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

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 (⌬G unf ) and osmotic second virial coefficient (B 22 ), respectively. Under solution conditions where rhGCSF conformational stability dominates (i.e., large ⌬G unf and negative B 22 ), 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 B 22 values) the second step is ratelimiting, 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.

Investigation of structural properties of clinoptilolite rich zeolites in simulated digestion conditions and their cytotoxicity against Caco-2 cells in vitro

“…Although the exact mechanism was hard to define, this complex mechanism with zeolite treatments might have resulted proteins to possess anisotropic charge distribution on their surfaces and this might have caused increase in dipoles. Thus protein-protein interactions might be attractive and lead aggregation to be energetically favorable [39,40].…”

Studying the formation of aggregates in recombinant human granulocyte-colony stimulating factor (rHuG-CSF), lenograstim, using size-exclusion chromatography and SDS-PAGE

“…The percent of aggregate was only 7%. Previous studies (10,11) relating to the aggregation of filgrastim reported that under the same aggregation conditions filgrastim was aggregated readily with 96% loss of monomer.…”

“…gregation is rapidly emerging as a key issue underlying multiple deleterious effects for protein-based therapeutics, including loss of efficacy and immunogenicity (10).…”

“…Many published studies for monitoring protein aggregation are available (12-15) and some of them referred to determination of aggregates formation in filgrastim (10,11,16,17). However, to our knowledge, the SEC-HPLC method for determination of intact protein and detection of aggregates of lenograstim has not been reported in the literature.…”

Lysozyme‐lysozyme self‐interactions as assessed by the osmotic second virial coefficient: Impact for physical protein stabilization

“…SIC allows rapid determination of B 22 under high throughput conditions using automated systems. A recent study [103] has presented a high-throughput self interaction chromatography application combined with chemometry (ANN: artifical is an indicator for physical protein stability (see Figure 10) [9,105,106].…”