Antibody therapeutics frequently require large doses which typically must be administered intravenously, requiring the patient to visit a healthcare professional. Subcutaneous injections, which could be self-administered, are preferred but require high concentrations of protein, often greater than 50 mg/mL. Because most biophysical techniques have been optimized for solutions with low concentrations of protein, there are relatively few techniques available to characterizing proteins at high concentrations. This study uses a combination of static light scattering (SLS), membrane osmometry and FTIR spectroscopy to analyze colloidal and structural non-idealities at high protein concentrations. In addition, isothermal titration calorimetry is used to quantify protein-excipient interactions.
The results have illuminated several unusual differences in the aggregation behaviors of the two mAbs. The less soluble of the two proteins exhibits unfolding intermediates during denaturant unfolding experiments as detected by intrinsic fluorescence and ANS binding studies, a reduced colloidal stability observed by SLS, a reversible association in solution and a reversible precipitation event similar to ‘salting out' observed with FTIR, SLS, and size exclusion chromatography. Furthermore all of these events are buffer type and salt concentration dependent. Addition of sodium chloride reduces the solution viscosity and increases the second osmotic virial coefficient at the protein's pI.