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Roles of conformational stability and colloidal stability in the aggregation of recombinant human granulocyte colony‐stimulating factor
Author(s) -
Chi Eva Y.,
Krishnan Sampathkumar,
Kendrick Brent S.,
Chang Byeong S.,
Carpenter John F.,
Randolph Theodore W.
Publication year - 2003
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.0235703
Subject(s) - ionic strength , chemistry , colloid , native state , solubility , protein aggregation , limiting , protein folding , crystallography , aqueous solution , biochemistry , mechanical engineering , engineering
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 ( Δ G unf ) and osmotic second virial coefficient ( B 22 ), respectively. Under solution conditions where rhGCSF conformational stability dominates (i.e., large Δ G unf and negative B 22 ), the first step is rate‐limiting, and increasing Δ G unf (e.g., by the addition of sucrose) decreases aggregation. In solutions where colloidal stability is high (i.e., large and positive B 22 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.