Table 2.
Timelines for experimental procedures
| Task | Section a | Duration |
|---|---|---|
| AAV production | 3 | |
| Cell seeding and transfection (small scale) | 3.1 | 1 h |
| Cell seeding and transfection (medium scale) | 3.2 | ≥1 h (depending on plate numbers) |
| Harvest of AAV particles | 30 min +1 h Benzonase treatment | |
| AAV purification | ||
| Sample preparation | 3.3 | 10 min |
| Column assembly and preparation | 3.4 | 5 min |
| Purification of AAV particles with a chromatography system | 3.5 | 30 min |
| Purification of AAV particles manually with a syringe | 3.6 | 15 min |
| Dialysis (optional) preferably with 300 kDa cutoff | 3.7 | ≥4 h or overnight |
| Analytics | ||
| Reporter assay for transgene expression | 3.8 | ≈5,000 cells per well for cell seeding; ≈16–24 h for cell attachment (note: it is also possible to directly add vector particles during cell seeding to save time) |
| Total viral titer determination by PCR | 3.9 | ≥3.5 h |
| dsDNA assay | 3.10 | 30 min |
| Total protein assay | 3.10 | 30 min |
| TEM | 3.11 | 40 min |
| Analytical SEC | 3.12 | 60 min |
dsDNA, double-stranded DNA; SEC, size-exclusion chromatography; TEM, transmission electron microscopy.
Refer to the following sections in the text: 3, Experimental Procedure; 3.1, AAV Vector Stock Production—Small Scale; 3.2, AAV Vector Stock Production—Medium Scale; 3.3, Sample Preparation for SXC; 3.4, Assembly of SXC Filter Unit; 3.5, SXC Using a Liquid Chromatography System; 3.6, SXC Performed Manually with a Syringe; 3.7, Dialysis of Purified AAV Particles (Optional); 3.8, Reporter Activity Assay to Monitor In Vitro Transgene Expression in Cells Transduced with SXC-Purified AAV; 3.9, Determination of AAV Genomes via Droplet Digital™ PCR; 3.10, Evaluation of Impurity Depletion of SXC-Purified AAV Particles; 3.11, Evaluation of Physical Integrity of SXC-Purified AAV by Negative Staining and Transmission Electron Microscopy; 3.12, Analytical Size-Exclusion Chromatography.