3.3.1 Light obscuration
Light obscuration is a compendial method for the quantification of subvisible particles within parenteral solutions. Depending on the system, size and number of particles between 1 µm and 600 µm can be quantified. A large sample volume of 25 mL is required by both Ph.Eur.11 and USP13 for the analysis of low volume parenterals (volume smaller than 100 mL), which is often not feasible in the case of therapeutic protein products.21 Approaches to reduce the volume for light obscuration measurements of pharmaceutical products have been made to overcome this drawback.60,61 Small volumes may come along with increased data variability,7 but allow at the same time the detection of vial-to-vial variations which are missed if the vials are pooled to obtain a larger measurement volume.
The maximum particle counts are defined in the Ph.Eur.11 and the USP13 as follows: For a total volume of 100 mL or less, the maximum particle count is specified as 6000 particles ≥ 10 µm and 600 particles ≥ 25 µm, each per container. For a total volume larger than 100 mL, the maximum particle count is 25 particles ≥ 10 µm and 3 particles ≥ 25 µm, each per mL. The discussion on the significance of these numbers for therapeutic protein formulations is ongoing.7-9,61 The USP is in the process of developing a biologics-specific chapter for particle analysis in the µm range, which will include appropriate sample handling and analysis of small volumes, and is also going to develop an
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instructional chapter discussing some of the other technologies for this size range.
In light obscuration, particles passing a laser beam block a certain amount of light proportional to their cross-sectional area, which is recorded by a photo diode detector. Light obscuration instruments are typically calibrated with polystyrene standards and based on this calibration the equivalent circular diameter (ECD) of the analyzed particles is obtained. However, for the interpretation of the results it has to be considered that the physico-chemical properties of protein particles, with respect to shape, transparency, and refractive index, are highly different from standard beads.29,62 Therefore, there is a need for standard particles that better represent the properties of protein particles.9,27,62 The simple measurement principle is certainly an advantage of light obscuration methods leading to straightforward and fast measurements.
Nevertheless, this simplicity comes along with some restrictions: the particles have to pass the laser beam individually to avoid overloading and coincidence, i.e. two particles being detected as one larger particle. Therefore, the particle concentration must not exceed a certain limit depending on the system. The following light obscuration systems are mainly used for the analysis of protein products: HIAC HRLD by Hach® (Loveland, CO)32,62,63 with a linear range up to 18,000 particles per mL, SVSS by PAMAS GmbH (Rutesheim, Germany)64-66 with a linear range up to 200,000 particles per mL and AccuSizer 780 by Particle Sizing Systems (Port Richey, FL)67 for particle concentrations up to 15,000 particles per mL. Further available systems are APSS2000/LiQuilaz® by Particle Measuring Systems (Boulder, CO) and Syringe® by Klotz GmbH (Bad Liebenzell, Germany).
Light obscuration cannot differentiate between proteinaceous particles and particles of other origin. Moreover, the technique is sensitive to air bubbles, which could be introduced during sample preparation or analysis. On this account, sample preparation, e.g. reconstitution of lyophilized products and handling of highly concentrated solutions of high viscosity, can have great influence on the result.20 Therefore, degassing of the sample is often performed prior to measurement, however, this procedure can also change sample properties.61 Furthermore, translucent protein particles could be underestimated
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in number and size as more light passes through such particles as compared to the polystyrene standards used for calibration.27,49 Similar to flow imaging microscopy, analysis of highly-concentrated protein solutions or formulations containing high concentrations of excipients such as sugars can be challenging due to low differences in refractive index between particle and solvent; thus, particle number and/or size could be underestimated.32 Despite these restrictions, light obscuration has been routinely used for lot release and has enabled the manufacturing and release of drugs that are safe and efficacious.9 It is also regularly used for the monitoring of subvisible particle counts in therapeutic protein formulations to compare various formulations or stress conditions.63,64,66,68
3.3.2 Nephelometry / turbidimetry
Nephelometry and turbidimetry are both light scattering-based methods that are listed in the Ph.Eur.69 and in the USP.70 Nephelometry is defined as the measurement of light scattered by the sample solution compared to a formazin reference suspension. The scattered light is measured in a nephelometer at a high wavelength, typically 850 or 860 nm, at a scattering angle of 90°. In contrast, turbidimetry is defined as the measurement of light transmitted through the sample solution compared to a formazin reference suspension. The transmitted light can be measured in a UV spectrophotometer at a wavelength where proteins do not absorb light, i.e. in the range of 320-800 nm. Ratio turbidimetry measures both light scattering and light transmission and thereby determines the ratio of scattered light to transmitted light typically at 860 nm.
Ratio turbidimetry is recommended by the Pharmacopeias for colored solutions as it compensates for the reduction of the transmitted light by absorption.
These measurements are simple and useful for a non-specific comparison of samples as limited sample preparation is required and the methods are non-destructive. The results are given in various synonymous units, e.g. NTU (nephelometric turbidity units), FNU (formazine nephelometric units) or FTU (formazine turbidity units). Although nephelometry and turbidimetry do not provide information about size, concentration or nature of protein aggregates or particles, the methods are often used to detect relative changes in the aggregate status.33,36,68 However, high turbidity values can also originate from other factors
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such as high protein concentration and do not necessarily reflect the presence of aggregates or particles.71