Sequencing of RNase A treated Pfizer bivalent vaccines reveals paired-end sequencing evidence of circular plasmids and an inter-vial 72bp variation in the SV40 promoter.
Sequencing of RNase A treated Pfizer bivalent vaccines reveals paired-end sequencing evidence of circular plasmids and an inter-vial 72bp variation in the SV40 promoter.
IntroductionNepetalactone Newsletter is a reader-supported publication. To receive new posts and support my work, consider becoming a free or paid subscriber. Previous work sequencing the Pfizer bivalent vaccines utilized a method that sequenced both the RNA and DNA in the sample. The RNA-Seq kit employed favored sequencing RNA and under estimated the DNA contamination levels in the vaccine. This made it difficult to assess the % of the plasmid DNA that was linear versus circular DNA. To address this problem, we RNase A treated the sample and sequenced the remaining DNA. RNase A digests RNA into small pieces that can be eliminated with purification. This enabled a deeper sampling of the DNA based molecules without the mRNA sequence obscuring and suppressing the depth of coverage for the plasmid vector.
Mar 12, 2023·edited Mar 12, 2023Liked by Anandamide
Anandamide, you says that the plasmid contamination is a proxy for LPS contamination.
As a dinosaur in age, I remember back to the 60's when the NCI scientists were making L-asparaginase to be used against cancer, primarily leukemia, lymphomas, melanomas, breast cancer etc...
Except it was causing huge problems for patients, including the liver, kidneys, blood clotting system and the brain, and central nervous system issues.... There were a lot of sudden deaths....
A bright spark suggested that it was E.coli LPS toxicity to which they laughed and said that "the amounts wouldn't hurt a monkey" Dr Gordon Zubrod was in charge...
Anyway, Zubrod wouldn't listen, until three years later Dr Tibor Borsos proved that 13 of 15 commercial lots of L-Asparaginase were contaminated with E.Coli Endotoxin.
all that time, they were still using it, and killing patients with it. All during that time, none of the oncologists could see the LPS toxic syndrome for it punching them on the nose.
At some point the light-bulb lit, and they changed the manufacturing process and used Erwinia Carotovora instead, and suddenly all the "mysterious" toxic deaths stopped.
Another story buried in the "no fishing" cupboard to remind us just what can happen when people don't appear to understand or take note of - or perhaps just ignore.... the basics of E.coli LPS toxicity.
Mar 12, 2023·edited Mar 12, 2023Liked by Anandamide
Well done, again! Not surprising you have confirmed the presence of circular plasmid by sequencing given your earlier results, but it did need to be done. Also nice to have an improved assembly of their vector sequence and solve the polyA "mystery".
A stand out for me is still the quantity of DNA you detected in your previous Substack. Circular or linear, I still think this will happily transfect human cells, in some cases permanently altering the genome.
I was left wondering how the heck it could be that there is that much DNA left there, and also what kind of QC parameters they have for residual DNA in the first place. After perusing some Australian TGA FOI documents yesterday, and quickly reading through the EMA document you've linked here, this is basic summary as I see it:
1) The manufacturing process uses a linearised DNA plasmid together with T7 polymerase and other reagents to generate the RNA.
2) The removal of the DNA is reliant on the DNase I step performed after RNA product is complete. The EMA document suggests sizable (10×) batch-to-batch variation in the amount of residual DNA present (10-220 ng/mg) in production batches, even if all of these were below their specification of 330 ng DNA/mg RNA. One development batch did have much higher template concentration (815 ng/mg) due to an "incorrect DNase I stock solution", as you noted.
3) The assay to detect residual DNA is a qPCR-based assay with primers on the T7 promoter and Kozak sequence. Presumably the motivation for this assay is high sensitivity to detect what is expected to be a rather trace impurity. This method will not quantify any DNA not containing the target sequence, but should detect the input plasmids sequenced here (I checked and the primers do match).
4) The residual template check is done after the Drug Substance production stage, prior to the LNP Fabrication stage.
6) The is an oversight in this process that I can see, which is there is no guarantee that the subsequent steps used in product will not effectively concentrate the DNA relative to the RNA. For example, the DNA will be obviously a lot more stable than RNA, so will be more resistant to degradation. In addition, what if the LNP is more efficient at packaging DNA than RNA?
