mRNA Chromatography Applications: Resin Types and Purification Efficiency

This content delves into mRNA chromatography applications, focusing on the mRNA purification process using dT affinity chromatography, IEX, HIC, and RP techniques. It discusses the application of mRNA affinity resins like Monomix dT20 and Proteomix POR50-dT20, highlighting their properties, purification results, and experimental schemes. Various case studies demonstrate the effectiveness of these resins in terms of yield, purity, and client feedback, showcasing their significance in mRNA purification processes.

• mRNA chromatography
• Resin types
• mRNA purification
• Affinity chromatography
• mRNA purification efficiency

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1. mRNA Chromatography Applications mRNA Purification Process: Step One dT Affinity Process related impurities (DNA, nucleotides, enzymes, buffers). Product related impurities (mRNA without PolyA tail) Step Two IEX/ HIC/ RP dsRNA and uncapped RNA / www.sepax-tech.com.cn

2. mRNA affinity resins Proteomix POR50-dT20 Resin type Monomix dT20 Matrix Particle size distribution D50 Ligand PMMA PS/DVB 30 m 50 m Oligo dT20-mer 2.0 mg/mL resin mRNA DBC Operating Temp. ( ) 4-65 4-65 pH range 2-12 Operating pressure Mobile phase compatibility 10 bar (1 MPa) 10 bar (1 MPa) Compatible with common salt buffers, organic/aqueous solutions (acetonitrile, ethanol, etc.), and commonly used additives for mRNA purification Store in a solution of 20% ethanol water and 2% benzyl alcohol at 2-8 ; Do not freeze fillers or columns 0.1-0.5 M NaOH It is recommended to start with 0.1 M NaOH to extend the life of the resin Storage Online CIP * DBC test on 1000 nt mRNA sample binding condition: 10 mM Tris-HCl, 1 mM EDTA, 5 mM DTT, 1.0 M NaCl, pH 7.5 2 / www.sepax-tech.com.cn

3. Case 1mRNA affinity-Monomix dT20 Sample mRNA 2000nt 1 mL prepacked column DBC:5.6 mg/mL Result analysis Collected volume (mL) concentration (ug/mL) Producti on(ug) SEC-HPLC (%) Sample yield (%) Flow through A01 5 10 0.90 NA 50 Flow through A02 9.2 544 4993.9 89.50 99.7 lost NA NA NA 9.62 NA 3 / www.sepax-tech.com.cn

4. Case 2mRNA affinity-Monomix dT20 Client feedback Monomix dT20 purification, the purity is over 95%, and the yield is over 90% 4 / www.sepax-tech.com.cn

5. mRNA Affinity resin Experimental Scheme Step Buffer RT CV Remarks CIP 0.1 M NaOH 5 min 5 Adjust salt concentration based on the balance between load and purity Load is related to molecular size and salt concentration balance 10 mM Tris, 5 mM EDTA, 0.5-1.0 M NaCl, pH7.0 5 min 6 Loading 5 min N/A balance 1 10 mM Tris, 5 mM EDTA, 0.5-1.0 M NaCl, pH7.0 5 min 3 Salt concentration can be adjusted according to the strength of the binding Adjust the elution volume according to the actual situation balance 2 10 mM Tris, 5 mM EDTA, 0.25 -0.5 M NaCl, pH7.0 5 min 3 Elution H2O 5 min 10 CIP 0.1 M NaOH 5 min 5 Clean H2O 5 min 5 Ethanol backpressure is high, and the flow rate can be appropriately reduced Storage 20%ethanol 5 min 3 5 / www.sepax-tech.com.cn

6. Case 3mRNA affinity-Proteomix POR50-dT20 Client feedback This mRNA sample was tested with three types of Resins: Sepax, Thermo Fisher, and another Chinese brand The results show: Similar yield (~65%) Load test: Sepax and Thermo are both>2.0 mg/mL, while another Chinese brand are ~1.7 mg/mL Sample: 2000 nt DBC 2.6 mg/mL yield 65% 6 / www.sepax-tech.com.cn

7. Case 4Proteomix POR50-dT20 VS. Poros dT25 mAU 2500 Proteomix POR50-dT20 Sample1 Poros dT25 Sample1 2000 1500 1000 500 0 CV -10 -5 0 5 10 15 20 25 30 35 Sample 4: mRNA 4300nt Load 2mg/mL Resin Resin Yield% Proteomix POR50-dT20 74.3 Thermo Poros dT25 61.1 7 / www.sepax-tech.com.cn

8. Case 5Proteomix POR50-dT20 VS. Poros dT25 mAU 2400 Proteomix POR50-dT20 Sample2 2200 2000 Poros dT25 Sample2 1800 1600 1400 1200 1000 800 600 400 200 0 -200 CV -10 -5 0 5 10 15 20 25 30 35 40 Sample 5: mRNA 4000nt Load 2mg/mL Resin Resin Yield% Proteomix POR50-dT20 85.5 Thermo Poros dT25 83.1 8 / www.sepax-tech.com.cn