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Join this webinar for key insights on using the SV-AUC assay for empty/full analysis of your AAV viral vector. We’ll cover the technical requirements for this assay, data interpretation, and finally how this assay fits into the larger picture of AAV characterization.
Recombinant adeno-associated viruses (AAV) are widely used as gene transfer vectors. However, AAV production generates mixed populations of viral capsids containing either complete viral vector genome (full capsids); partially filled, and those lacking the viral genome (empty capsids). Sedimentation Velocity Analytical Ultracentrifugation (SV-AUC) offers a robust, accurate, and consistent method for characterizing empty/full AAV capsid composition. In this webinar we will review the key technical requirements for performing an AUC assay as well as analysis and data interpretation of the results generated.
In this webinar, you will learn:
• Regulatory expectations for empty/full analysis
• Key technical requirements for running an AUC assay and how to interpret the data from the results generated
• How the AUC assay fits into the larger picture of AAV characterization
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Using SV-AUC to analyze empty and full AAV capsids
1. The life science business of Merck KGaA, Darmstadt, Germany
operates as MilliporeSigma in the U.S. and Canada.
See the whole
picture: Using SV-
AUC for empty/full
AAV capsid analysis
Kamran Anwar, PhD
Daryl-Anne Watson
2. The life science business
of Merck KGaA, Darmstadt,
Germany operates as
MilliporeSigma in the U.S.
and Canada
3. Agenda
An Introduction to AAV
Regulatory expectations
Key technical requirements and data interpretation
1
2
3
4
How Empty/Full fits into the larger picture of
AAV characterization
5. See the whole picture: Using SV-AUC for empty/full AAV capsid analysis5
Ongoing Clinical Trials for Cell and Gene
Therapeutic Areas in 2019
0
50
100
150
200
250
300
350
400
450
500
Gene-Modified & Cell-
Based IO
Gene Therapy Cell Therapy Tissue Engineering
Phase I Phase II Phase III
Source, Alliance for Regenerative Medicine 2019 Report
NumberofClinicalTrials
1,066
Total trials in 2019
6. See the whole picture: Using SV-AUC for empty/full AAV capsid analysis6
While Adeno-associated virus (AAV) has limited packaging
capacity, it’s versatility makes it a compelling choice
Parvovirus family
- First discovered as an Adenovirus contaminant in 1965
- Known as a dependovirus which means it requires helper genes
from another source to replicate
Relatively stable
- Non-enveloped capsid provides protection towards low pH and
temperature changes
- Insensitive to freeze-thaw cycles and dehydration
Tissue tropism
- The serotype used can impact the therapeutic effectiveness of the
product
- Promoters can also be used to expand or limit the tissue selectivity
- Chimeras (a hybrid of 2 or more different serotypes) can enhance
tissue selectivity
• Delivery mechanism
- Delivering the GOI episomally decreases the likelihood of
insertional mutagenesis
Features Adeno-Associated Virus
Typical Use In vivo
Genome ssDNA
Virus Coat Non-enveloped
Diameter 18-26nm
Packaging Size 4.7kb
Infection Range Mostly dividing cells
Charge Positive
Integration Mostly non-integrating
Main advantage Non-pathogenic
Main disadvantage Small packaging capacity
3’ITR
Promoter
Gene of
interest
VP1, VP2, VP35’ Inverted
terminal repeat
(ITR)
7. 7
Current manufacturing platforms being employed to generate rAAV
Transfection Stable cell line Co-Infection
See the whole picture: Using SV-AUC for empty/full AAV capsid analysis
Source, Penaud-Budloo et al. 2018
e.g. HEK293 Cells e.g. HEK293, HeLa, A549 Cells e.g. HEK293 or BHK Cells e.g. Sf9 Cells
e.g. HSV e.g. Baculovirus
e.g. wtAdPlasmids
8. Generation & separation of defective particles are two of the
greatest challenges facing AAV developers today
