Recap from the past 3 years: Update with some extra info, links and explanations

What do we know about the drug substance (mRNA)

I believe the multilayered, complex biosynthetic mRNA is coming into view. Much of what I will write was known or considered a risk at the time of authorization by the regulators. But now we have empirical data to assess and it is not a pretty picture. Even worse than I had imagined.

The Codon Optimization of the mRNA

1. Kevin McKernan first brought this to our attention in a preprint dated November 25, 2021 and has STILL not been accepted Differences between vaccine and SARS-CoV2 mRNA. The mRNA is highly codon optimized with increased GC content (process used to increase protein production using alternative but synonomous codons . This leads to

   1. mRNA secondary structure with G-quadraplex formation

      1. virus has 4; Pfizer has 19

      2. implications for folding and translation efficiency

   2. ribosomal pausing affecting protein folding

   3. GLYCOSOLATION: the S protein expressed in the cell after translation in the ribosomes undergo posttranslational modifications via glycosylation which (glycosylation implications for Covid vaccines):

      1. partially determines how immunogenic the epitopes are depending on folding and glycosylation

      2. influences cell uptake and T-cell priming

      3. glycosylation does NOT reflect the specific features of the glycosylation machinery of the host cell and will therefore VARY WITH EACH CELL TYPE, where viral replication takes place. Moreover, since glycosylation exhibits also inter-individual differences, the viral glycosylation pattern may differ among patients infected with the virus. See: glycosolation is key in Covid infection

      4. Therefore, are glycosolation sites etc the same as that of the native SARS-CoV2 virus? Can anyone determine that inside a living human?

      5. Does this partially explain response to vaccination and/or adverse effects? The study of glycosylation of the S protein produced by these vaccines is lacking. This is critical, and for therapeutic proteins, glycosolation sites are considered a critical quality attribute.

2. The use of N-1-methylpseudouridine

   1. there are 800+ of these in the biosynthetic mRNA (every uridine is replaced!)

   2. they have a high melting temperature and are not normally found in human mRNA (0.06%)

   3. is known to increase protein production +++ and decrease immunogenicity

   4. are prone to frame-shifting and lack of fidelity which has now been shown by Mulroney et al Ribosomal frame-shifting and our response to this paper Wiseman et al response to Mulroney

   5. we know this has caused a great deal of angst as the “'explainers” are out in full force Dont worry we can fix this

3. Stop codon read though, A good paper to read was published in Jun 2021 and should have been enough to have halted the mRNA right then. I read this many many times. At first I only understood about 20%, but now it is about 90%. When issues came up I would ask the same questions as Xia and then get sidelined. So I knew these issues were critical. Xia critical evaluation of the mRNA vaccines.

4. Segmented poly (A) tails which get polyadenylated only in vivo and other weird stuff. Note this paper has not been fully published Repolyadenylation in vivo

   This observation may affect that truncated mRNA are supposedly not translated, but unless you look for this effect IN VIVO you don’t know if it is happening or not. Think about this.

   1. CODON OPTIMIZATION INCREASES RISK OF MAKING ABERRANT PROTEINS (cancer, autoimmune disease, neurological disease). GLYCOSYLATION may affect protein folding and thus variations in immune response and possibly adverse effects. DESIGN of the mRNA INCREASES ITS DURATION OF ACTION, AND RESISTANCE TO BREAKDOWN

The making of the plasmid in E coli

1. The original master cell bank was made in Chesterfield Missouri in April 2020. Far away from prying eyes, especially those that know what to look for. This master cell bank is then frozen and shipped to various sites, as working cell banks to make the linear DNA template. The linear DNA template is what is used to make the mRNA (snapshots are from the EMA leak of their rolling review of the Pfizer’s manufacturing of the vaccine.)

2. Here you can see what was released and annotated to the regulators regarding the plasmid used to generate the linear DNA template.

