The International Conference on Harmonisation (ICH) requires that marketing applications include characterization of working cell banks (WCB’s) used for biotherapeutic production. One dimension of cell bank characterization includes cell substrate stability, which is intended to demonstrate consistent production of the intended product and production capacity retention during storage under defined conditions for a given cell substrate (ICH Q5B/Q5D).  In order to evaluate cell substrate stability, reconstituted cells are examined at a minimum of two time points, one at WCB vial thaw and another at or beyond the limit of in vitro cell age of the production process.  At Pfizer, our strategy incorporates the use of 15L fermentation cultures for cell substrate stability evaluation once the in vitro cell age has been established at production scale (1500+L).  The microbial cell line presented demonstrated cell substrate stability beyond the limit of in vitro cell age based on: 1) genetic stability through plasmid and product expression cassette integrity and retention; 2) biopharmaceutical production with consistent characteristics including titer, purity and specific productivity; and 3) additional quality characteristics such as culture growth and morphology.  Furthermore, this cell line evaluation represents a best practices approach for characterizing microbial cell substrates used for biotherapeutic production.