Morphologically-Directed Raman Spectroscopy used for the characterization of protein aggregates in suspension

Introduction

Optical microscopy has long been used to characterize particulates present in biotherapeutic formulations, providing their size, shape, and transparency characteristics, which can then be used to group particulates into distinct classes (i.e. aggregates, silicone oil, sundry contaminants).  However, the ability of a microscope to provide explicit identification is limited to the two-dimensional images it collects.  The addition of Raman spectroscopy to an automated microscopy system provides a robust primary identification method for the verification of particle chemistry, and together the techniques offer the potential to enumerate, characterize, and identify particulates in native formulations, as well as those immobilized on a filter substrate.  This range of capabilities directly addresses FDA quality requirements for drugs administered by injection, in terms of particulate count per unit volume.  These regulations are put into practice through USP <788>¹, which calls for enumeration of particulates in two size categories: larger than 10 μm and larger than 25 μm.  The more recent release of USP <787>² (and <1787>) has extended recommendations to include sizes below 10 μm.  This lower size range has drawn considerable attention from the FDA due to concerns about immunogenic response related to proteinaceous aggregates³, reduced product efficacy, and general safety.  Formulation stability can be affected by many factors that lead to the formation of aggregated material^4,5.