviations.
To confirm successful removal of host-cell DNA
according to regulatory requirements for lot-release
testing, AAV product developers should incorporate
a simple and reliable analytical kit that measures
residual DNA into their workflows. To monitor DNA
clearance, quantitation can be performed at
different stages throughout downstream processing,
from cell-culture harvest through to the final drug
substance.
The Applied Biosystems resDNASEQ™
quantitative DNA system provides an effective, fully
integrated, and all-inclusive approach to determining
levels of residual DNA. Sample preparation, a
sensitive and accurate DNA quantitation method,
highly characterized DNA standards, all necessary
reagents, and data analysis are included. Residual
DNA testing assays are available for a number of cell
lines, including two commonly used systems for AAV
manufacturing: human embryonic kidney (HEK) 293
host-cell DNA quantitation and simultaneous
quantitation of both Spodoptera frugiperda (Sf9)
host-cell DNA and baculovirus vector DNA.
To address the need to quantitate residual vector
DNA in AAV production using HEK293 cells, the
resDNASEQ kit was developed for quantitative
plasmid DNA with a kanamycin-resistance (KanR)
gene.
The resDNASEQ assays offer ultrahigh sensitivity
with a limit of quantitation (LoQ) down to 0.3 pg/
reaction for HEK293 DNA, Sf9–baculovirus, and 30
copies for plasmid DNA with the KanR gene. A rapid,
streamlined workflow with optional automated
sample preparation provides results in under five
hours. Table 2 summarizes the specifications of
resDNASEQ quantitative HEK293, Sf9, baculovirus
DNA, and quantitative plasmid DNA KanR gene kits.
Standard curves in Figure 7 demonstrate the high
sensitivity and broad dynamic range of two of those
assays as examples.
The resDNASEQ quantitative plasmid DNA
kanamycin-resistance–gene kit was designed to
detect and quantitate the vast majority of currently
known kanamycin-resistant–gene families. Careful
analysis of conserved regions led to creation of a
multiprimer assay to target all alleles with the same
sensitivity. A number of common commercially
relevant plasmids were spiked into the matrix in
quantities of either 100 or 100,000 copies, then
manually extracted and quantified using a
resDNASEQ quantitative plasmid DNA kanamycinresistance–gene kit. Each plasmid was recovered at
>85%, and similar results have been observed for
resDNASEQ quantitative HEK293 DNA kits.
To demonstrate specificity of the resDNASEQ
quantitative plasmid DNA kanamycin-resistance–
gene kit, a series of experiments used unrelated DNA
directly spiked into the PCR reaction (Table 3) and
Figure 7: Standard curves of (left) plasmid DNA with a kanamycin-resistance (KanR) gene and (right) human
embryonic kidney cell (HEK293) assays
KanR Standard HEK293 Standard
Slope –3.201
R2 = 0.997
Eciency 105.3%
Slope –3.284
R2 = 0.999
Eciency 101.6%
Quantity Quantity
1 10 100 103 104 105 106 10–4 10–3 0.1 1 10 100 103 104 105
Cycle Threshold
Cycle Threshold
35
30
25
20
15
35
30
25
20
Table 2: Residual DNA kit specifications used in common AAV production platforms
Specification Kanamycin-Resistance–Gene Plasmid DNA Kit HEK293 DNA Kit Sf9 Baculovirus DNA Kit
Linearity R2 > 0.99 R2 > 0.99 R2 > 0.99
PCR Efficiency 100% ± 10% 100% ± 10% 100% ± 10%
Precision ≤10% CV ≤10% CV ≤10% CV
LoD 15 copies 30 fg 30 fg
LoQ 30 copies 300 fg 300 fg
Range 300,000 copies to 30 copies 300 fg to 3 ng 300 fg to 3 ng
PCR = polymerase chain reaction Sf9 = Spodoptera frugiperda cell line 9 LoD = limit of detection LoQ = limit of quantitation33 thermofisher.com/aav-purification
8 BioProcess International 20(1–2)si January–February 2022 Sponsored
included an internal PCR control to monitor PCR
inhibition (Figure 8). All reactions provided the same
Ct value, which indicated that the PCRs performed as
intended. None of the DNA in the exclusion panel
was amplified by the resDNASEq kanamycin assay.
