The colored bit is yours. Comments after - mostly mine,

So the cells that they retained innitrogen can't be tested using the same process but with better QC?

Generally,in biology, we can store in liquid nitrogen vapour phase, (what Icall cryopreserve), pretty much any cells - (not neurons in their stretched out form), and embryos (early stage,because early stages are just clusters of 16 or 32 cells, like inIVF, also sperm and eggs, but we can’t store organs – be great ifwe could we be storing hearts and livers and kidneys all over theplace – but organs have a differentiated cell-based structure thatcells and embryos don’t have, this essentially means when waterexpands as it freezes the organs get damaged structuresget pushed apart by the water expanding between them– the cells areflexible enough and structured such that water expanding doesn’thurt their structures – not so organs.) In theory cells can bestored at the very cold temperatures of liquid nitrogen almostindefinately – probably decades at least. But the natural state ofhuman cells is about 37 degrees C, that is the temperature theynormally are in a healthy human body. Heating cells up, thawing them,will result in some of them dying, likewise cooling them down,likewise getting them to de-adhere or stop sticking to a surface bybreaking their attachments with trypsin can damage their surfacereceptors.

I see no reason that they can't use the same test but using a morereliably produced test on cryopreserved cells.

Sure, but acrosstime like years or a decade some of the biological tools likeantibodies used to measure aspects of interest about cells can ceaseto be available if the provider goes out of business for instance.Also some of the tools can be improved so the old less improved toolsare produced anymore.

Specificallythe reference was one of 5 reagents used in the potency assay waspoorly controlled.

Ok

Youseem pretty well informed on the methodologies used.

Ican understand papers or protocols relevant when I read them.

Whatis the exact process in the potency assays used and what reagents areused in each step?

That is a very bigquestion! As I understand where you are at in terms of presentunderstanding is that you don’t even know know the names of the twopotency assays components MSB has placed into the public domain andhow they work. I know that if I write ‘prohibition of IL2Ralpha onco-cultured T cells’ eyes will glaze over. It would take a longtime (I’d have to do a lot of work) to answer that unless you weresay a colleague in another lab in which case I could probably justlink or send you the protocol – we aren’t in that situation.Perhaps we can find papers or patent applications, almost certainly alot of details are in the ODAC papers (which include slides andtranscripts – and I’ve spent well over a hundred hours probablyover two hundred hours on that stuff in the past – but I don’tcurrently have even item of interest on a easy find index – I justhave my memory). I don’t have at hand the exact recipe MSB usesdown to the level of reagents provided by specific provider – but Ido think I could possibly reverse engineer something close if it wasimportant enough and I cared enough – but it would be a very bigjob.

Below in red – aresome things which could be called re-agents highlighted.

*********' video that I posted about at approx the 3 minute mark SI says.' Secondpotency assay for gvhd that was in place during the phase III trialbut wasn’t shown to fdahavenow been given to the fda. Will be discussed in the fda mtg march2024. msb have analysed the data of the potency assay that was inplace during the original trial in a different way that more thanprovides what the fda have been asking for.'

Troublefor me is that two matrix components TNFR1 and IL2Ralpha were bothshown to some extent to the FDA, but when SI has described orabbreviated what he’s done in less formal than ODAC contexts he hasused language that obscures. I believe prior to the BLA 2 SI and Cowere stressing, and folk like otherperspective here were seeminggetting excited about and amplifying, that there was a bunch of extradata about IL2Ralpha and that Dr J Kurtzberg’s follow up worth washighlighting that. So SI’s loose talk sometimes makes it hard toknow what he is actually specifically saying. I’m not sure he isnot just talking about more data (that at CRL1 or CRL2) to do withIL2Ralpha. He could be talking about something additional (andtreating TNFR1 plus IL@Ralpa as onepotency assay only though it has two parts) or he could be justswinging his emphasis onto the IL2Ralpha stuff again.

SImost certainly discussed with a group of people after the AGM thatthe new potency assay that the FDA accepted (perhaps an in principleacceptance rather than a formal one; I don't know) for use in theadult GvHD study was in his view inferior to one previously used inthe trial.He indicated thathe believed the new data they had generated would provide the FDAwhat they asked for.Guesswhat, he did just that, and the FDA did accept the new data.No clearer indication that I heard him correctly. BTW, as is mypractice, I take contemporaneous notes so I don't have to rely onmemory.

As for yourreference to correlation between each passage it seems that this isnot relevant as the Research Report states(https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8322819/)

'InvestigationalAgent
Remestemcel-L comprises healthy adult volunteer donorhuman bone marrow-derived MSCs that have been ex vivo cultured andcryopreserved in Plasma-Lyte A supplemented with humanserum albumin and dimethylsulfoxide. Eachremestemcel-L dose was stored in the vapor phase of liquid nitrogen,thawed, and reconstituted in Plasma-LyteA (Baxter International,Inc. USA) immediately prior to administration.

MSCs arenonhematopoietic cells that express low levels of majorhistocompatibility complex class I, are negative for majorhistocompatibility complex class II molecules, and are negative forcostimulatory molecules CD40, CD80, and CD86. Remestemcel-L cells areCD105+, CD156+, and CD45−; express tumor necrosis factor receptor1; and suppress IL-2Rα expression on activated lymphocytes.Remestemcel-L cells are harvested at passage5 and then cryopreserved asa final product. In thisstudy, 4 donors and multiple product lots were used. Most patientsreceived infusions from more than 1 lot, and some patients wereexposed to cells from more than 1 donor.'

I see no referencein the Kurzberg paper from 2020 to which you’ve referred to passage2 – but at ODAC (so in the ODAC papers and transcripts) its madeclear that cells were passaged to passage 2 at Lonza Walkerville inthe USA, then cryopreserved, then later thawed out at Lonza SingaporeI believe, then taken to passage 5. After passage 2 and after passage5 a release test is done such that levels of TNFR1 were taken onbatches that measured amounts of that substance. There can be yearsdifference between passage 2 and passage 5.

BTW. If you checkthe bit I underlined in what you excerpted you’ll see “Mostpatients received infusions from more than 1 lot, and some patientswere exposed to cells from more than 1 donor”and that is exactly abig problem I’ve stressed – and the FDA stressed, there are twomany confounding variables to link a specific dose of product with aparticular outcome in a particular patient.

Canyou please provide detail as to where you saw referenced all thepotency assays used to test Potency (included in the original datasent to the FDA and those provided later). I can't seem to findreference to it to further my learning.

Itis in the ODAC material. Do you have access to that stuff on-line?Its been archived but a few folks on here, including some holderslike phaedrus have linked to it previously.

Idon’t want to do too much work (sourcing stuff that I’ve sourcedover and over before and that is not controversial in that peoplewith as different views as phaedrus and myself would both agree thatthat is original source material) – unless there is something in itfor myself in terms of me learning something as well for my timespent.