A study of adeno-associated virus (AAV) empty and full capsid separation
Due to the challenging nature of adeno-associated virus (AAV) empty and full capsid separation, selecting a chromatography sorbent that is reproducible across manufacturing batches and ensures repeatable results, is essential.
In these studies, we show Mustang™ Q membrane to be:
- Reproducible across three different manufacturing batches with three different feedstreams using Mustang Q XT Acrodisc™ units.
- Repeatable across nine back-to-back experiments with the same Mustang Q XT5 capsule, enabling rapid cycling and short processing times.
Taken together, the data confirms Mustang Q membrane to be a robust and reliable solution for the manufacturing of AAV.
Introduction
AAV vectors have become one of the leading platforms for gene therapy delivery, however, the inherent formation of empty AAV capsids during upstream production poses a challenge during drug development. Lacking the DNA payload, empty AAV capsids have no therapeutic benefit. These capsids are considered a product repleted impurity and may have negative implications during treatment. Therefore, empty AAV capsid levels must be controlled to ensure safety and consistency of the drug product.
Anion exchange chromatography has become a popular tool for the separation of empty and full capsids. Mustang Q is an anion exchange (AIEX) membrane, capable of high chromatographic resolution at fast processing flow rates, and has shown to provide successful and scalable separation of empty and full AAV capsids across its XT product range (1). Additionally, Capto™ Q resin has also proven to be an effective solution to this challenging separation (2). The successful separation of empty and full AAV capsids at the manufacturing scale requires a consistent and robust process. In many cases, the AIEX operating space is narrow, therefore the reliability of processing equipment and reagents is critical. Having confidence in batch reproducibility of your AIEX membrane is essential.
In this study we showcase the reproducibility of Mustang Q membrane across three different manufacturing batches utilizing three different AAV5 feedstreams. We also highlight the repeatability of Mustang Q membrane through nine discrete experiments utilizing the same Mustang Q XT5 capsule. This demonstrates the rapid cycling capabilities of Mustang Q membrane capsules.
Materials and methods
Study 1: Mustang Q membrane reproducibility
Three distinct manufacturing batches were used to determine batch-to-batch variability on empty and full AAV capsid separation across membrane in the Mustang Q XT Acrodisc capsule (0.86 mL) format. Three AAV5 feedstreams from different upstream production techniques were utilized on all batches of Mustang Q membrane. The feedstreams were produced from the following cell lines and cell culture techniques: HEK 293F cell line with adherent cell culture, HEK 293F cell line with suspension cell culture, and SF9 cell line with suspension cell culture. Figure 1 shows the experimental design with the three different manufacturing batches and the three distinct feedstreams.
Fig 1. Experimental design.
To test the variability across batches we implemented an isocratic elution step strategy with 1 mS/cm conductivity increments. These conductivity steps resolve discrete elution peaks, which enable simple assessment and straightforward comparison of empty and full capsids separation performance across Mustang Q membrane batches.
Study 2: Mustang Q repeatability
The Mustang Q XT product line is offered in 0.86 mL, 5 mL, 140 mL, 450 mL, and 5000 mL sizes. A particular challenge we can encounter is having to process AAV feedstream past the capacity of a particular Mustang Q capsule size, without having enough to sufficiently challenge the next larger sized capsule (or only having one size Mustang Q XT capsule available). Here we show that cycling a Mustang Q XT capsule at high processing flowrates is a viable solution to this challenge. To test this, we performed nine back-to-back empty and full capsid separation processes with the same Mustang Q XT5 capsule, at a flowrate of 10 membrane volume/min (MV/min) MV/min. Here a two-step elution strategy was implemented where the conductivity of the first step is selected to elute mostly empty capsids, and on the following elution step, the conductivity is increased to elute the full capsid population.
Additionally, this test was performed with three distinct sets of buffers to showcase the practicality of cycling with distinct buffer sets, when there is a need for additional buffer formulation between experiments. Each buffer set was used for three experiments.
Results
Study 1: Mustang Q membrane reproducibility
Each experiment resulted in the elution of one main empty peak and one main full peak. For each empty and full peak the UV 260 to 280 nm peak area ratio was calculated. For each of the three feedstreams the 280nm signal was overlayed for all three membrane batches. This was repeated for the 260 nm signal. The chromatograms are shown in Figure 2.
