Massive Endotoxin Contamination introduced on move from Process 1 to Process 2 mRNA

Pfizer lied to EMA for its Emergency Use Authorization claiming it could control Endotoxin in industrial scale-up. Nobel Prize winner Drew Weissman and his team of 32 helpers failed to clean it up.

Many moons ago I reported what we could find in Pfizer documents released under Court Order, or leaked, on the filthy E. coli production of jabs that were not tested on more than 250 people before Billions of jabbed people suffered the Endotoxin consequences.¹²

I have previously referred to work performed at Duke University by Nobel Prize winner Drew Weissman³ on the problems involved but my friend Chris Edwards prompted me to write about one paper⁴ ⁵ that was published in 2021 that openly discussed the Endotoxin problem.

Here is the Plasmid they made.

Readers who have been following Kevin McKernan will be familiar with this type of figure. Note the transcriptional point of origin (ori in Yellow), the Kanamycin Resistance Gene (KanR in Green) and the sequence needed to generate the mRNA (Immunogen A, in Purple).

You can read the full paper or download online, but here I just highlight the fact that they mention Endotoxin 13 times.

We found that endotoxin control was the primary raw material risk due to its variability in raw material lots and the difficulty of its removal in subsequent downstream purification steps. Endotoxins can be introduced to the product primarily from IVT reaction components and large volume buffers used in purification.

and

Additional controls, such as release testing of incoming materials to detect endotoxin, vendor testing for nucleases, and sequencing to verify material prior to use are a few examples of outcomes derived and implemented from the risk assessments.

and in Table II which shows the 300ml scale results

and on “meeting regulatory expectations”

As with more established therapeutic modalities, the testing panel includes evaluation of quality attributes such as appearance, pH and osmolality, and safety tests for measuring endotoxin, bioburden and sterility.

On their Influenza jab safety for sacrificed Animals

For each antibody sequence to be tested, the mRNA heavy and light chain sequences were mixed at a 1:1 molar ratio, encapsulated into lipid nanoparticles by Acuitas Therapeutics (Vancouver, BC) and tested for acceptable endotoxin levels for testing in mice and non–human primates (NHP).

On moving to Human Jabbees

In our take on this approach, the impact to patient safety, operator safety, product quality, and regulatory hurdles can be assessed for each new material, along with adding additional controls such as release testing of incoming materials to detect endotoxin, vendor-based testing for RNase, and sequencing to verify materials prior to use.

Use of Endotoxin Removal Buffer and Chromatography

Plasmid production

The DNA plasmid is manufactured and purified by a commercial manufacturer. E. coli cells are transfected with 1–5 ng of cDNA plasmid. Transformed cells are inoculated into animal-derived component free growth media containing kanamycin. Following fermentation at 30°C for approximately 16 hours, bacterial cells are harvested by centrifugation. The cell paste is subjected to alkaline lysis with RNase A. The cell lysate is then clarified by centrifugation and adjusted to chromatography loading conditions through the addition of endotoxin removal buffer.

They saw massive increase of Endotoxin from 0.036 EU/ml, through 0.5 EU/ml, finally getting ≤30 EU/mg - note change of unit from ml to mg, for Plasmid release in their Table V.

Note that they fail to report Endotoxin measurements in Table VIII for mRNA drug substance, or Table IX for Release testing for mRNA-LNP drug product.

We know the LNP materials introduce additional Endotoxin.⁶

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4. Download pdf. Jill Whitley, Christopher Zwolinski, Christian Denis, Maureen Maughan, Leonie Hayles, David Clarke, Meghan Snare, Hong Liao, Sean Chiou, Tina Marmura, Holly Zoeller, Ben Hudson, John Peart, Monica Johnson, Amelia Karlsson, Yunfei Wang, Cynthia Nagle, Cherell Harris, Daniel Tonkin, Stephanie Fraser, Lieza Capiz, Christina L Zeno, Yvonne Meli, Diana Martik, Daniel A Ozaki, Amy Caparoni, Jason E Dickens, Drew Weissman, Kevin O Saunders, Barton F Haynes, Gregory D Sempowski, Thomas N Denny, Matthew R Johnson. 2021. Translational Research. 242:38–55. https://www.translationalres.com/article/S1931-5244(21)00282-6/fulltext

5. Read online. https://pmc.ncbi.nlm.nih.gov/articles/PMC8641981/

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