Miklavčič, Rok, Polona Megušar, Špela Meta Kodermac, Blaž Bakalar, Darko Dolenc, Rok Sekirnik, Aleš Štrancar, and Urh Černigoj
International Journal of Molecular Sciences 24, no. 18: 14267
Messenger RNA (mRNA) is becoming an increasingly important therapeutic modality due to its potential for fast development and platform production. New emerging RNA modalities, such as circular RNA, drive the need for the development of non-affinity purification approaches. Recently, the highly efficient chromatographic purification of mRNA was demonstrated with multimodal monolithic chromatography media (CIM® PrimaS), where efficient mRNA elution was achieved with an ascending pH gradient approach at pH 10.5. Here, we report that a newly developed chromatographic material enables the elution of mRNA at neutral pH and room temperature. This material demonstrates weak anion-exchanging properties and an isoelectric point of 5.3. It enables the baseline separation of mRNA (at least up to 10,000 nucleotides (nt) in size) from parental plasmid DNA (regardless of isoform composition) with both a NaCl gradient and ascending pH gradient approach, while mRNA elution is achieved in a pH range of 5–7. In addition, the basic structure of the novel material is a chromatographic monolith, enabling convection-assisted mass transfer of large RNA molecules to and from the active surface. This facilitates the elution of mRNA in 3–7 column volumes with more than 80% elution recovery and uncompromised integrity. This is demonstrated by the purification of a model mRNA (size 995 nt) from an in vitro transcription reaction mixture. The purified mRNA is stable for at least 34 days, stored in purified H2O at room temperature.
Research Outreach, 2023
The COVID-19 pandemic placed mRNA at the centre of biopharmaceutical research, as mRNA is now being developed for cancer therapy, protein replacement therapy, and infectious diseases. That is why, worldwide, the need to produce mRNA on a large scale has increased dramatically. The currently used method is quite costly, limiting the scale-up of mRNA production. Dr Rok Sekirnik and colleagues at Sartorius BIA Separations, Slovenia, found a way to monitor and analyse the production of mRNA in the laboratory while decreasing the cost by up to 50%.
- What is the impact of each reagent on in vitro transcription yield and kinetics?
- How can at-line analytics be implemented to monitor capping reaction?
- Does feeding NTPs to the IVT reaction affect capping efficiency?
- How to accurately quantify mRNA in a crude IVT reaction?
Individual IVT reaction components were optimised by understanding IVT reaction kinetics. The paper shows that development of fed-batch IVT requires more than just NTP addition, with Mg2+ being a critical component. Batch and fed-batch IVT were evaluated in terms of capping efficiency.
Domen Pregeljc, Janja Skok, Tina Vodopivec, Nina Mencin, Andreja Krušič, Jure Ličen, Kristina Šprinzar Nemec, Aleš Štrancar & Rok Sekirnik
Biotechnology and Bioengineering, December 2022
The COVID‐19 pandemic triggered an unprecedented rate of development of messenger ribonucleic acid (mRNA) vaccines, which are produced by in vitro transcription reactions. The latter has been the focus of intense development to increase productivity and decrease cost. Optimization of in vitro transcription (IVT) depends on understanding the impact of individual reagents on the kinetics of mRNA production and the consumption of building blocks, which is hampered by slow, low‐ throughput, end‐point analytics. We implemented a workflow based on rapid at‐line high pressure liquid chromatography (HPLC) monitoring of consumption of nucleoside triphosphates (NTPs) with concomitant production of mRNA, with a sub‐3 min read‐ out, allowing for adjustment of IVT reaction parameters with minimal time lag. IVT was converted to fed‐batch resulting in doubling the reaction yield compared to batch IVT protocol, reaching 10 mg/ml for multiple constructs. When coupled with exonuclease digestion, HPLC analytics for quantification of mRNA was extended to monitoring capping efficiency of produced mRNA. When HPLC monitoring was applied to production of an anti‐reverse cap analog (ARCA)‐capped mRNA construct, which requires an approximate 4:1 ARCA:guanidine triphosphate ratio, the optimized fed‐ batch approach achieved productivity of 9 mg/ml with 79% capping.
