Biologics and especially monoclonal antibodies (mAbs) represent the fastest growing segment in the pharmaceutical industry. Their therapeutic uses range from cancer to asthma and to chronic inflammatory diseases like rheumatoid arthritis. Often, mAbs need to be administered to patients in doses of several mg/kg. For diseases that are treated in clinical settings, the intravenous administration of more than 100 mg of a protein-based drug does not pose serious concentration problems. However, in chronic diseases and other conditions treated outside clinical settings, application of mAbs often requires self-administration of highly concentrated protein solutions, as the total injection volume for subcutaneous administration cannot exceed ~1.5 mL. Protein concentrations as high as 100 mg/mL or more are not unusual. Three immediate issues are associated with high protein concentration solutions: the structural stability of the protein, the tendency to aggregate and the viscosity of the solution. These biologics need to be formulated under conditions that ensure structural stability, lack of aggregation and adequate viscosity at high protein concentrations. 1,2
The optimization of formulation conditions for highly concentrated biologics necessitates stability evaluation at the required protein concentrations for administration (~100 mg/mL). Traditional techniques either lack the dynamic range to measure protein stability over a wide range of concentrations, or the physical perturbation by itself induces protein aggregation. Temperature denaturation, either differential scanning fluorimetry (DSF) or differential scanning calorimetry (DSC), has been the preferred way of evaluating protein stability in the biopharmaceutical industry. Most of the time, temperature denaturation of mAbs and other biologics is irreversible and is coupled with aggregation, conditions that are aggravated at high concentration. An attractive alternative is the measurement of protein stability at low temperature using the well-known approach of chemical denaturation.
Until recently, no automated instrumentation for chemical denaturation analysis was available, a situation that has changed with the recent introduction of the AVIA 2304, which is capable of automatically preparing formulation solutions and measuring up to 2304 chemical denaturation data points.3,4 The stability of proteins is determined through chemical denaturation by measuring the entire fluorescence spectrum (intrinsic tryptophan fluorescence or extrinsic fluorescence probes) as a function of the concentration of denaturant (urea or guanidinium chloride). The resulting data is analyzed in terms of well established two-state or multi-state thermodynamic models in order to get denaturant transition midpoint concentrations, m values and Gibbs energies related to stability.
As opposed to temperature denaturation, chemical denaturation induced by urea or guanidinium chloride is reversible for most proteins—including monoclonal antibodies—allowing for the determination of highly accurate and reliable protein stability data.3 Figure 1 shows the unfolding and refolding of an IgG monoclonal antibody using urea. Thermodynamic stability parameters (ΔG, m and C1/2 values) obtained in the denaturation and refolding experiments are identical within the experimental error. The experiment was performed at a protein concentration of 0.36 µg/mL.
Perhaps one of the key features of chemical denaturation is that the transition remains reversible even at concentrations similar or higher to those required for subcutaneous administration. Reversibility is necessary to rigorously measure the thermodynamics of protein stability. In addition, the dynamic range of the fluorescence signal can be modulated by the gain of the detector and/or slit width, which allows for measurements spanning over five orders of magnitude in concentration. Having access to protein stability data over a wide range of concentrations provides a way to identify concentration dependent trends that can otherwise be overlooked.
Figure 2 shows the chemical denaturation of trypsinogen measured at concentrations of 0.9, 9 and 90 mg/mL by the AVIA 2304 instrument. These experiments demonstrate the validity of using chemical denaturation in general and with this new technology in particular to evaluate protein stability at concentrations in the 100mg/mL range. For trypsinogen, it seems apparent that the stability shows very little variation with concentration under the solvent conditions studied. The three curves are very close to each other with a variation in C1/2 lower than 0.1M.
The new Model 2304 Automated Protein Denaturation System completely automates sample preparation, data collection, data analysis and stability report-generation for protein stability determinations using chemical denaturation and can evaluate up to 96 different formulations, process conditions or individual protein constructs.
- Connolly BD, Petry C, Yadav S, et al. Weak interactions govern the viscosity of concentrated antibody solutions: high-throughput analysis using the diffusion interaction parameter. Biophys J. 2012;103(1):69-78.
- Freire E, Schön A, Hutchins BM, Brown RK. Chemical denaturation as a tool in the formulation optimization of biologics. Drug Discov Today. 2013;18(19-20):1007-13.
- Schön A, Brown RK, Hutchins BM, Freire E. Ligand binding analysis and screening by chemical denaturation shift. Anal Biochem. 2013;443(1):52-7.
- Liu J, Nguyen MD, Andya JD, Shire SJ. Reversible self-association increases the viscosity of a concentrated monoclonal antibody in aqueous solution. J Pharm Sci. 2005;94(9):1928-40.
Ernesto Freire is the Henry Walters Professor of Biology at The Johns Hopkins University in Baltimore, MD and co-founder of AVIA Biosystems. Dr. Freire’s laboratory conducts research on protein stability, molecular recognition, and drug design.
Richard Brown is president and co-founder of AVIA Biosystems. Dr. Brown was formerly president of MicroCal.
Arne Schön is a research scientist at The Johns Hopkins University.