2021 FDA Science Forum
Processing of Lentiviral Vector Pseudotypes Using Anion Exchange and Affinity Chromatography
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Contributing OfficeCenter for Biologics Evaluation and Research
Abstract
In order to simplify the processing of large volumes of lentiviral vector (LVV) supernatants involving LVVs pseudotyped with VSV-G, the measles virus (MV) hemagglutinin (H), or the Tupaia Paramyxovirus (TPMV) H glycoprotein, we previously implemented a concentration and purification strategy based on anion exchange (AEX) membrane chromatography. LVV supernatants processed in this way showed enhanced purity and reduced toxicity compared to vectors concentrated using standard ultracentrifugation protocols.
To improve vector purity, we are currently exploring strategies based on immobilized metal ion affinity chromatography (IMAC) to concentrate LVV pseudotypes bearing hexahistidine (6 x His)-tagged envelope (Env) glycoproteins. Initial experiments focused on LVVs pseudotyped with a 6 x His-tagged, receptor-blind MV H glycoprotein displaying IL-13. Such pseudotypes were captured on HiTrap IMAC HP columns using the AKTA Pure L1 chromatography system and batch eluted using buffers containing imidazole. The recoveries observed were up to 37% of the input samples.
We also tested the ability of the IMAC strategy to capture lentiviral vectors pseudotyped with the commonly used VSV-G Env glycoprotein bearing a 6 x His tag at position 8 of the mature VSV-G glycoprotein. The recoveries observed were up to 7% of the input vector sample. The reduced recovery observed using VSV-G pseudotypes compared to MV H pseudotypes is possibly related to limited accessibility of the 6 x His tag present on VSV-G. To improve exposure of the 6 x His tag, we investigated additional sites within the VSV-G ectodomain that can tolerate 6 x His tags while retaining the function of the VSV-G protein. To do this we designed variants with 6 x His tags inserted at positions 2 and 352 of the mature VSV-G glycoprotein. The data obtained showed that the presence of single or double-6 x His tags inserted at position 2 resulted in vector titers comparable to those obtained using unmodified VSV-G.
Studies aimed at optimizing the conditions for IMAC purification of LVVs bearing 6 x His tagged Env glycoproteins and improving their recovery and stability are ongoing.
