Improvement of Precision in Recombinant Adeno-Associated Virus Infectious Titer Assay with Droplet Digital PCR as an Endpoint Measurement

Abstract

Recombinant adeno-associated virus (rAAV) has been utilized successfully for in vivo gene delivery for treatment of a variety of human diseases. To sustain the growth of recombinant AAV gene therapy products, there is a critical need for the development of accurate and robust analytical methods. Fifty percent tissue culture infectious dose (TCID₅₀) assay is an in vitro cell-based method widely used to determine AAV infectivity, and this assay is historically viewed as a challenge due to its high variability. Currently, quantitative PCR (qPCR) serves as the endpoint method to detect the amount of replicated viral genome after infection. In this study, we optimize the TCID₅₀ assay by adapting endpoint detection with droplet digital PCR (ddPCR). We performed TCID₅₀ assays using ATCC AAV-2 reference standard stock material across 18 independent runs. The cell lysate from TCID₅₀ assay was then analyzed using both qPCR and ddPCR endpoint to allow for direct comparison between the two methods. The long-term 1-year side-by-side comparison between qPCR and ddPCR as endpoint measurement demonstrated improved interassay precision when the ddPCR method was utilized. In particular, after the addition of a novel secondary set threshold for infectivity scoring of individual wells, the average infectious titer of 18 runs is 6.45E+08 with % coefficient of variation (CV) of 42.5 and 5.63E+08 with % CV of 34.9 by qPCR and ddPCR, respectively. In this study, we offer improvements of infectious titer assay with (1) higher interassay precision by adapting ddPCR as an endpoint method without the need of standard curve preparation; (2) identification of a second "set threshold" value in infectivity scoring that improves assay precision; and (3) application of statistical analysis to identify the acceptance range of infectious titer values. Taken together, we provide an optimized TCID₅₀ method with improved interassay precision that is important for rAAV infectious titer testing during process development and manufacturing.

Keywords: AAV; TCID50; ddPCR; infectious titer; infectivity scoring; qPCR; threshold.

Figure 1.

TCID₅₀ workflow with qPCR and ddPCR as an endpoint measurement. TCID₅₀ is a cell-based assay to measure infectious virus titer. The assay workflow is as follows: (A) Day 0: cell plating. HeLaRC32 cells were plated in 96-well plate at the density of 4.0E+05 cells/mL. (B) Day 1: infection. Twenty-four hours after cell plating, series of diluted viral vectors were added to the wells accordingly, along with Ad5 helper virus; the infection plate layout is explained in Supplementary Table S1 (gradient blue code is for serial dilution of AAV-2 co-infected with Ad5, green is for Ad5 control-only wells, and gray is for UI wells. (C) Day 3: cell lysis. Forty-eight hours after infection, cells were lysed using Proteinase K solution; Day 3–9: endpoint detection. Cell lysate was diluted 1:50 or 1:100 in TE buffer before use with ddPCR or qPCR runs. The results were then analyzed according to the Spearman-Kärber method to calculate the TCID₅₀ in terms of infectious titer (in IU/mL) and specific infectivity (vg/IU). AAV-2, adeno-associated virus-2; ddPCR, droplet digital PCR; IU, infectious unit; qPCR, quantitative PCR; TCID₅₀, 50% tissue culture infective dose; TE, Tris-EDTA buffer; vg, viral genome.

Figure 2.

Identification of AAV-2 viral genome replication in the limiting dilution using the ddPCR method. HelaRC32 cells were infected with 10-fold serial dilutions of AAV-2 rss along with Ad5 in 96-well plate format. Vector input amounts were 0.05, 0.5, 5, 50, 500, and 5,000 viral genomes per well (vg/well). Cells were lysed with Proteinase K mix, and 1/20 of diluted cell lysate (1:50) was used for ddPCR with the CMV primers and probe set to detect viral genome replication. A graph representing the input vector genome and replicated genome during infection provided a copy number linear response with an identified replication rate at 3,178 copies/vg (R = 0.99, slope = 3,178 copies/vg) (n = 8 independent runs).

Figure 3.

qPCR standard curve for TCID₅₀ analysis of AAV-2 rss. (A) The table shows the serial dilutions of linearized plasmid used for the standard curve. Seven standard points were used with the copy number of linearized plasmid, from 1.00E+07 to 1.00E+01. Standard curve slope, Y-Intercept, R², and qPCR efficiency were calculated. (B) The standard curve graph indicates the cycle threshold corresponding to the log (copy number) of seven standard points used. This standard curve is from one single run that represents our 18 monitored runs.

Figure 4.

ddPCR amplitude graphs for TCID₅₀ analysis of AAV-2 rss. (A–J) PCRs were partitioned into ∼20,000 droplets. The amplitude graphs display the separation of positive and negative droplets within 10 replicated wells of Dilution 1—D1, D2, D3, D4, D5, D6, D7, Ad5-only control, UI. and NTC. Based on the fluorescence amplitude, a single threshold line was drawn to separate the positive and negative droplets. The number of positive and negative droplets was used to calculate the viral genome concentration, using modeling as a Poisson distribution. These amplitude graphs are from one single run that represents our 18 monitored runs. NTC, nontemplate control.

Figure 5.

Infectious threshold logic test and 18 independent TCID₅₀ runs were analyzed with qPCR and ddPCR endpoint detection. (A) Infectious threshold logic test. For the qPCR method, the second set threshold was identified as 35 C_T. If [Ad5 only mean − 3 × SD] <35, then the [Ad5 only mean − 3 × SD] is used as threshold for infectious scoring. If [Ad5 only mean − 3 × SD] >35, then the second set threshold (35 C_T) is used for infectious scoring. For the ddPCR method, the second set threshold was identified as 1.64 copies/μL. If [Ad5 only mean + 3 × SD] >1.64, then the [Ad5 only mean + 3 × SD] is used as threshold for infectious scoring. If [Ad5 only mean + 3 × SD] <1.64, then the second set threshold (1.64 copies/μL) is used for infectious scoring. (B) The summary graph shows infectious titer values according to 18 independent runs using different detection methods (qPCR in green lines, and ddPCR in blue lines) and different threshold methods (with and without a second set threshold). (*) Runs that had higher infectious titers due to false positive infectious scoring in the highest dilution group, D7. C_T, cycle threshold; SD, standard deviation.

Figure 6.

TCID₅₀ statistical analysis. Tolerance intervals are calculated for the infectious titer assay using a set of 18 runs for four determinations. (A) qPCR_1 (no set threshold), qPCR_2 (set threshold as 35 C_T), (B) ddPCR_1 (no set threshold), and ddPCR_2 (set threshold as 1.64 copies/μL). Due to the nature of data, a logarithmic transformation is used to re-scale the data, resulting in lower limits that will remain positive.