International Journal of Biomedical Science and Research
New Human Fibrinogen Multi-Peptide Affinity Ligands Allowing High Temperature Virus Inactivation During Chromatographic Process
Abstract
Serhiy P. Havryliuk, Olena S. Havryliuk and Heorhii L. Volkov
According to the 8 algorithms approach it was developed the high affinity multi-peptide chromatographic gel for purification human fibrinogen – one of the basic components of the fibrin surgery glue or sealant. Given the next step of fibrinogen virus inactivation directly in the chromatographic column, the multi-point peptide-affinity gels had to bind this protein at several spatially spaced points in order to limit the protein’s degree of freedom for holding it at high buffer flow rates or elevated buffer temperature without denaturation.
Based on previous experiments, an multi-point peptide-affinity chromatographic gel had the following properties: the dynamic binding capacity with enough residence time 20 min was around 54-58 mg×mL-1 led to the >92% purity of isolated fibrinogen without virus-inactivation and to the 96,4% after in-column virus-inactivation. The virus inactivation of this protein (coupled by the gel) directly in chromatographic column shown a highly effective virus elimination (log10>9) for both nonenveloped and lipid enveloped viruses.
Using fibrinogen sequence from UniProt_KB and dates from more than 25 literature sources on the protein interaction, affinity peptides were calculated against several sequences or separate amino acid fragments. Then these peptides were modified to reach more affinity enhancement and multi-point affinity-peptide chromatographic gel was synthetized and tested.
By synthetized multi-point affinity-peptide chromatographic gel and one step chromatography the virus inactivated human fibrinogen was purified (96,4% pure) with acceptable quantity (71-76%) and ability to polymerize.