Anna Moberg, Research Associate
Adeno-associated virus (AAV) is an increasingly common vector for delivering nucleic acid into living cells, and scientists use it primarily for gene therapy.
Standard titer analysis uses ELISA, an assay that requires a lot of manual interaction and can present challenges for accuracy and reproducibility.
In this application note, we describe the performance of two assays for titer analysis of adeno-associated virus (AAV), serotype 2 and serotype 5, based on surface plasmon resonance (SPR). Both assays provide repeatable results and correlate well with established AAV total capsid titer assays using ELISA. Compared to ELISA, Biacore™ assays have a higher level of automation and accuracy, and are compatible with process samples from collection to final purified volume. Because of their power and ease of use, they are very suitable as process development tools.
We assessed the titer of AAV serotype 2 (AAV2) by allowing virus particles to bind to an AAV2-specific antibody covalently coupled to a Biacore™ sensor chip.
We assessed the AAV serotype 5 (AAV5) titer by allowing virus particles to bind to an AAV5-specific antibody captured on a prefunctionalized Biacore™ sensor chip. We developed both assays using a Biacore™ T200 SPR system, but you can run them on any Biacore™ SPR system that supports concentration analysis.
Virus particles can introduce foreign nucleic acids into living cells, making them a convenient vector for delivery of genetic material. Viral vectors have high potential for use in vaccines and therapeutic applications such as gene therapy, cell therapy, oncolytic cancer immunotherapies, and tumor vaccines. Common viral vector systems include adenovirus, adeno-associated virus, and lentivirus.
Adeno-associated virus (AAV) is an increasingly common vector in gene therapy. Scientists identified AAV as a distinct type of virus in the 1960s - previously thought to be a contaminant in adenovirus preparations. This type of virus is not known to cause disease, only mild infections. AAV is a small virus that limits its ability as a vector to smaller nucleotide sequences (<4.4 kb). However, there is ongoing research aimed at increasing the packaging capacity of AAV [Ref. 1].
This application note covers the analytical part of a complete adeno-associated virus production process that we have developed from initial production to a final purified product for AAV serotype 5. See Figure 1 below for a description of the production and purification scheme. See references 2, 3, 4 and 5 for more detailed information.
Illustration 1.AAV5 production and purification scheme.
During process development, final production, and purification, we monitor a variety of critical quality attributes, including infectious titers, viral genome titers, total viral particles, host cell protein, and DNA levels. Many of the techniques used for this are laborious, with low accuracy and a low degree of automation. In addition, analyzes are often time-consuming and expensive.
Optimized, high-precision analyzes such as SPR can improve today's workflows through a high degree of automation. Here we use SPR for AAV2 and AAV5 total titer analysis. Both assays are based on the binding of viral particles to an antibody specific for intact viral particles.
Materials and methods
AAV2 titer analysis
We developed the assay for the total titer of AAV2 virus particles using Biacore™ T200. However, you can run the assay on any Biacore™ system with software that supports concentration analysis. The test principle is shown in Figure 2.
Figure 2.Test principle Analysis of the AAV2 titer with Biacore™ systems. An anti-AAV2 antibody is coupled to amine in a flow cell on the CM5 sensor chip in Biacore™ T200. AAV2 samples diluted 5- to 1000-fold in running buffer supplemented with additional NaCl are injected across the surface and the virus titer is assessed using a standard curve.
We immobilized Progen's anti-AAV2 antibody (cat# 610298) on the CM5 sensor chip using standard amine coupling and HBS-EP+ as running buffer. We then diluted the antibody to 20 µg/ml in 10 mM acetate pH 5.0 and injected for 7 min, resulting in immobilization levels between 6,000 and 10,000 RU. We performed immobilization at 25 °C in a single flow cell.
