3.1.1 Deoxyribonucleic acid (DNA) is named for the sugar molecule, deoxyribose, that makes up the backbone of the double-strand helix. Most people will be familiar with the elegant, lightly twisting image of this nucleic acid. The two deoxyribose polymer strands bind to each other through the complementary pairing of bases between them, and the order of bases creates the digital code that is the information of life. While complementary bases in DNA are held together by only two or three weak hydrogen bonds, the large number of these bonds across the molecule makes DNA remarkably stable. The smallest human chromosome, 22, is a single chain containing 51 million bases. DNA is so stable that we have isolated relatively long fragments from long-dead plants, animals, and microbes. Stability of DNA has enabled forensic science and the study of ancient human family trees, to name just two applications. The imaginary basis of the fictional Jurassic Park movie franchise is ultimately due to this very real and simple trick of biochemistry.
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3.2.1 Messenger RNA intended for use as a vaccine or for therapeutic indications typically has certain structural features that ensure stability and efficacy ().An mRNA transcript is usually ~1,000 nucleotides long and always single-stranded. The core feature is the GOI that contains the open reading frame that will be translated into the functional protein. At the 5' end is an N7-methylguanosine nucleoside, which is linked via a 5'-5' triphosphate bond to the 5'-terminal nucleoside of the mRNA (Cap‑0). An additional 2'O-methylation at the +1 nucleotide enhances recognition of the molecule as self (Cap‑1) and thus is also desired in most instances. This is the same chemistry found in a natural eukaryotic mRNA and is required for translation, splicing, and affording protection from exonucleases.
3.2.2 A string of ~100 adenosine nucleotides at the 3' end of the transcript, (the poly-(A) tail) is also a feature of naturally occurring mRNA that imparts stability by preventing nuclease digestion and aids in protein translation. (Natural mRNA poly-(A) tails are about 250 nt long, but only 100 nucleotides are required for optimal translation efficiency for therapeutics.)[20] In between the 5' cap and poly-(A) tail are untranslated regions (UTRs) on each side of the actual coding sequence (CDS) for the gene of interest. These regions impart secondary structure that impacts translational efficiency.
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3.3.1 The manufacture of mRNA typically begins with a plasmid DNA (pDNA) template containing a DNA-dependent RNA polymerase promoter and the corresponding sequence for the mRNA construct.[20] The pDNA is most commonly produced via bacterial fermentation followed by purification of the plasmid DNA.[50] As with all therapeutics that are manufactured from a starting cell, mRNA developers will need to create a GMP master cell bank for the pDNA template.
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3.4.1 Conversion of pDNA to mRNA via IVT dominates large-scale mRNA manufacturing. RNA polymerases derived from bacteriophages are used in combination with chemically modified ribonucleoside triphosphate molecules (rNTPs), the RNA polymerase, pDNA template, and RNase inhibitors.[4] Other reaction components include the polymerase cofactor MgCl2 and a pH buffer containing polyamine and antioxidants.[49]
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3.5.1 Downstream processing of the mRNA bulk drug substance must be performed to remove impurities, including endotoxin, immunogenic dsRNA, residual DNA template, RNA polymerase, and elemental impurities, among others.[4][20][49] Design of the purification strategy depends largely on the capping strategy employed but generally will begin with tangential flow filtration (TFF) to exchange the buffer and concentrate the reaction solution. TFF also removes smaller impurities not retained by the membrane, with the size cutoff (typical range 30–300 kDa) depending on the size of the mRNA molecule. It is important, however, to be aware that, during TFF, small DNA fragments can hybridize with the mRNA, creating more impurities.[49]
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3.6.1 Four different types of lipids are currently used to form the LNPs: a cationic or ionizable lipid, polyethylene glycol (PEG), phospholipids, and cholesterol. The specific choice of lipids and their ratios is tailored for each mRNA to provide maximum protection from degradation, enhance delivery to target cells or tissues, and facilitate cellular entry.[20] A simple diagram of common LNP structures is presented in Figure 6.
3.6.2 The most common method for LNP formation involves continuous self-assembly.[4] In this process, lipids dissolved in ethanol or a similar solvent are rapidly mixed with an aqueous solution of the mRNA drug substance using crossflow or microfluidic mixing. Microfluidic mixing is advantageous because it allows for nonturbulent mixing, is scalable from lab to commercial production, and can be monitored using PAT.[4] A TFF step then provides the mRNA–LNP product in the desired final buffer solution at the desired concentration.[20]
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