Purification of IVT mRNA by 5'cap affinity chromatography
Despite the remarkable strides in mRNA therapeutics,exemplified by the development of COVID-19 vaccines,their widespread application faces various challenges.These obstacles encompass concerns related to immunogenicity,inefficient in-vitro synthesis,and instability within preparations.A prominent factor contributing to these challenges is the presence of uncapped mRNA molecules,lacking the essential 5'cap and its 7-methylguanosine(m7G)modification.This cap plays a pivotal role in enhancing stability,initiating translation,and evading the immune system.The absence of the cap leads to rapid degradation and potential immune activation.Current capping methods for in vitro transcribed mRNA(IVT mRNA)fall short of completely eliminating uncapped molecules.Both post-transcriptional capping and co-transcriptional approaches add the cap structure,but they cannot guarantee 100％efficiency.Due to the minor chemical distinction between capped and uncapped mRNA,there has been no established method for separating the two.Consequently,in the current pharmaceutical manufacturing of mRNA,uncapped RNA carries over from IVT reactions to the final drug products.This study bridges this critical gap by introducing a novel cap-affinity chromatography method for highly selective purification of IVT mRNA.Our approach harnesses the potent m G-binding capabilities of eukaryotic initiation factor 4E(eIF4E),a protein that plays a pivotal role in translation initiation by recognizing and binding the cap structure.We conducted a rigorous screening of 18 proteins,including eIF4E homologs from viruses,evaluating them for thermal stability,cap-binding affinity and specificity,and high-level recombinant expression.Three exceptional candidates emerged:(1)Ct.eIF4E from Chaetomium thermophilum,a thermophilic fungal eIF4E with excellent cap binding and thermal stability;(2)the La4E from Thermomyces lanuginosus,another thermophilic fungal eIF4E with strong cap affinity and stability;(3)human eIF4E mutant K119A,a modified human eIF4E exhibiting highly specific cap binding and good stability.These chosen proteins were immobilized on a solid resin for affinity purification of capped mRNA.This binding process is highly selective,effectively enriching capped molecules while leaving uncapped ones behind.Capped mRNA is then eluted using a simple and cost-effective sodium chloride(NaCl)gradient,eliminating the need for expensive and potentially denaturing cap analogs.Importantly,the NaCl elution preserves mRNA structure and functionality,ensuring high-quality preparations for downstream applications.This cap-affinity chromatography method represents a significant advancement in IVT mRNA purification.By selectively removing uncapped molecules,it unlocks several benefits:(1)Enhanced drug efficacy.A homogeneous mRNA population leads to more potent therapies,requiring lower doses and minimizing off-target effects.(2)Reduced production costs.Scalable and cost-effective purification facilitates wider accessibility of mRNA therapeutics.(3)Improved safety profiles.Elimination of uncapped mRNA minimizes potential immune activation and other adverse reactions.In conclusion,this paper introduces a novel cap-affinity chromatography technique for the efficient purification of IVT mRNA.This approach offers a groundbreaking solution for large-scale mRNA drug production,addressing a critical industry challenge of removing non-functional uncapped mRNA and representing a significant advancement in mRNA therapeutic technology.
m7G capcap-binding proteinaffinity chromatographymRNA purificationmRNA medicine
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