Purpose: USP <788> specifies the microscopic particle count test for the determination of subvisible particulate matter in parenteral products, in addition to light obscuration. For this test, the product is filtered through a membrane, and particles remaining on the filter surface are manually counted and sized under a microscope. Backgrounded Membrane Imaging (BMI) is a more automated, 96-well plate-based approach of this principle combined with automated image analysis.
The scope of our study was to critically evaluate BMI regarding its applicability for subvisible particle characterization during pharmaceutical development of biologics.
Methods: To assess the sizing accuracy of BMI, measurements of polystyrene standard particles (5, 10, 30 µm in diameter) were performed and compared to results obtained from Micro-Flow Imaging (MFI). As standard particles exhibited a marked tendency for agglomeration on the BMI membrane, linear concentration range of the technique was determined with the help of particles generated from a monoclonal antibody (mAb) by stir-stress. For the evaluation of the applicability of BMI in the characterization of biopharmaceuticals, comparative measurements of therapeutic protein products (two mAb products and one small protein) were performed with BMI, MFI, and imaging flow cytometry. The resulting concentrations and size distributions of particles of ECD above 2 µm were compared. To address the selectivity of BMI, measurements of varying concentrations of silicone oil (SO) spiked into mAb formulated without polysorbate and formulation buffer were performed.
BMI was found to show a trend towards an undersizing of polystyrene beads with a lower repeatability when compared to MFI. The upper limit of the linear concentration range of BMI was determined as 16,000 obj/well corresponding to particle concentrations between 1.3∙105 ml-1 and 8.0∙105 ml-1 for applied sample volumes from 125 µl down to 25 µl. Thus BMI linear concentration range lies in the same range as for MFI. Comparative measurements of unstressed therapeutic drug product from vials or pre-formulated bulk revealed a high similarity of particle concentrations and size distributions detected by BMI and MFI in the size range above 2 µm. BMI sizing exhibited a slight shift toward smaller particle diameters when compared to MFI, which may be attributed to the fragmentation of particles during image processing. In comparison to imaging flow cytometry, BMI detected lowe particle concentrations in the respective drug products. In contrast to the results obtained from the unstressed product in vial or unstressed mAb (without polyosrbate), particle levels detected in stressed mAb (without polysorbate) were lower for BMI than for MFI. In unstressed products from pre-filled syringes, particle concentrations (ECD≥2 µm) obtained from BMI were as well clearly lower than the concentrations retrieved from MFI. This was in agreement with a separation of silicone oil from protein in the BMI filtration step as indicated by the observation that spiking of mAb (without polysorbate) or formulation buffer with SO droplets did not lead to an increased detection of particles by BMI.
Our evaluation of BMI shows that overall, this technique yields similar results as MFI for the determination of subvisible particles of protein in unstressed products or in products from long-term storage. In conclusion, BMI appears to be a promising method for the high-throughput analysis of subvisible (protein) particles in biopharmaceutical formulations. Accordingly, BMI constitutes a useful tool for the screening and relative ranking of formulations, and as the technique only requires small sample volumes (approximately 25 µl to 125 µl), it is particularly suitable when only limited samples volumes are available.
Constanze Helbig– Coriolis Pharma Research GmbH
Gregor Ammann– Coriolis Pharma Research GmbH
Wolfgang Friess– Ludwig Maximilians University, Munich, Bayern
Klaus Wuchner– Janssen Research & Development, Pharmaceutical Development & Manufacturing Sciences