"Characterization of BNT162b2 mRNA to Evaluate Risk of Off-Target Antigen Translation"

This is must write-up...

Again, thank you to David Wiseman for throwing this into my email inbox this morning. It’s a big one guys.

Before I even start, all of these experiments need to be reproduced by a non-conflicted lab (or two) to confirm or deny their results.

No more of this ‘I am the criminal, jury and judge’ crap anymore. If my readers take nothing else from this Substack, this message, you should take.

This paper is hot-off-the-presses - published January 12, 2023 - and entitled: “Characterization of BNT162b2 mRNA to Evaluate Risk of Off-Target Antigen Translation”

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in the Journal of Pharmaceutical Sciences by Analytical Research and Development, Biotherapeutics Pharmaceutical Sciences, Pfizer Inc.. Pfizer, eh? Interesting. I wonder what they will report? Definitely something good, right. For them, I mean.

First of all, to be super litigious, I want to make a point about the language employed in the very first phrase of the abstract:

mRNA vaccines have been established as a safe and effective modality, thanks in large part to the expedited development and approval of COVID-19 vaccines.

Modality? I smell a CDO. Even though, by their own suggestions, “additional mRNA degradation mechanisms (e.g., through interactions with lipid excipients in the formulated drug product can potentially affect antigen translation”, they start the report of their findings by saying that these mRNA shots are ‘safe and effective’. But worse than that: they back this statement by stating that this ‘safety and efficacy’ is due to these products having been expedited through traditional safety testing procedures.

Oh, no. Just: no. Read the take home message of Slide 5 in my upcoming presentation below. It reads: “GENUINE SAFETY TESTING IMPOSSIBLE”. Yeah, it was impossible.

Figure 1: Slide 5 from Slide Deck for International conference on the COVID pandemic. Dr. Jessica Rose

This is also how they start the introduction as well: it’s almost as if they are trying to get themselves out of hot water with their ‘em, er, so… about that’ language. They claim in the introduction that after decades of failing to bring mRNA technology to humans, that the key thing needed to make it work was the replacement of uridines to pseudouridines in order to enable evasion of the innate immune system. Not by my understanding. By my understanding, the key thing needed to enable the use of mRNA technology was the enabling of the delivery vehicle that was very much dependent on the cationic lipid issue - magically, both Pfizer (ALC-0315) and Moderna (SM-102) found a cationic lipid that housed the mRNA inside the LNP in order to ensure cuddly and optimal transfer to cells for eventual translation by host cell machinery.

Bottom line: Without the LNP, there is no mRNA technology, pseudouridines or not!

They write:

mRNA fragment species are a product-related impurity that can be generated during the in vitro transcription (IVT) process through degradation of the full-length mRNA transcript.

Yes, we know. That was revealed in EMA leaked documents. You were meant to be addressing this issue and fixing it if possible. I guess this paper is that address. Good on ya. Timing’s interesting though. Right after #blotgate started. I’ll return to this.

They go on:

mRNA fragments generated during the IVT process can be present in the drug substance; however, fragment levels are controlled by an appropriate RNA integrity acceptance criterion.

Right. But we also know that the acceptance criterion was lowered to accommodate this low %RNA integrity issue.

Figure 2: Slide 41 from Slide Deck for International conference on the COVID pandemic. Dr. Jessica Rose

They definitively state at the end of the introduction that there’s no problem with the truncated species of RNA because they aren’t translated to proteins, according to their investigation. I would very much like to see that raw data and those blots.

Thus, no undesired protein by products derived from these fragments can be expected for the BNT162b2 vaccine.

‘Can be expected’. Well, I’m convinced.

Before I go on, I must give you guys some background on what they did here to generate the figures in the paper. The Western Blots, I have covered. Links below as well. All you need to know is that is a long, annoying methodology - whose endpoint are those little ‘bands’ on a membrane representative of expressed proteins of certain sizes - is the essence of the Western Blot. The HPLC, not so much easy. So here’s a crash course.

On High performance (or Pressure) Liquid Chromatography (HPLC)

HPLC is a neat way to separate, identify and/or measure things is a sample. The Pfizer lab rats used what is known as Ion Pair Reverse-Phase HPLC (IP-RP-HPLC). RP-HPLC relies on hydrophobic interactions → more water = higher retention times for less polar molecules → polar molecules elute more readily. RP-HPLC allows the measurement of these interactive forces. IP-RP-HPLC adds the element of enhanced peak shapes and retention times.

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There. That’s all you need to know. You can also watch this video for a visual description.

The output of IP-RP-HPLC is a graph that represents the components in a sample, separated by elution time. The output looks like Figure 3, whereby each of those ‘peaks’ represents the retention time of the individual components.

