title: High concentration formulations of recombinant human interleukin-1 receptor antagonist: II. Aggregation kinetics.
abstract: At high protein concentrations (i.e., 50-100 mg/mL) and 37 degrees C, low solution ionic strength accelerates aggregation of recombinant human interleukin-1 receptor antagonist (rhIL-1ra). We have used a variety of physical and spectroscopic techniques to explain this observation. A population balance model was applied to a continuous mixed suspension, mixed product removal (MSMPR) reactor at steady-state to determine aggregate nucleation and growth rates. Nucleation rates increase at low ionic strength, while growth rates are unaffected. At low rhIL-1ra concentrations (i.e., <1 mg/mL), no conformational changes or differences in free energies of unfolding (DeltaG(unf)) were observed at 37 degrees C over the solution ionic strength range of 0.025-0.184 molal used for aggregation studies. However, increasing the protein concentration to 100 mg/mL shifts the rhIL-1ra monomer-dimer equilibrium significantly at low ionic strength to favor dimerization, which is reflected in subtle conformational changes in the circular dichroism and second-derivative FTIR spectra. In addition to a reversible dimer, an irreversible dimer forms by second-order kinetics during incubation at 37 degrees C. This noncovalent dimer does not significantly participate in further aggregation. The loss of native protein due to aggregation at 37 degrees C was third order in protein thermodynamic activity due to the rate-limiting formation of an aggregation-prone trimer. This trimer forms from irreversible attractive monomer-reversible dimer interactions, which were quantified using second osmotic cross virial coefficients. Lastly, the activity coefficient of rhIL-1ra estimated from aggregation rates is 50% higher at 100 mg/mL protein concentration than at 50 mg/mL, in close agreement with predictions from a hard-sphere model for activity coefficients.
authors.affiliation: Department of Chemical and Biological Engineering, University of Colorado, Boulder, Colorado 80309, USA.
authors.forename: John R
authors.initials: JR
authors.lastname: Alford
authors.forename: Brent S
authors.initials: BS
authors.lastname: Kendrick
authors.forename: John F
authors.initials: JF
authors.lastname: Carpenter
authors.forename: Theodore W
authors.initials: TW
authors.lastname: Randolph
mesh_terms: D002626:Chemistry, Pharmaceutical; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D003102:Colloids; D004591:Electrophoresis, Polyacrylamide Gel; D053590:Interleukin 1 Receptor Antagonist Protein / Q000737:chemistry*; D007700:Kinetics; D009994:Osmolar Concentration; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D013057:Spectrum Analysis / Q000379:methods; D013816:Thermodynamics
chemical_list: D003102:Colloids; D053590:Interleukin 1 Receptor Antagonist Protein; D011994:Recombinant Proteins