For titer analysis, we used HBS-EP+ buffer supplemented with additional NaCl to a final salt concentration of 0.3 M as running buffer and sample diluent. Additional NaCl eliminates non-specific binding of crude samples. Samples are generally diluted between 5 and 1000 times depending on the expected titer and we include two different dilutions of each process sample in each experiment. We inject the samples with a contact time of 400 s at 10 L/min and perform regeneration with a 60 s injection of 3 M MgCl2. We then performed the analysis at 25°C with the sample compartment temperature set at 10°C to preserve sample integrity. Titer analysis was performed without a reference surface as recommended for Biacore™ concentration analysis.
We constructed the calibration curve using an AAV2 ATCC standard (ATCC AAV2 reference standard VR-1616) and ranged from 3.6 × 108for 9.2×1010VP/mL in a 2-fold dilution series. A first calibration curve is inserted before the samples and a second calibration curve is inserted after the samples. The test includes 5-10 buffer and regeneration cycles.
The anti-AAV2 antibody used in the titration assay is specific for intact virus particles only. This means you can use the assay to monitor AAV2 titer in raw cell culture samples without interference from free virus and host cell proteins. According to the manufacturer, this antibody is specific for AAV3, so you can apply the assay to this serotype after optimizing standards, regeneration, contact times, calibration range and other parameters.
AAV5 Titer Analysis
We also used the Biacore™ T200 for AAV5 virus titer analysis. This assay is performed using sensor chip protein A capture and does not require separate immobilization. Because of antibody binding for this assay, we use a capture format rather than covalent coupling. Our initial testing showed that the antibody was sensitive to acidic pH, ruling out amine coupling as this method requires dilution in acidic buffer. Capture takes place at neutral pH, which is much gentler on the antibody. The test principle is shown in Figure 3.
Figure 3.Test principle Analysis of the AAV5 titer with Biacore™ systems. An anti-AAV5 antibody is captured in a flow cell on the Protein A sensor chip in Biacore™ T200. AAV5 samples diluted 5- to 1000-fold in additional salt-supplemented running buffer are then injected across the surface and the virus titer is assessed using a standard curve.
We diluted Progen's anti-AAV5 antibody (cat# 610148) to 16 µg/mL in running buffer and captured it on the sensor chip in a single flow cell before injecting AAV5 virus samples. We performed the acquisition with a contact time of 240 s and a flow rate of 5 L/min.
We inject samples with a contact time of 120 s and a flow rate of 10 µL/min. Regeneration with 30 s 10 mM glycine-HCl pH 1.5 removes captured antibodies and viruses from the sensor surface. A new antibody is captured in each cycle. In order to preserve the samples, we set the sample room temperature to 10 °C and the analysis temperature to 25 °C. We used HBS-EP+ supplemented with additional NaCl to a final salt concentration of 500 mM as running buffer and sample diluent. Samples are diluted between 10- and 1000-fold depending on the expected titer, and typically two different dilutions of each sample are performed. We constructed the calibration curve in the 1.7 × 10 range10for 5.8×1011VP/mL using Vigene Bio pAV-CMV-GFP (Full) AAV5 (Art.#CV10005) as standard. We performed the assay with 5-10 cycles of buffer priming and regeneration, running a first calibration curve before the samples and a second calibration curve after the samples.
The anti-AAV5 antibody is specific for intact virus particles, making this assay suitable for the analysis of cell culture samples as there is no interference from free virus and host cell protein.
AAV upstream and downstream samples
We tested different steps in upstream and downstream workflows with wide variations in example matrices and expected titles. See Figure 1 for more details.
results and discussion
AAV2 titer analysis
Figure 4 shows the stability of the AAV2 titer assay. The figure shows a perfect overlap of two calibration curves with 66 samples run between them.
Figure 4.AAV2 titer analysis with Biacore™ T200. The figure shows an overlay of two calibration curves run with 66 samples in between. The span of the calibration curve is 3.6 × 108for 9.2×1010PV/ml.
