Genetic Stability Testing

Genetic Stability Analysis: Characterization and Stability Testing 

Genetic stability is a specific characterization of the cells used in the production of a biologic. Genetically modified cells contain transgene insertion sequences that code for the protein of interest. Transgenes have the potential to mutate, which can impact the quality of the product or the proper execution of processes.  

ICH Guidelines state the stability and integrity of the insertion sequence needs to be characterized to ensure stable production of the correct protein. This is just one of the required steps ensure the cell line and production system is acceptable. 

There are multiple techniques to determine the genetic stability of a cell line. These tests take place during the characterization of a production cell line, and to assess the genetic stability of the Master Cell Bank and the End of Production Cell Bank. Testing can also be carried out at other times, but is particularly useful during phases where there are changes to the fermentation process. 

Sartorius offers the following genetic stability assays and can advise on the most appropriate approach for your product: 

  • Cell Line Identity Testing (DNA Barcoding, RAPD) 

  • Gene Copy Number 

  • Sequencing of the transgene 

  • Southern Blot 

Navigate the regulations

The Sartorius team will help guide through the regulations to the most appropriate assay for your application

Build a customized testing plan

Our experts can help develop custom genetic sequencing assays to test the stability of your product using our platform approach

Regulatory compliance

Our cGMP-compliant assays align with European Pharmacopeia (EP) and United States Pharmacopeia (USP) guidelines

Sartorius Solutions for Genetic Stability Analysis

Sartorius Solutions for
Genetic Stability and


DNA Barcoding assay 

Using information from a few gene regions, this barcoding can confirm the species-level cell line identity.

Random Amplified Polymorphic DNA analysis (RAPD)

RAPD identifies mutations or contaminating cells, and is typically used to compare Research, Master, Working or End of Production cell banks.

Gene copy number

Performed on recombinant cell banks to confirm transgene copy number per cell.

Sanger sequencing using the ABI 3500 platform

Confirming cell line stability based on the nucleic acid sequence of the transgene used to produce the protein of interest and the flanking region.

Southern blot analysis

Southern blots identify the presence of the transgene in a digest of the production cellular DNA, demonstrating its integrity.


Cell Line Identity Testing

Sartorius offer two methods for confirming cell line identity:

  • DNA barcoding assay
  • Random Amplified Polymorphic DNA analysis (RAPD)

DNA barcoding assay for cell line identity

Sartorius offers DNA barcoding for cGMP cell line characterization testing where species-level identity for mammalian derived cell lines can be determined according to ICH and CBER/FDA regulatory guidelines. A tried and true method already known and trusted by regulatory authorities, DNA Barcoding is now considered the preferred method for species determination.

The Sartorius DNA barcoding assay has been validated for the following cell lines; HeLa, HEK-293, MRC-5, CHO-K1, Vero, A9, MA104 and MDBK.

Random Amplified Polymorphic DNA analysis (RAPD)

An alternative cell line identity testing method to the DNA barcode assay is the Random Amplified Polymorphic DNA analysis (RAPD). In this procedure, 6 different primer sets are used to amplify regions of the target genome.

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Sanger Sequencing

Determining the nucleic acid sequence of the transgene used to produce the protein of interest is one of the critical tests carried out to determine the genetic stability of the cell line.

During early stages of the development of a recombinant protein drug, the messenger RNA (mRNA) sequence of the transgene is determined for both the Master Cell Bank (MCB) and End of Production Cell (EOP) Bank. This reveals evidence confirming there are no changes to the genetic code and the cells have not undergone any significant changes.

In addition to the coding region, the flanking region of the transgene is also an important consideration as this can affect the productivity and expression levels. Sequencing of the flanking region (at the DNA level) is recommended by ICH guidelines and should be considered prior to phase 3 clinical trials.

Sartorius uses the ABI 3500 platform for traditional Sanger sequencing building on more than 25 years’ experience in nucleic acid sequencing with a wide array of different organisms and sample types including recombinant proteins from both human and CHO cells.

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Sequencing Services include:

  • High throughput sequencing
  • Re-sequencing of genes and or genomic regions
  • Single/Multiple read sequencing
  • Primer design
  • PCR amplification and/or purification

Southern Blot

As one of the oldest methods to analyze DNA, the Southern Blot offers a digest of a sample of DNA separated by gel electrophoresis based on size of the fragments generated. Specific DNA target regions are then identified using a nucleic acid probe.

Southern Blot testing identifies the presence of the transgene in a digest of production cellular DNA. In cases when there are multiple copies of the transgene in a production cell line, the Southern Blot will identify each target sequence as they are dispersed along the genome or will yield a more intense band where more than one insertional sequence are positioned adjacent to one another.

This enables the Southern Blot to reveal approximate information on the copy number of the transgene and samples from Master Cell Banks and End of Production Cell Banks can be compared on the same blot to demonstrate similarity.

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Gene Copy Number

Identifying the transgene copy number is regarded as a critical stability indicator for productivity in CHO and other cell lines used in recombinant protein production.

Cell lines that maintain consistent levels of productivity have a constant transgene copy number, while cell lines exhibiting a loss of productivity demonstrated drift with passaging. Quantitative PCR is the method of choice to determine the copy number of a clone. Sartorius offers a complete service from assay design and validation through sample qualification.

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