Cell Line Identification – The Applicability of Cytochrome C Oxidase 1 Barcoding in Cell Bank Authentication

• RMC Pharmaceutical Solutions, Inc

The use of a PCR and sequencing based cytochrome c oxidase subunit 1 (CO1) barcode assay for establishing the species level identity of animal cells is discussed below. The methodology targets a semiconserved region of the mitochondrial CO1 gene for species-level identity testing of animal (mammalian and insect) cells. CO1 barcoding is intended as an alternative to the isoenzyme analysis assay, for which reagents are no longer commercially available. This white paper provides the opinion of a subjectmatter expert on the suitability of the CO1 barcode platform for species-level identity testing and as a replacement for isoenzyme analysis testing for animal cells.

The following is an excerpt of a white paper published on 5th August 2015. To download the full version, and find out more about the CO1 barcode assay available at BioReliance please visit www.bioreliance.com/CO1-barcode

Introduction

In regulated spaces, such as biologics manufacturing and safety testing, cell substrate characterization guidance documents mandating identity testing have been in place for more than two decades^1-5. Some of these guidance documents mention specific methodologies for establishing identity, while others do not. The primary requirement is, and still remains, demonstration that the production (or testing) substrate is the same cell line identified in the regulatory submission documents, and not some cross-contaminant or mis-identified cell. Historically, diploid cells have been identified by karyotyping and isoenzyme analysis. Aneuploid (i.e., transformed/ continuous) cells have been identified most commonly by isoenzyme analysis. The reason for this is that the methods were in place during the time frame covered by the earliest guidance documents (for example, references 1 and 2). It is important to remember that these methods provide species-level identity only. 

As a result of the unexpected non-availability of isoenzyme reagent kits from the de facto sole supplier, it has been necessary to identify suitable replacement methods for that cell identity assay. The replacement assays for identifying human cells include short tandem repeat (STR) profiling^6-8 and single nucleotide polymorphism (SNP) profiling⁹ . The replacement technologies now being considered for animal cell identity testing include DNA fingerprinting and CO1 barcoding. Will these methods also be acceptable to the regulatory agencies? The answer is that a science-driven rationale for replacing isoenzyme analysis with one of these newer candidate technologies should be acceptable.

Isoenzyme Analysis

Isoenzyme analysis is specifically mentioned as an appropriate identity test in ICH Q5D² , WHO³ , and European Pharmacopoeia 5.2.3⁴ , although in each case it is clearly indicated that other technologies can be employed for this purpose. For instance, in the 2010 WHO guidance³ , it is stated that: 

“Other tests that may be used but tend to be less specific include isoenzyme analysis and karyology, which may be particularly useful where there are characteristic marker chromosomes. However, where more specific genetic markers are available, they should be considered.”

Isoenzyme analysis provides a verification of species identity by electrophoretic comparison of various cytosolic enzymes against known mobility information¹⁰. In some instances, closely migrating bands for a given enzyme may leave two or more animal species unresolved. For this reason, an assignment of species of origin for a test sample is made on the basis of mobility information obtained through analysis of four or more target enzymes^11,12. The reagents required to perform this assay were available only from a single supplier, and the recent non-availability of reagents has severely limited the use of this method worldwide.

The CO1 Barcoding Platform

DNA barcoding has been used within the zoological community for years and has now been proposed for animal cell identity testing¹³. It is useful since the target (mitochondrial CO1 gene) varies sufficiently between species in order to allow discrimination. Mitochondrial genes are considered preferable over nuclear genes for DNA barcoding.

For animal species identity testing, the CO1 barcoding platform is superior to the isoenzyme analysis platform in terms of specificity (i.e., able to resolve cells derived from different species). The various factors determining specificity are displayed in Table 1.

As displayed in Table 1, the specificity advantages of the CO1 barcoding platform include not only the much greater number of reference species for which information is available, but also the minimal subjectivity for this platform relative to the isoenzyme analysis method and the ability of the barcoding assay to definitively establish the animal species of origin for the test sample.

