THE PBS BUFFER CAUSES HIGHLY STABLE FOLDED FORM OF RNA

This is a reason why the BioNTech changed the PBS buffer to the Tris buffer.

We discovered a further new BioNTech patent.

We call for verification of this patent by experts and volunteers.

The patent publication number is US20230414747A1 and the applicant is BioNTech SE. The US20230414747A1 was published on Desember 28, 2023. This patent application claims priority to the provisional application 63/115,128 filed in the USPTO on November 18, 2020. This patent application has not been registered yet.

The filing date of this patent application is deemed to be November 18, 2020.

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BioNTech (Pfizer) changed the PBS buffer to the Tris buffer at the beginning of the introduction of the mRNA-LNP vaccine into the society.

This patent discloses the basis why BioNTech changed the PBS buffer to the Tris buffer in the paras [1023] to [1026] as reproduced below.

[1023]

RNA LNPs were prepared by the aqueous-ethanol mixing protocol using 20 mM Tris added to the organic phase. LNPs were generated in 50 mM Tris:acetate pH 4, pH 5.5 or pH 6.8 and the resulting primary LNPs were split: one portion was subjected to dialysis against PBS (A); the other portion was subjected to dialysis against 50 mM Tris:acetate pH 7.4 (B). For comparison, the organic phase did not receive Tris, LNP were generated in 50 mM Na-acetate buffer pH 5.5 and the material was dialysed against 50 mM Tris:acetate pH 7.4. All samples were stored for 50 h at room temperature. The RNA integrity was measured as described above using capillary electrophoresis. The results are shown in FIGS. 2A and B.

[1024]

RNA LNP compositions containing a cationically ionizable lipid, in particular lipid I-3, and PBS adopt a highly stable folded form of RNA (detectable as tailing of the main peak at about 2190 sec). This is also true if the LNPs were prepared in buffer other than PBS (such as Tris), i.e., in the absence of PBS, and during the dialysis the buffer was exchanged to PBS; cf., FIG. 2A. In all these samples, the amount of this highly stable folded form of RNA was between 18% and 21%.

[1025]

However, using the monovalent buffer substance Tris (instead of the polyvalent PBS) in the composition (i.e., in the preparation in which the drug product is stored, shipped and administered, when formulated as ready-to to-use composition) inhibits the formation of the highly stable folded form of RNA; cf., FIG. 2B.

[1026]

Thus, from these results, one can conclude that it is sufficient to add Tris during dialysis in order to inhibit the formation of the highly stable folded form of RNA. In contrast, the addition of Tris only in the upstream parts of the LNP preparation process does not protect from the formation of the highly stable folded form of RNA when the primary LNP formulation is subjected to dialysis against PBS.

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BioNTech found it problematic that the PBS buffer produced a highly stable folded structure in the LNP, and solved the problem by changing the PBS buffer to the Tris buffer (see para [0912]).

[0912]

The present inventors have surprisingly found that using a buffer based on Tris, Bis-Tris-methane or TEA, in particular Tris, instead of PBS in a composition comprising LNPs inhibits the formation of a very stable folded form of RNA.

In other words, this patent is a corroborating evidence that BioNTech was aware of the risks of the highly stable folded structure being released in the human body.

According to the Drug Product document and the EMA document, this problem does not occur at the DS (Drug Substance) stage but at the DP (Drug Product) stage.

Therefore, this is a problem caused by the mRNA wrapped by the  LNP. We focused on this fact and arrived at this patent and that paras [1023] to [1026] by carefully examining multiple patents.

Pfizer remarks "conformationally folded or reversibly aggregated RNA" in the Drug Product documentation, while remarking "highly stable folded form of RNA" in this patent. In other words, Pfizer is attempting to trivialize the risks of "the highly stable folded form of RNA" by replacing to the other term, in the Drug Product document.

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Kevin McKernan has disclosed the research related to this on the Substack and discovered the long mRNA with the RNA seq. We must not forget the fact that most of the expression vectors have been identified through this process.

Moderna has used the Tris buffer since the beginning.

On the other hand, there are no studies on Pfizer vials after changing to the Tris buffer. If electrophoresis is performed using a Pfizer's vial after changing to the Tris buffer and no improvement was observed, then that could also be a reason for a lawsuit.

This patent newly raises one possibility that Moderna adopted the Tris buffer since they could not remove the late-migrating species.

The mRNA-LNP vaccines including the PBS buffer before changing to the Tris buffer were actually introduced into the human body early on. Therefore, there is some concerns that this may cause adverse reactions.

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The hint that led me to investigate this issue from a patent perspective was the following post by 苦労人の改 (@5rHxIhQGQnnRSOe).

https://x.com/5rHxIhQGQnnRSOe/status/1751436200332997011?s=20

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Many people focus on this issue and feel that the mRNA-LNP vaccines are in full of deception.

We hope that this disclosure of this patent will be further scrutinized by experts, and various issues including the mechanism of adverse reactions caused by the mRNA-LNP vaccines will be elucidated.

Thank you.

Cultivate 2

mbi

Patent SUN

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Reference lists

US20230414747A1 (USPTO)

https://image-ppubs.uspto.gov/dirsearch-public/print/downloadPdf/20230414747　

US20230414747A1 (Google)　

https://patents.google.com/patent/US20230414747A1/en?oq=US20230414747A1　

EMA document 

https://docs.google.com/document/d/1K-dWmwzH-gbsPstj3bTfYgS1DUTEqIn1uA2T5-4jZr0/edit 

Annex 1 - Draft 3.2.P.2.2 Drug Product

https://drive.google.com/file/d/1rxVfN5_eItZFUkFFg6Ansl53Mnijsjqr/view

Substack/ANANDAMIDE

Deep sequencing of the Moderna and Pfizer bivalent vaccines identifies contamination of expression vectors designed for plasmid amplification in bacteria