Implementation of the mille-feuille nanofilter paper in the virus removal filtration of IgY purified from chicken egg yolk

Abstract: The purpose of this thesis was to test the mille-feuille nanofilter paper for a virus removal filtration from a potential protein-based nutraceutical. Therefore, chicken IgY was purified from egg yolk, with PEG precipitation for crude fractionation followed by thiophilic adsorption chromatography. The biuret total protein assay was done for quantitative analysis, and for qualitative analyses, sodium dodecyl sulphate- polyacrylamide gel electrophoresis, dynamic light scattering and size exclusion-high performance liquid chromatography were performed. The protein characterization results revealed that the final preparation of IgY was a complex mixture of proteins and aggregates. Two variants of the mille-feuille filter paper were produced for the two-step nanofiltration: 11 m pre-filters to remove aggregates and other high molecular weight impurities, as well as dedicated 31 m virus removal filters. The thickness, basis weight and hydraulic flux of the produced filters were measured for quality control, and the pore size distributions of the filters were estimated with the use of cryoporometry by differential scanning calorimetry and the BJH method derived from nitrogen gas sorption isotherms. Finally, the IgY preparation was spiked with the model virus ΦX174 and tested for virus removal with the mille feuille filter paper, which resulted in an LRV over 5 as determined with the plaque forming units assay. Flux measurements as well as following pre-filtrate and filtrate characterizations with dynamic light scattering revealed that the pre-filtration had a critical impact on the quality of the final product. The final filtrate was gone through tandem mass spectrometry, upon which IgY and several other egg yolk proteins were identified.  The results of this thesis show that the mille-feuille nanofilter paper has a high potential for virus removal of nutraceuticals.