Fig 2. Overlayed 280 nm and 260 nm chromatograms across three membrane batches and three feedstreams.
Despite the significant differences in empty and full capsid separation across the different AAV5 feedstreams, as seen in Figure 2, the performance across Mustang Q membrane batches is highly comparable. The UV 280 and 260 nm area ratios for each empty and full peak were calculated and are shown in Table 1.
Table 1. UV 260:280 empty and full peak area ratios, averages, and standard deviation for batches 1-3, on all feedstreams.
| Feedstream | Empty peak UV 260/280 Ratios: Batch 1, Batch 2, Batch 3, (Mustang Q average, STD, RSD) |
Full peak UV 260/280 Ratios: Batch 1, Batch 2, Batch 3, (Mustang Q average, STD, RSD) |
| Suspension HEK | 0.56 0.58 0.59 (0.57 0.016, 3%) | 1.08 1.09 1.11 (1.09 0.014, 1%) |
| Adherent HEK | 0.61 0.59 0.58 (0.59 0.017, 3%) | 0.83 0.79 0.83 (0.82 0.023, 3%) |
| Suspension SF9 | 0.68 0.69 0.82 (0.73 0.077, 11%) | 1.18 1.15 1.18 (1.17 0.026, 2%) |
The data in Table 1 shows that Mustang Q membrane batch-to-batch variability for empty and full AAV capsid separation is insignificant. For the full peak across all serotypes a standard deviation of less than 3% across batches was observed. These results confirm that Mustang Q membrane is a robust, reproducible solution for the manufacturing of AAV vectors.
All Mustang Q XT units are produced from the same membrane manufacturing process across the entire product range. Therefore, the robustness in batch variability shown here with Mustang Q XT Acrodisc capsule (0.86 mL) format, translates to the entire Mustang Q XT product range.
Study 2: Mustang Q membrane repeatability
An overlayed chromatogram of the 9 experiments with the three buffer sets is shown in Figure 3. It can be seen that highly similar chromatograms are obtained from the nine back-to-back experiments, indicating the repeatability of results.
Fig 3. Overlayed chromatograms of all nine experiments: 260 nm.
The capsids and vector genomes for peak 2 were quantitated utilizing a capsid ELISA and ddPCR assays respectively. A ratio of vector genomes to total capsids was obtained to calculate the percent full capsids for the second peak population of each experiment. The results are shown in Figure 4.
Fig 4. Capsid and VG concentration on second peak, percent full capsids on second peak.
The data shown confirms the highly repeatable performance of Mustang Q XT products for empty and full AAV capsid separation over several cycles. Here we can see that a full capsid population percentage (of over 50%) were achieved across all three buffer sets. This showcases that the independent preparation of buffers for empty and full capsid has no impact on the chromatographic performance of Mustang Q (note: buffer was prepared carefully and systematically).
A total of 4.5 × 1016 capsids over 675 mL of feedstream were processed over these nine back-to-back experiments in 180 minutes. This is a short total processing time, enabled by the fast flow rate capabilities of Mustang Q membrane technology. This data shows how cycling Mustang Q XT capsules can be an effective avenue to process high amounts of AAV feed with an "undersized" capsule.
Conclusion
Our studies have confirmed that Mustang Q membrane is reproducible across manufacturing batches enabling a robust empty and full AAV capsid separation solution, from process development to manufacturing scale. The reproducibility of manufacturing batches is proven across several feedstreams produced from different sources and techniques. Mustang Q membrane in XT capsule format is also shown to produce highly repeatable results providing a pathway to process large amounts of AAV feedstream by rapid cycling of the Mustang Q XT .
REFERENCES
- Hejmowski, A. L., Boenning, K., Huato, J., Kavara, A., Schofield, M. Novel Anion Exchange Membrane Chromatography Method for the Separation of Empty and Full Adeno-Associated Virus. Biotechnology Journal. 2022;17(e2100219). doi:10.1002/biot.202100219
- Hagner-McWhirter, A. From cells to purified capsids: How to develop a scalable rAAV process. Cell Gene Ther. Insights. 2022;8:611-619. doi:10.18609/cgti.2022.094