Virus Counter® Reagents Rapid, Direct, BiologicallyRelevant Virus Quantification

Infective Particle Empty Particle Defective Particle Unassociated Nucleic Acid Unassociated Protein

Heterogeneity within Virus Samples

Historical Challenges with Virus Quantification

Traditional methods of virus enumeration, including TCID50 or plaque titer assays, quantify infectious particles or units. These methods require considerable time and are burdened with high variability. Other methods for virus quantification, like qPCR and ELISA, quantify a particular component of the virus of interest (nucleic acid or viral protein, respectively) and result in derived titers versus direct quantification.

Quantification of a single virus sample with multiple methods often demontsrates the high degree of variability among methods. This is a result of the inherent heterogeneity of virus samples, which can contain a mixture of infectious particles, empty particles, defective particles, or unassosciated nucleic acid and proteins that derive from unassembled virus and cell components. Different quantification methods may enumerate a subpopu-lation or component within the sample, explaining the disparity among the results.

ELISA assays enumerate the total protein content in the sample and qPCR delivers a readout of total nucleic acid. These techniques can result in a falsely elevated result by the amplification of viral components, rather than intact virus, within the sample. While these methods are generally less time consuming and more reproducible than traditional functional assays, they may fail in effectively relating their readout to total virus counts. In contrast, functional assays quantify the subpopulation of viruses that can successfully infect cells but they cannot be used to investigate non-infectious particles in the sample. These non-infectious particles may be large portion of the total virus popluation, with a potential impact on the safety of virus-derived products and treatments.

The Virus Counter® 3100 Platform

The Virus Counter® 3100 platform is a powerful analytical tool that provides real-time virus counts and can be used throughout virus growth and purification processes. This approach allows differentiation among unassociated proteins and nucleic acid in the sample, as well as discrete particle types, providing a clear advantage of this technology over quantification approaches that only measure one subcomponent of the virus.

The platform enables users to identify optimal harvesting times, validate processing steps, and define final formulations with speed and confidence. The Virus Counter® platform features multiple detection methods, offering the right solution for individual quantification needs.

Speed – Real-time quantification for process monitoring and production optimization

Biologically-relevant readout – Total viral particle quantification

Versatility – High-specificity and universal quantification reagents

Ease of use – software-assisted system operation and data analysis (including 21 CFR Part 11 capabilities)

Reliability – Rugged design for industrial settings, easy system maintenance

The Virus Counter® Platform offers Real-Time Insights into Virus Titers• Assists in optimization of each processing step.

• Allows increased viral vector yield by comparing growth conditions and recovery during process development.

• Enhances protein expression yields and shortened timelines by harvesting with pinpoint precision.

• Provides early identification of problems during manufacturing, minimizing time and cost impacts.

• Increases safety and efficiency by facilitating in-depth characterization of total particle concentration in a final product.

The Virus Counter® instrument detects viral particles in a fluid stream.

Integrated software directs instrument function and enables data analysis.

ViroTag® Assay and Combo Dye® Assay Kits label virus particles using direct, no-wash assays.

Total Particle Quantification Matters

Non-infectious particles can represent a large portion of a virus sample (Figure 1). For this reason, enumerating infectious particle counts alone is not sufficient for in-depth virus sample characterization.

Severe setbacks in early gene therapy clinical trials are illustrative of the importance of total particle quantification. These events induced the FDA to issue new guidelines to minimize the risk of adverse outcomes when using viral vectors: “Given the potential toxicity of the adenoviral particles themselves, CBER recommends that patient dosing be based on particle number” (Guidance for Human Somatic Cell Therapy & Gene Therapy, FDA Centers for Biologics Evaluation and Research, 1998).

In-depth sample characterization of viral vectors is essential to ensure therapy is safe and efficient. This depends not only on the number of infectious particles, but, also on an understanding of potentially adverse side-effects that may be caused by the total viral particles in a dose. Non-infectious particles can represent a large portion of a virus sample, underlining the fact that infectious particle counts alone are not sufficient for thorough virus sample characterization. In the rapidly growing area of viral vector therapy, where the industry struggles to meet demand, optimization of virus manu-facturing is crucial. Rapid and reliable virus quantification allows timely identification of optimal growth conditions and harvesting times, and can increase the productivity of viral vector manufacturing.

