TY - JOUR

T1 - Purification of recombinant human basic fibroblast growth factor

T2 - stability of selective sorbents under cleaning in place conditions

AU - Birger Anspach, F.

AU - Spille, Henning

AU - Rinas, Ursula

PY - 1995/9/8

Y1 - 1995/9/8

N2 - 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%.

AB - 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%.

UR - http://www.scopus.com/inward/record.url?scp=0029120857&partnerID=8YFLogxK

U2 - 10.1016/0021-9673(95)00102-S

DO - 10.1016/0021-9673(95)00102-S

M3 - Article

C2 - 7496484

AN - SCOPUS:0029120857

VL - 711

SP - 129

EP - 139

JO - Journal of Chromatography A

JF - Journal of Chromatography A

SN - 0021-9673

IS - 1

ER -