Lentivirus are becoming a largely used tool in gene therapy for transferring of therapeutic genes into the patient's cells. However, there are several limitations on current downstream methods for purification of lentivirus, especially removal of contaminants generated by HEK cells during lentivirus production. An example of contaminant is host cell proteins (HCPs) that can trigger immune response on patients. Because peptides can be used as affinity ligands, due to their ability to selectively bind a target protein, it was created libraries of peptides on polymer-based beads and lentivirus and HEK proteins were labeled with green and red fluorescent dyes, respectively. The libraries were screened against free lentivirus proteins in competitive mode via dual fluorescence selection using a microfluidic device that enables the selection of ligands with controlled binding and elution behavior. Elution conditions were further optimized by conducting stability assays of lentivirus in different buffers and salt concentration. At high salt concentrations magnesium chloride in citrate buffer showed no reduction of virus titer; however, the same result was not observed when sodium chloride was used. Following library screening, selected beads were sequenced via Edman degradation to determine amino acid composition. Therefore, these results show that the selected peptides could be used for functionalization of commercial resins for affinity purification of lentivirus in bind-and-elute mode, producing more efficient methods for lentivirus downstream processes.

Design and selection of lentivirus-targeting ligands

ACS Spring 2022

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.