Comparison of Infectious Titers with Total Particle Count Samples of Influenza H1N1 (FLU) cytomegalovirus (CMV), respiratory syncytial virus (RSV), and rubella virus (rubella) were measured by 50% tissue culture infective dose assay (TCID50), Virus Counter® platform and quantitative trans-mission electron microscopy (TEM). Total particle counts determined by either TEM or the Virus Counter® platform were statistically undistinguishable. Titer measured by TCID50 was a fraction of total particles, with counts ranging from 2 – 4 orders of magnitude lower than TEM or Virus Counter® platform values.

3

1.00E+05

1.00E+06

1.00E+07

1.00E+09

1.00E+11

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FLU CMV RSV RUBELLA

1.00E+08

ViroCyt Virus Counter TEM TCID50

Difference = NoninfectiveParticles

Tite

r (v

p/m

L)

Virus Counter® platform TEM TCID50

Figure 1: This comparison of infectious titer with total particle counts shows that noninfective particles can be a large portion of the total particle concentration of a virus sample.

30 mins

No Wash AssayBoth Combo Dye® and ViroTag® labeling systems use a rapid, no-wash workflow.

Pipet reagent … … into sample vials con-taining sample with virus within the dynamic range of the instrument.

Mix and incubate protected from light for 30 minutes.

Analyze.

Simple and Quick No-Wash Assay

Utilizing a no-wash assay, virus samples are mixed with Combo Dye® or ViroTag® reagents and incubated for 30 minutes*, protected from light.

* A few ViroTag® reagents might require longer incubation times. Please refer to the Product Information Sheet

Combo Dye® Reagent Virus Sample Sample Analysis Sample Signal

Single Channel SignalUnassociated Nucleic Acid

Single Channel SignalEmpty Particles | Fragments

Simultaneous SignalIntact Virus Particle

Protein Dye

Nucleic Acid Dye

Combo Dye® Reagent: Virus Quantification using a Dual Stain Approach

Combo Dye® reagent is a versatile staining system used in the Virus Counter® platform to rapidly quantify total virus particles. The reagent is based on a dual fluorogenic stain approach: a protein binding dye and a nucleic acid binding dye added simultaneously to virus samples. Upon binding to their respective targets, the quantum yield of the dyes increases significantly. Washing steps to remove unbound dye are unnecessary due to their low, unbound background signal.

Stained samples are guided through the fluidic system of the Virus Counter® instrument where virus bound fluorochromes emit light in response to laser excitation. The emitted light from protein and nucleic acide dyes is measured in two separate channels. Detection of simultaneous signals is interpreted as an intact virus particle. Unassociated nucleic acid or empty capsid are detected as signals in a single channel and are not counted as viruses. The number of events counted during the analysis time is used in combination with sample flow rate to calculate the concentration of virus particles per milliliter of sample.

Laser

+

Versatility of Combo Dye® Reagent

Combo Dye® is suited for enveloped viruses with single- or double-stranded DNA or RNA genomes. Examples of viruses compatible with the Virus Counter® Platform and Combo Dye® reagent:

ArenaviridaePichinde virus

FiloviridaeEbola virusMarburg virus

PoxviridaePox virusVaccinia virus

BaculoviridaeBaculovirus

HerpesviridaeHerpes Simplex Virus ICytomegalovirus

RetroviridaeLentivirusy-Retrovirus

CoronaviridaeCoronavirusInfecious bronchitis virus

OrthomyxoviridaeInfluenza virus

RhabdoviridaeRabies virusVesicular Stomatitis virus

FlaviviridaeDengue virusZika virus

ParamyxoviridaeMeasles virusNewcastle disease virus

TogaviridaeChikungunya

For a complete list please visit: www.sartorius.com/virus-analytics

" We have basically stopped running plaque assays on our P0 and P1 virus stocks because the accuracy of the titers obtained with the Virus Counter® leads to better virus amplifications than those obtained using plaque assay titers. The instrument saves one to two weeks on our virus production time-line and it is very helpful to know within a day or two that a transfection or co-transfection has yielded virus particles."

Kempbio

Titer Determination with High Precision

When analyzing a virus sample for the first time, it is recommended to determine the linear range of the assay (Figure 2) before measuring the titer at an optimal dilution (Figure 3). This protocol ensures measurements in the optimal range of the sample and instrument, and provides high quality data with little variability. Sample titers are determined with high precision and low sample-to-sample (Figure 3) and day-to-day variability (Figure 4).

