Roles of conformational stability and colloidal stability in the aggregation of recombinant human granulocyte colony‐stimulating factor

Protein Science - Tập 12 Số 5 - Trang 903-913 - 2003
Eva Y.1, Sampathkumar Krishnan2, Brent S. Kendrick3, Byeong S. Chang2, John F. Carpenter4, Theodore W. Randolph1
1Department of Chemical Engineering, Center for Pharmaceutical Biotechnology, University of Colorado, Boulder, Colorado 80309-0242, USA
2Amgen Inc., Amgen Center, Thousand Oaks, California 91320 USA
3Amgen, Inc., Longmont, Colorado 80503, USA
4Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA

Tóm tắt

Abstract

We studied the non‐native aggregation of recombinant human granulocyte stimulating factor (rhGCSF) in solution conditions where native rhGCSF is both conformationally stable compared to its unfolded state and at concentrations well below its solubility limit. Aggregation of rhGCSF first involves the perturbation of its native structure to form a structurally expanded transition state, followed by assembly process to form an irreversible aggregate. The energy barriers of the two steps are reflected in the experimentally measured values of free energy of unfolding (ΔGunf) and osmotic second virial coefficient (B22), respectively. Under solution conditions where rhGCSF conformational stability dominates (i.e., large ΔGunf and negative B22), the first step is rate‐limiting, and increasing ΔGunf (e.g., by the addition of sucrose) decreases aggregation. In solutions where colloidal stability is high (i.e., large and positive B22 values) the second step is rate‐limiting, and solution conditions (e.g., low pH and low ionic strength) that increase repulsive interactions between protein molecules are effective at reducing aggregation. rhGCSF aggregation is thus controlled by both conformational stability and colloidal stability, and depending on the solution conditions, either could be rate‐limiting.

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Protein Science

Tập 12 Số 5

903-913

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