Tools and methods for providing assurance of clonality for legacy cell lines

Over the last several years demonstration of cell line clonality has been a topic of many industry and regulatory presentations and papers. Guidance has been provided by the regulatory authorities, especially the FDA, on a path forward for providing evidence of clonality with high probability. It has been recommended that two-rounds of limiting dilution cloning (LDC) at sufficiently low seeding densities (≤0.5 cells/well) provides sufficient evidence that a cell line is clonal. Furthermore, one-round of LDC may also suffice if supplemental data from a characterized FACS or plate-imaging workflow are also included in the package. Cell lines generated by methods that do not demonstrate high probability of clonal derivation, including legacy cell lines, may require additional studies to provide assurance and/or process control strategies to satisfy regulatory expectations. Within the Biologics function of the IQ Consortium the “Clonality” Working Group is focusing on methods and tools which could be utilized to provide a high assurance of clonality for legacy cell lines. The presentation will outline a three tier approach to address legacy cell line clonality assurance: standard practices already used in industry to support limit of in vitro cell age studies, enhanced control strategies to ensure process consistency, and emerging technologies that could be used to further support cell line clonality

A widespread family of bacterial cell wall assembly proteins

Teichoic acids and acidic capsular polysaccharides are major anionic cell wall polymers (APs) in many bacteria, with various critical cell functions, including maintenance of cell shape and structural integrity, charge and cation homeostasis, and multiple aspects of pathogenesis. We have identified the widespread LytR–Cps2A–Psr (LCP) protein family, of previously unknown function, as novel enzymes required for AP synthesis. Structural and biochemical analysis of several LCP proteins suggest that they carry out the final step of transferring APs from their lipid-linked precursor to cell wall peptidoglycan (PG). In Bacillus subtilis, LCP proteins are found in association with the MreB cytoskeleton, suggesting that MreB proteins coordinate the insertion of the major polymers, PG and AP, into the cell wall

Advancing Biologics Development Programs with Legacy Cell Lines: Advantages and Limitations of Genetic Testing for Addressing Clonality Concerns Prior to Availability of Late Stage Process and Product Consistency Data

The bioprocessing industry uses recombinant mammalian cell lines to generate therapeutic biologic drugs. To ensure consistent product quality of the therapeutic proteins it is imperative to have a controlled production process. Regulatory agencies and the biotechnology industry consider cell line ″clonal origin″ an important aspect of maintaining process control. Demonstration of clonal origin of the cell substrate, or production cell line, has received considerable attention in the past few years and the industry has improved methods and devised standards to increase the probability and/or assurance of clonal-derivation 1-4. However, older production cell lines developed before the implementation of these methods, herein referred to as ″legacy cell lines″, may not meet current regulatory expectations for demonstration of clonal-derivation. In this article, the members of the IQ Consortium ″Working Group on Clonality″ present our position that the demonstration of process consistency and product comparability of critical quality attributes (CQAs) throughout the development life cycle should be sufficient to approve a license application without additional genetic analysis to support clonal origin, even for legacy cell lines that may not meet current day clonal-derivation standards. With this commentary we discuss advantages and limitations of genetic testing methods to support clonal-derivation of legacy cell lines and wish to promote a mutual understanding with the regulatory authorities regarding their optional use during early drug development, subsequent to IND application and prior to demonstration of product and process consistency at BLA submission