High concentration formulations of recombinant human interleukin-1 receptor antagonist: II. aggregation kinetics

✍ Scribed by John R. Alford; Brent S. Kendrick; John F. Carpenter; Theodore W. Randolph

• Publisher: John Wiley and Sons
• Year: 2008
• Tongue: English
• Weight: 309 KB
• Volume: 97
• Category: Article
• ISSN: 0022-3549
• DOI: 10.1002/jps.21205

✦ Synopsis

At high protein concentrations (i.e., 50-100 mg/mL) and 378C, 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 (DG unf ) were observed at 378C 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 378C. This noncovalent dimer does not significantly participate in further aggregation. The loss of native protein due to aggregation at 378C 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.