Affinity adsorption of viruses using small peptide ligands

The removal of viruses from process streams is an extremely important problem to the pharmaceutical industry, as more biotherapeutic products are being produced from human or cell-based sources. Commonly, nanofiltration or chemical inactivation is used to remove viruses from protein therapeutic products. Nanofiltration efficiently removes large viruses, and chemical inactivation renders many enveloped viruses noninfectious. Yet there remains a group of small, nonenveloped viruses whose removal from process streams still pose serious challenges.;In this work, porcine parvovirus (PPV) was produced, radiolabeled with 35S and purified. The purified PPV was examined for many different properties, including infectivity, protein content, purity, and the ability to bind to small peptide ligands. The titration of the virus to determine the amount of infectivity was examined using three different assays, leading to the conclusion that detection of viable cells using the tetrazolium salt MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) was the most quantitative and could handle a large number of samples.;Trimeric peptides that bound to PPV were found from the screening of a solid phase combinatorial library that was synthesized on a chromatographic support. The resin WRW with a peptide density of 0.1 mmol/g was found to bind all of the PPV in solution from saline, and was able to clear all detectable viruses in the first three column volumes from 7.5% human blood plasma. The removal of PPV by WRW was slightly improved by reducing the peptide density to 0.008 mmol/g. The binding of this ligand was also affected by the presence of an ethylene oxide spacer arm. When the spacer arm was removed, the binding of WRW at either peptide density was reduced. Hexamer ligands that bind to PPV were also identified by screening trimer libraries constructed on the best trimers. The hexamer ligands did not perform as well as the trimer ligands.;This work lays the foundation for additional studies with other viruses and ligands in the future.