Abstract
Producing mesenchymal stem cell (MSC)-secretome for dose escalation studies and clinical practice requires scalable and good manufacturing practice (GMP)-compliant production procedures and formulation into a standardized medicinal product. Starting from a method that combines ultrafiltration and freeze-drying to transform MSC-secretome into a pharmaceutical product, the lyosecretome, this work aims to: (i) optimize the lyosecretome formulation; (ii) investigate sources of variability that can affect the robustness of the manufacturing process; (iii) modify the ultrafiltration step to obtain a more standardized final product. Design of experiments and principal component analysis of the data were used to study the influence of batch production, lyophilization, mannitol (M)/sucrose (S) binary mixture, selected as cryoprotectant excipients, and the total amount of excipients on the extracellular vesicles (EV) particle size, the protein and lipid content and the in vitro anti-elastase. The different excipients ratios did not affect residual moisture or EV particle size; simultaneously, proteins and lipids were better preserved in the freeze-dried product using the maximum total concentration of excipients (1.5% w/v) with a M:S ratio of about 60% w/w. The anti-elastase activity was instead better preserved using 0.5% w/w of M as excipient. The secretome batch showed to be the primary source of variability; therefore, the manufacturing process has been modified and then validated: the final product is now concentrated to reach a specific protein (and lipid) concentration instead of cell equivalent concentration. The new standardization approach led to a final product with more reproducible quali-quantitative composition and higher biological activity.
Highlights
In the 1990s, the multipotency of mesenchymal stem cells (MSCs) was identified as a promise for developing new and more effective cell therapies intended to revolutionize the clinical practice in regenerative medicine and improve the patient quality of life [1,2]
Many challenges still need to be faced in the transition of MSC-secretome therapies into the clinic
Three primary defects were noted in cake appearance evaluation: collapse, cake shrinkage and cracked cake (Figure S1)
Summary
In the 1990s, the multipotency of mesenchymal stem cells (MSCs) was identified as a promise for developing new and more effective cell therapies intended to revolutionize the clinical practice in regenerative medicine and improve the patient quality of life [1,2]. MSC-secretome can reproduce the therapeutic effects of stem cells themselves and cell-free therapies should provide numerous advantages compared with whole-cell MSC infusions in terms of safety and technological advantages [9]. It remains a significant challenge translating this therapy into the clinic: with the focus on therapeutic applications, conventional manufacturing processes (such as ultracentrifugation or chromatography) limit secretome applications and the ability to evaluate safety and efficacy at high doses on a large animal or clinical trials [10,11]. Producing MSC-secretome for dose escalation studies and clinical practice requires a scalable production procedure, including raw materials or consumables, compatible with current good manufacturing practice (GMP) procedures [12]
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