Backgrounded Membrane Microscopy, a novel technique for the determination of contaminating particles in cell therapy products.

Background & Aim

Distinguishing between different subvisible particle (SVP) species within a cell therapy product has proven challenging using standard techniques such as flow cytometry. This is in part due to the cell therapy product itself contributing to the subvisible particle population. This has been exemplified by the recent FDA issuance of a Form 483 concerning particle contamination in Kymriah® with wood, cellulose, brass, and steel, attributed to the cryopreservation bags. This has highlighted a need for a new approach in the determination of sub-visible particles within cell therapies.

Methodology

The combination of Backgrounded Membrane Imaging (BMI) and Side Illumination Membrane Imaging (SIMI), a form of darkfield microscopy, provides unique and definitive insights into the nature of different particle species. The presence of biological and non-biological (plastic, wood, metal) particles can be identified and counted using this combination of imaging approaches. Non-biological materials exhibit a strong SIMI signature when compared to biological matter, due in part to the relative refractive index and rigidity when examined on a filter membrane.

Results

Here in, we demonstrate the utility of the combined imaging modes for the identification of different non-biological materials mixed with CAR T cells at two loaded volumes 40uL and 400uL using a 96-well plate and 24-well white membrane plate, respectively.

The specific determination of biological matter, be it protein, DNA or lipid, is facile when stained with appropriate fluorescent dyes on a 96-well black membrane plate. The specific determination of biological matter, be it protein, DNA or lipid, is facile when stained with appropriate fluorescent dyes on black membrane plates.

Conclusion

This approach provides additional utility for the identification of protein aggregates, cell clumps, cell-bound microspheres, viable cell populations within a rapid high-throughput assay.