Figure 3: HPLC plot. https://en.wikipedia.org/wiki/High-performance_liquid_chromatography

The presence of a peak means the presence of something based on its time to elute (elution is like ‘letting go’ of the component from the adsorbent it’s clinging to - kind of like that annoying person you used to know in high school who never grew up), as compared to a standard curve - a predetermined established curve. So if you have different peaks, you have different components. These components are always compared to a standard so that you can know what component you’re looking at.

Back to the paper…

The Pfizer lab rats checked RNA fragments (by a couple of different means - which is awesome), and intact RNA by assessing the presence (or absence) of the 5’ cap, the poly-A tail, along with protein (antigen) by-products. They used cell-based and cell-free methodologies combined with Western Blotting (both old-school and Robo-Jess techniques) to assess the presence of the latter, and IP-RP-HPLC to assess the former.

They hypothesize in the results that the low %RNA integrity bits, derived from the 5’ end of the mRNA, were, in fact low integrity, because of problems in the IVT step, or possibly from hydrolysis of full-length transcripts.

Based on manufacturing process knowledge and the directional 5’-to-3’ polymerase transcription, these low-level species were hypothesized to be 5’-end BNT162b2 fragments generated from premature transcription stops during the IVT reaction.

They found that both the full-length BNT stuff and the fragments were 5’ capped but that only the BNT stuff had the poly-A tail. I had written about this and suspected as much a while ago. You can read about that here, or watch here.

Figure 4: Comparative HPLC plots from the paper and from a FOIA-requested data release from the TGA. https://jpharmsci.org/article/S0022-3549(23)00009-6/fulltext; https://twitter.com/Jikkyleaks/status/1610888191342710787

Jikkyleaks wrote about the problematic issue with the extra hump (indicated by the 3 red stars) in a post on Twitter on January 5, 2022, and as you can see from the side-by-side screenshots in Figure 4, these plots are indeed referring to the same peaks found as per the fragmented species (peak 1 - red trace) and the intact RNA (peak 2 - black trace) referred to in Figure 2 in the paper. Jikky had raised some very good questions pertaining to the natures of the fragmented species.

The Pfizer guys claim that this peak is not a problem and that these fragments will never make it to protein production. They write (Discussion)":

Low level fragments observed in “Peak 2” are thought to either originate from carry-over during “Peak 1” fractionation or from low levels of mRNA hydrolysis that could occur during sample handling.

Here are the ‘blots’ to show that the BNT stuffs were in-tact - with S1 and S2 domains (Domain 1 and 2). Wow! What Western Blot perfection is this!? If only this was relevant to reality. I wrote this up as part #Blotgate: an ongoing saga to reveal the shenanigans of corrupt, greedy corporations whose interests lie in profits not people. These Western Blot results are the product of Robo-Jess (not kidding) and I, and many other serious lab rats, are not impressed. Please do read my Substack on this entitled “A follow-up about Western Blots”. Figure 5 are the ‘Western Blot results’ from the cell-based study they did.

Figure 5: Expression analysis of mRNA transfected HEK-293 cells demonstrates the requirement for 5’-cap and poly(A) tail for protein translation. Figure 4 from https://jpharmsci.org/article/S0022-3549(23)00009-6/fulltext

Their perfect results show that without the 5’ cap and poly-A tail, no expression of protein.

Ok so let’s assume that their Western blots done by Robo-Jess are legitimate. Where are the Westerns for the questionable RNAs that lack poly-A tails? Oh it’s coming. Don’t worry.

So they took the full-length BNT stuff and made truncated species of RNA without the head (5’ cap) or the tail (Poly-A tail) and tried to make proteins out of them. What happened? Did they succeed? Well, no. They found that in order for spike protein to be expressed (as determined by protein bands on their Western Blots), you need both the head and the tail. Good enough. They also checked out the original fragmented RNAs against the original full-length template as per peak 1 and peak 2 from above, and also, tremendously consistently, as demonstrated using Robo-Jess, that there was zero point zero zero expression in the context of the former, as shown in Figure 6 below.

Figure 6: Figure 5 from the paper showing Robo-Jess WB results for the fragmented mRNA lack of expression and full-on expression in peak 2 - representative of the BNT crap. https://jpharmsci.org/article/S0022-3549(23)00009-6/fulltext

They also checked out fragments that they made themselves from the full-length template using temperature modifications. Figure 7 below is the blot from an actual Western Blot showing the protein by-products of the various ‘fragments’ produced as a result of heating up the BNT stuff. My first question is, why did they use the old-school Western Blot technique (complete with air bubble! - bad rolling) to demonstrate the decreasing expression of proteins according to increasing degraded RNA?