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Influenza B/Brisbane

Lentivirus

Baculovirusy = -1.00x + 8.84 R² = 1.00

y = -0.93x + 8.63 R² = 0.99

y = -0.76x + 7.71 R² = 1.00

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Influenza B | Brisbane Lentivirus Baculovirus

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Baculovirus

Figure 2: Determination of linear range of Baculovirus, Influenza virus B/Brisbane and Lentivirus samples using the Virus Counter® Platform.

Figure 3: Optimal dilution within the linear range of the instrument was chosen to determine sample counts in replicates of five. Measured coefficients of variance (CVs) are 10.3% (Influenza B), 10.1% (Lentivirus) and 5.7% (Baculovirus).

Figure 4: Baculovirus titer was determined over a four day period. The measured CV of day-to-day variablity was 6.6%

ViroTag® Reagents: Virus Quantification with Antibody-Based Reagents

Antibody-based ViroTag® reagents are conjugated to fluorophores and bind to their respective targets with high specificity. Viruses that are labeled with ViroTag® reagents are guided through the Virus Counter® instrument and interrogated by a laser, where the conjugated dyes emit light upon excitation. The Virus Counter® instrument detects the emitted light and uses it together with the sample flow rate to calculate total particle concen tration. ViroTag® reagents allow quantification of viruses in crude and purified samples making them the optimal tool to follow virus titer during the whole manufacturing process. ViroTag® reagents are also compatible with VLPs and other particles that do not contain nucleic acids.

Comparison of Quantification Methods Virus titers obtained with ViroTag® reagents using Virus Counter® Platform show a very tight standard deviation compared to many other quantification methods (Figures 5 – 8). Discrepancies among quantification methods are due to the heterogeneity of virus samples. While ELISA and qPCR enumerate total protein or nucleic acid content of a sample, these methods can lead to an overestimation of viral titer due to inclusion of unassociated particles in the quantification process (Figure 5 and 6). Methods such as plaque titer assays and TCID50 quantify only infectious units, while the Virus Counter® platform generates total particle counts (Figure 7 and 8).

3

1.00E+08

1.00E+09

1.00E+10

1.00E+12

1.00E+13

1.00E+11

Aver

age

Tite

r (v

p/m

l)ViroTag® AAV2 – 3 TCID50/ml Transducing

units/mlqPCR

(genomes/ml)ELISA (vp/ml)

5.61E+094.37E+09

5.09E+08

3.28E+10

9.18E+11

3

1.00E+08

1.00E+09

1.00E+10

1.00E+12

1.00E+13

1.00E+11

ViroTag® AAV2/3T CID50/ml Transducingunits/ml

qPCR(genomes/ml)

ELISA (vp/ml)

5.61E+094.37E+09

5.09E+08

3.28E+10

9.18E+11

7.90E+081.49E+09

1.00E+11 6.41E+10

1.00E+00

1.00E+02

1.00E+04

1.00E+06

1.00E+08

1.00E+10

1.00E+12

1.00E+14

Crude Sample Purified Sample

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ViroTag® VSV G

ELISA p24

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ViroTag® INVA TCID50

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Figure 5: ViroTag® VSVG reagent was used to quantify to VSV-G pseudotyped Lentivirus samples (crude and purified). CV for Virus Counter® platform measurements were 9 % and 14 %, for p24 ELISA CVs were >50 % for crude and purified sample.

Figure 6: ViroTag® AAV 2/3 was used with other quantification methods to quantify AAV 2 virus standard (ATCC). CV for Virus Counter® platform measurement 2.79 %. CVs measured for other methods from left to right 374 % c.v., 192 % c.v., 109 % c.v., 29.0 % c.v. Data from Lock et al.

Figure 7: Quantification of influenza A strains with ViroTag® INVA reagent and TCID50. ViroTag® result show high precision. ViroTag® reagents quantify total virus particles in the sample compared to TCID50 which enumerates only the infectious subpopulation.

Figure 8: Quantification of influenza B strains with ViroTag® INVB reagent and TCID50. ViroTag® result show high precision and higher quantification results than TCID50. This is due to the quantification of total virus particles with ViroTag® compared to infectious units enumerated with TCID50.

