Ligands from Combinatorial Peptide Libraries for Virus Removal

Abstract

Small peptides were investigated as affinity ligands for virus removal from human blood plasma. Sindbis virus (SV) was radiolabeled with ³⁵S and screened against a solid phase peptide library. The screening identified 9 hexapeptides that were synthesized on a TosoBioseparations Toyopearl 650M Amino Resin. The peptide sequences SGKPVA and IATDGG were found to remove approximately 3 logs and 2.4 logs of SV from buffer respectively and approximately 1.5 logs of SV from 50% human blood plasma (HBP). Toyopearl Amino resin was used as a control and bound only 0.6 logs of SV in both buffer and 50% HBP. Good agreement was seen between infectious quantification methods and quantification of the virus using radiation. Injections of several batches of SV showed variations between the batches.

A similar screening procedure was also applied to radiolabeled canine parvovirus (CPV). Screening against a solid phase library identified 25 leads through to be specific to CPV. Tests with a portion of these leads found less than 0.5 logs of CPV clearance in both buffer and 50% HBP. Electron microscopy of the CPV used in these experiments showed that large aggregates of virus were present in the radiolabeled virus. Infectious assays for CPV were also problematic and inconsistent.

Work done in collaboration with the American Red Cross (ARC) identified several leads capable of binding porcine parvovirus (PPV) and human B-19 parvovirus (B-19). Selected leads were tested for viral clearance from 1 ml samples of spiked buffer and 50% HBP. The resins were shown to bind at least 4 logs of PPV and B-19 but less than 0.5 logs of CPV. Experiments with 10 ml samples of spiked buffer and 7.5% HBP showed that the resins were capable of 6 logs of PPV clearance. 10 ml experiments performed in 50% HBP showed an initial clearance of 6 logs of virus. Subsequent fractions showed decreasing clearance with complete breakthrough occurring by the 9th 1 ml fraction. Experiments that compared three different column systems showed that each system produced identical log clearance results.

This work shows that screening a solid phase library can identify peptide ligands and that the leads can be used for viral clearance from HBP.