Physical inspection

The solution appearance and colour needed to be assessed throughout the stability study by visual inspection⁴⁹ of samples withdrawn from the containers. Its evaluation was performed in a systematic and standardized way, incorporating more sensitive and specific techniques that complement visual control.

In the present work, the particle size of freshly reconstituted and stored reconstituted preparations (vials and syringes) was measured with a laser diffraction particle size analyser, considering the medium of the samples (water or/and reconstituted solution matrix). For this case, the samples were analysed using a Microtrac^® SRA 150 Laser Diffraction Particle Size Analyser, U.S.A. (measuring range 0.7 µm to 700 µm)⁴⁸. Since Velcade contains 35 mg of mannitol, a blank sample was prepared with mannitol (Merck, M0200000). The reference sample was polystyrene latex beads with a narrow size distribution and a certified particle diameter (EPRUI Biotech Co., Ltd.). The measurement duration was 60 s. Particle size assays were performed immediately after the samples were collected.

When particles ≥ 10 µm were not detected for the analysed samples (freshly reconstituted samples and stable samples), the potential particle growth needed to be monitors, especially if the reconstituted solution was stored in a refrigerator due to the possibility of precipitation of subvisible particles. Then, the histograms obtained by dynamic light scattering⁴⁸ from freshly and stored reconstituted products (vials and syringes) were compared. For this case, the samples were analysed by dynamic light scattering using Microtrac MRB’s NANOTRAC Flex, U.S.A. (measuring range 0.3 nm—10 µm)⁴⁸.

For both Microtrac^® SRA 150 Laser Diffraction Particle Size Analyser and Microtrac MRB’s NANOTRAC Flex, information regarding the limit of detection (the minimum number of particles needed to be present in the solution to be detected) was unavailable; this is a limitation of our study.