3. And here is how the plasmid ITSELF is purified. Fro this description, it appears to be adequate, and you can see they use chromatographic methods. This will be important later. Endotoxin is measured as a contaminant (specifications of <20EU/mL)

The IVT of the linear DNA template in producing the mRNA

1. Making the mRNA in vitro from the linear DNA template from Process 2 results in:

   1. only about 60% full intact mRNA with the 5’cap, and intact poly (A) tail

   2. a large amount of truncated and fragmented mRNA

2. Risks of process related impurities from the IVT itself include residual T7 polymerase, proteinase K, and nucleotides themselves, like extra N-1-methylpseudouridine

   1. THESE process related impurities may be as important as that of residual DNA such as:

      1. RNA polymerase which could affect both the biosynthetic mRNA and our natural mRNA

      2. metal ion coenzyme factors used in the IVT…effects unknown

      3. endotoxins and proteins discussed by many

      4. enzymes used such as the T7 polymerase, proteinase K (and DNase 1) can introduce pollutants which can induce inflammation. Details on this is sketchy but were identified by the EMA and recommendations placed on improving the purity of the enzymes used in the IVT

      5. nucleoside triphosphate substrates (such as N-1-methylpseudoridine). Leftover substrates not used to make the mRNA could be left in the mRNA itself and persist and how can it be extracted? These may activate the neuroinflammatory mechanism in the CNS. HAS ANYONE THOUGHT OF THIS? extracellular nucleotides and neuroinflammation

3. The effects of large amounts of truncated and fragmented synthetic mRNA in the cell is unknown. I don’t think it just sits there, even if it is not translated. This article reviews the risks of synthetic mRNA in the cell. Potential health risks of mRNA vaccine Plus, these ARE likely being polyadenylated and translated contrary to what Pfizer’s scientists have published. If you don’t look, you won’t find it especially if you dont look IN VIVO.

4. The measurement of the mRNA at the end of the IVT (bulk drug substance) uses spectroscopy which overestimates RNA especially if there are DNA/RNA hybrids. But it is quick and easy and what Pfizer proposed to the regulators. More later.

The mRNA is a PRO-DRUG

1. this is key to understanding the risks of this therapy. The mRNA is a prodrug in that it has no intrinsic activity to elicit the desired pharmacological response (i.e. the stimulation of a specific immune response). The modRNA is unchanged and provides the instructions for the production of the active form (spike protein) in ribosomes. What proportion of the mRNA (intact mRNA) is translated to a spike protein with fidelity? 80%? So 80% of 60% is about 50% is translated faithfully. In likelihood it is much less as it is dependent on cell type and metabolic function of the cell.

2. Do we have any evidence of spike protein production from the vaccine mRNA? This was the first and most important Specific Obligation imposed by the EMA on both manufacturers. And the bases for #blotgate

3. This was the Western blot provided by Pfizer to the regulators

The regulators were not happy, and stated

a.     The presence of the two bands for BNT162b2 mRNA (at 100 kD and 190 kD respectively)

b.     The 76.5 kD bands are not observed in both BNT162b2 nor in S1 control lanes, and the full S protein at 141.14 kD is also not observed in BNT162b2 lane.

c.     The lack of an important expression for the S1 protein control at 76.5 kD”

But what is REALLY IMPORTANT IS THAT THE SAMPLE USED WAS FROM A PROCESS 1 BATCH.

4. Did Pfizer provide a proper Western blot from a Process 2 batch to the regulators at the time of renewal in November 2021 as was required? This is still an open question in my mind. This is a very complicated and multilayer question and reporting from Sonia Elijah has been the most complete on this.

   Sonia Elijah and blot gate

5. this means that frame shifting is a given, and that this mRNA is making proteins of which we have no clue what they do or how much is actually made. I think the spike protein hypotheses of harm is hanging on by the thread, though it maybe spike-LIKE proteins that are even more harmful than spike protein itself are one of the causes of harm from these products. Plus glycosolation. Plus polyadenylation. Plus other stuff WE HAVEN’T EVEN THOUGHT ABOUT.

