Virus Counter® Reagents Rapid, Direct, Biologically Relevant Quantification 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 ( or , 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 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.

Heterogeneity within Virus Samples

Infective Particle Empty Particle Defective Particle Unassociated Nucleic Acid Unassociated Protein 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® instrument detects Integrated software directs instrument function ViroTag® and Combo Dye® Assay Kits label viral particles in a fluid stream. and enables data analysis. virus particles using direct, no-wash assays.

The Virus Counter® Platform offers Real-Time Insights into Virus Titers • Assists in optimization of each processing step. • Allows increased 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. 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 H1N1 (FLU) Virus ViroCyt Counter Vi® platformrus Count TEMer TCID TEM TCID cytomegalovirus (CMV), respiratory 50 50 syncytial virus (RSV), and rubella virus 1.00E+11 (rubella) were measured by 50% tissue 1.00E+10 culture infective dose assay (TCID50), Virus ® 1.00E+09 Counter platform and quantitative trans- Difference = Noninfective mission electron microscopy (TEM). Total Particles 1.00E+08 particle counts determined by either TEM Titer (vp/mL) ® or the Virus Counter platform were 1.00E+07 statistically undistinguishable. Titer 1.00E+06 measured by TCID50 was a fraction of total particles, with counts ranging from 2 – 4 1.00E+05 orders of magnitude lower than TEM or FLU CMV RSV RUBELLA Virus Counter® platform values. 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.

3 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

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

30 mins

Pipet reagent … … into sample vials con- Mix and incubate Analyze. taining sample with virus protected from light within the dynamic range for 30 minutes. of the instrument. 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 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.

Combo Dye® Reagent Virus Sample Sample Analysis Sample Signal

Laser

Single Channel ­Signal Unassociated Nucleic Acid

Protein Dye Single Channel ­Signal Empty Particles | + Fragments

Nucleic Acid Dye

Simultaneous ­Signal Intact Virus Particle 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:

Arenaviridae Baculoviridae Coronaviridae Flaviviridae Pichinde virus Baculovirus Coronavirus Dengue virus Infecious bronchitis virus Zika virus Filoviridae Herpesviridae Ebola virus Herpes Simplex Virus I Paramyxoviridae Marburg virus Cytomegalovirus Influenza virus Measles virus Newcastle disease virus Retroviridae Rhabdoviridae Pox virus Lentivirus Rabies virus Togaviridae Vaccinia virus y- Vesicular Stomatitis virus Chikungunya

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).

8.0 4.50E+07

Baculovirus 4.00E+07 y = -1.00x + 8.84 R² = 1.00 7.5 Lentivirus 3.50E+07 y = -0.93x + 8.63 R² = 0.99

) 3.00E+07 7.0 Influenza B/Brisbane y = -0.76x + 7.71 R² = 1.00 2.50E+07

6.5 2.00E+07

Log counts (vp/ml) 1.50E+07 Average Titer (vp/ml 6.0 1.00E+07

5.00E+06 5.5 0.51 .0 1.52 .0 2.53 .0 3.5 0.00E+00 Log dilution factor Influenza B | BrisbaneLentivirusBaculovirus

Figure 2: Determination of linear range of Baculovirus, Influenza virus B/Brisbane and Figure 3: Optimal dilution within the linear range of the instrument was chosen to Lentivirus samples using the Virus Counter® Platform. ­determine sample counts in replicates of five. Measured coefficients of variance (CVs) are 10.3% (Influenza B), 10.1% (Lentivirus) and 5.7% (Baculovirus).

1.00E+10 Baculovirus

1.00E+09 ) Titer (vp/mL 1.00E+08

1.00E+07 12 34 Time (days)

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

3 3

3 ViroTag® Reagents: Virus Quantification with -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 concen­tration. 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). Titer Determination with High Specificity

1.00E+14

1.00E+11 6.41E+10 1.00E+13 1.00E+12

9.18E+11 1.00E+10 1.49E+09 1.00E+12 7.90E+08 ViroTag® VSV G 1.00E+08 1.00E+11 ELISA p24 3.28E+10 1.00E+06 4.37E+09 1.00E+10 5.61E+09 Average Titer (vp/ml) 1.00E+04

Average Titer (vp/ml) 5.09E+08 1.00E+09 1.00E+02

1.00E+00 1.00E+08

Crude Sample Purified Sample ViroTag® AAV2 – 3 TCID50/ml Transducing qPCR ELISA (vp/ml) units/ml (genomes/ml)

Figure 5: ViroTag® VSVG reagent was used to quantify to VSV-G pseudotyped Lentivirus Figure 6: ViroTag® AAV 2/3 was used with other quantification methods to quantify samples (crude and purified). CV for Virus Counter® platform measurements were 9 % AAV 2 virus standard (ATCC). CV for Virus Counter® platform measurement 2.79 %. CVs and 14 %, for p24 ELISA CVs were >50 % for crude and purified sample. measured for other methods from left to right 374 % c.v., 192 % c.v., 109 % c.v., 29.0 % c.v. Data from1.00E+13 Lock et al.

