Purification of recombinant human basic fibroblast growth factor: stability of selective sorbents under cleaning in place conditions.
Human basic fibroblast growth factor (bFGF) was produced from recombinant Escherichia coli by high-cell-density cultivation. In order to develop a purification strategy for large-scale purification, chromatographic sorbents with different anionic functional groups were compared in terms of selectivity for bFGF and stability under cleaning in place (CIP) conditions. Heparin-Sepharose CL-6B, Fractogel EMD-SO3- 650 (S) and SP-Sepharose (high performance) were found suitable for this purpose with decreasing selectivity in that order. Each sorbent was treated eight times under CIP conditions employing both 0.2 and 1.0 M NaOH, in order to study modifications of these sorbents. Heparin-Sepharose displayed more than 50% loss of capacity after the first CIP treatment and decreasing selectivity with each cycle. Both cation exchangers displayed almost constant results regarding selectivity and capacity. The Fractogel EMD-SO3- exhibited only slightly lower selectivity for bFGF than Heparin-Sepharose and the highest capacity of all sorbents tested. Agglomeration of bFGF at low salt concentrations was a serious problem. By direct application of pooled fractions from Fractogel EMD-SO3- onto Heparin-Sepharose a highly pure product was obtained; however, the recovery after Heparin-Sepharose was only 30%.
D000837:Anion Exchange Resins / Q000737:chemistry*; D002411:Cation Exchange Resins / Q000737:chemistry*; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D016222:Fibroblast Growth Factor 2 / Q000737:chemistry / Q000302:isolation & purification*; D006801:Humans; D009113:Muramidase / Q000737:chemistry / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D013056:Spectrophotometry, Ultraviolet
D000837:Anion Exchange Resins; D002411:Cation Exchange Resins; D011994:Recombinant Proteins; D016222:Fibroblast Growth Factor 2; D009113:Muramidase