Insights into product and process related challenges of lentiviral vector bioprocessing

• Published in: Biotechnology and bioengineering 2024-08, Vol.121 (8), p.2466-2481
• Main Authors: Perry, Christopher, Mujahid, Noor, Takeuchi, Yasu, Rayat, Andrea C. M. E.
• Format: Article
• Language: English
• Subjects: Anion exchange; Anion exchanging; bioprocess; Bioprocessing; envelope proteins; Expression vectors; Filtration; Filtration - methods; Gene expression; Genetic Vectors - genetics; HEK293 Cells; Humans; lentiviral vector; Lentivirus - genetics; Manufacturing; Membrane filtration; Membranes; Protein purification; Proteins; pseudotype; Recovery; scale‐down; Shear flow; Temperature perception; Tropism; vector stability; Viral envelope proteins; Viral Envelope Proteins - genetics
• ISSN: 0006-3592; 1097-0290; 1097-0290
• DOI: 10.1002/bit.28498

Lentiviral vectors (LVs) are used in advanced therapies to transduce recipient cells for long term gene expression for therapeutic benefit. The vector is commonly pseudotyped with alternative viral envelope proteins to improve tropism and is selected for enhanced functional titers. However, their impact on manufacturing and the success of individual bioprocessing unit operations is seldom demonstrated. To the best of our knowledge, this is the first study on the processability of different Lentiviral vector pseudotypes. In this work, we compared three envelope proteins commonly pseudotyped with LVs across manufacturing conditions such as temperature and pump flow and across steps common to downstream processing. We have shown impact of filter membrane chemistry on vector recoveries with differing envelopes during clarification and observed complete vector robustness in high shear manufacturing environments using ultra scale‐down technologies. The impact of shear during membrane filtration in a tangential flow filtration‐mimic showed the benefit of employing higher shear rates, than currently used in LV production, to increase vector recovery. Likewise, optimized anion exchange chromatography purification in monolith format was determined. The results contradict a common perception that lentiviral vectors are susceptible to shear or high salt concentration (up to 1.7 M). This highlights the prospects of improving LV recovery by evaluating manufacturing conditions that contribute to vector losses for specific production systems.