Like a string and nail art project from the 1970’s, an evolutionary depiction of the “tree of life” is a complex pattern, drawn to classify organisms based on everything from physiological traits to in depth genetic analysis. This process reflects the beautiful diversity of life all around us. But in the world of biopharmaceutical drug manufacturing, untangling the strings causes one of the challenges in cell line identification.

There are several regulatory guidelines related to full characterization of recombinant cell banks used to generate biological drug products. Ultimately, however, the task of the cell bank researcher often comes down to answering one question: Is my organism what I think it is? This of course leads to the question: What tests can I use to identify my cell bank/bioreactor contents and will the end of production material be a match?

To begin to answer these questions, it is best to take a step back and determine what level of identification and how many layers of identification are really needed. Genus? Species? Sub-species? And then if the strain is recombinant, how much further characterization is required? These are manageable yet could become time consuming and potentially costly projects.

If a genotypic approach is taken (as opposed to phenotypic/proteotypic analysis), then cell line characterization for recombinant or non-recombinant cell lines can be accomplished using: nucleic acid sequencing (whole genome, multi-locus, recombinant gene) or genome fragment analysis (e.g., STR) as tools. STR analysis, the amplification of Short Tandem Repeat elements (example: repeats of ACGT) found in the genome of mammalian cells allows for a unique fingerprint of the organism. A multiplex PCR reaction with fluorescently-labeled oligonucleotide primers that flank multiple regions (genetic loci) of repeats is performed with subsequent analysis and scoring using a genetic analyzer. The level of discrimination is increased with the increased number of loci examined. The data gathered by STR analysis is being incorporated into cell line databases to show relatedness between cell lines.

In the case of bacterial and fungal cell lines, analysis using ribosomal gene sequence database profiling is “Not your Father’s Gram Stain” but is a simple yet powerful sequence-based identification tool, using the sequences as a “molecular clock” (evolutionary diversity has been theorized to occur at a certain rate over time). Another straightforward “molecular clock” tool for characterization of animal cell lines could include a PCR-sequencing-based determination of the 648-bp Cytochrome C oxidase I (COI) sequence, often referred to as the “Barcode of Life” region. The term “DNA Barcoding” refers to the use of UPCs and barcode scanners to uniquely identify consumer products.  The power of the Barcode of Life Project (BOLD) relies on both complexity as well as close sequence similarities of all organisms. DNA barcoding is also a practical classification/identification system due to the simplicity of the experimental work. Just one PCR amplicon is needed to generate sequence, with subsequent database searches. A key goal of the International Barcode of Life Project is the assembly of a DNA barcode reference library with at least 500K species by 2015.

Expanding on one gene analysis systems, as in the BOLD efforts,  the Open Tree of Life project (http://opentreeoflife.org), incorporates multilocus sequencing and lateral gene transfers (as in a recent article in Microbe (Vol 8:6, June 2013). This comprehensive overview of biodiversity is part of the National Science Foundation’s (NSF) Assembling, Visualizing, and Analyzing the Tree of Life (AVAToL) initiative and will provide better insights into the subtle and not-so-subtle influences on genetic drift and diversity.

From development to biological drug manufacture, cell substrate characterization is an important factor in bringing safe and effective drugs to the public. Researchers need to decide on the level of detail they really need and what the advantages and disadvantages are to the multiple methods. Partnering with experts in this field can help with the design and execution of a complete testing program for mammalian, insect, avian and stem cell or microbial cell line that is customized to meet specifications as well as regulatory guidelines.