The need to verify the purity of cell lines on an ongoing basis is critical.1 In some cases it may be possible to visually identify a contaminating colony of cells in an otherwise pure culture. In many cases, however, an unintentional co-culture cannot be determined by visual inspection. It is therefore necessary to repeatedly verify the identity and purity of cell cultures when used as substrates for the manufacture of biotechnology products. This is commonly done at the master and working cell bank levels. Pre-bank and end-of-production cultures may also be evaluated.
METHOD OVERVIEW The isoenzyme analysis method utilizes electrophoretic banding patterns to examine slight differences from species to species in the structure and mobility of individual isoforms for a number of intracellular enzymes. We perform assays with a commercially available kit, the AuthentiKit system, manufactured by Innovative Chemistry, Inc.6 The intracellular enzymes typically evaluated in speciation are nucleoside phosphorylase (NP), malate dehydrogenase (MD), glucose-6-phosphate dehydrogenase (G6PD), lactate dehydrogenase (LD), peptidase B (PepB), aspartate amino transferase (AST), and mannose 6-phosphate isomerase (MPI). Reagents for detection of these enzymes are provided as part of the kit. Also provided is a standard reagent, murine L929 cell extract, to be loaded onto each gel, which allows the migration data of the test sample to be corrected for day-to-day test variability.
The kit manufacturer offers a chart of standardized electrophoretic migration distances for various intracellular enzymes. This listing contains values for 25 animal species. Using this chart and a systematic process of elimination, species assignments for test samples can be made on the basis of the corrected electrophoretic migration distances.