Application Note

Do Your AAVs Contain DNA?
Rapid and Sensitive Empty/Full Capsid
Quantification with Simple Western

Empty AAV Particles Are Unwanted
Bioprocess Impurities for Gene Therapies
Adeno-associated viruses (AAVs) are frequently used asviral vectors in gene therapies to address human diseases,
with over 200 studies around the world conducting
active clinical Phase 1-3 trials. As gene delivery systems,
AAVs include a gene of interest encoded in plasmid
DNA that can be up to 5kb in length. AAVs can exist
as a heterogeneous population, giving a final sample
that is, for example, 30% full (including the desired
plasmid DNA) and 70% empty or partially empty(devoid of the desired plasmid DNA). These empty or
partially empty AAV particles can impact potency andimmunogenicity and thus are unwanted byproducts of
the AAV manufacturing bioprocess. Traditional analytical
tools such as transmission electron microscopy (TEM),
analytical ultracentrifugation (AUC), and ion-exchange
chromatography (IEX) can be used to characterize
capsid content but are complex, labor-intensive, andpose challenges in data reproducibility, throughput,and scalability.1-4 Therefore, there exists a need for bettermethods and systems for the efficient and sensitive
quantification of empty/full status of AAV samples to be
used for gene delivery.

All-In-One Assays for Titer, Capsid Protein
Ratio, and Empty/Full Ratio
Simple Western™ is the only platform that provides thecomplete picture of your AAVs in complex lysates to guide
downstream process decisions and optimization. With theseparative performance of capillary electrophoresis by size
(SW-Size) or charge (SW-Charge) and the specificity of an
immunoassay, Simple Western resolves individual capsid
proteins and DNA for high-specificity multi-attributeanalysis of titer, capsid protein ratio, and empty/fullstatus. With hands-free automation, you get quantitativeand reproducible measurements in your sample from any
process step without laborious purification.

Here, we describe straightforward assay development
to measure titer and capsid protein ratio with 2 Simple
Western runs and empty/full ratio* with 2 additional runs.
Once the titer and DNA assay standards are optimized
for a given serotype, it is possible to get multi-attributemeasurements with 2 runs, one titer and one DNA, forany sample in that given program. These assays provide
reproducible quantification with CVs of 15% or less fortiter and capsid protein ratio and 15% or less for empty/
full ratio measurements. In addition, the assays describedhere apply to other AAV serotypes and require 3 μL
of diluted unprocessed or processed sample. We also
show proof of concept that AAV Empty/Full status
can be characterized on Simple Western Charge foranalysis of intact AAVs by isoelectric focusing (IEF) andimmunodetection as an orthogonal assay.
As a result, Simple Western is meeting industry needs forrobust protein characterization with advantages criticalto gene therapy manufacturing, including speed, simple
assay development, and scalable automation. And you
don’t have to take our word for it – scientists at Merck
separately developed Simple Western assays for titer,
capsid protein ratio, and empty/full ratio analysis ofcoxsackievirus, stating:“In short, the Simple Western method was an invaluable
analytical tool providing timely information to help guide
downstream process decisions and optimization.”5

Materials and Methods
All size Simple Western Size experiments (SW-Size)in this study were performed on Jess™ and all Charge
experiments (SW-Charge) were performed on Peggy Sue™.
*Methods for determining viral empty/full ratios are protected by patents,
including U.S. Pat. Nos. 11,535,900 and 11,827,946.