Table 1. Comparison of determinants of specificity

Discussion

Isoenzyme analysis has served as the prototypic identity test for continuous animal cell lines for a number of decades. Advances in analytical science have paved the way for replacement of isoenzyme analysis with STR or SNP profiling for human cells. The specificity gains associated with the molecular methods, as well as the existence since 2012 of an ANSI standard for STR profiling⁸ , have helped to overcome the inertia associated with longstanding use of the electrophoretic method. The fact that the newer technologies have not already replaced isoenzyme analysis for animal cell identity testing should not be interpreted to mean that the latter technology is better suited for the purpose – the opposite is true. A method update has not occurred previously due to the inertia present in the regulated industries (i.e., the need for revision of release specifications for cell banks, or the need to file for approval of method changes, etc.) and the fact that there has been essentially no regulatory impetus for moving to the newer methods.

In conclusion, CO1 barcoding for animal cell identity testing clearly represents an improvement over the historical methods, including isoenzyme analysis, in terms of specificity. There is therefore no reason to expect that the regulatory authorities will have any objection to replacing isoenzyme analysis with CO1 barcoding for the identity component of cell line characterization.

References

1. US FDA Points to Consider in the Characterization of Cell Lines used to Produce Biologicals. 1993
2. International Council on Harmonisation (ICH) Q5D Derivation and Characterisation of Cell Substrates used for Production for Biotechnological/Biological Products.
3. World Health Organization (WHO) Recommendations for the evaluation of animal cell cultures as substrates for the manufacture of biological medicinal products and for the characterization of cell banks.
4. European Pharmacopoeia 5.2.3 Cell substrates for production of vaccines for human use
5. US FDA Characterization and Qualification of Cell Substrates and Other Biological Materials Used in the Production of Viral Vaccines for Infectious Disease Indications. 2010.
6. Barallon R, et al. Recommendation of short tandem repeat profiling for authenticating human cell lines, stem cells, and tissues. In Vitro Cell Dev Biol Anim 46:727-732, 2010.
7. Kerrigan L and Nims RW. Authentication of human cell-based products: the role of a new consensus standard. Regen Med. 6:255-260, 2011.
8. ANSI/ATCC ASN-0002-2012 Authentication of human cell lines: Standardization of STR profiling. ANSI, 2012.
9. Pakstis AJ, et al. SNPs for a universal individual identification panel. Human Genet. 127: 315-24; 2010.
10. Ziegenmeyer J. The AuthentiKit™ System Handbook for Cell Authentication and Identification. Innovative Chemistry, Inc., 1988.
11. Nims RW, et al. Sensitivity of isoenzyme analysis for the detection of interspecies cell line cross-contamination. In Vitro Cell Dev Biol Anim. 34, 35-39, 1998.
12. Nims RW and Herbstritt CJ. Cell line authentication using isoenzyme analysis. BioPharm Int 18:76-82, 2005.
13. ANSI/ATCC ASN-0003 Species-Level Identification of Animal Cells through Mitochondrial Cytochrome c Oxidase Subunit 1 (CO1) DNA Barcodes. Currently under review by the ATCC SDO Steering Committee.
14. Hebert PD, et al. Identification of Birds through DNA Barcodes. PLoS Biol 2(10): p. e312., 2004
15. Savolainen V, et al. Towards writing the encyclopedia of life: an introduction to DNA barcoding. Philos Trans R Soc Lond B Biol Sci. 360(1462): p. 1805-11, 2005.
16. http://www.boldsystems.org/

Author Biography

About the Author: Raymond Nims has provided consulting for the pharmaceutical/ biologics industries as an employee of RMC Pharmaceutical Solutions, Inc since 2009. Ray is a founding member of the International Cell Line Authentication Committee (ICLAC), and has been or is currently a participant in development of three ATCC SDO standards relating to cell line authentication.