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 (DeltaG(unf)) and osmotic second virial coefficient (B(22)), respectively. Under solution conditions where rhGCSF conformational stability dominates (i.e., large DeltaG(unf) and negative B(22)), the first step is rate-limiting, and increasing DeltaG(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.

Figure 1.

Structural changes of rhGCSF accompanying aggregation during incubation at 37°C. Area normalized second derivative FTIR spectra of native rhGCSF (solid line, pH 3.5 HCl with 150 mM NaCl; dashed line, pH 7 PBS) show predominantly α-helical structures and aggregated rhGCSF (dotted line, pH 3.5 HCl with 150 mM NaCl; dashed and dotted line, pH 7 PBS) exhibit high levels of intermolecular β-sheet structures. Spectra of soluble and aggregated rhGCSF in pH 7 PBS were taken from Krishnan et al. 2002.

Figure 2.

B₂₂/B₂₂^HS values (A) and extrapolated mass averaged molecular weights (B) of rhGCSF from static light scattering experiments in pH 7 PBS at various sucrose concentrations. Concomitant with the large increase in B₂₂/B₂₂^HS when 0.25 M or more sucrose was added, the apparent rhGCSF molecular weight was higher, indicating the presence of multimeric species. Extrapolated molecular weights for these samples correspond to 40–60 mole % dimers. Thus, B₂₂/B₂₂^HS values measured in these solutions reflect overall two-body interactions, not just from aggregating monomeric species.

Figure 3.

Schematic reaction profile for the aggregation of rhGCSF in pH 7 PBS on an arbitrary free energy y-axis. Curved lines illustrate kinetic energy barriers. M* is a structurally expanded transition state species and ΔG^‡_MM* is the activation free energy of aggregation. The initial rate of irreversible dimer (M₂) formation is second order in native state monomer (M) concentration. Dotted arrows illustrate, relative to M, shifts in the free energies of native dimers (D), unfolded monomer (U), and M* when sucrose is added. The off-path reaction that generates D is depicted to the left of the aggregation reaction coordinate.

Figure 4.

Schematic DLVO interaction energy of two spherical particles interacting at constant and uniform surface potential. (A) Total interaction energy is the sum of electric double-layer repulsion (∝ e^−κD, where κ is the inverse Debye length) and van der Waals attraction (1/6 D; Israelachvili 1992). ΔW₁ represents the maximum interaction energy barrier of the two particles. (B) Increasing salt concentration screens double layer repulsion, resulting in a decrease of ΔW₁. When ΔW₁ < 0 (curve iii), particles become unstable and coagulation occurs. Decreases in ΔW₁ could also be resulted by decreasing the absolute value of the difference between solution pH and the isoelectric point of a protein. (C) Schematic reaction profile for the aggregation of rhGCSF in pH 3.5 HCl on an arbitrary free energy y-axis. Curved lines illustrate kinetic energy barriers. M₂* is the dimeric transition state species and ΔG^‡_MM2* is the activation free energy of aggregation. Dotted arrows illustrate that increases in solution ionic strength (or decreases in |pH-pI|) decrease ΔG^‡_MM2*. At low ionic strength (i), ΔW₁ is large and positive, resulting in a high ΔG^‡_MM2*. Increasing ionic strength sufficiently led to a negative ΔW₁ (iii), lowering ΔG^‡_MM2* enough that M₂* is no longer the transition state of the aggregation reactions. At high ionic strength, M* is expected to be the transition state of aggregation.

Figure 5.

Comparison between experimental and theoretical B₂₂/B₂₂^HS of rhGCSF at pH 3.5 as a function of ionic strength. Degree of acetic acid ionization was accounted for in calculating the ionic strength of sodium acetate buffers. Error bars on experimental B₂₂/B₂₂^HS (filled diamond) are linear regression standard errors of SLS data using equation 2. Theoretical B₂₂/B₂₂^HS (solid line) was calculated taking into account excluded volume and Coulombic charge–charge repulsion treating proteins as point charges (Petsev et al. 2000).

Scheme 1:

Aggregation pathway

Scheme 2:

Equilibrium reactions