Quantification of full and empty particles of adeno-associated virus vectors via a novel dual fluorescence-linked immunosorbent assay

The adeno-associated virus (AAV) vector is one of the most advanced platforms for gene therapy because of its low immunogenicity and non-pathogenicity. The concentrations of both AAV vector empty particles, which do not contain DNA and do not show any efficacy, and AAV vector full particles (FPs), which contain DNA, are important quality attributes. In this study, a dual fluorescence-linked immunosorbent assay (dFLISA), which uses two fluorescent dyes to quantify capsid and genome titers in a single analysis, was established. In dFLISA, capture of AAV particles, detection of capsid proteins, and release and detection of the viral genome are performed in the same well. We demonstrated that the capsid and genomic titers determined by dFLISA were comparable with those of analytical ultracentrifugation. The FP ratios determined by dFLISA were in good agreement with the expected values. In addition, we showed that dFLISA can quantify the genomic and capsid titers of crude samples. dFLISA can be easily modified for measuring other AAV vector serotypes and AAV vectors with different genome lengths. These features make dFLISA a valuable tool for the future development of AAV-based gene therapies.

(A) Sedimentation coefficient distribution of AAV8-Lot1 vector sample, which is used as the standard for dFLISA analysis.
(B) Sedimentation coefficient distribution of AAV8-Lot2 vector sample, which is used as sample for dFLISA analysis.
(C) Sedimentation coefficient distribution of AAV8-Lot3 vector sample, which is used as sample for dFLISA analysis.
The observed peaks are assigned as empty particle (EP), full particle (FP), or extra filled (ExP). 1,2 is important to note that the unknown peak was not counted as particle.S2).
(A) Sedimentation coefficient distribution of AAV2-Lot3 vector sample, which is used as the Standard for dFLISA analysis.
(B) Sedimentation coefficient distribution of AAV2-Lot4 vector sample, which is used as the sample for dFLISA analysis.
(C) Sedimentation coefficient distribution of AAV8-Lot5 vector sample, which is used as the standard for dFLISA analysis.
(D) Sedimentation coefficient distribution of AAV8-Lot6 vector sample, which is used as the sample for dFLISA analysis.

D C
The observed peaks are assigned as empty particle (EP), full particle (FP), extra filled particle (ExP) or partial particle (PP).
(A) Sedimentation coefficient distribution of AAV2-Lot1 vector sample, which is used as the standard for dFLISA analysis.The observed peaks were identified as EP, PP, FP and PP dinner.PP is higher than LOQ, and the PP dimer is lower than LOQ of BS-AUC.
(B) Sedimentation coefficient distribution of AAV2-Lot2 vector sample, which is used as standard for dFLISA analysis.The observed peaks are assigned as empty particle (EP), full particle (FP); partial particle (PP) as shown in the figures.Observed Peak1 (red) with mass corresponding to FP, while the observed Peak 2 (blue) with mass corresponding to EP.For each AAV8 sample, a single representative mass histogram is displayed.

FP
Gaussian distribution fit was applied to the histogram peaks.From these Gaussian fits, the percentage of full and empty AAV8 capsids were extracted.

Figure S6. Spike-recovery test of dPCR.
Genomic titer quantification of crude samples by dPCR [7][8][9] were evaluated by spike-recovery test.The spike-recovery test was conducted as described in the method section: Quantification of crude sample by dFLISA and other method.
The different dilution factors for spike recovery were assessed by comparing the experimental values, as determined by the dPCR (dark green), with the expected values obtained by dFLISA (dark blue).
The standard deviation (SD) of each parameter was obtained from the triplicated experiments.Analysis Studio software (ver.12.2) was used for data analysis.The interaction between anti-AAV VHH antibody and AAV2 was analyzed by 1:1 fitting.
According to the manufacture's website of the 96-well plate, the maximum coating amount is 650 ng/cm 2 .The bottom area of 96-well plates is approximately 0.33 cm 2 , and the volume of AAV solution was 100 microliters.Thus, the coated concentration of VHH, whose molecular weight is 14 kDa, would be 150 nM.Using these values and the result of BLI, we calculated the binding efficiency of AAV based on the following formula: 10 The binding efficiency of VHH antibody and AAV2 was >99% over the entire range of the standard curve.Although there is no information about the affinity of VHH for AAV8, the binding efficiency would be >98% even if KD value of VHH for AAV8 is 100 times larger than that for AAV2.Table S1

Figure S4 .
Figure S4.Linear correlation of total capsid titers (cp/mL) and genomic titer (vg/mL) of six mixed

Figure S5 .
Figure S5.Histogram from MP analysis 3,5 of ssDNA packaged AAV8 capsids obtained from six mixed
Constructed mass histogram of 0 % full capsid.

. Summary of in-house AAV8 vectors used in this study, as determined by BS-AUC
GOI, gene of interest; CMV, cytomegalovirus promoter; HCRhAAT, hepatic control region and human a1 antitrypsin promoter; EGFP, enhanced green fluorescent protein; FIX, factor IX. cp, capsid particle; vg, viral genome; EP, empty particle; ExP, extra filled particle; FP, full particle; PP, partial particle; LOQ, Limit of quantification; na, not applicable.

Table S3 . Precision and accuracy of the dFLISA of capsid titer (cp/mL)
Results represent the mean values from 3-day experiments, in which each sample was analyzed in duplicate wells.The samples were initially diluted 400-fold, followed by a 2-fold serial dilution.CV , coefficient of variation; cp, capsid particle; SD, standard deviation.

Table S5 . Determination LOQ of dFLISA for capsid titer detection
LOQ) of capsid quantification are estimated from fluorescence intensities of blank.Results represent the mean results of 3-day experiments in which each sample was analyzed in duplicate wells.cp, capsid particle; SD, standard deviation.

Table S6 . Determination LOQ of dFLISA for genomic titer detection
LOQ) of genome quantification are estimated from fluorescence intensities of blank.Results represent the mean results of 3-day experiments in which each sample was analyzed in duplicate wells.

Table S8 . Comparison of genomic titers (vg/mL) of six mixed spike samples using orthogonal techniques: dFLISA, BS-AUC, and dPCR
nd, not detected; vg, viral genome. a

Table S9 . Comparison of fluorescence intensity of AAV2 with different genome lengths by dFLISA
Calculated from standard curve of AAV vectors with scDNA and ssDNA.SD, standard deviation.