Characterization of pEVs purified by LEAP
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STERILE, STERILITY
Platelet releasate from activated platelet packs was processed through a chromatography‐based EV isolation protocol, LEAP, to produce purified pEVs (Law et al., The product passed tests for sterility and for endotoxin (data not shown). Protein content was measured by BCA protein assay, and 1.41 ± 0.34 mg total protein was derived from each pooled platelet pack. Each vial of drug product contained 0.3 ± 0.1 mg/mL of protein and a total volume of 0.5 mL. Placebo vials were produced containing sterile filtered formulation buffer. Particle analysis was performed by Spectradyne and the total number of pEVs isolated from each pack was determined to be 8.71 ± 2.75 × 10Characterization of LEAP‐purified pEVs. (a) Size distribution of clinical pEVs measured by Spectradyne, detecting pEVs from 65 to 400 nm. (b) Percentage of pEVs in clinical pEVs present within the size ranges of 65–100, 100–200, and 200–400 nm. (c) Cryo‐TEM images (left zoomed out, right zoomed in) of clinical pEVs, confirming the size distribution and innate morphology of the pEVs. Scale bar: 500 nm (left), 200 nm (right). (d) Western blot analysis of clinical pEVs showed enrichment of known EV markers CD9, CD63, and syntenin relative to platelet lysate and showed the absence of the negative EV marker, calnexin.Size distribution and consistent morphology were confirmed by cryo‐TEM (Figure 