In developing the assay, we found that the immobilized chip is stable enough to be used in multiple runs for at least 10 consecutive days under the recommended running conditions. High stability also supports a master calibration curve - a calibration curve that is run on the first day and then used to evaluate samples in subsequent runs. We monitor the performance of the immobilized chip by including an internal positive control sample with each run. Figure 5 shows the positive control response levels from three separate runs performed over ten days on the same immobilized sensor surface. The relative standard deviation of the control response level is less than 4%. A master calibration curve helps eliminate expensive standards and reduce time to results.
Figure 5.Positive control response from three separate analysis occasions using the same sensor chip immobilized for ten days. The relative standard deviation for replicate measurements of the control is less than 4%.
The AAV2 ATCC standard is shipped on dry ice in frozen 500 µl aliquots and stored at -80°C upon receipt. On first use, we thawed the ATCC vial and dispensed the volume in 15 µl aliquots into Protein LoBind Eppendorf vials. We then stored the aliquots at -80 °C. We thawed fresh 15 µl aliquots for each analysis occasion.
Despite careful handling, ATCC aliquots gradually lost activity over time, which was observed as a reduction in the response range for the calibration curve. We saw no difference in the level of response for the internal positive control over the corresponding period. The reduced activity and lower response level of the standard resulted in an overestimation of the virus titer for the samples. We compared the aliquoted batch of ATCC standard to a freshly thawed batch of standard in a test run. The response level of the new lot was twice that of the old lot, confirming the loss of activity of the old lot.
The stability problem was likely caused by poor handling with accidental freezing and thawing associated with removing the standard from the freezer for analysis. Tighter handling with minimal exposure to ambient temperature has greatly increased the longevity of the pattern. Proper handling of the ATCC standard at all times is essential for high-performance analysis.
LOD and LOQ for the AAV2 titer assay
We calculated the limit of detection (LOD) and the limit of quantification (LOQ) from the background signal of negative bulk samples. We calculated the mean response and standard deviation of ten replicate measurements of a negative sample in three consecutive runs. We then calculate LOD (RU) and LOQ (RU) as Average+3*Stdev and Average+10*Stdev respectively. By reading the standard curve, we estimate the LOD and LOQ to be approximately 2 × 109VP/mL e 5 × 109PV/ml
Comparison with ELISA
We compared the Biacore™ AAV2 titration assay to ELISA (Progen AAV2 Titration ELISA, Art. No. PRATV). We perform analyzes on upstream harvest samples and downstream process samples. The results showed a good correlation between the Biacore™ assay and the ELISA. The intra-assay precision of the Biacore™ assay is 3%, well below the ≥20% precision of the ELISA.
Figure 6.Comparison between ELISA and Biacore™ assay determination of AAV2 titer in upstream and downstream samples.
AAV2 titer analysis with Biacore™ 8K
We set up the assay on a Biacore™ 8K system, which significantly increases sample throughput.
The Biacore™ 8K is equipped with eight channels with two serial flow cells and eight hypodermic needles working in parallel. This parallel configuration allows parallel concentration analysis to be used. In a parallel concentration experiment, the calibration curve is run on the channels with one calibration point per channel. This means scientists only need one cycle to run the calibration curve, as opposed to a serial setup where calibration points are run in separate cycles. The parallel test uses the same span for the calibration curve and the same contact times. A normalization cycle is added to the sweep to compensate for small differences between channels. For practical reasons, the normalization solution can be one of the calibration points. With the parallel calibration curve and eight samples running in parallel, the system significantly reduces the total running time. For a full 96-well plate of samples, the system reduces run time to less than 3 hours compared to 19 hours for a single-needle system.
Figure 7.With Biacore™ 8K you can analyze eight samples in parallel, which significantly reduces runtime.
AAV5 Titer Analysis
The AAV5 titer assay is set up and performed as a capture assay, meaning that the anti-AAV5 antibody is captured in each cycle. This way the surface is seen as new on each cycle, preventing the use of a master calibration curve like for the AAV2 title. However, the sensor chip used to capture the anti-AAV5 antibody, Sensor Chip Protein A, is very stable and can be reused for multiple experiments without affecting surface performance. A calibration curve using the full Vigene standard is shown in Figure 8.
