Cell Line Authentication Using Isoenzyme Analysis

Strategies for accurate speciation and case studies for the detection of cell line cross-contamination using a commercial kit.
Jun 01, 2005
Volume 18, Issue 6

Raymond W. Nims, Ph.D
Confirmation of purity and identity of cell cultures is a necessary step in the production of biotherapeutics. Manipulation of multiple cell lines in the same facility introduces the possibility that cross-contamination may occur. Different cell lines may proliferate with varying growth rates such that a single cell from a rapidly growing line, introduced into a culture of slower growing cells, can overtake the original culture in the course of a few passages. In addition, cultures may be mislabeled during manipulation, again resulting in a misidentification of the culture.

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.

Isoenzyme analysis is commonly performed as part of cell line authentication and characterization. It often is part of an overall testing battery for authentication of seed stocks submitted to cell repositories.2,3 In addition, authentication is typically performed on master and working cell banks as part of US and EU's mandatory overall characterization testing.4,5 Because isoenzyme analysis is technically simple, robust, and rapid, it is used in conjunction with, or often in place of, karyotyping or DNA fingerprinting. With commercially available kits, it only takes a few hours for an investigator to confirm a cell line's purity and species of origin.

Karyotyping and DNA fingerprinting are useful for these purposes but can be expensive, time consuming, and may require comparison with a karyotype or fingerprint taken from the original culture. These techniques may be required to establish identity at the individual cell line level. For routine speciation and assessment of purity, however, the relatively rapid and inexpensive isoenzyme analysis method is commonly employed as a useful alternate.

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.

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