Obtaining enough synthetic mRNA for clinical use has long been a hurdle in treating conditions like cancer and infectious diseases. Current mRNA production methods involve complex purification steps and chemicals, limiting large-scale manufacturing.
This poster highlights an advanced chromatography resin that simplifies the mRNA purification process, maximizing efficiency and yield while reducing complexity.
Download this poster to learn how to help:
- Simplify your mRNA process using AT base pairing
- Increase purity and yield
- Perform a scalable mRNA purification process without the use of toxic chemicals
INTRODUCTION ThediversityofpotentialmRNA-basedtherapieshasledtoincreasedinterestinusing syntheticmRNAasatoolinthetreatmentofmultiplediseases,suchascancer,stemcell therapiesandinfectiousdiseases.Nevertheless,obtaininglargerquantitiesofsynthetic mRNAforclinicaltreatmentremainsachallenge.CurrentmRNAproductionmethods requiremultiplepurificationstepsandvariouschemicals,therebycreatingabottleneck forlarge-scalemanufacturingof invitrotranscribed(IVT)mRNA.Toovercomethese challenges,anewaffinitychromatographyresinfortheisolationandpurificationof mRNAwasdeveloped. Kelly Flook1, Brian Sim1, Jim Molinari1, Julia Baek2, Sirat Sikka1, Alejandro Becerra1 1.Thermo Fisher Scientific, Bedford, MA 2.Thermo Fisher Scientific, Sunnyvale, CA Accelerating mRNA-based therapy development with scalable purification of in vitrotranscribed mRNA TRADEMARKS/LICENSING © 2020 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and itssubsidiaries unless otherwise specified. This information is not intended to encourage use of these products in any manner that might infringe the intellectual property rights of others. THERMO SCIENTIFIC™ POROS™ OLIGO (dT)25 AFFINITY RESIN Designed for the isolation and purification of mRNA from in vitro transcription manufacturing processes Simplified workflow to maximize efficiency and reduce complexity of subsequent polish steps LINKER A A A A A A A AAA A A A A A A Poly-A mRNA Poly-dTligand Fig.1POROSOligo(dT)25 affinityresinconsistsofapolydeoxythymidine(dT-25)ligand attachedtoa50µmrigid,porous beadthroughaproprietary linker.Thepoly-dTligandallows bindingwithpoly-AtailedmRNA moleculesthroughATbasepairing. Simple mRNA capture through AT base pairing Easy to use:load in NaCland elute in water Dynamic Binding Capacity up to 5 mg/mL for 4000nt mRNA > 90% recovery Excellent scalability Non-animal derived OPTIMIZING PROCESS CONDITIONS FOR RESIN USE Precipitation point determination Salt type and concentration The POROS Oligo (dT)25 affinity resin demonstrates: Efficient elution at different load concentrations Excellent recovery with high purity independent of sample type used RESIN PERFORMANCE –ELUTION, RECOVERY & PURITY Below detection limit Fig.2mRNAsalttolerancetoprecipitation. Precipitationof2000ntmRNAat>1.4MNaCl. SalttoleranceisdependentonmRNAsizeand sequence,typeofsaltandconcentration. Fig.3Effectofsalttypeandconcentration onmRNAbindingduringscreening. This graphshowsrecoverydataafterstaticbinding ofa2000ntmRNAmolecule. Purification with POROS Oligo (dT)25 resin leads to significant reduction of impurities Fig.4Chromatogramshowingefficientseparationofa2000ntmRNAfromanIVTmixtureata loadconcentrationof2mg/mL.ElutionwasperformedusingH2Oandyieldedin>95%recovery. Fig.5Highrecoveryandpurityindependentofsampletypeused.RecoveryofmRNAfrompure mRNAandunpurifiedIVTmixture,showednodifferences(left).Amountofproteinwasdeterminedin load,flowthrough(FT)andelutionpools(right).Noproteinsweredetectedintheelutionpool,indicating excellentimpurityremovalofIVTmixtureproducts. Determine precipitation point to optimize recovery Recovery and yield (% load in elution pool) are dependent on the type of salt and concentration used. Compatible with a range of buffer and elution salts CONCLUSIONS The POROS Oligo (dT)25 resin addresses the current challenges involved with large scale mRNA purification used in potential mRNA-based therapies. Use of this resin will: Simplify your mRNAdownstream process Increase purity and yield Allow for scalable mRNA purification process without the use of toxic chemicals Customer testimonial