EMPTY
FULL
• Adeno-associated virus (AAV) production gives rise to a
mixed population of viral particles
• The impact of these defective particles is largely
unknown, however, they are thought to:
Diminish product efficacy
Compete for receptor binding sites
Provoke an immune response
• However, a recent study suggested that the empty
particles could act as a decoy to overcome pre-existing
immunity towards AAV
• As the risk or benefit remains unclear, it is imperative to
monitor product composition and quality
See the whole picture: Using SV-AUC for empty/full AAV capsid analysis8
Empty Partial Full Other
10. Increase in Regulations for Cell and Gene Therapy Observed in the
Past Two Years
FDA: Content and review of CMC
information for
Human gene therapy INDs
Human somatic cell therapy INDs
EMA: Guideline on human cell-based,
gene therapy medicinal products
EMA: GMP for ATMPs
EU Draft : Annex 1 revision for
sterile medicinal products
PIC/S Annex 2A Draft guidance for
the manufacture of ATMPs
NIFDC. China: Quality Control of
CAR-T Cell Therapy Products and
Consideration for Non-clinical
Research
FDA: Potency tests for cellular and
gene therapy products
FDA Draft guidance on CMC and Retroviral
testing guidance
EMA Guideline on quality, non-clinical and
clinical aspects of medicinal products
containing genetically modified cells
FDA: Finalized guidance
Testing of retroviral vector-based
human gene therapy products for
replication competent retrovirus during
product manufacture and patient
follow-up.
Chemistry, manufacturing and control
(CMC) for human gene therapy INDs
ChP: General Chapter of Gene Therapy
Products for Human Use (draft)
2008 2011 2017 2018 2019 2020
10 See the whole picture: Using SV-AUC for empty/full AAV capsid analysis
11. • US FDA Guidance for Industry: Chemistry, manufacturing and control (CMC) for human gene
therapy Investigational New Drug Applications (INDs). (2020)
• US FDA Guidance for Industry: Testing of retroviral vector-based human gene therapy products for
replication competent retrovirus during product manufacture and patient follow-up. (2020)
• Draft guideline on quality, non-clinical and clinical requirements for investigational advanced
therapy medicinal products in clinical trials, EMA/CAT January 2019
• EMA Guideline on the quality, non-clinical and clinical aspects of gene therapy medicinal products.
EMA/CAT/80183/2014 March 2018
• European Commission Guideline on Good Manufacturing Practice specific to Advanced Therapy
Medicinal Products (2017)
• ICH Q5D: Derivation and Characterization of Cell Substrates used for Production of
Biotechnological /Biological Products (1997)
• ICH Q5A: Viral safety evaluation of biotechnology products derived from cell lines of human or
animal origin. (1997)
• WHO Recommendations for the evaluation of animal cell cultures as substrates for the manufacture
of biological medicinal products and for the characterization of cell banks. TRS 978, Annex 3 (2011)
Regulatory Guidance of note for Cell & Gene Therapy Products
11
FDA
EMA
ICH
WHO
See the whole picture: Using SV-AUC for empty/full AAV capsid analysis
12. FDA CMC Information for Human Gene
Therapy IND Applications, January 2020
(p30)
“For viral vectors, typical product-related impurities may
include defective interfering particles, non-infectious particles,
empty capsid particles, or replicating recombinant virus
contaminants. These impurities should be measured and
may be reported as a ratio, for example, full:empty particles or
virus particles:infectious units.”
Guideline on the quality, non-clinical and
clinical aspects of gene therapy medicinal
products (p17)
“For viral vectors, infectious titre should be quantified; the
number of particles (infectious/non-infectious, empty/genome
containing) should also be determined. Particle to infectivity
ratio should be included to define the content of the drug
substance.”
