This study explores the use of ultra-wide pore size exclusion chromatography (SEC) columns for the analysis of messenger RNA (mRNA) samples. The research focuses on optimizing SEC conditions to effectively characterize mRNA products, particularly those ranging in size from 500 to 5000 nucleotides (nt). The study evaluates the performance of SEC columns with pore sizes of 1000 and 2500 Å, finding that the 1000 Å column is optimal for mRNA analysis. The addition of 10 mM magnesium chloride (MgCl₂) to the mobile phase was shown to improve resolution and recovery of large size variants for some mRNA samples. However, increasing column length or decreasing flow rate can lead to an increase in low-molecular-weight species (LMWS) and monomer peak tailing due to prolonged residence time inside the column. The optimal SEC method was successfully applied to a wide range of mRNA products, including those from different suppliers and stressed/unstressed samples. The study also demonstrates that SEC can be a stability-indicating method for mRNA samples, as it can detect changes in LMWS and HMWS under different conditions. The results indicate that MgCl₂ can improve the separation of some mRNA samples, although its effectiveness may depend on the sequence and size of the mRNA. The study concludes that SEC is a valuable tool for the analysis of mRNA, with the potential to improve the quality and safety assessment of mRNA-based drugs.This study explores the use of ultra-wide pore size exclusion chromatography (SEC) columns for the analysis of messenger RNA (mRNA) samples. The research focuses on optimizing SEC conditions to effectively characterize mRNA products, particularly those ranging in size from 500 to 5000 nucleotides (nt). The study evaluates the performance of SEC columns with pore sizes of 1000 and 2500 Å, finding that the 1000 Å column is optimal for mRNA analysis. The addition of 10 mM magnesium chloride (MgCl₂) to the mobile phase was shown to improve resolution and recovery of large size variants for some mRNA samples. However, increasing column length or decreasing flow rate can lead to an increase in low-molecular-weight species (LMWS) and monomer peak tailing due to prolonged residence time inside the column. The optimal SEC method was successfully applied to a wide range of mRNA products, including those from different suppliers and stressed/unstressed samples. The study also demonstrates that SEC can be a stability-indicating method for mRNA samples, as it can detect changes in LMWS and HMWS under different conditions. The results indicate that MgCl₂ can improve the separation of some mRNA samples, although its effectiveness may depend on the sequence and size of the mRNA. The study concludes that SEC is a valuable tool for the analysis of mRNA, with the potential to improve the quality and safety assessment of mRNA-based drugs.