Graduate project

Validation of Leukopaks for Manufacturing and Quality Control Testing

Thermo Fisher Scientific was established with the goal to enable customers to make the world healthier, cleaner, and safer by providing high quality scientific products and laboratory services. The global team of 75,000 employees ensure that researchers have the capacity to accomplish scientific aims and create an environment of discovery and innovation. With the emergence of the SARS-CoV-2 global pandemic, San Diego County has witnessed a decline in blood donations and patient availability, this situation has a direct effect on Thermo Fisher product development and the verification processes needed for research reagents for flow cytometry. Whole blood is required for the testing of human antigens for Thermo Fisher antibodies and with San Diego having a blood supply shortage, alternative blood sources are needed to keep pace with testing demands. The purpose of this project is to validate the use of leukopak-derived peripheral blood mononuclear cells (PBMC) as an alternative or replacement for PBMC derived from whole blood. The leukopak validation plan was accomplished through flow cytometry experimentation and addresses the criteria of yield, viability, surface staining, intracellular staining, and nuclear staining. The plan utilizes a range of different reagent properties and antigen targets in order to achieve the ultimate goal of a comprehensive comparison to the whole blood standard. Leukopaks have become the starting material of choice for cell therapy research and drug development and are expected to meet all passing criteria. The cellular profile of the leukopaks was expected to mirror that of whole blood given that they are the same mononuclear cells present in whole blood but have been isolated by apheresis. Analysis was conducted through the usage of Median Fluorescence Intensities (MFI) to generate a robust quantitative statistical measurement to test the different donor parameters. The results showed that leukopak yield was greater than that of whole blood leading to a decrease in cost per mL of tissue from $0.94 for whole blood to $0.88 for the HemaCare leukopak and $0.83 for the San Diego Blood Bank leukopak. Leukopak viability was as robust as whole blood leading to the conclusion that the leukapheresis process and transportation of the tissue did not compromise cell integrity. No aberrant results were observed for the surface staining of non-stimulated mononuclear cells but discrepancies in staining patterns and percentages of intracellular and nuclear antigens were observed. This variation was attributed to current stimulation protocols not being tailored to the differences in mononuclear cell concentrations in the leukopaks versus the PBMCs from whole blood. Further refinement of protocols surrounding isolation of mononuclear cells from the leukopaks will need to be addressed; high yield was observed but the cells were not as concentrated as expected. The Thermo Fisher established optimal concentrations of the monoclonal antibodies were identical across the different PBMC donors. The LogMFI values of those from two different lots from the various donors were within +/- 5% of each other meeting manufacturing specifications. Although not all passing criteria for nuclear and intracellular antigens were met at this current time, the leukopaks were approved for surface antigens that do not require mononuclear cell stimulation. The availability of an alternative source of PBMC will no doubt enable the R&D and QC departments to have little to no disruptions in flow cytometry reagent releases and decrease both the time of mononuclear cell isolation and sample costs dramatically.