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PharmTech.com The Real Message Behind Commercial mRNA Products April eBook 2023 Pharmaceutical Technology ® 43 AnAly tics therapy medicinal products (ATMPs) by the European Medicines Agency and as biologicals by the World Health Organization (4,9,10). As such, they are subject to quality regulations related to biotechnological products in ICH guidelines, for example, ICH Q5C (stability) or ICH Q6B (specifications) (9). Regulatory considerations for mRNA therapeutics are similar to biologics in the sense of starting material integrity, good manufacturing prac‑ tice (GMP) manufacture consistency, and quality control of the drug substance/product. Chemistry, manufactur‑ ing, and controls management of enzymes, nucleotides, and linear DNA templates used for IVT synthesis is cru‑ cial for maintaining quality. CQAs of the DS are mRNA purity, capping efficiency, and heterogeneity of the poly A tail or base sequence. CQAs of the DP are mRNA en‑ capsulation, size distribution and heterogeneity of the delivery nanoparticle, stability, and sterility (11). Purification Initial removal of residual double‑stranded DNA tem‑ plate, un‑incorporated ribo‑nucleoside triphosphates, or enzyme is required to increase DS purity, as their presence can inhibit protein translation, cause immu‑ notoxicity, and affect manufacturing variability and quality (12,13). Although electrophoretic methods can enable purification of the mRNA, the employed dena‑ turing reagents and acrylamide may cause covalent modifications or affect the molecular integrity of the mRNA therapeutic. An alternative approach is the use of size‑exclusion chromatography (SEC), which facilitates purification of native state RNA. To remove IVT contam‑ inants, such as the plasmid template, an agarose‑based gel matrix was used in conjunction with a mobile phase consisting of 150 mM sodium chloride, 50 mM sodium phosphate, and 0.1 mM EDTA (pH 6.5) (14). Size exclu‑ sion columns are also compatible with fast performance liquid chromatography (FPLC) systems for rapid purifi‑ cation (15). However, SEC is limited by low resolution of similarly sized polynucleotides and may not be able to purify all possible fragment contaminants. Ion‑pair (IP) reversed‑phase chromatography has also been demonstrated as an efficient approach to remove IVT contaminants (16). mRNAs were purified using a polymeric alkylated, non‑porous polystyrene‑divinyl‑ benzene stationary phase and a mobile phase consisting of 0.1 M triethylammonium acetate, pH 7 with 25% v/v acetonitrile as eluting solvent. The effective purification of mRNAs in this way resulted in a 1000‑fold increase in the translation efficiency, indicating the critical na‑ ture of the purification step. Other chromatographic approaches may involve affinity‑based purification where the mRNA is captured on the solid phase using embedded poly‑dT to hybridize with the mRNA poly‑A tail (17) or anion exchange (AEC). Chromatographic pu‑ rification of mRNA is a widely accepted gold standard due to ease of scale‑up, and may be coupled with other filtration methods, such as tangential flow filtration or precipitation, to further remove contaminants (12). Analysis of drug substance mRNA is composed of multiple structural elements that require characterization to determine the DS identity, consistency, and heterogeneity. The 5' cap structure en‑ sures that the mRNA is recognized and stable against enzymatic degradation. The 5' cap can be attached to the IVT mRNA transcript during or after synthesis using capping enzymes or by including a dinucleotide cap analogue to the IVT reagent mixture, respectively. As the latter capping approach can lead to mRNA with the analogue in the reverse orientation, the yield can DNA plasmid Linearized DNA template + RNA polymerase + nucleotide triphosphates + cap analogues + capping enzyme 5' Cap 5' UTR ORF 3' UTR Poly A tail FIGURE 2. In vitro transcription. A DNA plasmid is linearized and used as the template for RNA polymerase to build a transcribed mRNA from nucleotide triphosphate monomers. The mRNA consists of a 5' Cap, untranslated regions (UTR), and a polyadenylated tail.