RESUMO
Background: Natural Killer (NK) cell-based therapies represent a ground-breaking opportunity for the treatment of solid tumors and hematological malignancies. NK cell manufacturing under good manufacturing practice (GMP) is complex and requires attentive assessment the product's safety and efficacy through quality control (QC). Release testing includes monitoring of in vitro cell expansion, differentiation, purity, phenotype, and cytotoxicity. As NK cells are biologically active products, the establishment of potency methods is particularly relevant; surrogate or improper assays can lead to rejection of qualifiable batches or to release of products that falsely meet potency specifications, potentially causing low efficacy during clinical trials. As cell-based therapeutics are highly heterogeneous, no universal guidelines for product characterization are available, and developers must invest significant effort in establishing and validating robust and fit-to-purpose assays. In this study, we describe the qualification procedure of a flow cytometry-based analytical method to assess in vitro potency of GTA002 NK cells, to be applied to oNKord®/inaleucel allogeneic off-the-shelf NK cell product from Glycostem Therapeutics, undergoing a Phase I/IIa clinical trial in acute myeloid leukemia (AML) patients (NCT04632316). Methods: First, we established multi-color flow cytometry panels to quantitatively determine the count of effector (E) GTA002 cells and leukemia target (T) K562 cells alone and in co-culture at different E:T ratios (10:1, 3:1, 1:1). Effector potency was then qualitatively expressed as percentage of cytotoxicity. Next, we defined protocols for method qualification to assess the pivotal features of the assays, including accuracy, precision, linearity, range, specificity, robustness, and carryover; quantitative acceptance criteria were determined for all parameters. Results of the qualification procedure are reported and discussed against pre-defined acceptance criteria. Results: Overall, our methods show robust performance across all parameters, ensuring QC-compliant assessment of NK cell potency as part of the release test panel for clinical batches. Notably, we identified relevant aspects to address when progressing towards method validation to support pivotal clinical studies. Conclusions: This article provides a "case-study" of how analytical method development for cell therapeutics is planned and executed from early clinical stages, anticipating the need to establish robust procedures to overcome scientific and regulatory challenges during method validation.
RESUMO
Trillium govanianum Wall. (Melanthiaceae alt. Trilliaceae), commonly known as 'nag chhatri' or 'teen patra', is a native species of the Himalayas. It is used in various traditional medicines containing both steroids and sex hormones. In folk medicine, the rhizomes of T. govanianum are used to treat boils, dysentery, inflammation, menstrual and sexual disorders, as an antiseptic and in wound healing. With the only exception of the recent report on the isolation of a new steroidal saponin, govanoside A, together with three known steroidal compounds with antifungal property from this plant, there has been no systematic pharmacological and phytochemical work performed on T. govanianum. This paper reports, for the first time, on the cytotoxicity of the methanol extract of the roots of T. govanianum and its solid-phase extraction (SPE) fractions against four human carcinoma cell lines: breast (MCF7), liver (HEPG2), lung (A549) and urinary bladder (EJ138), using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide cytotoxicity assay and liquid chromatography and electrospray ionization quadrupole time-of-flight mass spectrometry analysis of the SPE fractions. The methanol extract and all SPE fractions exhibited considerable levels of cytotoxicity against all cell lines, with the IC50 values ranging between 5 and 16 µg/mL. Like other Trillium species, presence of saponins and sapogenins in the SPE fractions was evident in the liquid chromatography mass spectrometry data. Copyright © 2016 John Wiley & Sons, Ltd.