Titer and Capsid Protein Ratio by SW-Size
Materials required for measuring titer and capsid protein
ratio using SW-Size are provided in TABLE 1. If you
are also running the empty/full assay, the Anti-Rabbit
Detection Module includes Luminol-S, Peroxide, Antibody
Diluent 2, and Streptavidin HRP (TABLE 2). They may bepurchased a la carte (TABLE 1) if not.
Additional materials required but not provided by BioTechne:• Anti-AAV VP1/VP2/VP3 mouse monoclonal, B1,
liquid, purified (PROGEN, 690058)• Pluronic F-68

Item

Vendor66-440 kDa Separation
ModuleAnti-Rabbit Detection
ModuleSM-W005
ProteinSimple, a
Bio-Techne Brand

Milk-free Antibody
Diluent
dsDNA Antibody

Part Number

DM-001
043-524

Novus Biologicals,a Bio-Techne Brand

NBP3-07302

TABLE 2. Materials for empty/full assays using SW-Size.

Additional materials required but not provided by BioTechne:• Pluronic F-68

• 1X PBS• Fully characterized AAV standards with known titerfrom a matching cell line, e.g. HEK293.
The standards and unknown samples are diluted to
1.25X final concentration in 1X PBS containing 0.001%
Pluronic-F68, then diluted in 5X Master Mix (1 part sample
to 4 parts 5X Master Mix), creating 1X Master Mix andsample concentrations. The standards and samples arethen denatured at 95 °C for 5 minutes. The anti-AAV
VP1/2/3 antibody is diluted to 20 µg/mL in Antibody
Diluent 2, and the Anti-Mouse Secondary HRP is diluted
to 1X in Antibody Diluent 2. Note that the Anti-Mouse
Secondary Antibody (040-655) is used for VP detection
despite being specified for Charge assays on Peggy Sue™
and NanoPro™ 1000 (TABLE 1). The AAV standards andsamples are loaded on the Jess plate according to the66-440 kDa Separation Module Product Insert (PL3-0005)
with default instrument settings, except the sample loadtime was set to 6 seconds.

Item

Empty/Full Ratio by SW-Size

VendorPart
Number

• 1X PBS• AAV standards with known empty/full ratios from amatching cell line, e.g. HEK293. The same standard forthe titer analysis may be used.
The 5X Master Mix is denatured at 95 °C for 5 minutes,
cooled on ice, centrifuged, vortexed, and stored on ice.
The samples are diluted to 1.25X final concentration in
PBS containing 0.001% Pluronic F-68 and mixed with thedenatured 5X Master Mix to create a 1X Master Mix andsample concentration. It is important not to heat denature
the samples.
The anti-DNA antibody is diluted to 2 µg/mL in
Milk-Free Antibody Diluent and the Anti-Rabbit
Secondary Antibody is used at ready-to-use (RTU) stock
concentration. The samples and antibodies are loadedon the Jess plate according to the 66-440 kDa Separation
Module Product Insert (PL3-0005), except Milk-Free
Antibody is used in place of Antibody Diluent 2 forblocking. Instrument settings are default, except thestacking load time is set to 36 seconds.

Empty/Full Ratio by SW-Charge

66-440 kDa Separation Module

SM-W005

Goat-Anti-Mouse-HRP
Secondary Antibody

040-655

Materials required for measuring empty/full ratio using
SW-Charge are provided in TABLE 3. Additional materials
required but not provided by Bio-Techne:043-311• Mouse anti-VP1/2/3 antibody (PROGEN, 61058-647)

Luminol-S
Peroxide

ProteinSimple, a
Bio-Techne Brand043-379

Antibody Diluent 2

042-203

Streptavidin HRP

042-414TABLE 1. Materials needed for titer and capsid protein ratio assays.

• Nuclease (Benzonase) (Sigma, E1014)• Biolyte 5-8 (Bio-Rad, 1631192)• Biolyte 8-10 (Bio-Rad, 1631182)

Item

VendorPart Number

Amplified Mouse
Secondary Antibody
Detection Kit

041-127

SimpleSol
500 mM Arginine
pI Standard Ladder 3046-575
ProteinSimple, a
Bio-Techne Brand

042-691040-646

pI Standard 8.4

041-036

pI Standard 9.7

040-790

1% Methyl cellulose

101876

Mouse anti-ssDNA
antibody

Novus Biologicals,a Bio-Techne Brand

NBP2-29849

unknown so that all titers are determined on the samerun. For example, if you are measuring titer with fullreference particles, treat empty reference particles as
unknown samples.
In the initial run, build a 5-point, 5-fold serial dilution
series from 1011 VP/mL to 1.6 x 108 VP/mL that will
guide the optimization of a standard curve for titerquantification in the second run. The reference sample(empty or full) not in the standard curve that will betreated as an unknown should be diluted to an initial
dilution equal to approximately the 2nd standard point(approximately top half of the curve).

