Virus clearance is required for pharmaceutical preparations derived from animal or human sources such as blood products, vaccines, recombinant proteins produced in mammalian cell lines, etc. High cost and substantial protein losses during virus inactivation are significant problems for protein/peptide manufacturing. The goal of this project was to develop a method to perform virus inactivation in a course of protein chromatographic purification. Another goal was to show that the chromatographic adsorbent can serve as reliable "sieva" for mechanical washing away of infecting viruses. Using chromatographic, photometric, IFA, and RT-PCR approaches, it was discovered that high temperature-depending dynamic capacity of adsorbent allowed to perform a virus inactivation directly in a chromatographic column by solvent/detergent treatment. The peptide/protein biological activity was completely preserved. Using this new approach enveloped and nonenveloped viruses were effectively removed protein preparation. In addition, it was shown that RT-PCR method demonstrates more precise and reproducible results and robust properties for assessment of virus reduction than virus titer followed by infectivity studies. Presented method allowed to obtain the factor of virus concentration decrease (FVD) values that were higher than those provided by known technologies and was sufficient for a full inactivation of viruses. The method is recommended to use in pharmaceutical industry.

According to "The Proteome Code" concept introduced by J. Biro and our early development of affinity peptide calculation method it was studied the possibility of high affinity peptide chromatographic gels development for IgG1-4 separation from the donor plasma. Given the next step of virus inactivation of IgG directly in the chromatographic column, the affinity gel had bind IgG at several spatially spaced points in order to limit the degree of freedom of the protein for retention IgG at high buffer flow rate or elevated buffer temperatures without denaturation. In addition, the possibility of creating highly specific affinity sense-antisense peptides against Rubella virus in order to increase the titer of aRIgG in plasma or even its isolation in highly purified form was studied. Based on previous experiments, an affinity multi-peptide chromatographic gel with the following properties was developed: the DBC with enough residence time 10 min was around 50-54 mg × mL-1 of total 98.0% purity of IgG with natural proportion of the 1-4 subclasses, any other immunoglobulins were not found. The virus inactivation/elimination on this gel directly in chromatographic column shown a highly effective virus elimination (log10>9) for both nonenveloped and lipid enveloped viruses. Using RV sequence from UniProt_KB and dates from more than 20 literature sources on the virus proteins interaction, affinity peptides were calculated against virus proteins C and E1,2. Then these peptides were modified to reach more affinity enhancement and affinity-peptide chromatographic gel was synthetized. By this gel from total mass IgG1-4 contained 6644 IU anti-Rubella IgG with specificity 6.64 IU × mg-1 were isolated 5382 IU aRIgG (> 80%) with a specificity of 791 IU × mg-1.