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Sedimentation Velocity Analytical Ultracentrifugation as a Method for Quantifying the Amount of Empty Virion Particles in AdenoAssociated Virus Preparations
Christopher Sucato, Libo Wang, and Mario DiPaola
Blue Stream Laboratories, 8 Henshaw Street, Woburn, MA 01801 [email protected] 617-234-0001
ABSTRACT
Adeno-associated virus (AAV) is a small, benign virus which
infects humans, and is a promising delivery system for gene
therapy applications. While AAV vector systems have been
used in many clinical trials worldwide, and promising
results have been obtained for a number of diseases,
certain aspects of the efficiency of vector transduction
remain to be well characterized. During the production of
recombinant AAV vectors for gene therapy, empty virions
are present after the packaging process, which can result
in vector lots with mixtures of full and empty virions at
variable ratios. Further, it has been shown that the
presence of empty virions correlates with suppression of
transduction in human tissue. In this presentation we show
that analytical ultracentrifugation (AUC) is a powerful
analytical assay for the quantitation of structural
heterogeneity in recombinant AAV preparations, allowing
for the characterization of empty-to-full virion ratios. The
results of sedimentation velocity AUC are presented here
on AAV therapeutic product samples and on control empty
capsids. The resulting size distributions are evaluated in
the calculation of empty-to-full ratios.
Figure 1, below, depicts the AAV virion and its mechanism
of action as a therapeutic vector. The presence of empty
virions in a therapeutic drug product has been shown to
correlate with suppression of transduction in human tissue.
In order to eliminate any possible buffer mismatch effects, a
portion of the two samples were also dialyzed against the DP buffer
and diluted in the AV997 buffer for testing. The SV AUC assay was
performed by taking radial scans of the concentration profile,
sequentially by absorbance at 220 nm, until no further
sedimentation was observed. The resulting data sets were analysed
using the program SEDFIT with a continuous c(s) distribution model,
yielding best-fit distributions for the number of sedimenting
species and the effective molecular weights.
Figure 2: Present methods for quantifying the amount of AAV protein and
nucleic acid, and therefore the empty-full ratio. Protein is often quantified by
ELISA, while nucleic acid by qPCR. A more recent method utilizes the
A260/A280 ratio of processed AAV samples [1]. Image courtesy of www.progen.de.
Figure 6: Comparison of three independent SV AUC assays on the AAV DP
material, and another assay after dialysis. The empty-full ratios, as shown in
the data of Table 1, indicate that the method has good reproducibility.
Figure 5A: Size distribution profile by SEDFIT c(s) fitting of the SV AUC data of
a sample of empty AAV2 virus particles. Sample was obtained commercially
from Applied Viromics, and the centrifugation was conducted at 20 C and
20000 rpm until sedimentation had ceased. Sample test concentration was
approximately 2×1010 vp/mL.
Figure 4: Sedimentation velocity analytical ultracentrifugation (SV AUC) is a
promising method as applied to measurement of empty-full AAV ratios,
because both empty and full particles are in principle resolved in the same
experiment, and requires minimal sample manipulation. Image courtesy of Beckman
Coulter.
Figure 1: Left: space filling illustration of the AAV virion. Three proteins
(VP1, VP2, and VP3) make up the capsid, which packages a single-stranded
DNA genome. Right: illustration of therapeutic strategy involving the AAV
virus as a gene vector. Image courtesy of boettcher.bio.ed.ac.uk.
BACKGROUND
Several methods for the quantitation of vector genome and/or
capsid particle titers have been developed, which can be used to
calculate the ratio of empty-to-full virions. Such methods include
measurement of capsid protein concentrations by ELISA, vector
genome levels by quantitative PCR, and recently a method of
determining the empty-to-full virion levels by the ratio of
absorbance at 260 nm versus 280 nm. These strategies are shown
schematically in Figure 2. Because the empty-to-full virion ratio is
a critical parameter for assessing the quality of an AAV drug
product, a method which is simple, rapid, and accurate is highly
desirable. The strategies depicted in Figure 2, for measurement of
the empty-to-full virion ratio, while useful, can have some
drawbacks that leaves open the possibility for improved methods. A
recent study, wherein the A60/A280 method is compared to
quantitation by ELISA and qPCR, illustrates some of these
drawbacks (refer to Figure 3).
RESEARCH POSTER PRESENTATION DESIGN © 2012
www.PosterPresentations.com
In order to investigate whether a method is possible that can more
simply, rapidly, and accurately measure the empty-to-full virion
ratio in AAV samples, we turned to sedimentation velocity
analytical ultracentrifugation (SV AUC). The SV AUC method is
widely used as a characterization of higher order structure in
protein drug products, and is well suited to resolving species by
mass, in the size regime covered by empty and full AAV virions
(Figure 4). The details of our SV AUC method as applied to AAV
samples is shown below.