TABLE 3. Materials for empty/full assays using SW-Charge.

AAV samples were diluted stepwise in SimpleSol before
final mixing into IEF master mixture. The final sample
solutions contained: 50% SimpleSol, 1% Biolyte 5-8 and
1% Biolyte 8-10, 0.35% methylcellulose, 10 mM arginine, pI
marker ladder 3 (pI 4.9 – 7.3), pI markers 8.4 and 9.7. The
focusing conditions were set to 35 minutes at a constant
power of 21000 microwatts and the immobilization timewas 220 seconds.

Results
The standard curve should be built with an empty or
full reference sample with a matching serotype and cellline and with a known titer specified by the vendor or
determined by an orthogonal method. If you are runningthe empty/full assay after the titer assay, include thereference sample (either empty or full) that will not
be used in the standard curve of the titer assay as an

Chemi. (RLU x 10,000)

20

15

Sample Type

Recommended Starting Dilution

In process

1:1.25

Purified

1:10

Purified/Concentrated

1:100

Drug Substance

1:1000TABLE 4. Recommended starting dilution by common AAV manufacturing
sample types.

Signal Too High

Acceptable Signal

VP3
10
1-4s
Chemi. (RLU x 10,000)

25

Exposure(secs)
1
2
4
8
16
32
64
128
512

Overlaid electropherograms using the All Exposures
option resulting from an acceptable standard curve and astandard curve with signals that are too high are provided
in FIGURE 1. The top point of the curve should have 4
short exposures overlaid (1-8s).
For the second run, build an optimized 5-point, 3- or
4-fold standard curve, using the bottom of the curve fromTiter and Capsid Protein Ratio by SW-Size

FIGURE 1. Optimizing standard dilution
to calculate titer. The top standard pointshould have at least 4 short exposures(1-8s) overlaid, as shown on the right.
When the signal is too high, only 1-4s (orless) are overlaid, as shown on the left.

Depending on the number of samples and the process step(in process, purified, etc.), the samples should be diluted
to a starting dilution and titrated 3-fold if 3 dilutions per
sample are required or 5-fold if 2 dilutions per sample are
required. General guidance for starting sample dilutions
is provided in TABLE 4.

8s

10

VP2

5

8

6

Exposure(secs)
1
2
4
8
16
32
64
128
512

VP3
All Exposures
1-8s

16s

4

2

VP1

0

VP2

VP1

0
66

116
MW (kDa)

200

280

66

116
MW (kDa)

200

280

7

AAV8 Empty(VP/mL)VP1/2/3 Sum Peak Area (log)

Chemi. (RLU x 10,000)

12

5 x 1010
10

1.67 x 1010
5.56 x 109

86

dilutions fall within the standard curve, then average
their corrected titers to establish the final titer (example
provided in TABLE 6).

VP3

14

1.85 x 109
6.17 x 108

R² = 0.9765

6

5
4

3
8

4
2

VP2

9
10
AAV8 Empty (log VP/mL)

11

VP1

Capsid ratios should be determined from the 2nd
optimized run. The capsid ratio should not be determinedusing a signal where the VP3 signal has less than 4
exposures overlaid (FIGURE 3, top panels). Conversely,
the capsid ratio should not be determined from VP1 and
VP2 peaks that are not clearly defined and above thebackground noise (FIGURE 3, bottom panels).

