Fig. 3: Selection of unused plasma industrial fractionation intermediates as source material and purification and characterization of CP from FIV_1-4.

a FIV_1-4 is the most suitable starting material for CP purification. CP Immunoblotting analysis of CP in commercially available plasma-derived CP, FIV_1-4, FIII and FI. Intact CP has a predicted MW of 132 kDa (black arrowhead), and FIV_1-4 contains the highest proportion of intact protein. Known protease cleavage sites likely result in the production of smaller fragments of 70, 90 and 116 kDa respectively²⁸. b–d FIV_1-4 contains the highest levels of CP enzymatic activity (oxidase assay, b), of CP protein c, and the highest CP levels relative to total protein d. e–i Biochemical characterization of CP purified from FIV_1-4 (kCP). e Relative protein composition in FIV_1-4 and in kCP as analyzed by biochemical methods (top panels) and mass spectrometry (bottom panels; see also Supplementary Data 3, ceruloplasmin characterization). f–i Comparison between FIV_1-4 and kCP. Enrichment in CP content f, oxidase activity g and purity h in kCP vs FIV_1-4. Specific oxidase activity is preserved during kCP purification i. j Immunoblotting illustrating preservation of CP content and integrity from the starting material (FIV_1-4), through purification intermediates and to the final product, kCP, with commercially available CP as a reference. k, l kCP concentrate specific activity as measured by both oxidase k and ferroxidase l assays. Data are presented as means ± SEM; each dot corresponds to one analysis in a–d and to one batch in e–l. Numerosity: N = 9 for FI, N = 15 for FIII and FIV_1-4 in b; N = 3 for FI, FIII and FIV_1-4 in c, d; N = 4, three analyses for each batch in panels f–i; N = 3 for CP, N = 5 for kCp, three analyses for each batch in k and l. Statistical P values were evaluated by one-way ANOVA followed by Tukeys’s multiple comparisons test in panels b-d and by unpaired t-test in panels f-i and k-l.