An off the shelf, GMP compliant,fully closed and semi-automated large-scale production system for allogeneic NK cells

Abstract

Background & Aim Natural killer cells (NK) have been on the mainstay in cancer treatment for over two decades. Most of the clinical trials till date, have either used autologous or allogeneic NK cells that were often produced in limited quantities to tailor the needs of a single patient. With the highly unmet need to treat large cohort of patients and with multiple doses, a true off the shelf approach is the only way. Glycostem began to envision this, by setting up a world's first completely closed manufacturing platform for allogeneic NK cells (oNKord®) from fresh umbilical cord blood stem cells. A closed system is a process where the cells are never exposed to the open environment to avoid a potential risk for contamination. Methods, Results & Conclusion Glycostem manufactures oNKord® at its own state of the art Class C/D GMP clean room, using PVC Sterile Connection Device and Sealer (Terumo), C-Flex Biowelder and Sealer (Sartorius), to connect and seal two tubes aseptically and keeping the system closed. These equipments are key elements in the routine functioning of the closed system, while cells are transferred, or medium is added. In our process, fresh cord blood is used as a starting material, using fully automated CliniMACS Prodigy to select CD34+ cells, which are then expanded using static cultures in incubators and further differentiated into mature NK cells in Xuri bioreactors. After 40 days of culture, NK cells are harvested, washed and aliquoted into cryobags and cryopreserved in controlled rate freezing device. Optimised freezing and thawing conditions, which result in high recovery and product functionality, enabled cell banking of multiple infusion doses produced from one manufacturing batch. Frozen doses are shipped to clinical centres in temperature controlled dry shippers and thawed using waterbath at the time for infusion. The NK cell product is scalable resulting in a 2000 - 3000-fold expansion, averaging up to 11 billion NK cells from a single cord blood unit (n=3). Validated QC testing methods, in process controls using flow cytometry assays and standardised potency assays are in place to address product safety, identity and functionality for release. Setting up of a completely closed system faces many challenges, and during this journey over the last two years, Glycostem has successfully resolved several bottlenecks and worked closely with the regulatory authorities in achieving an “universal off the shelf” NK cell therapy product.