0
66

116

200
MW (kDa)

280

440

FIGURE 2. Example standard curve to measure titer.

Standards (VP/mL)

Rep 1

Rep 2

Average

5 x 1010

100.6%

115.8%

108.2%

10

1.67 x 10

98.4%

98.7%

98.6%

5.56 x 109

90.3%

90.4%

90.3%

1.85 x 10

83.7%

97.4%

90.5%

108.7%

115.2%

112.0%

9

6.17 x 108

TABLE 5. Recovery percentages from each point of the standard curve tomeasure titer and capsid protein ratios.

Sample

VP/mL

% CV

1

1.52 x 10

14.0

25.90 x 1012

5.1

3

12

4.73 x 10

0.1

45.14 x 1012

2.5

13

TABLE 6. Average titer measurements and CV percentages of unknown
samples were interpolated using the standard curve.

the first run to choose between a 3- or 4-fold standard
curve resulting in an identical or similar top calibrator
as determined in the first run. Sometimes, a 2.5-foldstandard curve is necessary. Run the unknowns in 2-folddilutions based on the initial run, aiming to fall in the
middle range of the standard curve with 3 dilutions per
sample, if possible, and a minimum of 2 dilutions.
Create the standard curve from the optimized 2nd run
by plotting the Total VP area (VP1+VP2+VP3) against
the known concentration (VP/mL) with a log-log 1/y²
weighted fit. An example standard curve is provided
in FIGURE 2. You may choose to use only the VP3 peakarea if the standards and unknowns are from the same
program. Interpolate the standard curve values against
the standard curve fit to ensure you have a valid standard
curve; the average % recovery of the standard curve
points should be +/- 20% (example provided in TABLE 5).
Interpolate the titer of the unknown total VP peak area or
VP3-only peak area from the standard curve and correctfor dilution to determine titer. If multiple unknown

If the above criteria are met, then the capsid ratio is
determined by simply dividing the VP3 and VP2 areaby the VP1 area; the VP1 ratio will always be 1.0, so
dilution correction is not necessary. If multiple sample
dilutions meet the criteria, determine the capsid ratio at
each dilution, then average the individual capsid ratios toestablish the final capsid ratio (TABLE 7).

Empty/Full Ratio by SW-Size
Ideally, assay development is performed as described in“Setting Up Your Assay” before running the empty/fullassay below. The typical titer range for a well-developed
empty/full assay using Simple Western is between 1010
and 109 VP/mL. It is important to note that all standards
and samples need to be run at the same titer for successful
Sample

Std.

1

2

3

4

Concentration or Dilution

VP2

VP3

5.00 x 1010 (VP/mL)

1.21

14.67

10

1.67 x 10 (VP/mL)

1.00

14.91

5.56 x 109 (VP/mL)

1.03

11.93

Average

1.1

13.8

1:500

0.79

13.48

1:1000

0.84

12.13

1:20000.78

9.24

Average

0.8

11.6

1:250

0.59

11.28

1:500

0.57

8.88

1:1000

0.61

8.94

Avg.

0.6

9.7

1:250

0.61

9.31

1:500

0.63

8.07

1:1000

0.89

8.01

Avg.

0.7

8.5

1:250

0.71

8.84

1:500

0.71

7.35

1:1000

0.70

6.45

Average

0.7

7.5

TABLE 7. VP1:2:3 ratio measurements generated from the optimized 2nd runof the titer assay. Measurements are reported relative to VP1, so VP1 is equal
to 1 in all samples.

empty/full determination of unknown samples with equalloading, so the only variable should be the % DNA. If an
initial DNA assay, to determine what VP/mL to run thestandards and samples, cannot be run before the complete
empty/full assay, we recommend using a titer of 3 x 109

15

A 5-point % empty/full standard curve should be built
as follows: full only, 75% full/25% empty, 50% full/50%

Signal Too HighVP3

Acceptable Signal
Exposure(secs)
1
2
4
8
16
32
64
128
512

4
1-4s

Chemi. (RLU x 10,000)

Chemi. (RLU x 10,000)

20

Exposure(secs)
1
2
4
8
16
32
64
128
512

VP/mL based on the titers of various serotypes we haveevaluated by Simple Western.