Figure 7: Made fragments apparently have bad expression levels as shown in cell-free expression system. https://jpharmsci.org/article/S0022-3549(23)00009-6/fulltext

The part that bothers me about this particular result is that their conclusion that the BNT fragments lack translational viability, is based on their positive and negative control definitions and I wasn’t really satisfied with what those were. What were the positive and negative controls? Did I miss that. I might have.

They write:

…no truncated or other protein species were detected beyond the background bands observed in the negative control sample.

The negative control was what now? If someone can find where they wrote what they used for positive and negative controls, let me know. I might be having a ‘forest for the trees moment’.

Just to remind everyone, it has been reported here that the human enzyme neutrophil elastase (NE) can cut spike up into bits and that some of those bits have amyoidogenic properties. So the question about amyloid protein production is a very important question to answer.

Figure 8: Slide 44 from Slide Deck for International conference on the COVID pandemic. Dr. Jessica Rose

So that’s about it for the paper’s results. Their Discussion is revealing as are their conflicts of interest.

Here are my questions to Pfizer for now:

1. Why did you use Robo-Jess to show the BNT protein products and regular old-school Western Blotting technique for the cell-free fragment experiment where you replaced the lysines with biotinylated lysines. I mean, is that why, because of the lack of requirement of detection antibodies?

2. You’ve ‘demonstrated’ here that ‘fragments generated either during in vitro transcription or via mRNA hydrolysis lack capacity for protein translation’, but the question remains - and you cannot ignore it - what happens in vivo? Are you willing to bet the lives of millions of recipients of this product on this finding?

3. And perhaps the most important point is raised by you guys at the end and I wonder if you will follow-up on the question of hidden open reading frames?

They write in the Discussion:

It is, however, important to assess the off-target protein translation risk for new construct designs, for example to evaluate novel sequence elements (e.g. alternative poly(A) tail designs or the potential introduction of hidden open reading frames within the gene of interest or during construct design and optimization.

Yes. It is. And it was. Did you evaluate potential introduction of hidden open reading frames with the gene of interest during codon optimization? It seems like you didn’t to me. There are many peptides of interest with potential for immunological dysfunction discovered in the spike protein; from amyloidogenic peptides to the furin cleavage site, and a large array of pentapeptides that are implicated as molecular mimics with potential to induce autoimmunity in vivo.

The authors claim that it was ‘outside of the scope of this study’ but that “additional mRNA degradation mechanisms (e.g., through interactions with lipid excipients in the formulated drug product) can potentially affect antigen translation”. Really? Through interactions with lipid excipients in the formulated drug product? Can you be more specific? Which lipid excipients? PEG? ALC-0315?

They conclude by repeating, once again, that they ‘needed’ to rush this shit into billions of people’s bodies.

And oh, yes, this study was completed and written up by Pfizer employees. You have to ask yourselves then, why wouldn’t this company - that has paid the most in fines in the history of fines in the world for ‘improperly marketing 13 medicines’ - paint themselves in a good light in the context of a product that’s already in billions of people? Other people bound by contracts with Pfizer are legally obligated to write their product up in a good light, in fact.

Figure 9: Conflict of Interest statement made by authors. https://jpharmsci.org/article/S0022-3549(23)00009-6/fulltext

All in all, I am not impressed and I have three final summary points

1. This paper was published (whatever that means - I honestly don’t know anymore) on January 12, 2022 - a mere 7 days after Jikkyleaks went public with his TGA FOIA-requested data revelations. Then we united to announce #blotgate.

2. Why are the results so bloody perfect? This doesn’t happen in biology. I don’t care how sponsored by Pfizer you are: there are always imperfections. That’s the joy of doing things with your hands whilst thinking.

3. Why would Pfizer lab rats ever be allowed to present results in the form of a publication like this that wouldn’t paint their product in a good light? That would mean very bad things for them, wouldn’t it.

All they can do now is keep lying. They have nothing to fall back on but bankruptcy and criminal charges if they admit to any negligence or fraud on their part. It’s pretty simple really. The people have been duped.

1. Patel HK, Zhang K, Utegg R, Stephens E, Salem S, Welch H, Grobe S, Schlereth J, Kuhn AN, Ryczek J, Cirelli DJ, Lerch TF. Characterization of BNT162b2 mRNA to Evaluate Risk of Off-Target Antigen Translation. J Pharm Sci. 2023 Jan 12:S0022-3549(23)00009-6. doi: 10.1016/j.xphs.2023.01.007. Epub ahead of print. PMID: 36642376.

2. https://lab-training.com/ion-pair-chromatography-a-viable-alternative-for-separation-of-high-polarity-molecules-by-reverse-phase-hplc/