By Gail Dutton
Lentiviral vectors (LVs) used to transfect cells for gene expression can withstand shear rates that are twice as high as those normally used and still maintain comparable productivity. That insight from researchers at University College London removes some of the hurdles that, so far, have constrained gene therapies from advancing in clinical trials.
Reporting in Biotechnology & Bioengineering, researchers led by Andrea C.M.E. Rayat, PhD, associate professor, bioseparations and downstream processing, University College London, evaluated the manufacturing processes various vectors experience and the conditions that lead to vector loss. They assessed a three-plasmid system and a four-plasmid system (both pseudotyped with the RDpro, Cocal-G, and VSV-G envelope proteins) in terms of process shear, manufacturing duration and temperature, pump flow, the impact of membrane materials, and the impact of chromatography buffers on the lentiviral vectors.
Functional titer increases with shear
To evaluate shear forces during diafiltration, the researchers used an ultra-scale-down (USD) tangential flow filtration (TFF) mimic. Many researchers perform TFF at flow rates of less than 500 s−1 and shear rates of less than 2000 s−1. Rayat’s team, however, began with a flow rate of approximately 800 s−1 and a shear rate of 1000 RPM.
“For Cocal-G and RDpro enveloped LVs, the overall functional titers appear to increase with higher shear. VSV-g envelope protein has a mild decrease from initial to 3,000 RPM, but titers from this point onwards are maintained,” the authors noted. Using the higher shear rate during TFF, they explain, maintains high permeate flux rates and reduces membrane fouling.
In terms of processability, clarification by membrane filtration had the greatest effect on the lentiviral vectors, followed by the pump, which affects shear. They found “a weak correlation of lower functional titers with pump flow.”
Of the four filter materials tested—polyvinylidene difluoride (PVDF), polyethersulfone (PES), mixed cellulose esters (MCE) and nylon—the PVDF filters consistently recovered more lentiviral vector than the others, while PES filters recovered the least.
“Separate, larger scale studies, using commercial hollow fiber TFF systems, have demonstrated comparable results to the USD system,” Rayat’s team reported. “These results reinforce the need to…test new LV products in terms of their robustness for manufacture,” and to apply rational bioprocess development to lentiviral production. The benefits, they suggest, may be like those experienced by the rational design of recombinant protein production.