Scaling-up production of lentiviral vectors (LV)-based therapies is a persistent challenge for biomanufacturers, and one that threatens their development unless commercial-scale production methodologies are developed.
McGill University researchers say they have a solution: a semi-continuous manufacturing system that integrates upstream and downstream bioprocesses. This novel approach generates a significantly higher quantity of LVs, enhances recovery, reduces instability, and improves product quantity. Compared to batch processing, it cuts processing time by four-fold.
Led by Amine A. Kamen, PhD, professor, bioprocessing of viral vaccines, McGill University scientists combined clarification and capture-step loading processes and operated them in parallel with capture-step purification. This enhances the recovery of functional vector particles by 26% and total vector particles by 18%. The protein clearance level of 25% is higher in semi-continuous mode, while the 15% DNA removal is similar to that of batch processing.
Potency, measured as the ratio of vector genome units to transducing units, was 75 in semi-continuous mode. The ratio was 130 for batch, 168 for batch with one-day hold, and 360 for batch with six-day hold.
The downstream semicontinuous system works by rotating three membranes among two systems: System One, for clarification and loading, and System Two, for purification.
Once the first membrane (A) is loaded in System One, it is transferred to System Two for the wash, elution, and regeneration steps. The second and third membranes (B and C) move into position for loading and clarification in System One. Once B is loaded, it moves to System Two, C moves forward to loading, and A returns to System One in position behind C. The cycle continues until each membrane has completed two full cycles.
“The Mustang Q membranes are moved manually in the downstream setup,” the team wrote, hence the method is semi-continuous.
Major time savings
Because the loading time for the Mustang Q membrane and purification steps for the AKTA Avant chromatography system the researchers used are comparable, they found “the two systems could run in parallel.”
This semi-continuous system cut the loading and eluting time for 516 mL of LV harvest to 1.75 hours. That became 2.25 hours when nuclease treatment was included. In contrast, processing the same quantity of LVs in batch mode, using the same membrane size, takes about 8.75 hours.
At commercial-scale using a 2-L working volume perfusion reactor, overall manufacturing time using perfusion upstream and semi-continuous downstream processing takes an average of 12.75 days, the team reported. In contrast, perfusion upstream/ batch downstream processing takes 18.25 days. Using batch processing for both up- and downstream processing takes 84.5 days.