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 material4,5.The Morphologi G3-ID, a hybrid optical microscope-Raman spectrometer from Malvern Instruments, provides the imaging power of a high-resolution optical microscope with the chemical identification capabilities of a benchtop Raman spectrometer, and is completely automatable. This application note explores the use of the Morphologi G3-ID for Morphologically-Directed Raman spectroscopy (MDRS) to identify and analyze contaminants including protein aggregates present in a stressed sample of lysozyme, and to compare particles held in suspension within a thin path wet cell with those collected on a filter membrane.
Materials and Methods
A Morphologi G3-ID from Malvern Instruments was used to locate particulates for morphological characterization (size, shape, and transparency), and identify the species chemically using Raman spectroscopy. The Morphologi G3-ID was run with a magnification of 10x, providing an effective lower size limit of 2 μm for particle count. The practical upper size limit at this magnification is around 200 μm, although this can be extended significantly with the included particle stitching routine, which is capable of “reassembling” the images of particulates that span more than one field of view. Raman spectra were collected using a 3 μm spot at 50x magnification of 785 nm excitation (~20 mW) from 150 cm-1 to 1850 cm-1 at 6 cm-1 resolution. Two separate 100 mg/mL, pH 7.1 solutions of lysozyme were stressed to produce aggregates: one was used for the filter-based analysis and the other for the suspension-phase analysis. Membrane Filter One of the lysozyme preparations was stirred rapidly at room temperature for 16 hours. Aliquots (1 mL) of this aggregated suspension, spiked with a small amount of polystyrene latex spheres as a reference species, were vacuum filtered using a 47 mm cellulose-based Millipore GSWP filter. The filter was taped to a glass slide and allowed to dry before examination. A circular scan area of 15 mm radius was scanned at 10x magnification in order to characterize particulates and record their locations. The particulates were then divided into groups according to their size and morphology, and a subset of each group was chosen for Raman spectral analysis. Suspended Sample The second lysozyme preparation was stirred at 45°C for four hours and then analyzed in suspension-phase. An aliquot (55 μL) of suspension was delivered to a proprietary thin-path wet cell (25 μm path length) with quartz windows. The cell was allowed to reach thermal equilibrium for 15 minutes before examination. A 20 mm radius scan area was investigated, corresponding to 31.4 μL in volume.Results
The size groups defined for particle counting included 2 μm to 10 μm (small), 10 μm to 25 μm (medium), and greater than 25 μm (large), with the addition of a specific group for the 40 μm polystyrene latex spheres. A selection of images from each group of particles detected on the membrane filter sample is shown below. >> Download the full Application Note as PDFMalvern provides the materials and biophysical characterization technology and expertise that enables scientists and engineers to investigate, understand and control the properties of dispersed systems. These systems range from proteins and polymers in solution, particle and nanoparticle suspensions and emulsions, through to sprays and aerosols, industrial bulk powders and high concentration slurries. Used at all stages of research, development and manufacturing, Malvern’s instruments provide critical information that helps accelerate research and product development, enhance and maintain product quality and optimize process efficiency. Our products reflect Malvern’s drive to exploit the latest technological innovations. They are used by both industry and academia, in sectors ranging from pharmaceuticals and biopharmaceuticals to bulk chemicals, cement, plastics and polymers, energy and the environment. Malvern systems are used to measure particle size, particle shape, zeta potential, protein charge, molecular weight, mass, size and conformation, rheological properties and for chemical identification, advancing the understanding of dispersed systems across many different industries and applications. www.malvern.com Material relationships http://www.malvern.com/en/ portal@malvern.com
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