dsDNA contamination Process 1 vs Process 2 (Pfizer): Part 1

Was it intentional?

Upscaling the manufacturing: Process 1 vs Process 2

The manufacturing process of the modRNA was changed substantially for the commercial scale-up lots (Process 2) from the pilot-scale process used to produce the BNT162b2 vaccine candidate for the clinical trials (Process 1). This had implications for Good Manufacturing Practices (GMP), risked Marketing Authorization, and has implications clinically.

Process 1

Process 1 was used to produce modRNA evaluated in the clinical trials. Using a cell free method, linear DNA was amplified using the polymerase chain reaction (PCR). This results in a linear template including the open reading frame for the S1S2 protein, and the 3’ and 5’ UTRs. The 5’ cap is added enzymatically as is the poly (A) tail. However, this technique does not produce sufficient modRNA for commercialization for billions of doses in the time frame and fidelity required as is possible from plasmid DNA.

Process 2

Process 2 used a DNA plasmid in E coli which includes the poly (A) tail to multiply the DNA. Then it is linearized and the IVT processes are virtually the same. The steps in purification differ however between the 2 processes as seen below.

DNase 1 digestion

An enzyme used to break up the DNA. Commercially available. Both processes need to do this. Small fragments are then left (supposedly but now we know different).

see here

Magnetic Bead Purification

This is really neat. And they can purify and extract the mRNA very well. The beads are treated with ligands to attract mRNA specifically. you add the bead and place a magnet to the tube. Wash everything well and then remove the magnetic field. Voila! It’s mostly a manual process so it cant be scaled up well. Therefore this was not used for Process 2 jabs.

magnetic bead purification for mRNA

Proteinase K Digestion

So for Process 2 they used Proteinase K Digestion which break down proteins, these are the proteins from the E coli.

proteinase K

Ultrafiltarion/Diafiltration

THEN they have to do a series of filtration steps to get rid of all the bits and pieces of proteins from E coli, the DNA template, endotoxin, the chemicals used to make the mRNA etc etc.

Often tangential flow filtration is combined with chromatographic techniques for better purification. One of my favourite papers explains the difference between process 1 and 2 very well. Hope this gives you a general idea of the overall purification steps used between the 2 processes. There were also issues with the IVT and poly (A) tails and capping, but we are concentrating on dsDNA here

Sousa-Rosa et al