The first lane in the graph shows amplification of
the DNA standard included in the kit at 15 copies
(Ct � 34). Most of the other reactions provided a
nonpurification curve; others provided a range of Ct
values well above the limit of detection (LoD).
Enabling Technologies
In addition to the AAV gene therapies approved thus
far, a robust pipeline of clinical candidates
reinforces the potential of this modality to treat a
wide range of diseases caused by single-gene defects
and more complex conditions such as cancer,
neurological, cardiovascular, and infectious
diseases. AAV vectors are likely to remain a genedelivery mechanism of choice for many such
treatments.
Sustained growth of the AAV industry sector and
the safety of gene therapies both depend on a
combination of high-quality purification tools and
analytical methods that are orthogonal to titer and
recovery testing and are capable of meeting evolving
regulatory requirements. Here, we have outlined
analytical methods that work in conjunction with
both capture and polish chromatography steps to
create a simplified and streamlined AAV downstream
process. Workflows that incorporate these advanced
technologies will help ensure the quality and safety
of gene therapies for their intended recipients and
build confidence in this powerful therapeutic
modality throughout the healthcare infrastructure.
References
1 Wang C, et al. Developing an Anion Exchange
Chromatography Assay for Determining Empty and Full
Capsid Contents in AAV6.2. Mol. Ther. Methods Clin. Dev. 15,
26 September 2019: 257–263; https://doi.org/10.1016/j.
omtm.2019.09.006.
2 Füssl F, et al. Cracking Proteoform Complexity of
Ovalbumin with Anion-Exchange Chromatography–HighResolution Mass Spectrometry under Native Conditions. J.
Proteome Res. 18(10) 2019: 3689–3702; https://doi.
org/10.1021/acs.jproteome.9b00375.
3 Qu G, et al. Separation of Adeno-Associated Virus
Type 2 Empty Particles from Genome Containing Vectors By
Anion-Exchange Column Chromatography. J. Vir. Methods
140(1–2) 2007: 183–192; https://doi.org/10.1016/j.
jviromet.2006.11.019. c
Chantelle Gaskin and Ilaria Scarfone, PhD, are field applications
specialists in the purification and pharma analytics group; and
Julia Beck, PhD, is a staff scientist in the chromatography and
mass spectrometry division at Thermo Fisher Scientific.
Applied Biosystems, MycoSEQ, AccuSEQ, POROS, CaptureSelect,
ProPac, and resDNASEQ are registered trademarks of Thermo
Fisher Scientific.
Figure 8: DNA specificity exclusion-panel test results
from a kit for plasmid DNA with a kanamycin resistance
gene, including standard at limit of detection (LoD) of 15
copies
Ct
40
35
30
25
20
Targets
IPC_OP Kan_OP
Crossreactant
Samples H–W
Standard
Standard
NTC
NTC
Crossreactant
Samples H–W
Table 3: Standards and reagents tested in the
kanamycin-resistant plasmid kit exclusion panel (Figure 8)
Crossreactant H 3-ng spike Escherichia coli DNA
Crossreactant I 3-ng spike human embryonic kidney
(HEK293) cell DNA
Crossreactant J 3-ng spike adenovirus 2
Crossreactant K 3-ng spike murine leukemia virus (MuLV)
Crossreactant L 3-ng spike rabbit antibody
Crossreactant M 3-ng spike bovine antibody
Crossreactant N 3-ng spike chicken antibody
Crossreactant O 3-ng spike pAV1 (AAV genome)
Crossreactant P 3-ng spike Madin–Darby Canine Kidney
(MDCK) cells
Crossreactant Q 3-ng spike Chinese hamster ovary (CHO)
cells
Crossreactant R 3-ng spike murine myeloma (NS0) cells
Crossreactant S 3-ng spike Pichia pastoris
Crossreactant T Ampicillin (300,000 copies)
Crossreactant U Blasticidin (300,000 copies)
Crossreactant V Hygromycin (300,000 copies)
Crossreactant W Puromycin (300,000 copies)
Standard 428std6 15 copies
NTC No template control
Originally cited and published by Bioprocess International.AAV Purification Solving
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