The mRNA reacts with the LNPs

1. because of a tertiary amine in the LNPs, the backbone of the mRNA can form adducts with the LNPs. This renders the mRNA untranslatable. But what we don’t know is whether these adducts can also affect mRNA in the cell, or whether LNPs are forming adducts with our own mRNA

Here Moderna in its Science Day 2022 explains the adducts. The whole presentation is fascinating but the adducts are discussed starting on slide 50 to slide 83. This is a big deal. It may give an explanation of LNP toxicity to DNA and mRNA in our cells as well.

Moderna Science Day and Adducts

Process-Related Impurities: DNA

1. large amounts of residual DNA from the plasmid used to generate the mRNA. Kevin McKernan has discussed this in his substack.expression dsDNA vector is mRNA vaccines And of course, our work in Canada with 27 vials verified the SV40 elements and levels of residual DNA as well. 27 vial analysis

2. Regulatory elements which are known to ferry the DNA into the nucleus is present in the vials but were HIDDEN FROM THE REGULATORS, specifically the SV40 promoter/enhancer/ori.

3. The 72bp tandem repeat of the SV40 promoter is a nucleus location signal used to ferry DNA into the cells. It is USED IN GENE THERAPY EXPERIMENTS. See the work of Deans et al. Sequence requirements for plasmid nuclear import

4. there is a hidden gene with some homology to spidrion silk not disclosed to the regulators.

5. risk of insertional mutagenesis as well as oncological risks appear likely

6. the amount of residual DNA was underestimated by using qPCR

7. the role of small <200bp pieces of plasmid DNA needs to be elucidated

8. DNA fragments can also be making adducts as discussed above

9. polymerases and other chemicals and nucleotides could be present in the LNPs with unknown effects as discussed above.

10. dsRNA appears to be the common process related impurities that is well controlled by Pfizer. WHY? It is much harder to eliminate dsRNA than plasmid DNA fragments. Pfizers is much much cleaner on dsRNA compared to Moderna.

Purification of the mRNA drug substance

1. used DNase1 which was not certified/verified by the EMA and was suboptimal. This was identified by the EMA but little was done for 2 years despite the EMA citing this every 6 months or thereabouts. Plus the purity of other enzymes used in IVT delineated above. It is not just about DNase1. I think we are just scratching the surface of what process-related impurities ARE in these LNPs.

2. There were no chromatographic techniques used to purify the mRNA (considered standard and used by Moderna). WHY NOT? Pfizer used chromatographic techniques to purity the plasmid itself once it was linearized.

3. the filters for dia- and ultrafiltration used to filter out the DNA fragments were reused several times after sterilization in NaOH. EMA accepted this.(!!!) WTH?

4. contaminants such as endotoxin are not measured accurately and are likely underestimated with possibility for serious and synergestic effects.

See this review purifying the mRNA

Analytical Methods

1. What methods are used to analyze the purity of the mRNA. What methods are used to analyze the efficacy of these vaccines? And potential adverse effects. These are the critical quality attributes. Please read my substack on this issue because it is critical in understanding this mess.

   How is quality and purity measured

2. Lets look first at the “purity” and “identity” of the mRNA. What is tested?

   1. clarity, coloration, pH (used for all IV drug products)

   2. RNA integrity (% intact mRNA) capillary gel electrophoresis

   3. 5’ cap (RP-HPLC)

   4. poly (A) tail (ddPCR)

   5. RNA concentration for content measured using spectroscopy

   6. “Identity” of RNA sequence using RT-PCR to confirm the RNA is what it is

   7. In vitro expression of the spike protein: what percent of cells translate the mRNA vaccine? This is REQUIRED to establish the actual potency of the vaccine…is it doing what it is supposed to be doing?

3. Safety (considered standard for all IV drug products)

   1. endotoxin

   2. bioburden

4. Product related impurities

   1. dsRNA using immunoblot. This is a very important impurity to measure and control.

5. Process related impurities

   1. dsDNA using qPCR

OK. Based on the above discussion what is MISSING to determine safety, efficacy or purity?

