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Use of the Cyto-Mine for Rapid Generation of Clonal Cell Lines
Janssen R&D, Inc.
Rapid Generation of High Titre Stable Producer Cell Lines for Lentiviral Vector Manufacture
Assuring and Proving Monoclonality
It is a regulatory requirement (ICH Q5D) that cells used for the production of therapeutic antibodies have to be clonal, i.e. derived from a single cell progenitor. The FDA has recently provided guidance on acceptable procedures for the generation of clonal cell populations (e.g. two subsequent rounds of limiting dilution). In case cell lines were generated using a process not fully compliant with the procedures suggested by the FDA, additional data need to be generated. Glenmark has developed a method using the statistical analysis of presence and absence of specific genetic features in a population of sub-clones to assess the monoclonality status of a cell population. The method can be applied to existing cell lines with unknown monoclonality status and may allow to avoid changing the production clone or adapting the control strategy (if no secondary population can be detected).
The Cyto-Mine is a microfluidic instrument designed for encapsulating single-cells in picodroplets and FRET-based productivity screening in a single-use cartridge. We have tested this instrument and method with mAb and non-mAb projects and found it can generate cell lines with high productivity, high viability, and a high assurance of clonality after a single round of screening, all in shorter timelines.
Along with productivity, PTMs, process robustness and expression stability proven clonality is key for pharmaceutical cell line development. Automated Lab Solutions (ALS) has developed a novel technique to reliably image individual cells and automatically select and isolate resulting colonies from liquid medium based on its automated cell picking platform ALS CellCelector. The approach provides unique in-process validation of clonality and allows parallel assessment of growth and productivity for thousands of colonies. We will demonstrate its performance within cell line development workflow with single cloning round.
Cellular whole genome analysis by NGS can be used to assess the genomic integration locus of the transgenes, as well as the propagation of single-nucleotide variants that result from genome-wide mutations. This presentation will illustrate how this can be used to assess the monoclonal origin of CHO cell populations, and how this can reliably detect even minor population contaminants in non-clonal populations.
Increasing Expression Efficiency and Product Quality in Cell Line Development for Novel Modalities and Difficult to Express Proteins
Timely generation of good quality CHO cell lines that can be seamlessly transferred to a CDMO is a key activity during IND-enabling studies for an immuno-oncology candidate. In this talk, we describe the selection process for in-licensing a CLD technology, optimization of CLD platform, and development of cell lines for model molecules. The host cell line, vector configuration, medium and utilization of a scale-down model are all factors in expediting development timelines.
cytena’s single-cell printer uses an imaging system and object recognition algorithms to detect cells in a single-use dispenser cartridge. Droplets are produced similar to inkjet printing. Cells are classified in the nozzle and subsequently dispensed directly into well plates. Image sequences of the printing process enabling assurance of clonality. High viability has been observed for several cell lines (CHO: >85 %; HEK: >90 %; L292: >80 %) and furthermore customer data will be presented.
DART and TRIDENT molecules have been designed to achieve multiple mechanisms of action and clinical applications. They are composed of two to four peptide chains which need to assemble correctly for functional activity. The appropriate expression of each component by the production cell line is important in achieving high expression levels and product quality in order to successfully manufacture the clinical product. By incorporating molecule engineering strategies, optimizing vector selection, and cell line screening, we have been able to achieve correct molecule assembly, high expression levels, and biological activity of these complex, multi-chain molecules.