Integration Site Analysis

Characterisation of genetic modifications for clone selection, genetic stability and lot release

What is INTEGRATION SITE ANALYSIS and why is it important?

As part of the development of a cell line for manufacturing, candidate clones will be selected based on several attributes including production yield, number of transgene copies, as well as the structure and location of the transgene in the host cell genome. Indeed, a certain level of genetic characterization of the cell line is expected by the regulatory authorities (as outlined in Cell Line Characterization).

However, developers can benefit from a deeper understanding of the genome integration site of the transgene when making clone selection. For example, transgenes located in areas of instability or regions prone to epigenetic silencing may lead to a significant reduction in manufacturing yields over the cell line’s manufacturing lifetime.

Employing long read sequencing techniques, such as nanopore long read sequencing, can provide detailed insights into the genetic site of transgene integration. This genome sequence analysis allows for the identification of potential instability or silencing issues that might not be detected with shorter read methods. By using long read sequencing, developers can ensure that transgenes are integrated into stable regions of the genome, thereby optimizing production yields, and maintaining consistency throughout the cell line’s manufacturing lifecycle.

Classical integration site analysis methods such as fluorescence in situ hybridization (FISH) or Southern blotting can provide information on the integration location in the genome but still offer very low resolution. Sequencing the integration site offers single nucleotide resolution; however, it can be challenging, especially if the transgene is randomly integrated into the host genome. Methods such as long-range PCR combined with Sanger sequencing are also employed, but these are cumbersome and require prior knowledge of the integration site. More recently, NGS whole genome sequencing (WGS) has been used to characterize the integration site. However, WGS can be complex for cells with unstable genomes such as CHO. Additionally, WGS tends to provide a lower level of sequence coverage, making conclusive identification of the integration site and its epigenetic status difficult.

A targeted short-read NGS method utilizes whole genome crosslinking to generate circular DNA fragments up to 100kb either side of a known primer site from the gene of interest. This method does not require any prior sequence information of the insertion site, and good information is obtained on the junction site. However, due to the nature of short-read sequencing, this approach can struggle to identify concatemers, truncations, and inverted repeats.

With iDTECT® INTEGRATION SITE ANALYSIS, PathoQuest has validated an enhanced integration site analysis approach that combines long read nanopore sequencing with transgene expression cassette-targeted cleavage, called long-read ISA. By targeting cleavage to the transgene expression cassette, both the transgene and the junction site can be comprehensively sequenced. Long read sequencing has the advantage over short-read NGS methods as it better characterizes concatemers, truncations, and inverted repeats. This method provides detailed insights into the genetic site of transgene integration, allowing for comprehensive genome sequence analysis and a more accurate understanding of genome integration.

What we do

Modalities Tested:

  • Cell banks for mAbs and recombinant biologics, Viral vectors, Cell therapies, Vaccines
  • Cell therapy lots
  • Gene therapy
  • Cultured meat banks

Benefits of Long-Read iDTECT® NGS assay

Validated method
Single nucleotide precision
Resolve concatemers, truncations and inverted repeats more easily than short-read NGS

Our Assay:

  • iDTECT® Integration Site Analysis

INTEGRATION SITE ANALYSIS METHODS

WHY THIS APPROACH

ICH: Scientific guideline Q5B analysis of the expression construct in cell lines

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FDA: Characterization and Qualification of Cell Substrates and Other Biological Materials Used in the Production of Viral Vaccines for Infectious Disease Indications

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Publication: Nanopore Cas9‐targeted sequencing enables accurate and simultaneous identification of transgene integration sites, their structure and epigenetic status.

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PathoQuest Webinar

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SAMPLE REQUIREMENTS

The specific sample requirements can depend on the source material and the desired outputs.  We would therefore recommend having a consultation with one of our experts to discuss your exact requirements.

In general our sample requirements are:

  • 5×10⁶ cells recommended (≥10µg DNA per sample)
  • Storage and shipment at -80°C or on dry ice
  • Cell pellets or cryo-preservation buffer
  • Back up sample is recommended

Challenges solved

  • Understanding complex transgene arrangements
  • Low sample volumes
  • Tight clinical timelines
  • Reducing risk in cell banking with more information on integration site

“We acquired a production cell bank that had minimal information on the integration site other than the sequence of the transgene. PathoQuest’s approach allowed us to fully sequence the insertion site giving us the confidence to take our bank forward for GMP manufacturing”

OTHER SERVICES

Adventitious Virus Testing

Detection of viral contamination within the manufacturing process and beyond.

READ MORE

In Vivo Replacement

NGS as an ethical alternative to animals in biosafety testing and characterization.

READ MORE

Identity Confirmation

Genetic characterization of viral and plasmid products for release.

READ MORE

Cell Line Characterization

Biosafety screening and stability testing of manufacturing cells.

READ MORE

HLA Genotyping

Characterizing and screening for novel and emerging cell therapies.

READ MORE

Raw Material Testing

Screening of high-risk inputs such as animal products and media.

READ MORE

Contact us

U.S.

+1 484 212 9360

466 Devon Park Dr
Wayne, PA 19087
United States

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France

+33 (0)1 70 82 17 90

Biopark -Bâtiment B,
11, rue Watt
75013 Paris, France

How can PathoQuest help?

Name(Required)
This field is for validation purposes and should be left unchanged.

U.S.

+1 484 212 9360

466 Devon Park Dr
Wayne, PA 19087
United States

France

+33 (0)1 70 82 17 90

Biopark -Bâtiment B,
11, rue Watt
75013 Paris, France

How can PathoQuest help?

Name(Required)
This field is for validation purposes and should be left unchanged.

Sign up for our latest news