Rapid Quantification of Adeno-Associated (AAV) and Lentiviral Vectors with the Virus Counter® Platform: Near-Real-Time Monitoring of Production

Tyler Gates, Rebecca Montange Ph.D., Antje Schickert Ph.D., Katherine D. Shives Ph.D., Jeff Steaffens M.S. | Sartorius Stedim North America Inc., 6542 Fig St., Arvada, CO 80004, 720-480-6390

Abstract Results

Adeno-Associated Virus (AAV) and Lentivirus are commonly used vectors in modern gene and cell therapies. History has shown that in-depth sample characterization and precise enumeration of total viral vectors in final 1.00E+10 ® formulations are critical for the safety of these promising new therapies. Real-time monitoring of complex viral 1.00E+14 Virotag VSVG ELISA p24 Figure 6: Virotag® VSVG vector manufacturing processes is needed to address the rapidly-growing demand for viral vectors. The Figure 3: Virotag® AAV2-3 reagent reagent and ELISA were ® 1.00E+12 Virus Counter platform offers an emerging method to quantify total intact particles for AAV and Lentiviral quantifies AAV2 and AAV3 virus used to quantify crude and ® vectors with high speed and precision. The Virus Counter platform achieves this by utilizing -based samples with high speed and purified Lentivirus samples. ® ® 1.00E+10 reagents (Virotag AB), Virotag AAV2-3 and Virotag VSVG, to measure virus titers with high specificity for 1.00E+09 precision. Final titers (triplicate Virotag® reagent shows high measurements) were 1.16 × 109 vp/ml precision in enumerating samples. their respective targets. Using a patented no-wash assay, biologically relevant total particle counts can be ob- (vp/ml) Titer (1.33 % c.v.) for AAV2 (customer 1.00E+08 tained in three minutes following a 30 minute incubation. Virotag® AAV 2-3 demonstrated rapid and precise Both lentivirus samples were sample), 5.61 x 109 vp/ml (2.79 % c.v.) ® ® measured with Virotag VSVG in enumeration of AAV2 and AAV3 . Virotag VSVG is capable of detecting the VSV-G -pseudotype in for AAV2 reference material (ATCC) 1.00E+06 Titer (vp/ml) Titer triplicate, resulting in average titers Lentivirus and and 4.39 × 109 vp/ml (1.90 % c.v.) for of 1.00 × 108 vp/ml (9 % c.v.) for the ® AAV3 sample. 1.00E+04 BacMam expression vectors. The results of the Virus Counter method for total particle quantification was 1.00E+08 crude sample and 1.49 × 109 vp/ml AAV2 AAV2 reference AAV3 compared to other enumeration methods. Many quantification methods rely on the enumeration of a single 1.00E+02 (14 % c.v.) for the purified sample. virus building block to calculate virus titer. This can lead to overestimated virus concentrations. Virus Counter® (customer sample) (ATCC) ELISA results for both Lentivirus samples had >50 % c.v. assay results tend to be less variable and exhibit little to no statistically significant cross-reactivity against Strain 1.00E+00 related viruses. The Virus Counter® Platform, a rapid and dependable method for virus quantification, can Crude Sample Purified Sample accelerate process development and increase the efficiency of manufacturing potent viral vectors.

Introduction 1.00E+09 AAV2 BacMam Baculovirus Baculovirus 1.00E+09 Adenovirus 5 Quantification Limit Metric Infectious Particles Total Particles Total Protein Total 1.00E+08 Quantification Limit 1.00E+08

1.00E+07 Figure 7: Virotag® VSVG reagent 1.00E+07 is highly specific VSV-G epitope. Figure 4: Virotag® AAV2-3 Quantified Only BacMam particles are 1.00E+06 reagent is highly specific for AAV recognized and quantified while ® 1.00E+06 Titer (vp/ml) Titer serotypes 2 and 3. The Virotag (vp/ml) Titer wildtype Baculovirus, that does reagent binds specifically to an not express the VSV-G epitope, is epitope on these viruses, enabling excluded. BacMam is specifically 1.00E+05 1.00E+05 quantification, while excluding recognized and measured, titers Plaque Titer other viruses (Baculovirus and are linearly correlated with dilution Transducing Units Virus Counter® ELISA qPCR 1.00E+04 Adenovirus data shown). 1.00E+04 factor. Methods Flow Cytometry 0 20 40 60 80 100 0 20 40 60 80 100 TEM HPLC A260 Absorbance TCID50 Dilution Factor Dilution Factor

Functional Assays Non-Functional Assays

1.00E+13 Figure 1: Virus quantification methods measure different subpopulations or viral building blocks to determine viral titers. Figure 5: Comparison of AAV2 Discussion & Conclusion The icons shown in the quantified population box refer to intact viral particles, empty particles, defective particles, free protein reference strain (ATCC) quantifica- 29.0 % c.v. and unassociated nucleic acid. The quantified subpopulation box summarizes which fractions different methods measure in tion with different methods. These Traditional quantification methods such as TCID and ELISA can be time-consuming and variable, while also their titer determination. This explains the divergence in titers measured for a single virus sample with different methology. 1.00E+12 50 results emphasize the importance yielding indirect data in regard to total particle counts. As an emerging state-of-the art the Virus Counter® of understanding which subpopula- tions or viral building blocks within Platform provides unique insight into the levels of non-infective particles in virus preparations. 1.00E+11 109 % c.v. a sample are enumerated. ® ® Virotag® reagents quantify total The use of Virotag reagents AAV2-3 and VSVG with the Virus Counter instrument provides more precise 374 % c.v. ® 1.00E+10 2.79 % c.v. viral particles with high precision uantification of total particles. Both reagents exhibit high specificity for their respective targets. Virotag VSVG

Titer (vp/ml) Titer while other methods measure reagent demonstrates that VSV-G-expressing BacMam particles can be detected, while excluding native infectious units (TCID and TU) ® 192 % c.v. 50 Baculovirus and other negative controls. Virotag reagents demonstrate lower counts in comparison with 1.00E+09 or virus building blocks (qPCR, ELISA and qPCR methods, suggesting overestimation of total particle counts by these latter approaches . ELISA) to calculate the total virus concentration of a sample. Data from This is likely due to recognition of unassociated proteins, virus fragments and free nucleic acid in the assay. 1.00E+08 Lock M et. al. ® Virotag TCID50/ml Transducing qPCR ELISA Antibody-based quantification delivers biologically relevant and highly specific read-out of total particles AAV2-3 units/ml (genomes/ml) (vp/ml) concentration in a viral sample. The Virus Counter® instrument Integrated software directs Virotag® AB and Virotag® DY detects and measures viral instrument function and en- assay kits label virus particles Dilution Factor Citations: Lock, M. et al, Rapid, Simple, and Versatile Manufacturing of Recombinant Adeno-Associated Viral particles in a fluid stream. ables data analysis. using direct, no-wash assays. Vectors at Scale, Human Gene Therapy (2010)

Figure 2: The Virus Counter® platform offers an integrated solution to measure virus titers specifically and in near-real-time.