See the whole picture: Using SV-AUC for empty/full AAV capsid analysis12
Regulatory agencies consider empty particles as product
impurities
FDA
EMA
14. 14
Recombinant AAV Heterogeneity: Empty:Full by Transmission
Electron Microscopy (TEM)
See the whole picture: Using SV-AUC for empty/full AAV capsid analysis
AAV2 mixture of
empty and full capsids
Full
Empty
15. See the whole picture: Using SV-AUC for empty/full AAV capsid analysis15
A multi-faceted or “package” approach is recommended to
determine product composition
Analytical Ultracentrifugation
Electron Microscopy
ELISA + PCR
Anion-exchange chromatography
1
2
3
4
Theoretical calculation of empty capsids based on quantitation of
viral capsid proteins to determine virus particles/ml with viral
genome content assessment by PCR. Variability seen
Allows visualization of the particles but analysis is subjective.
Capsids with partial genomes can be challenging to identify
Determine percentage of empty/full capsids due to ability to
separate particles by mass, shape and size. Serotype-
independent
Separates viral particles based on capsid surface charge.
Needs to be optimized depending on capsid used.
Empty Partial Full
16. See the whole picture: Using SV-AUC for empty/full AAV capsid analysis16
Comparison of different analytical methods in determining
rAAV purity
17. OptimaTM AUC System
17
Analytical Ultracentrifugation (AUC)
Sedimentation velocity (SV) measures how fast macromolecules move in response to centrifugal force
Shape
Size
Mass
Moving molecules are scanned simultaneously by 2 independent optical systems:
UV Absorbance (selective)
Rayleigh Interference (RI)detection (non-selective)
SEDFIT analysis software
Cell
Rotor AN-50Ti
See the whole picture: Using SV-AUC for empty/full AAV capsid analysis
18. Measuring changes in sedimentation boundary movement gives us information about the mass and
shape of macromolecules.
Analytical Ultracentrifugation (AUC)
18
6.2 6.3 6.4 6.5 6.6 6.7 6.8 6.9 7
-0.1 -0.1
0 0
0.1 0.1
0.2 0.2
0.3 0.3
absorbance[OD]
Radial Distance (d)
Meniscus Boundaries (t,d)
Initial
Conc
See the whole picture: Using SV-AUC for empty/full AAV capsid analysis
19. 19
Requirements for Sedimentation Velocity AUC
Formulation buffer “recipe”
Sample volume of 400 µL
Sample concentration of 1E12 vp/ml minimum
Formulation buffer 1-2 mL
1
2
3
4
Avoid sugars and high concentrations of certain
buffering agents in the formulation buffer5
See the whole picture: Using SV-AUC for empty/full AAV capsid analysis
20. 20
Sedimentation Velocity Data Analysis
SEDFIT: Typical AAV profile containing different composition of capsids
empty partial full aggregates
See the whole picture: Using SV-AUC for empty/full AAV capsid analysis
21. See the whole picture: Using SV-AUC for empty/full AAV capsid analysis21
Sedimentation Velocity Data Analysis
Direct relationship with peak area and viral concentration
AAV8-LacZ (Rayleigh Interference) AAV8 empty (Rayleigh Interference)
22. 22
Sedimentation Velocity Data Analysis
Integration by SEDFIT helps calculate the different capsid
content by quantifying the area under the peaks
Initial integration of area containing all peaks of interest for total area
See the whole picture: Using SV-AUC for empty/full AAV capsid analysis
empty partial full aggregates
23. 23
Sedimentation Velocity Data Analysis
Followed by integration of individual peaks of interest
See the whole picture: Using SV-AUC for empty/full AAV capsid analysis
empty
24. 24
Sedimentation Velocity Data Analysis
Integration of individual peaks of interest
See the whole picture: Using SV-AUC for empty/full AAV capsid analysis
full
25. 25
Sedimentation Velocity Data Analysis
Area of peak
of interest
Total
area
Percent of AAV capsid in
mixture
Once the area of all peaks
of interest is determined by
integration, a simple
calculation then determines
the peak percentage
See the whole picture: Using SV-AUC for empty/full AAV capsid analysis
28. Characterization helps to answer crucial questions surrounding product
and process quality
Structural information
• Identity
• Product related impurities:
empty/full capsids
Aggregates
Degraded vector
AUC, SEC, electrophoresis, DLS
Biological activity
• Does it transduce cells efficiently?
