Thanks for your input. In your experience, what is the LLOQ in matrix? I.e. what do you consider BQL? This is critical as we are expected to collect 3 consecutive timepoints after BQL. BQL differs from matrix to matrix and DNA extraction efficiency needs to factor into the calculation. I don't ever see any mention of these aspects shared in publications. Comparability of assays is not possible unless we know the assay sensitivity (BQL). - Lena
Disclaimer: Opinions expressed are solely my own and do not express the views or opinions of my employer.
Original Message:
Sent: 03-16-2023 11:43
From: Amanda Hays
Subject: Vector shedding for GT products
Hi Lena,
I agree with Johannes' comments! I also tend to agree that the guidance on LOD of 50 copies/ug genomic DNA is called out for preclincial biodistribution and not necessarily applicable to viral shedding assays.
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Amanda Hays Ph.D.
Scientific Officer
BioAgilytix
Olathe KS
[email protected]
Disclaimer: Opinions expressed are solely my own and do not express the views or opinions of my employer.
Original Message:
Sent: 03-16-2023 09:08
From: Johannes Stanta
Subject: Vector shedding for GT products
Hi Lena.
This is a great question and there is indeed a need to harmonise our approaches. The 50 copies/ug level is for the old biodistribution guideline and in my view irrelevant for shedding.
For a shedding assay we focus on the low-copy part of the qPCR range and optimise the assay to detect (and quantify) low copy numbers.
In my experience we can find primers/probes and master mix combinations that achieve a LLOQ of between 20-50 copies/reaction and a LOD between 3-10 copies/reaction for the qPCR.
To achieve this we design 3-5 primer/probe pairs and test them in 3-5 master mixes to find the best combination for the low copy number detection.
The extraction recovery is assessed separately and for semen it is usually good. For saliva and urine it's usually less good (~20%). However, this recovery isn't factored into the overall concentration calculation. The amount of matrix taken into the extraction will give you the concentration (copies/uL).
I hope this is helpful and the start of a good discussion and harmonisation approach.
Best wishes.
Johannes Stanta, PhD
Director of Molecular and Cellular Biology
Celerion
Lincoln, NE
[email protected]
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Johannes Stanta
Global Director Molecular and Cell Biology
Celerion
Lincoln NE
[email protected]
Disclaimer: Opinions expressed are solely my own and do not express the views or opinions of my employer.
Original Message:
Sent: 03-15-2023 09:54
From: Lena Hofer
Subject: Vector shedding for GT products
Hi All, I was wondering what kind of sensitivity you achieve in your vector shedding assays in matrices like semen, saliva, urine. There is no clear regulatory guidance that spells out the expectation. The only guidance I am aware of is for preclincial biodistribution and mentions 50 copies /ug genomic DNA (LOD). This level is hard to achieve in clinical matrices. Thanks!
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Lena Hofer
Spark Therapeutics
Philadelphia PA
[email protected]
Disclaimer: Opinions expressed are solely my own and do not express the views or opinions of my employer.
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