The study provides a methodological platform for optimization of factors influencing IVT reactions, converting the reaction from batch to fed‐batch mode, determining reaction kinetics, which are critical for optimization of continuous addition of reagents, thus in principle enabling continuous manufacturing of mRNA.
- How does Oligo dT18 compare to Oligo dT24?
- Does flow rate affect binding capacity for mRNA?
- Does variability in ligand density affect binding capacity?
- How scalable is purification by Oligo dT18?
A comprehensive overview of development and optimisation of Oligo dT monoliths describes multiple factors affecting its chromatographic performance. Flow rate, ligand density, size of mRNA are discussed in the paper.
Nina Mencin, Dona Štepec, Alja Margon, Jana Vidič, Darko Dolenc, Tina Simčič, Sara Rotar, Rok Sekirnik, Aleš Štrancar, Urh Černigoj
Separation and Purification Technology, Volume 304, 1 January 2023
Oligo-deoxythymidilic acid (OdT) probes conjugated to solid-phase supports exhibit high affinity for poly-adenylated mRNA (target) through high-affinity base-pairing interactions. Here we report the development of a OdT-functionalized chromatographic monolith for purification of polyadenylated mRNA and development of purification methods to support large-scale purification of mRNA-based therapeutics. We report the development of a chromatographic assay based on a synthetic oligo-deoxyadenylic acid chain of 10 or 20 nucleotides (OdA10 and OdA20) as a surrogate for polyadenylated mRNA, which was used for optimization of the OdT affinity column (i.e. the amount and structure of OdT immobilized and monolithic channel size). OdA hybridization to OdT monoliths correlated well with the amount of immobilized OdT, while an in-depth analysis revealed that hybridization yield decreased with increasing size of the target, temperature and probe surface coverage. OdA hybridization kinetics was flow rate-independent, confirming convection-based mass transport within the monolith’s channels. The demonstrated steep adsorption isotherms enable chromatographic capture of even highly diluted OdA-containing molecules. Dynamic binding capacity for model mRNA was independent of OdT length and amount of immobilized OdT probes above a critical threshold but was highly influenced by the composition of the binding buffer and mRNA residence time. We demonstrated the scalability of the mRNA purification process with OdT monoliths from 0.1 mL to at least 800 mL bed volume, paving the way for manufacturing processes on OdT monoliths with 40 L bed volume.
- Can IVT yields be increased beyond 5-8 g/L?
- Does feeding nucleotides into the IVT reaction increase its yield?
- Is there a fast analytical method to quantify NTPs in IVT in real-time?
- Can production of mRNA be automated?
Transitioning from batch to fed-batch IVT can increase IVT yield to 12 g/L resulting in 50 % reduction in cost per gram of mRNA. Integrating HPLC monitoring of IVT reaction can allow real-time decisions on feed additions.
Janja Skok, Polona Megušar, Tina Vodopivec, Domen Pregeljc, Nina Mencin, Matevž Korenč, Andreja Krušič, Anže Martinčič Celjar, Nejc Pavlin, Jana Krušič, Matthias Mueller, Kevin McHugh, Aleš Štrancar, and Rok Sekirnik
Chemie Ingenieur Techik, October 2022
The COVID-19 pandemic triggered an unprecedented surge in development of mRNA-based vaccines. Despite the need to increase process productivity and thus decrease the cost of mRNA vaccines, limited scientific literature is available on strategies to increase the yield of in vitro transcription (IVT) reaction, the unit operation with highest cost of goods, which has traditionally been performed as a batch reaction. Single-use bioreactors are traditionally used for cell-based production of biopharmaceuticals, but some core functionalities, such as controlled and automated feed addition, are potentially useful for cell-free mRNA processes. We report the production of 2 g mRNA in an Ambr® 250 Modular bioreactor system with a starting volume of 100 mL, reaching a maximum mRNA concentration of 12 g L−1 by a fed-batch IVT approach, and demonstrate the feasibility of continuous fed-batch production, paving the way towards continuous manufacturing of mRNA.
