To diagnose patients suffering from blood clotting disorders latex immununoassays (LIA) can be used. A time consuming manual tangential flow filtration (TFF) process suggests the implementation of an automatic TFF system to improve the efficiency, profitability, and expandability of the production facility of LIA at Nordic Biomarker. Tests were made of the automatic TFF system's ability to perform the desired steps of concentration, dilution and diafiltration, both with purified water and mimicked product. The mimicked product of micro particles (MP) mixed with monoclonal antibodies (mAb) was also used to further test the system's pressure control, safety alarms and stops, and to determine a permeate flux by a critical flux experiment. The results imply a functional TFF system able to automatically concentrate the process fluid and maintain a stable volume during diafiltration, although an additional permeate pump was ordered to be able to attain a fully functional performance of the automatic TFF process. The final part of the implementation was to initiate a validation draft including a risk assessment, OQ plan and PQ plan that resulted in a plan of the main tests to be performed. To conclude, the essential part of the implementation of a high quality and efficient automatic TFF process was conducted to facilitate future expansion of the production of LIA.

Background: The focus of this study was to investigate how producing human bone marrow (hBM) derived mesenchymal stromal cell (MSC) extracellular vehicles (EVs) in a high purity isolation system would affect their established characterization criteria and address the validity of these methods of EV production. Additionally, we set out to functionally characterize the hBM-MSC-EVs for their identified immunomodulatory ability while also assessing the presence of novel MSC-EV marker NRP1 identified by our group to further affirm its validity as a functional MSC-EV identity marker. Methods: Each hBM-MSC-EV donor was cultured in a hollow-fiber bioreactor system in non-stimulating serum/xeno-free conditions for 25 days to produce EVs persistently under quiescent conditions to characterize the hBM-MSC-EVs in their native form. EVs were isolated by traditional PEG-based precipitation for preliminary characterization to monitor bioreactor production wherein they were characterized using multimodal tangential flow filtration coupled with fast protein liquid chromatograph (FPLC) size exclusion/high-affinity purifications to obtain the final highly purified EV sample. Additionally, functional analysis of their immunomodulatory ability, EVs and MSCs were incubated with activated peripheral blood mononuclear cells (PBMCs) as an in-vitro model to evaluate their potency. Results: The hBM-MSC-EVs produced from the bioreactor system showed consistent characterization in accordance with the MISEV2018 establish criteria. We were also able to demonstrate their functional ability by observing statistically significantly immunomodulatory ability of activated PBMCs equivalent to native MSC ability. We were also able to validate the present of NRP1 on all hBM-MSC-EV samples produced confirming its validity as a MSC-EV marker. Conclusion: The significance of the results obtained from this study confirms the production of MSC-EV using a bioreactor and high purity isolation for obtaining consistent MSC-EVs for downstream investigation. Additionally, we were able to demonstrate the significance of MSC-EVs on MSC signaling for immunomodulation by showing equivalent functional potency when tested in-vitro. These results contribute to further understanding the biological attributes of MSC-EVs and contribute to the validation of currently established characterization guidelines.

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