8s

10

3

VP3

All Exposures

1-8s

16s

2

1

5
VP2

57 kDax-reactivity

VP1

0

VP2

280 kDax-reactivity

VP1

0
66

0.5

116

MW (kDa)

200

280

440

66

VP1/2 Signal Too Low

VP3

4

116

MW (kDa)

200

280

440

Acceptable Signal

VP3

3.5

Chemi. (RLU x 10,000)

Chemi. (RLU x 10,000)

0.4

0.3

0.2

0.1

57 kDax-reactivity

280 kDax-reactivity
VP2

3
2.5
2
1.5
1
57 kDax-reactivity

0.5

VP1

0

VP2

280 kDax-reactivity

VP1

0
66

116

MW (kDa)

200

280

440

66

116

MW (kDa)

200

280

440

FIGURE 3. Examples of standards or samples from which capsid ratio can be determined. The guidance applies to standards and samples. VP3 should have atleast 4 short exposures (1-8s) overlaid (top right). Do not calculate VP3 peak area from too high signal where only 1-4s or less exposures are overlaid (top left). The
VP1/2 peaks should be clearly defined above background noise (bottom right). Note that cross-reactivity may occur at 280 kDa and 57 kDa.

Sf9

10

5

Chemi. (RLU x 10,000)

Chemi. (RLU x 10,000)

8

6

4

4

3

2

2

1

0

0

116

HEK293

6

280

440

MW (kDa)

116

FIGURE 4. Example DNA profiles from AAVs produced in Sf9 (left) and HEK293 (right) cells.

280

440

MW (kDa)empty, 25% empty/75% full, and empty only. Eachstandard’s final % empty/full will be determined based on
the known % empty/full of the empty and full Standards.

the standard curve fit to ensure you have a valid standard
curve; the average % recovery of the standard curve
points should be +/- 20% (example provided in TABLE 8).

Note that the DNA peak profile can vary, as shown by
DNA detected in AAVs produced in Sf9 and HEK293(FIGURE 4). It is, therefore, important to use standards
produced from the same cell line as the unknown
samples. The same standard may be used for the titeranalysis.
As with the titer standards, the top standard point shouldhave at least 4 short exposures overlay (FIGURE 5). Run
the unknowns at the same VP/mL as the % empty/fullstandard curve (FIGURE 6). The only variable should be
the % DNA that is to be determined from the standard
curve.

20

15

Average88.46%

99.2%

108.9%

98.4%

102.1%

68.14%

99.3%

106.2%

102.3%

102.6%

47.81%

93.7%

91.5%

104.2%

96.4%

27.49%

88.5%

104.4%

97.4%

96.8%

7.16%

98.1%

101.1%

107.1%

102.1%

Sample% Empty/Full

% CV

1

90.7%

3.7

2

55.8%

10.0

3

0.8%

3.9

Acceptable Signal

Exposure(secs)
1
2
4
8
16
32
64
128
512

10
1-4s

8s

10

5

8

6

1-8s

All Exposures

16s

4

2

0
116

Rep 3

12

Chemi. (RLU x 10,000)

Chemi. (RLU x 10,000)

25

Rep 2

TABLE 9. Average empty/full measurements and CV percentages of unknown
samples were interpolated using the standard curve.

Signal Too High

Exposure(secs)
1
2
4
8
16
32
64
128
512

Rep 1

TABLE 8. Recovery percentages from each point of the % full standard curve.