Figure 8.AAV5 titer analysis with Biacore™ T200. The figure shows an overlay of two calibration curves run with 16 samples in between. The span of the calibration curve is 9.1 × 109for 5.8×1011PV/ml.
We used Vigene Full Standard as the standard for the AAV5 assay. We prepared 25 µL aliquots of the standard and stored them at -80 °C. New aliquots were removed from the freezer for each new analysis. During the project we had no issues with stability and drop in activity for this pattern. The Vigene standard appeared less temperature sensitive than the AAV2 ATCC standard.
LOD and LOQ for the AAV5 titer assay
We calculated the mean response and standard deviation of a negative sample of ten replicate measurements in three consecutive runs. We then calculate LOD (RU) and LOQ (RU) as Average+3*Stdev and Average+10*Stdev respectively. We estimate the LOD and LOQ to be 9.8 × 109VP/mL e 1.7 × 1010VP/ml.
Comparison with ELISA
We performed Biacore™ analysis on a pool of samples from the upstream and downstream AAV5 process and compared the results to ELISA (Progen AAV5 titration ELISA, art.no. PRAAV5). Our results correlate well with a slightly lower titer for the Biacore™ assay (Figure 9). However, the titer difference between the assays is small and probably due to differences in the running conditions since the Biacore™ assay is optimized to reduce non-specific binding and the ELISA is run under standard buffer conditions. This means that non-specific binding can occur in the ELISA.
For this set of samples, the Biacore™ assay determined the mean intra-assay precision to be 2%, while the ELISA determined it to be 15%.
Figure 9.Comparison between ELISA and Biacore™ virus titer determination in different types of upstream and downstream AAV5 samples. We got slightly higher titers with ELISA which could be due to non-specific binding to the ELISA plates.
Biacore™ AAV titration assays produce robust, repeatable results that correlate well with established methods using ELISA. Compared to the ELISA, the Biacore™ titration assays show significantly fewer assay variations. Biacore™ assays are more automated with minimal labor, simple sample preparation, and automated data analysis. Another benefit is that you can reuse Biacore™ sensor surfaces multiple times. For AAV2 titer analysis you can use the same sensor chip immobilized for up to at least 10 days with a master calibration curve to save standards and costs. The sensor surface we used in the AAV5 assay, Sensor Chip Protein A, is also very stable and can be reused for multiple analyses.
You can run both assays on any Biacore™ SPR system that supports concentration analysis. Running the assay on the Biacore™ 8K or Biacore™ 8K+ systems significantly increases sample throughput and reduces overall runtime. Biacore™ assays are excellent tools not only for quality control but also for process optimization.
- Choi JH, Yu NK, Baek GC. et al., Optimization of AAV Expression Cassettes to Improve Packaging Capacity and Transgene Expression in Neurons. MolBrain 7, 17 (2014).https://doi.org/10.1186/1756-6606-7-17
- Application note:Development of a cell culture method for the production of the AAV vector in cells in suspension
- Application note:Production of adeno-associated virus in HEK293 cells in suspension using disposable bioreactors
- Application note: AAV5 capture and polish optimization
- Application note: AAV5 purification including analysis
- Article:Develop and refine your AAV production process
|Product information||Information||Product Code|
|Sensorchip der CM5 S-Serie, 3er-Pack||3er-Pack||BR100530|
|Protein A of the S series sensor chip||3er-Pack||29127556|
|amine coupling kit||Immobilization reagents||BR100050|
|Puffer HBS-EP+ 10x||Analysepuffer||BR100669|
|Glycine 1.5||regeneration solution||BR100354|
|Acetate 5.0||coupling plug||BR100351|
|Biacore™ T200||SPR system for characterizing molecular interactions||28975001|
|Biacore™ 8K||SPR system for characterizing molecular interactions||29337763|
|Biacore™ 8K+||SPR system for characterizing molecular interactions||29283382|