The document discusses the life science business of Merck KGaA, Darmstadt, Germany, operating as MilliporeSigma in the U.S. and Canada, with a focus on the regulatory expectations and technical requirements for the analysis of adeno-associated virus (AAV) capsids. It highlights the importance of characterizing AAV particles, including measuring the composition and quality of the viral preparations to comply with evolving regulatory standards. Additionally, the use of analytical ultracentrifugation (AUC) is recommended for the quantitative assessment of AAV capsid content and impurity monitoring.

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

4. An Introduction to
AAV

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

9. Regulatory
expectations

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

13. Key technical
requirements and
data interpretation

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

26. How Empty/Full fits
into the larger
picture of AAV
characterization

27. Identity Purity
Strength Safety
Product
Quality
27
Characterization and safety testing of gene therapy products
follows the basic tenets of all biologics
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

34. Head of Commercial Development,
Cell & Gene Therapy
daryl-anne.watson@milliporesigma.com
Virology Development Services
kamran.anwar@milliporesigma.com
Daryl-Anne Watson
Kamran Anwar, Ph.D.
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