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

¹ Department of Chemical Engineering, Center for Pharmaceutical Biotechnology, University of Colorado, Boulder, Colorado 80309-0242, USA
² Amgen, Inc., Amgen Center, Thousand Oaks, California 91320, USA
³ Amgen, Inc., Longmont, Colorado 80503, USA
⁴ Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA

Reprint requests to: Theodore W. Randolph, Department of Chemical Engineering, Center for Pharmaceutical Biotechnology, ECCH 111, Campus Box 424, University of Colorado, Boulder, CO 80309-0242, USA; e-mail: randolph{at}pressure3.colorado.edu; fax: (303)492-4341.

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 (G_unf) and osmotic second virial coefficient (B₂₂), respectively. Under solution conditions where rhGCSF conformational stability dominates (i.e., large G_unf and negative B₂₂), the first step is rate-limiting, and increasing G_unf (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.

Keywords: Non-native protein aggregation; rhGCSF; protein stability; protein formulation strategy; osmotic second virial coefficient; light scattering; protein interactions; activation energy

Abbreviations: rhGCSF, recombinant human granulocyte colony stimulating factor • SE-HPLC, size-exclusion high-performance liquid chromatography • FTIR, Fourier transform infrared spectroscopy • SLS, static light scattering • CD, circular dichroism • PBS, phosphate-buffered saline • DLVO, Derjaguin-Landau-Verwey-Overbeek theory

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