ABSTRACT
The need to monitor, measure, and control sub-visible proteinaceous particulates in biopharmaceutical formulations has been emphasized in recent publications and commentaries. Some of these particulates can be highly transparent, fragile, and unstable. In addition, for much of the size range of concern, no practical measurement method with adequate sensitivity and repeatability has been available. A complication in measuring protein particulates in many formulations is the simultaneous presence of other particle types such as silicone micro-droplets, air bubbles, and extrinsic contaminants. The need has therefore been identified for new analytical methods which can accurately measure and characterize sub-visible particulates in formulations. Micro-flow imaging has been shown to provide high sensitivity in detecting and imaging transparent protein particles and a unique capability to independently analyze such populations even when other particle types are present.
Subject(s)
Biological Products/chemistry , Microscopy/methods , Proteins/chemistry , Limit of Detection , Refractometry , Reproducibility of Results , ViscosityABSTRACT
Micro-flow imaging (MFI) 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 (approximately 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 (approximately 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 microm size range. The less opaque proteinaceous particles were relatively more underestimated by the LO method in opalescent solutions.
Subject(s)
Chemistry, Pharmaceutical/methods , Proteins/chemistry , Antibodies, Monoclonal , Diagnostic Imaging/methods , Dosage Forms , Dust , Glass , Light , Physical Phenomena , SolutionsABSTRACT
The U.S. and European Pharmacopeias require subvisible (> or =10 and > or =25 microm) and visible particulate testing of therapeutics to ensure their safety and suitability for clinical use. The objective of this article is to compare the sizing and counting accuracies of light obscuration, which is the standard technique used to measure subvisible particulate matter, and Micro-Flow Imaging (MFI), a new imaging-based technology. An immunoconjugate was selected as the model protein for this study since it could be induced to form particulate matter in PBS. Light obscuration was performed as described in USP chapter <788> while MFI measurements were conducted per the manufacturer's procedures. The two techniques yielded similar results when polystyrene standards were analyzed. However, the MFI measurements indicated the presence of significantly more particles in the protein-containing solution compared to the light obscuration measurements. The presence of nonspherical protein particles as well as particles that possess a refractive index similar to the solvent that they are in appear to be detected by MFI, but not by light obscuration, leading to the difference in the results. Imaging-based technologies could aid in developing formulations and processes that would minimize the formation of protein particulates.
