Key considerations for processing lentiviral vectors

Lentiviral feedstocks vary considerably in production method, lentiviral vector titer, genetic payload, and contamination profile. These factors can influence the performance of chromatography steps. Therefore, optimizing the process for any new feedstock is crucial to achieving the best results. Here are the key steps to consider, along examples of successful strategies we’ve seen in practice:

1) Quality of feed (the ratio of physical titer to functional titer):

• Larger volumes of the lower quality feed will be required to load the appropriate number of functional particles. As a result, it’s important to consider the feedstock quality to ensure that the volume of feed available for characterization is adequate.

• Due to variability in the biological batches of LVV and the TU assay, perform loading experiments and optimization in triplicate.

2) Endonuclease treatment:

• To reduce host cell nucleic acid levels, we recommend 50 units of endonuclease per mL with 2 mM MgCl2 for at least 1 hour at 37 oC.

3) Clarification:

• For primary clarification, we recommend centrifugation, or a DEPTH filtration step with a pore size of 1 µM, followed by a 0.45 µM PES filter.

4) Loading:

• Aim to load 1E 10 TU per mL of LentiHERO® adsorbent. Collect fractions at 1/10 of total load volume and analyze each fraction.

• It is preferable to measure functional particles in the flowthrough if that assay is available. Alternatively, calculate the physical titer using qPCR for genome copies, or with a p24 assay. Be aware that free p24 may break through immediately, so adjust the breakthrough capacity accordingly.

• The process load should be to 80% of the TU value where 10% of the functional particles breakthrough. Calculate recovery from the elution pool of this process load.

5) Optimization of elution buffers:

• We have observed that functional particles of LVV elute earlier from the LentiHERO® than from commercially available Q membranes.

• As a reminder, the most relevant measure of recovery is the functional assay. The ratio of functional particles to physical particles should improve in the elution pool.

• Since different pseudotypes and payloads or genes of interest (GOI) can influence the particle’s surface charge—and thus the conductivity at which the LVV elutes— we recommend conducting an initial study with stepwise elution to establish the appropriate elution range. Collect each step immediately into a diluent to achieve a final concentration of 200 mM NaCl in the final pool.

Example of elution buffer optimization study:

Should further optimization of elution, and the ongoing stability of viral vector through buffer exchange and formulation for fill and finish be required, these aspects of the process can be explored by adjusting the elution or dilution buffer composition. Consider the following suggestions:

• Lowering elution pH to 6.5-7 (e.g., by using BTP at pH 6.5, or HEPES at pH 6.8 for the elution buffer), since the weak anion exchange of the Lenti HERO® allows elution at even lower conductivity at lower pH.

• Screening additives to increase freeze-thaw stability (e.g., sucrose or trehalose up to 10%).

• Matching the dilution buffers for the elution buffer, but raising the pH to 7.5, and matching the additives to achieve a final concentration of 10% of the sucrose or trehalose.

Key considerations for processing lentiviral vectors