| Abstrakt: |
Purpose: The presence of visible and sub-visible particles is a critical quality attribute of biotherapeutics that needs to be monitored closely. Backgrounded membrane imaging (BMI) is an alternative technique to determine the size and concentration of particles in liquid formulations. The particles are imaged in a dry state on a membrane, as the liquid phase is removed during the preparation of BMI samples. Therefore, the refractive index difference (ΔRI) of the particle to the background is drastically increased and the detection of translucent particles improved. In this study, we compared BMI to established methods such as microflow imaging (MFI) and light obscuration (LO). Method: We have evaluated several experimental parameters, such as the sample volume and the washing routine, to determine a suitable handling procedure for optimizing the accuracy and reproducibility of the measurements. Particle concentrations of polystyrene and protein samples were compared using BMI, MFI, and LO. Furthermore, the influence of various additives, as well as hard- and software parameters on BMI measurements, was evaluated. Results: BMI is a high-throughput microscopic method that requires a sample volume of only 25 µl to monitor particles as small as 2 µm. This enables particle detection in case of limited sample availability, but the final particle concentrations are calculated by data extrapolation to particle counts in 1 ml. During the verification of the system's performance, we have observed a clustering of the hydrophobic polystyrene standard beads on the hydrophilic membrane. This can be avoided by using negatively charged polystyrene particle standard beads. For stressed monoclonal antibody (mAb) samples, the particle concentrations determined by BMI were about one to two orders of magnitude increased in comparison to MFI and LO. Furthermore, the influence of various additives such as sucrose, polysorbate 20, silicone oil, and glycerol (to mimic high viscous samples) was evaluated. Critical hard- and software parameters such as the camera focus, the particle detection algorithm, and the sample efficiency, which have impaired the measurements, as well as accuracy of BMI measurements were discussed. Conclusion: We have addressed several new aspects to improve the experimental procedure and accuracy of BMI measurements. In general, BMI is an alternative method to determine particle size and concentration in pharmaceutical products, especially if the sample volume is limited. [ABSTRACT FROM AUTHOR] |
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