Light obscuration (LO)
Light obscuration (LO) is the compendial technique for the analysis of subvisible particles in parenteral and ophthalmic products.
LO is listed in the pharmacopoeias Ph. Eur. 2.9.19, USP <788>, USP <787> and USP<789>. During measurement, the sample is drawn into the system through a needle and particles passing a laser beam block a certain amount of light and produce a “shadow” on a light-sensitive detector. The area of this shadow is then converted to an equivalent circular diameter of the particle, based on a calibration curve generated with polystyrene standard beads.
LO allows counting and sizing of subvisible particles in the size range from about 1 to 200 µm. The upper particle concentration limit is system dependent and usually about 10^5 and 10^6 particles/mL. The pharmacopoeias give specifications for particles >10 µm (less than 6,000 particles per container for low-volume parenterals of 100 mL or less) and particles >25 µm (less than 600 particles per container for low-volume parenterals of 100 mL or less). However, the quantification of smaller subvisible particles (>1 µm) is nowadays also expected by the regulatory authorities. At Coriolis, we have already many years of experience in using low volume methods for LO analysis of numerous different biopharmaceutical products (see our publication “Pharmaceutical feasibility of sub-visible particle analysis in parenterals with reduced volume light obscuration methods").
The LO technique is used during all stages of development, from early research to batch release testing for the quantification of subvisible particles. At Coriolis, LO can be offered in full GMP compliance.
Main advantages of LO are the high sampling efficiency, as the whole sample volume can be analyzed during the measurement, and the short measurement and data handling times. However, the presence of air bubbles and the formation of Schlieren patterns can result in erroneously high particle levels in LO measurements. But, a well-established sample preparation procedure can avoid these issues. Like all light-based techniques, LO depends on a difference in refractive index between particle and solvent. The validity of LO results should thus be validated by using orthogonal methods.
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S. Zölls, R. Tantipolphan, M. Wiggenhorn, G. Winter, W. Jiskoot, W. Friess, A. Hawe, Particles in therapeutic protein formulations, Part 1: overview of analytical methods., J. Pharm. Sci. 101  914–35. doi:10.1002/jps.23001.
Hawe A, Schaubhut F, Geidobler R, Wiggenhorn M, Friess W, Rast M, de Muynck C, Winter G., Pharmaceutical feasibility of sub-visible particle analysis in parenterals with reduced volume light obscuration methods., Eur J Pharm Biopharm. 2013 Nov