1. NO SEQUENCING FOR EACH MRNA BATCH

2. NO SIZE DETERMINATION OF EACH MRNA BATCH (just because the proportion of intact mRNA is measured, that is measured indirected from the number of transcripts without a 5”cap)

3. THERE IS NO CORRELATE OF PROTECTION. JUST PROPORTION OF CELLS TRANSFECTED AND PRODUCING PROTEIN

4. THERE IS NO VERIFICATION OF THE PROTEIN PRODUCED (ie what the EMA would say is biological characterization)

5. PURITY INCLUDING

   1. aggregates such as the mRNA adducts

   2. percent fragmented mRNA

   3. quantification of free nucleotides

   4. residual T7 polymerase and other enzymes

6. dsRNA need MULTIPLE METHODS to determine quantity and size

Putting it all together.

1. EVERYTHING about the mRNA (except for the adducts) were known before they received their EUA

   1. codon optimization

   2. frame shifting

   3. excess mRNA

   4. refusing to provide an appropriate western blot

   5. using process 1 batches instead of process 2

   6. hiding the SV40 and reverse ORF (hidden gene)

   7. high levels of DNA

   8. use of analytical techniques to underestimate DNA and overestimate RNA

   9. endotoxin contamination etc

   10. possibility of nucleotide and enzyme contamination

2. All of the risks delineated above were known before the EUA (except the adducts, guess that was a real surprise). The regulators knew and asked for changes. It fell on deaf ears. Pfizer prevaricated and did not provide required data and analysis and that is strange as well.

3. It is instructive of what Pfizer improved with respect to the mRNA and what they refused to do

   1. what they did to improve the mRNA itself

      1. addressed the adduct issue

      2. removed the dsRNA to a very high degree

   2. what they refused to do despite Specific Obligation or Recommendations

      1. provide a true Process 2 Western blot of various batches

      2. improve the DNase 1 purity

      3. improve the purity of RNAse, Proteinase K etc

      4. inappropriate endotoxin measurement and mitigation

   3. what they did with aforethought

      1. used known codon optimization and other genetic engineering techniques to make a durable, long lasting mRNA with known issues for fidelity on translation and on cell signalling

      2. did not use state of the art purification techniques such as chromatography to purify the mRNA

      3. excluded the presence of the SV40 promoter/enhancer/ori

      4. excluded the presence of other regulatory sequences

      5. exclude the presence of another ORF or hidden gene

      6. used unusual analytical methods or non comparable analytical methods

         1. spectroscopy for bulk RNA, flummery for RNA in the LNPs of the drug product itself, qPCR for dsRNA

         2. a weird ddPCR for measuring poly (A) tails

      7. no sequencing of the mRNA batches

      8. made batches of mRNA with widely differing levels of dsDNA but low levels of dsRNA

Although I am not totally certain, the development and manufacturing of the Pfizer mRNA product appears to be a DNA vaccine with the distinct risk of DNA integration. The amount and proportion of actual S protein produced by the mRNA itself is unknown. The quality of this S protein is unknown. It does not appear to me, that the primary objective of this product is to produce neutralizing antibodies to the virus. This may occur secondarily but it is, in my opinion, not the primary purpose. To what purpose, then? We can speculate, but there is a lot here that points to aforethought of harm or potential harm. Even Pfizer, at least in the past, would not allow such egregious harmful elements. Elements with potential harm which could be explained as unknown or weakly known effects but this mRNA has many KNOWN harmful issues prior to manufacturing which was compounded by the poor and suboptimal manufacturing and testing itself.

I am of the opinion, that neither Pfizer/BioNTech nor the regulators are in charge of this operation and I hope I have delineated why I think this is the case. This product would have been limited, improved in many aspects if things worked as they should, even in a “pandemic’ situation, especially after Omicron. Most of these findings show harm or intent to harm.

I believe this Christmas, hold on to your dear ones, forgive them for any past harms and ask that our Lord and the Blessed Virgin Mary to mitigate any further harm.

And pray the rosary every day. Just in case