METHODS
The samples used in this study were as follows: AAV2-Empty virions
from Applied Viromics, 4x1011 vp/mL in 1 x PBS, 0.001% Pluronic F68, pH 7.4; and an AAV drug product (DP) under development, at a
concentration of 5x1012 DRP/mL and formulated in Lactated
Ringers Solution, 0.3 M NaCl, 0.001% Pluronic F-68. AAV2-Empty
sample was diluted by the AAV2-Empty buffer 20-fold to 2 x 1010
vp/mL, and the AAV drug product was diluted by the DP Buffer 3fold to about 1.7 x 1012 DRP/mL for SV AUC testing.
The SV AUC assay provides a series of sedimentation profiles, which
are then deconvoluted to produce a size distribution plot. The
fitting algorithm used in our analysis is from the program SEDFIT,
making use of a c(s) distribution model. The c(s) distribution
functions for empty virions and an AAV drug product are shown in
Figure 5. These results indicate that the completely empty capsids
are correctly identified by the AUC assay, as only a single main
peak. The AAV drug product sample studied here is shown to have a
majority species of higher molecular weight than the putative
empty species, at about 60% main peak abundance and roughly 40%
empty species. These results are notable, because when currently
employed methods for measuring the empty-full ratio---such as
ELISA combined with qPCR---are employed on the same AAV sample
type, a much higher abundance of empty capsids are found.
However, indications from the manufacturer are that the amount of
empty capsids found by the standard methods are overestimated
(data not shown, personal communication). Therefore the AUC
method we present here has potential as a more accurate means of
quantifying the empty-full ratio. Furthermore, as compared to the
assays illustrated in Figures 2 and 3, the AUC method is relatively
simple to perform on a single instrument with minimal sample
manipulation.
AAV DP
Sample
Major Peak 2 (P2)
ff0
P2 /P1
ratio
s-value
% area
MW
(MDa)
s-value
% area
MW
(MDa)
Rep 1
60.41
36.84
4.40
93.80
51.37
8.28
1.55
1.39
Rep 2
60.65
34.47
2.54
93.67
57.03
4.85
1.09
1.65
Rep 3
60.41
33.81
4.88
94.08
53.96
8.93
1.62
1.60
Rep 4 (after
dialysis)
59.52
37.03
3.84
91.84
59.30
7.29
1.45
1.60
Average
60.25
35.54
3.92
93.35
55.42
7.34
1.43
1.56
Std. Dev.
0.50
1.64
1.01
1.02
3.47
1.79
0.24
0.12
Table 1: SV AUC assay results on AAV DP material. The ratio of peak areas, as
shown at the far right, is the full-empty ratio.
RESULTS AND DISCUSSION
Figure 3: The current most frequently employed assays for determining the
empty-full ratio in AAV preparations suffer from some drawbacks. The
combination of ELISA and qPCR can be susceptible to large errors as shown
plot. Also, the newer A260/A280 method cannot be applied above a certain
ratio, due to inherent limits in the method [1].
Major Peak 1 (P1)
CONCLUSIONS
Figure 5B: Size distribution profile by SEDFIT c(s) fitting of the SV AUC data of
an AAV drug product preparation. Sample test condition are the same as in
Figure 5A, allowing for direct comparison with the empty AAV control. Test
concentration of the DP material was 1.7×1012 DRP/mL.
We have shown here that sedimentation velocity analytical ultracentrifugation
can be used to measure the ratio of empty to full AAV virus particles in a method
that is relatively simple to perform, requires minimal sample manipulation, and is
reproducible and may be more accurate than current best practices for
determining values for this important variable in AAV therapeutic vector
development. The next steps in moving this method forward for use in
characterizing AAV drug products will be to fully assess the performance of the
assay to ensure that the method is accurate, specific, reproducible and rugged
over the specified range that a target analyte will be analysed.
REFERENCES
To further investigate the utility of our SV AUC assay on AAV virions,
we have looked at the day-to-day reproducibility of the results,
and also the effect on the results from small changes to the
method (robustness), in this case from dialysis of the samples into
a common buffer followed by subsequent re-analysis by AUC. The
data in Figure 6 indicates that results remain consistent in terms of
the empty-full ratio, after day-to-day reproducibility challenge and
after dialysis.
[1] Sommer, Smith, Parthasarathy, et al. (2003) Quantification of
Adeno-Associated Virus Particles and Empty Capsids by Optical Density
Measurement, Molecular Therapy, 7, 1, 123.
ACKNOWLEDGMENTS
We would like to acknowledge all the members of Blue Stream Laboratories for
their support.
The work described herein is intended for research purposes only; not intended for
any human or animal therapeutic or diagnostic use, unless otherwise stated. The
trademarks or copyrights mentioned herein are the property of their respective
owners.