The last point could alone potentially bring the residual amount of DNA to above the specification of 330 ng/mg RNA, if it was already close to the spec. I was trying to dig up some figures on what the RNA losses are going from Drug Substance production to final product to estimate worst case scenario but haven't found any yet. Nevertheless, I assume the losses are <25% at worse case, so in order for a large amount of DNA to be present the DNAse would have to fail, and the residual DNA assay would have to fail or be ignored, implying extremely shoddy standards. In any case, residual DNA testing should really by part of the QC for the final product, but it's not. That's lax in itself.
As for the specification itself, the EMA document has this justification:
"The specification for residual DNA template was based on the WHO recommendation of not more than 10 ng DNA/dose. Based on these considerations, and assuming a maximum dose of 30μg, the commercial acceptance criterion at release is ≤330 ng DNA/mg RNA. The approach is endorsed. "
I haven't looked at the WHO justification for this and I'm not sure I really agree 10 ng is acceptable. This is more than enough DNA to transfect some cells, with potential genomic integration, when multiplied over enough cells exposed and enough recipients.
So glad I've subscribed to this substack, amazing detailed work (much beyond my limited uni biology studies two decades ago!) ... A few substacks have mentioned the DNA plasmids, but not in the detail you have explored... Do you think these plasmids are replication competent due to the sheer amount of linearized DNA? Given they are less replication competent than circular plasmids, is there still enough potential replication possible, at the high volumes measures? There are indications of circular plasmids detected as mentioned, in which case is this a potential long term expression of spike for the vaccinated? As they appeared to be replication competent in E.Coli in the lab... and therefore also could go onto contribute high endotoxin loads in the jab if there's any contamination at the production stage of the vaccine (anaphalxis potential/toxic shock) due to E.Coli based plasmids detected in the Pfizer jab ?
Excuse my poor dropout level of understanding, but this article compliments and refined the other substack from Jessica Rose I'd read on the subject.
Anandamide, you says that the plasmid contamination is a proxy for LPS contamination.
As a dinosaur in age, I remember back to the 60's when the NCI scientists were making L-asparaginase to be used against cancer, primarily leukemia, lymphomas, melanomas, breast cancer etc...
Except it was causing huge problems for patients, including the liver, kidneys, blood clotting system and the brain, and central nervous system issues.... There were a lot of sudden deaths....
A bright spark suggested that it was E.coli LPS toxicity to which they laughed and said that "the amounts wouldn't hurt a monkey" Dr Gordon Zubrod was in charge...
Anyway, Zubrod wouldn't listen, until three years later Dr Tibor Borsos proved that 13 of 15 commercial lots of L-Asparaginase were contaminated with E.Coli Endotoxin.
all that time, they were still using it, and killing patients with it. All during that time, none of the oncologists could see the LPS toxic syndrome for it punching them on the nose.
At some point the light-bulb lit, and they changed the manufacturing process and used Erwinia Carotovora instead, and suddenly all the "mysterious" toxic deaths stopped.
Another story buried in the "no fishing" cupboard to remind us just what can happen when people don't appear to understand or take note of - or perhaps just ignore.... the basics of E.coli LPS toxicity.
Well done, again! Not surprising you have confirmed the presence of circular plasmid by sequencing given your earlier results, but it did need to be done. Also nice to have an improved assembly of their vector sequence and solve the polyA "mystery".
A stand out for me is still the quantity of DNA you detected in your previous Substack. Circular or linear, I still think this will happily transfect human cells, in some cases permanently altering the genome.
I was left wondering how the heck it could be that there is that much DNA left there, and also what kind of QC parameters they have for residual DNA in the first place. After perusing some Australian TGA FOI documents yesterday, and quickly reading through the EMA document you've linked here, this is basic summary as I see it:
1) The manufacturing process uses a linearised DNA plasmid together with T7 polymerase and other reagents to generate the RNA.
