Freeze-Dried Mesenchymal Stem Cell-Secretome Pharmaceuticalization: Optimization of Formulation and Manufacturing Process Robustness

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.

New idea to promote the clinical applications of stem cells or their extracellular vesicles in central nervous system disorders: Combining with intranasal delivery

“…Lyophilization with cryoprotectants could avoid aggregation of EVs and extend the storage time of EVs 179 . Mannitol 180 and trehalose 181 , 182 were effective cryoprotectants for EVs and may enable EVs to adapt to powder IN-delivery devices.…”

Regenerative medicine 2.0: extracellular vesicle–based therapeutics for musculoskeletal tissue regeneration

“…18,40 Rehydrated lyophilized EVs maintained their protein and RNA cargo and biological potency following IV injection into mice. 18 Lyophilized EV products have recently been developed and characterized for possible human, 69,70 canine, 40,42 and equine 71 applications in which EV morphology, surface biomarkers, and biological activity were preserved following rehydration. These studies support the feasibility of developing freeze-dried MSC-derived EV therapeutics that can be stored for long periods of time and shipped without the need for cold chain conditions.…”

Potential of extracellular space for tissue regeneration in dentistry

“…Secretome can be easily harvested, freeze dried and transported. As a cell free approach, secretome therapy does not induce the adverse effects of cell therapies such as tumorgenicity and host rejection ( Song et al, 2019 ; Mocchi et al, 2021 ). An important and biologically active component of the secretome are Extracellular vesicles.…”