Quantification and Characterization of Subvisible Proteinaceous Particles in Opalescent mAb Formulations Using Micro-Flow Imaging

Micro-flow imaging (MFI (TM)) has been shown to be more sensitive than light obscuration (LO) methods for measuring subvisible proteinaceous particles in protein formulations. Given the potential challenges in detecting particulates in opalescent mAb formulations, the accuracy of MFI to size and count particles in opalescent solutions was investigated and compared to LO and membrane microscopy methods. Proteinaceous monoclonal antibody (mAb) particles, generated either by chemical denaturation or agitation stress, polystyrene and glass particles were used as model systems for measurements in opalescent mAb solutions. The sizing and counting accuracies of MFI were unaffected by the opalescence of the medium. Using glass particles as a model system for proteinaceous particles, MFI was able to detect relatively low particle concentrations (similar to 10/mL) in opalescent solutions. MFI showed excellent linearity (R-2 = 0.9969) for quantifying proteinaceous particles in opalescent solutions over a wide range of particle concentrations (similar to 20-160,000/mL). Analyses of MFI particle image intensities revealed significant differences in the transparency of proteinaceous particles as a function of their size and mode of generation. LO method significantly underestimated proteinaceous particles, particularly those in the 2-10 mu m size range. The less opaque proteinaceous particles were relatively more underestimated by the LO method in opalescent solutions. (C) 2010 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:2628-2642 2010