2) The removal of the DNA is reliant on the DNase I step performed after RNA product is complete. The EMA document suggests sizable (10×) batch-to-batch variation in the amount of residual DNA present (10-220 ng/mg) in production batches, even if all of these were below their specification of 330 ng DNA/mg RNA. One development batch did have much higher template concentration (815 ng/mg) due to an "incorrect DNase I stock solution", as you noted.
3) The assay to detect residual DNA is a qPCR-based assay with primers on the T7 promoter and Kozak sequence. Presumably the motivation for this assay is high sensitivity to detect what is expected to be a rather trace impurity. This method will not quantify any DNA not containing the target sequence, but should detect the input plasmids sequenced here (I checked and the primers do match).
4) The residual template check is done after the Drug Substance production stage, prior to the LNP Fabrication stage.
5) Key point: After this stage, no further testing for residual DNA is performed. The amount of residual DNA is assumed to be within spec after that in the initial test. Many of the methods used to check RNA will happily detect DNA as well, such as the RT-PCR you highlighted. I have found no information on the amount of DNA present in the batch QCs reported in the TGA FOI requests (e.g. FOI 3471 from https://www.tga.gov.au/resources/publication/publications/documents-released-under-section-11c-freedom-information-act-1982-jul-2021-jun-2022).
6) The is an oversight in this process that I can see, which is there is no guarantee that the subsequent steps used in product will not effectively concentrate the DNA relative to the RNA. For example, the DNA will be obviously a lot more stable than RNA, so will be more resistant to degradation. In addition, what if the LNP is more efficient at packaging DNA than RNA?
The last point could alone potentially bring the residual amount of DNA to above the specification of 330 ng/mg RNA, if it was already close to the spec. I was trying to dig up some figures on what the RNA losses are going from Drug Substance production to final product to estimate worst case scenario but haven't found any yet. Nevertheless, I assume the losses are <25% at worse case, so in order for a large amount of DNA to be present the DNAse would have to fail, and the residual DNA assay would have to fail or be ignored, implying extremely shoddy standards. In any case, residual DNA testing should really by part of the QC for the final product, but it's not. That's lax in itself.
As for the specification itself, the EMA document has this justification:
"The specification for residual DNA template was based on the WHO recommendation of not more than 10 ng DNA/dose. Based on these considerations, and assuming a maximum dose of 30μg, the commercial acceptance criterion at release is ≤330 ng DNA/mg RNA. The approach is endorsed. "
I haven't looked at the WHO justification for this and I'm not sure I really agree 10 ng is acceptable. This is more than enough DNA to transfect some cells, with potential genomic integration, when multiplied over enough cells exposed and enough recipients.
wow, quite good work.
Excellent work, thanks very much.
Wow. Amazing work.
Something tells me the methylpseudouridine replacement is going to have many many far reaching, yet to be revealed ripples. Yikes.
From the EMA doc
“Neither the source and generation of the pST4-1525 plasmid used are documented ”
So glad I've subscribed to this substack, amazing detailed work (much beyond my limited uni biology studies two decades ago!) ... A few substacks have mentioned the DNA plasmids, but not in the detail you have explored... Do you think these plasmids are replication competent due to the sheer amount of linearized DNA? Given they are less replication competent than circular plasmids, is there still enough potential replication possible, at the high volumes measures? There are indications of circular plasmids detected as mentioned, in which case is this a potential long term expression of spike for the vaccinated? As they appeared to be replication competent in E.Coli in the lab... and therefore also could go onto contribute high endotoxin loads in the jab if there's any contamination at the production stage of the vaccine (anaphalxis potential/toxic shock) due to E.Coli based plasmids detected in the Pfizer jab ?
Excuse my poor dropout level of understanding, but this article compliments and refined the other substack from Jessica Rose I'd read on the subject.
https://open.substack.com/pub/jessicar/p/follow-up-on-dna-contamination-of?utm_source=share&utm_medium=android
Apologies again for being almost kindergarten level compared to your knowledge base, hopefully my questions make sense...
Aaaauuugh! (Charlie Brown gif here)
Thanks for all your persistent work Kevin. In my head I now call you Hercule Poirot, for solving one of the greatest mysteries of our time.
The work you are doing is of historic significance and important to humanity. #TheLittleGreyCells