Plot the total DNA area by the % empty/full of thestandard curve and apply a simple linear regression(FIGURE 6). See the Appendix for recommended peak fit
settings. Interpolate the standard curve values against
30% Full Std

0
280

440

116

MW (kDa)

280

440

MW (kDa)FIGURE 5. Optimizing standard dilution to calculate empty/full ratios. The top standard point should have at least 4 short exposures (1-8s) overlaid, as
shown on the right. When the signal is too high, only 1-4s (or less) are overlaid, as shown on the left.

12

AAV5 Empty/Full Standard Curve

12

8

Full
75/25 (Full/Empty)
50/50 (Full/Empty)
25/75 (Full/Empty)
Empty

6

6×106
4×106
2×106
0

4

R2 = 0.9925

8×106

Chemi. (RLU x 10,000)

10DNA Peak Area

Chemi. (RLU x 10,000)

1×107

0

2

20

40
60
80
AAV5 E/F Standards (% Full)

100

S1
S2
S3

10
8
6
4
2

0

0
66 116280

440

MW (kDa)66 116

FIGURE 6. Example standard curve (left) to measure empty/full ratios and unknown samples S1-3 (right).

280

440

MW (kDa)We compared the peak areas of VP1/2/3 and DNA byoverlaying their respective electropherograms from the
77% and 9% full AAV9 samples (FIGURE 8A). When wequantified the VP1/2/3 and DNA peak areas from thiscomparison, we found that the fold change in VP1/2/3
was 1.8X while the fold change in DNA was 15.8X(FIGURE 8B). Therefore, the fold increase in DNA content
when normalized to VP1/2/3 was 8.9X, which closely
matches the manufacturer’s specifications of these AAV9
samples (9% and 77% full).

anti-VP1/2/3
anti-DNA

5
0
10
8
6

9% Fullanti-VP1/2/3

4

anti-DNA
2
0
4.5

B

5

5.5

6

6.5

7

7.5

pI

12
10
8

DNA

6

Untreated

4

Buffer-Only Treatment

2

Nuclease Treatment

0
40
30

VP1/2/3

20

Untreated
Buffer-Only Treatment

10

Nuclease Treatment

0
4

5

6

7

pI

8

9

FIGURE 7. SW-Charge Empty/Full Assay of AAV9 samples. (A) Signals weredetected using anti-DNA and anti-VP1/23 antibodies on 77% and 9% full
AAV9 samples. (B) Nuclease treatment of 77% full AAV9 particles probed with
anti-DNA and anti-VP1/2/3 antibodies.

A

20
15

VP1/2/3

10
Chemi. (RLU x 1,000)

We tested if treatment of AAV9 viral particles with
nuclease resulted in a decrease in DNA as indicated bya diminished signal from the anti-DNA antibody. When
we compared untreated AAV viral particles with viralparticles treated with nuclease buffer alone or particles
treated with nuclease, only the sample containing
nuclease resulted in a virtually undetectable anti-DNA
signal (FIGURE 7B, top panel). Meanwhile, the signal
from the anti-VP1/2/3 antibody remained relatively stable
across all three sample treatments (FIGURE 7B, bottom
panel). These data indicate that nuclease can dissolve the
DNA content of AAV9 particles, and they confirm that thesignal resulting from the anti-DNA antibody is the resultof DNA present in the viral capsid.

77% Full

10

77% Full
59% Full

0
6
5

DNA

4

77% Full

3

9% Full

2
1
0

B

4.5
8

5

5.5

pI

6

6.5

7

7.5

1.8X

7
Peak Area (x 100,000)

With the Empty/Full AAV assay established on Simple
Western Size, we lastly tested if this Empty/Full assay
could be extended to SW-Charge, which separates nondenatured (intact) particles by IEF followed seamlessly
by immunodetection with conventional Western blot
antibodies. From this experimental analysis, strong
signals corresponding to intact AAV9 particles wereobserved in the 77% full AAV9 sample with both
antibodies (FIGURE 7A, top panel). In contrast, only arobust signal was detected with the anti-VP1/2/3 antibody
in the 9% full AA9 sample, whereas little to no signal
was observed with the anti-DNA antibody (FIGURE 7A,
bottom panel). Interestingly, the charge separation profileof the empty AAV9 particles detected with the antiVP1/2/3 antibody was differently distributed comparedto full particles, indicating that the nucleic acid content of
the capsid may have an impact on the stability of the viralparticle. Nonetheless, these results demonstrate that the
Simple Western Empty/Full AAV assay can be performed
on SW-Charge in addition to SW-Size.

