Accelerated stability studies of abatacept formulations: comparison of freeze-thawing- and agitation-induced stresses.

Accelerated degradation studies are frequently used to screen for formulation conditions that confer adequate shelf life for therapeutic proteins. To speed development cycles, degradation is often accelerated by application of conditions that expose proteins to elevated temperatures, dynamic air-water interfaces created by agitation, or stresses induced by freeze-thaw cycling. The purpose of this case study was to compare freeze-thaw- and agitation-induced aggregations with aggregation previously studied at elevated temperatures (Fast J, Cordes AA, Carpenter JF, Randolph TW. 2009. Biochemistry 48:11724-11736) using the therapeutic fusion protein abatacept as a model. The stability of abatacept against aggregation induced by the freeze-thaw and agitation degradation methods was assessed by size-exclusion chromatography (SEC) and microflow imaging (MFI) analysis. pH conditions that were previously found to increase conformational stability of abatacept and reduce aggregation during incubation at elevated temperature (Fast J, Cordes AA, Carpenter JF, Randolph TW. 2009. Biochemistry 48:11724-11736) also reduced aggregation induced by freeze-thaw cycling and by agitation in this study. Especially in the case of the freeze-thaw cycling, wherein the formation of aggregates was not readily detectable by SEC, MFI proved to be a useful method to characterize the stability of the formulations against aggregation.

Selective domain stabilization as a strategy to reduce human serum albumin-human granulocyte colony stimulating factor aggregation rate.

Therapeutic proteins must be generally formulated to reduce unwanted aggregation. Fusion proteins, which comprise domains assembled from separate proteins, may require unique formulation strategies in order to maximize their stability. A fusion protein of human serum albumin (HSA) and human granulocyte colony stimulating factor (GCSF; HSA-GCSF) was used as a model to test the hypothesis that formulations that increase the thermodynamic conformational stability of the least stable domain of a fusion protein will stabilize the entire fusion protein against aggregation. Conformational stability of HSA-GCSF was modulated by addition of octanoic acid, which was previously shown to increase the conformational stability of HSA, the least stable domain. Contrary to our hypothesis, increased conformational stability of the HSA domain did not result in increased resistance to aggregation of HSA-GCSF. These results for HSA-GCSF were also compared with similar studies conducted previously on a therapeutic protein formed by the fusion of HSA and human growth hormone (hGH; HSA-hGH).

Selective domain stabilization as a strategy to reduce fusion protein aggregation.

A human serum albumin-human growth hormone (HSA-hGH) fusion protein was used as a model to understand the contributions of individual domains to the aggregation behavior of the overall fusion protein. Aggregation of HSA-hGH was studied at two different pH conditions, pH 5 and pH 7. Conformational stability of the HSA domain was modulated by addition of octanoic acid, a binding ligand. Conformational stability of the fusion protein and the HSA domain were determined from experimentally measured values for free energies of unfolding (ΔG(unf) ) with midpoint of apparent unfolding temperatures (T(m) ) used as surrogate in some cases. Apparent T(m) s of both HSA and HSA-hGH were increased by octanoic acid binding. Osmotic second virial coefficients were measured to monitor protein-protein interactions in solution. Reductions in rates of aggregation were observed under solution conditions that increased protein-protein repulsive interactions even when no changes in conformational stability were detected. The results indicate that colloidal instabilities are responsible for HSA-hGH aggregation and that conformational stability of the HSA domain does not play a dominant role in the aggregation of HSA-hGH.

Physical instability of a therapeutic Fc fusion protein: domain contributions to conformational and colloidal stability.

Protein therapeutics made up of artificially combined proteins or protein domains, so-called fusion proteins, are a novel and growing class of biopharmaceuticals. We have studied abatacept (Orencia), a fusion protein that is constructed of a modified IgG Fc domain and the soluble part of the T-cell receptor CTLA-4. In accelerated degradation studies conducted at 40 degrees C, a pH shift from 7.5 to 6.0 yields significantly faster aggregation kinetics, as measured by size-exclusion chromatography. To understand how the fusion domains and their interactions contribute to this result, we considered aggregation in light of the modified Lumry-Eyring reaction pathway. Protein conformational stabilities against chaotropes and temperature were measured. The structural consequences of these perturbations were observed by a variety of experimental techniques, including differential scanning calorimetry, circular dichroism, and intrinsic fluorescence. Abatacept's colloidal stability was studied by measuring zeta potentials and osmotic second virial coefficients, as well as by modeling electrostatic potentials on the protein's surface. The domains of abatacept exhibit different conformational stabilities that are highly pH dependent, whereas abatacept was weakly colloidally unstable at pH 6 or 7.5. These results are ascribed to conformational instability of the CTLA-4 and C(H)2 domains, which unfold to form a molten globule-like structure that is aggregation-prone. We suggest the instability against aggregation is determined by the least stable domains.