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FELICIANO, G. P. Innovation in human plasma fractionation process using liquid pseudo-affinity chromatography. 2016. 88 p. Masters thesis (Biotechnology) Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, 2016. In this study we showed that the proposed purification method, employing an anion exchange column and a wash with CaCl2 (pseudoaffinity chromatography) and washes with NaCl (traditional anion exchange chromatography) can be successfully used for the purification of coagulation factors. This strategy allows us to obtain concentrates of vitamin K dependent and factor VIII (FVIII) coagulation factors in a single chromatographic step. We tested four anion exchange resins (ANX Sepharose FF, Q Sepharose FF, QA monolithic column and Sartobind Q membrane) and three buffers (citrate, 2-(N-morpholino)ethanesulfonic acid (MES) and bis(2-hydroxyethyl)aminotris(hydroxymethyl)methane (Bis-Tris). The ANX Sepharose FF column and the citrate buffer showed better protein separation profile. We designed a protocol in which vitamin K dependent proteins elute with 25 mM CaCl2 and FVIII elute with 500 mM NaCl chloride. Factor II (FII) and factor IX (FIX) activity recovery was almost total and Protein C was 55%. FVIII activity recovery was approximately 60% and purification factor of 220 times. In the fraction containing vitamin K dependent proteins, it was identified through mass spectrometry analysis presence of Fibrinogen, complement C4 and C4b Binding Protein. Analysis by SDS-PAGE showed that the presence of these proteins wasn't calcium chloride concentration dependent. This result and the fact that they are high molecular mass proteins (>200 kDa) and are present in plasma at high concentrations, especially Fibrinogen (3 g/mL), indicate that their presence on vitamin K dependent proteins fraction was due to slowly diffusion through the resin and not by calcium chloride elution. More than 99% of the plasma proteins are not adsorbed in the column, including albumin and IgG and can be purified in following chromatographic steps.

Inovação no processo de fracionamento de plasma humano através do uso de cromatografia líquida de pseudoafinidade.