• Transgene expression levels?
• Is the protein expressed functionally
active?
Structure
Binding/
infection of
target cells
Biological
Activity
How does my
vector
function?
What are the
physical/
structural
attributes of
my vector?
28 See the whole picture: Using SV-AUC for empty/full AAV capsid analysis
29. “Uniquely identify” a
product & distinguish it
from others. Good practice
to use different test
methods, e.g. peptide
mapping, LC-MS, ELISA,
PCR
Elucidation of structure and
other characteristics –
sequence analysis and
confirmation of the primary,
secondary, or higher order
structure; post-translational
modifications, e.g. CD, LC-
MS, RP-HPLC
Key characteristics of the
DS that can influence the
performance of the DP:
concentration, viability,
aggregation infectivity
should be listed in the CTD,
e.g. ELISA, SEC-HPLC
- Capsid protein purity, e.g.
proteins, DNA, cell debris,
reagents
- Defective or empty capsid
particles should be measured &
reported
- Aggregated, oxidated, degraded
vector, e.g. DLS, SEC-HPLC
1 3
2 4
Capsid Characterization, as defined by the Regulatory Agencies
Capsid Identity
Capsid titer
Capsid Characterization
Capsid Purity
Sources, https://www.fda.gov/vaccines-blood-biologics/biologics-guidances/cellular-gene-therapy-guidances
Rumachik N G et al (2020) bioRxiv, doi.org/10.1101/640169 ; C Muck, BASG (Austrian Agency for Health & Food Safety)
29
30. 30
Genome characterization takes a similar approach
1 3
2 4
Genome Identity
Genome titer
Genome Characterization
Genome Purity
“Uniquely identify” a
product & distinguish it
from others. Good practice
to use different test
methods, e.g. sequencing,
PCR
Ratio of positive to negative
DNA strands & vector
genome size, e.g. Agarose
alkaline electrophoresis,
Size variants distribution
Key characteristics of the
DS that can influence the
performance of the DP:
concentration, viability,
aggregation infectivity
should be listed in the CTD,
e.g. PCR
- Unwanted packaged sequences,
e.g. NGS, PCR
- Ratio of positive to negative
DNA strands
- Empty/Full genome content,
e.g. AUC
Sources, https://www.fda.gov/vaccines-blood-biologics/biologics-guidances/cellular-gene-therapy-guidances
Rumachik N G et al (2020) bioRxiv, doi.org/10.1101/640169 ; C Muck, BASG (Austrian Agency for Health & Food Safety)
31. HeLaRC32
Genomic copies
Measurement of the
number of virus
genomes in a
preparation
PCR based assay
Number of particles
ELISA based assays to
measure virus capsid
proteins
Concentration of
viral particles that
can transduce cells
Infectivity assays
Require appropriate
cell substrate for
propagation
Virus measurement
using PCR, ELISA, flow
cytometry, plaque/foci
formation.
Total Intact Viral
Particles
Infectious Titer Transgene Expression Functional Activity
Flow cytometry
Can also be used to
deduce transduction
efficiency
ELISA
Other (e.g.HPLC)
Biological effect
related to MOA in
physiologically
relevant system
A multi-faceted approach is recommended to assess Biological
Activity
See the whole picture: Using SV-AUC for empty/full AAV capsid analysis31
32. See the whole picture: Using SV-AUC for empty/full AAV capsid analysis32
Monitoring and controlling process & product impurities
decreases safety risk
Impurity
Host cell protein
Host cell DNA
Cell culture related components
Process reagents
Residual plasmid DNA
Empty or defective particles
33. 33
Summary
Regulatory agencies specify that AAV particle
composition should be measured, monitored and
reported
We recommend a package approach to determine the
viral preparation
The AUC assay provides a quantitative, serotype-
independent analysis and can be validated to GMP
with a product-specific validation approach
AAV characterization expectations are evolving as the
impact of less understood attributes of AAV are being
analyzed