Human dialyzable leukocyte extracts (DLEs) are heterogeneous mixtures of low-molecular-weight peptides that modulate immune responses in various diseases. Due their complexity, standardized methods to identify their physicochemical properties and determine that production batches are biologically active must be established. We aimed to develop and validate a size exclusion ultra performance chromatographic (SE-UPLC) method to characterize Transferon™, a DLE that is produced under good manufacturing practices (GMPs). We analyzed an internal human DLE standard and 10 representative batches of Transferon™, all of which had a chromatographic profile characterized by 8 main peaks and a molecular weight range between 17.0 and 0.2kDa. There was high homogeneity between batches with regard to retention times and area percentages, varying by less than 0.2% and 30%, respectively, and the control chart was within 3 standard deviations. To analyze the biological activity of the batches, we studied the ability of Transferon™ to stimulate IFN-γ production in vitro. Transferon™ consistently induced IFN-γ production in Jurkat cells, demonstrating that this method can be included as a quality control step in releasing Transferon™ batches. Because all analyzed batches complied with the quality attributes that were evaluated, we conclude that the DLE Transferon™ is produced with high homogeneity.
Keywords: BCA; DLE; DTH; Da; Dialyzable leukocyte extracts; FDA; Food and Drug Administration; GMPs; Glu; Gly; HPLC; ICH; IFN-gamma; IFN-γ; IL-6; International Conference of Harmonization; NF-κB; Quality specifications; RP; RSD; SD; SE; Ser; TNF-α; Transferon; UPLC; bicinconinic acid; cAMP; cyclic adenosine monophosphate; daltons; delayed-type hypersensitivity; dialyzable leukocyte extract; glutamine; glycine; good manufacturing practices; high-performance liquid chromatography; interferon gamma; interleukin 6; nuclear factor kappa B; relative standard deviation; reverse-phase; serine; size exclusion; standard deviation; tumor necrosis factor alpha; ultra-performance liquid chromatography.
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