Identity Confirmation

Genetic characterization of viral and plasmid products for release.

What is Identity confirmation and why is it important?

Identity testing is a critical testing component to release vector products, including viral and non-viral cell and gene therapies; as well as vaccine and oncolytic applications. Determination of the nucleic acid sequence of the vector and gene of interest is a regulatory requirement, with full nucleic acid sequence provision for vectors under 40kb. Therefore, direct sequencing is a standard and well accepted approach.#contact

Sanger sequencing has traditionally been employed as a method for nucleic acid sequence determination, and is therefore well accepted by the regulator. However, an NGS approach provides advantages over the traditional Sanger method:

  • Identify ultra-rare genetic variants <<5% (~20% variant detection limit for Sanger).
  • More cost and time efficient over a sequential Sanger sequencing.
  • Full coverage can be challenging with Sanger sequencing

As there is a regulatory expectation for appropriate characterization of critical raw materials, an aligned testing strategy should also be employed for any plasmid input materials. This mitigates the risk that non-clonal variation within the manufacturing plasmids are carried through to affect the quality of the final product.

What we do

Modalities Tested:

  • Plasmids
  • Vaccines (Viral and RNA)
  • Gene Therapy Vectors
  • Oncolytic Virus
  • Bacteria

Benefits of NGS for Identity Testing

GMP Validated
Subpopulation Identification
Can handle sequence motifs that are challenging to PCR/sequence

    – e.g. AAV ITR, lentivirus LTR or certain promoters

Sanger VS NGS for identity testing

WHY THIS APPROACH

EMA – Guideline on the quality, non-clinical and clinical aspects of gene therapy medicinal products

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FDA – Information for Human Gene Therapy IND Applications – Guidance for Industry

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ICH Q7 – Good Manufacturing Practice for Active Pharmaceutical Ingredients

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FDA – Code of Federal Regulations, Title 21 Volume 7. Part 610 – General Biological Products Standards

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Sample requirements

Sample requirements

Shipment & storage Standard turnaround time Fasttrack turnaround time Sensitivity** Output

Min sample vol of 200μl at titres of 1×1013 for AAV, 1×1010 for lentivirus, 1×108 for adenovirus.

Minimum 1ng at 0.5ng/μl for plasmids.

Backup sample required

BSL1 or 2*

Dry Ice /
-80°C
28 days 14 days

Detection of variants validated at 5% abundance

(Lower occurrence variants can be reported if required)

GMP CofA
Consensus sequence

Variant sequences at >5% abundance

*Biosafety level classifications can vary between regulatory authorities – contact PathoQuest to discuss.

**NGS should be considered as semi-quantitative in this application. Abundance figures are provided at the occurrence rate of the variant as it appears within the read data. Detection of variants is possible below the validated 5% level. Please discuss with our experts if this is required.

*Biosafety level classifications can vary between regulatory authorities – contact PathoQuest to discuss.

**NGS should be considered as semi-quantitative in this application. Abundance figures are provided at the occurrence rate of the variant as it appears within the read data. Detection of variants is possible below the validated 5% level. Please discuss with our experts if this is required.

Challenges solved

  • AAV ITR and lentivirus LTR sequencing by Sanger
  • Difficult to sequence promoter regions within vector
  • Reducing number of Sanger Sequencing runs
  • Alignment of methods between plasmid and vector release

“We were having some issues in sequencing our AAV ITR regions with a Sanger approach requiring multiple GMP runs. PathoQuest were able to provide us a full vector sequence in a single run without any issues with the ITRs.”

OTHER SERVICES

Adventitious Virus Testing

Detection of viral contamination within the manufacturing process and beyond.

READ MORE

nCats Integration Site Analysis

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

In Vivo Replacement

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

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

E: contact@pathoquest.com

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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

E: contact@pathoquest.com

How can PathoQuest help?

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

Sign up for our latest news