20
15

Chemil. (RLU x 1,000)

Empty/Full Ratio by SW-Charge

A

Chemi. (RLU x 1,000)

Interpolate the % empty/full of unknown total DNA areafrom the standard curve for each replicate and determine
the average % empty/full of each unknown (example
provided in TABLE 9).

77% Full
69% Full

5
4

15.8X

3
2
1
0
VP1/2/3DNA

FIGURE 8. Comparison of VP1/2/3 and DNA signals resulting from
SW-Charge. (A) Signals were detected using anti-VP1/2/3 (top panel) andanti-DNA (bottom panel) antibodies on 77% full (red) and 9% full (black) AAV9
samples. (B) Quantification of VP1/2/3 and DNA peak areas.

Sensitive, Reproducible, & Scalable
Empty/Full Ratio Measurements
In cell and gene therapy applications, the purity ofthe viral vector is critical for the efficacy and safety oftreatment. Empty viral particles resulting from bioprocess
manufacturing can only reduce efficacy and contributeto toxicity. Methods for the rapid determination of thepercentage of empty viral particles in a sample havehistorically been lacking. Here, we developed new
methods for the quantitative characterization of thefull and empty viral capsid content in a sample using
Simple Western Size and Charge assays with antibodies
that target the viral capsid and the nucleic acid content
individually. The results obtained by this method
showed a linear correlation between the signal targetingthe nucleic acid content and the percentage of full AAV
capsids in each sample, enabling a quantitative assessmentof the percentage of empty or partially empty viral capsids
in a sample.
A major advantage of this method is that Simple Westernis a fully automated immunodetection platform with athroughput of up to 96 samples that can be processed
overnight. This automation not only decreases laborcosts but also enables faster iterations during in-processdevelopment. Equally advantageous is the tiny sample
volume requirement of Simple Western (as little as 3 µL
sample) that minimizes impact on final product titerand allows for the analysis of as few as 7.2 x 107 VP per
well. Plus, Simple Western is an immunoassay that is
capable of specifically detecting proteins even in highly
complex samples like cell lysates. This contrasts withother methods for assessing capsid content like CE-SDS,
cIEF, SEC-MALS, and HPLC, which rely on direct UVor fluorimetric detection, limiting their use on complex

Appendix
Recommended Total DNA Assay Settings
1. Under Edit -> Analysis, select Peak Fit
2. In the Analysis Groups section, add a fit 2 group
3. Under Apply Override, add two groups and update as
shown below
a. fit settings for Total DNA
b. fit 2 settings for the Biotin Ladder (default settings)
4. Update the analysis parameters for fit as shown below for
Range, Baseline and Peak Find, respectively
a. It is important to select Full View, this will allow
the user to appreciate the length of the capillary forproper baseline fittingb. Note: These are recommended analysis settings someadjustment may be necessary
5. Select Apply

Based on how the recommended settings work, it is best
to fine tune the settings by adjusting parameters in the
Baseline section, like Threshold and Window. You may
need to adjust the Maximum Range slightly dependingon where the cap end bump appears.

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sample types.

References

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1. Fu et al. (2019) Hum Gene Ther Methods 30 144–52
2. Lock et al. (2012) Hum Gene Ther Methods 23 56–64

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3. Yang et al. Waters Applications Note
4. Wang et al. (2019) Mol Ther Methods Clin Dev 15 257–263
5. Gillespie et al. (2023) Hum Gene Ther 34 68–77

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