Titer Determination with High Specificity

Which Reagent is Optimal for my Sample?

Enveloped Virus Non-Enveloped Virus

ViroTag® reagent available?

Use Combo Dye® reagent

Use ViroTag® reagent

Particle contains a genome?

ViroTag® reagent available?

Use ViroTag® reagent

Visit website for updates

Crude sample?

Salt concentration over 150 mM?

Serum concentra-tion above 5%?

Sample is compatible with Combo Dye® reagent and ViroTag® reagent

Use ViroTag® reagent

No

No

No

Yes

Yes Yes

No

Ordering Information

Reagent Target Order Number Quantity

AAV2-3 Intact AAV2 and AAV3 capsids VIR-92117 200 samples per kit

BCVB Budded Baculovirus VIR-92108 200 samples per kit

INVA Influenza A Viruses (H1 and H3 subtype) VIR-91151 200 samples per kit

INVB Influenza B Viruses VIR-91152 200 samples per kit

VSVG VSV-G Protein VIR-92332 200 samples per kit

Combo Dye® Universal stain for enveloped viruses VIR-92333 200 samples per kit

Replacement ConsumablesSheath Fluid (Virus Counter® 3100) VIR-92302 1 L bottle

Inter-Sample Wash (ISW) VIR-92303 6 vials

Cleanliness Verification Fluid (CVF) VIR-92295 6 vials

Startup | Shutdown Rinse (SSR) VIR-92338 6 vials

Yes

3

1.00E+08

1.00E+09

1.00E+10

1.00E+12

1.00E+13

1.00E+11

Aver

age

Tite

r (v

p/m

l)

ViroTag® AAV2 – 3 TCID50/ml Transducingunits/ml

qPCR(genomes/ml)

ELISA (vp/ml)

5.61E+094.37E+09

5.09E+08

3.28E+10

9.18E+11

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www.sartorius-stedim.com Spec

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Learn why leaders in vaccine development, protein expression, and viral therapeutics choose the Virus Counter® 3100 Platform as their tool of choice for virus quantitation. Please visit: www.sartorius.com/virus-analytics

www.sartorius.com/virus-analytics

The Virus Counter® Platform is for research use or further manufacturing use only – not for use in thera-peutic or diagnostic procedures. They are not for in vitro diagnostic use nor are they medical devices. Drug manufacturers and clinicians are responsible for obtaining the appropriate IND/BLA/NDA approvals for clinical applications.

Sartorius Stedim Biotech GmbHAugust-Spindler-Strasse 1137079 Goettingen, GermanyPhone +49.551.308.0www.sartorius-stedim.com

USA Toll-Free +1.800.368.7178Argentina +54.11.4721.0505Brazil +55.11.4362.8900Mexico +52.55.5562.1102UK +44.1372.737159France +33.442.845600Italy +39.055.63.40.41Spain +34.913.586.098Russian Federation +7.812.327.53.27Japan +81.3.4331.4300China +86.21.6878.2300

Specifications subject to change without notice. Printed and copyrighted by Sartorius Stedim Biotech GmbH. | WPublication No.: Order No.: Ver. 01 | 2012

Selected References

• Evaluation of the Virus Counter® for Rapid Baculovirus Quantitation. Journal of Virological Methods, 2011, 171(1); Ferris et al.

• Evaluation of ViroCyt® Virus Counter® for Rapid Filovirus Quantitation. Viruses, 2015, 7; pp 857-872; Rossi et al.

• Non-plaque-forming virions of Modified Vaccinia virus Ankara express viral genes. Virology, 2016, 499, pp 322-330; Lülf et al.

• Rapid and Effective Monitoring of Baculovirus Concentrations in Bioprocess Fluid Using the ViroCyt® Virus Counter®. BioProcessing Journal, 2014, 13(2), pp32-39; Birch et al.

• Characterization of Human Influenza Viruses Propagated by Madin-Darby Canine Kidney Cells or Embryonated Eggs. International Journal of Virology and AIDS 2014; Plant et al.

• Virotherapy Process Optimization. Bioprocessing Journal, 2015, 14(1); Artinger et al.

• Rapid, Simple, and Versatile Manufacturing of Recombinant Adeno-Associated Viral Vectors at Scale. Human Gene Therapy (2010); Lock, M. et al.