9.18E+11 1.00E+12

1.00E+11 1.00E+10 3.28E+10 ® 1.00E+10 ViroTag INVA TCID50 ViroTag® INVB 1.00E+09 4.37E+09 1.00E+10 5.61E+09 TCID 1.00E+09 50 5.09E+08 1.00E+08 1.00E+09 1.00E+08 1.00E+07 1.00E+08

ViroTag® AAV2/3T CID50/ml Transducing qPCR ELISA (vp/ml)

Average Titer (vp/ml) 1.00E+07 1.00E+06 units/ml (genomes/ml) Average Titer (vp/ml)

1.00E+05 1.00E+06 3 9 5 2

A/Perth/2009 A/Texas/50/2012 A/Victoria/361/2011 A/California/7/200 A/Michigan/45/201 B/Phuket/3073/2013 B/Brisbane/60/2008 A/Switzerland/971529 A/HongKong/4801/2015 B/Massachusetts/2/201

® ® Figure 7: Quantification of influenza A strains with ViroTag INVA reagent and TCID50. Figure 8: Quantification of influenza B strains with ViroTag INVB reagent and TCID50. ® ® ® ViroTag result show high precision. ViroTag reagents quantify total virus particles in ViroTag result show high precision and higher quantification results than TCID50. ® the sample compared to TCID50 which enumerates only the infectious subpopulation. This is due to the quantification of total virus particles with ViroTag compared to ­infectious units enumerated with TCID50.

3 3

3 3 Which Reagent is Optimal for my Sample?

1.00E+13 Enveloped Virus Non-Enveloped Virus

9.18E+11 1.00E+12

1.00E+11 3.28E+10 Use Combo Dye® No ViroTag® reagent ViroTag® reagent No Visit website 4.37E+09 1.00E+10 5.61E+09 reagent available? available? for updates

Average Titer (vp/ml) 5.09E+08 1.00E+09 Yes Yes

1.00E+08 ViroTag® AAV2 – 3 TCID /ml Transducing qPCR ELISA (vp/ml) 50 ® ® units/ml (genomes/ml) Use ViroTag No Particle contains Use ViroTag reagent a genome? reagent

Yes

Crude sample? Sample is

compatible with No Salt concentration Combo Dye® over 150 mM? reagent and Serum concentra- ViroTag® reagent tion above 5%?

Yes

Use ViroTag® reagent

Ordering Information Reagent Target Order Number Quantity AAV2-3 Intact AAV2 and AAV3 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 Consumables Sheath 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 3 Startup | Shutdown Rinse (SSR) VIR-92338 6 vials Sales and Service Contacts For further contacts, visit www.sartorius-stedim.com

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B1605ECS Munro 3rd Floor, North Wing, Tower 1 Phone +49.5665.407.0 [email protected] Buenos Aires No. 4560 Jinke Road • Non-plaque-forming virions of Modified Vaccinia virus Ankara Zhangjiang Hi-Tech Park Phone +54.11.4721.0505 Franceexpress viral genes. Virology, 2016, 499, pp 322-330; Lülf et al. Pudong District Poland Sartorius Stedim FMT S.A.S. Shanghai 201210, P.R. China Sartorius Stedim Poland Sp. z o.o. ZI des Paluds Brazil • Rapid and Effective Monitoring oful. Baculovirus Wrzesinska 70 Concentrations in Phone +86.21.6878.2300 Avenue de Jouques – CS 91051 Sartorius do Brasil Ltda 62-025® Kostrzyn ® 13781Bioprocess Aubagne CedexFluid Using the ViroCyt Virus Counter . BioProcessing Avenida Senador Vergueiro 2962 Phone +48.61.647.38.40 São Bernardo do Campo Sartorius Stedim (Shanghai) PhoneJournal, +33.442.845600 2014, 13(2), pp32-39; Birch et al. CEP 09600-000 - SP- Brasil Trading Co., Ltd. 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C.P. 54605 Guangzhou Branch Office Modecenterstrasse 22 Avda. de la Industria, 32 Room 1105 Adeno-Associated Viral Vectors at Scale. Human Gene Therapy Phone +52.55.5562.1102 1030 Vienna Edificio PAYMA Xing Guang Ying Jing Building (2010); Lock, M. et al. 28108 Alcobendas (Madrid) [email protected] No. 119, Shui Yin Road Phone +43.1.7965763.18 Yue Xiu District, Guangzhou 510075 Phone +34.913.586.098 Phone +86.20.3836.4193 Belgium Sartorius Stedim Belgium N.V. Switzerland Rue Colonel Bourg 105 Sartorius Stedim Switzerland AG India 1030 Bruxelles Ringstrasse 24 a Sartorius Stedim India Pvt. Ltd. 8317 Tagelswangen #69/2-69/3, NH 48, Jakkasandra Phone +32.2.756.06.80 Nelamangala Tq Phone +41.52.354.36.36 562 123 Bangalore, India Hungary Phone +91.80.4350.5250 Sartorius Stedim Hungária Kft. U.K. Kagyló u. 5 Sartorius Stedim UK Ltd. 2092 Budakeszi Longmead Business Centre Japan Blenheim Road, Epsom Sartorius Stedim Japan K.K. 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Ltd. ® The Virus Counter Platform is for research use or further manufacturing use only – not for use in thera- USA10 Science Toll-Free Park Rd+1.800.368.7178 peutic or diagnostic procedures. They are not for in vitro diagnostic use nor are they medical devices. Drug ArgentinaThe Alpha #02-13/14 +54.11.4721.0505 manufacturers and clinicians are responsible for obtaining the appropriate IND/BLA/NDA approvals for Singapore Science Park II BrazilSingapore +55.11.4362.8900 117684 clinical applications. Mexico +52.55.5562.1102 UKPhone +44.1372.737159 +65.6872.3966 France +33.442.845600 South Korea ItalySartorius +39.055.63.40.41 Korea Biotech Co., Ltd. Spain8th Floor, +34.913.586.098 Solid Space B/D, RussianPanGyoYeok-Ro Federation 220, BunDang-Gu +7.812.327.53.27 JapanSeongNam-Si, +81.3.4331.4300 GyeongGi-Do, 463-400 ChinaPhone +82.31.622.5700+86.21.6878.2300

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