Ana Ferjančič Budihna, Nejc Pavlin, Anže Martinčič Celjar, Andreja Gramc Livk and Aleš Štrancar
BioProcess International eBook, September 14, 2022
Robust and precise chromatographic analytical methods are key for the efficient development of the mRNA production process.
Three different analytical methods, which utilize three different column chemistries, are embedded in a ready-to-use PATfix™ analytical platform to support mRNA process development and product quantification and characterization.
Tingting Cui, Kareem Fakhfakh, Hannah Turney, Gülin Güler-Gane, Aleksandra Toloczko, Martyn Hulley, Richard Turner
American Institute of Chemical Engineers, September 2022
In recent years, mRNA-based therapeutics have been a fast-growing new class of biologics that can, in principle, encode any protein(s) directly in patients to treat various diseases. mRNA vaccines have been proven to work efficiently, have high potency, and can be rapidly developed and deployed, which is critical for a quick responses in the case of a pandemic. Such agile development is enabled by rapid synthesis of RNA in vitro using recombinant enzymes rather than relying on lengthy and complex cell culture processes. mRNA exhibits physical and chemical properties differing from protein-based therapeutics. It is highly negatively charged and the hydroxyl group makes mRNA less stable and more susceptible to hydrolysis and nucleophilic cleavage. This novel work shares comprehensive studies carried out to compare the performance of various mRNA purification strategies by considering its scalability and critical quality attributes. In addition, the paper provides insights on how to establish a scalable mRNA purification process that consists of ultrafiltration/diafiltration and chromatography steps with good recoveries. Alternative Oligo(dT) based columns were further explored aiming to improve total process recovery. With Oligo(dT) as a capture step, overall recoveries of 70% can be achieved for mRNAs studied here that encode anti-influenza immunoglobulin G monoclonal antibodies.
Nina Mencin, Andreja Krušic, Jure Ličen, Sebastijan Peljhan, Jana Vidič, Urh Černigoj, Tomas Kostelec, Aleš Štrancar and Rok Sekirnik
BioProcess International's Special Report, June 2022
Messenger RNA (mRNA) emerged as a powerful therapeutic tool for treatments in gene therapy, oncology, and infectious diseases, as recently demonstrated by vaccines against Covid-19. mRNA is produced by an enzymatic reaction that can be rapidly designed and scaled-up, and the platform is highly adaptable to different targets. One of the greatest challenges in mRNA production is the removal of process-related impurities stemming from in vitro transcription (IVT) reaction, such as residual nucleotide triphosphates, DNA template, enzymes, abortive transcripts.
Affinity-based chromatographic isolation of mRNA is robust and simple, lending itself as a useful industrial platform. mRNA constructs typically contain a 3’ polyA tail to increase stability in vivo, thereby enabling affinity purification using oligo-deoxythymidinic acid (Oligo dT) probes covalently coupled to a solid support. Macro-porous polymethacrylate monoliths offer high binding capacity and resolution for mRNA due to the convective nature of interconnected flow-through channels (>1.5 μm) modified with ligands that are easily accessible for mRNA. Typical binding capacity for CIMmultus™ Oligo dT for mRNA is 2-4 mg/mL, depending on construct length and loading concentration of NaCl.
Due to an increasing productivity of IVT reaction protocols, which routinely reach 5-10 mg/mL, elucidation of conditions that increase binding capacity of Oligo dT has been an intense focus of development. CIM® Oligo dT 0.05 mL Monolithic 96-well Plates were used for multi-parallel screening of binding conditions. Binding capacity could be significantly increased if NaCl is replaced with Gu-HCl, with DBC values of >6 mg/mL demonstrated, and scalability of binding capacity shown on CIMmultusTM Oligo dT preparative scale, which spans bed volume range 1 mL – 40 L, thereby theoretically supporting the purification of >200 g mRNA in a single run.
- At which scale should chromatographic purification be introduced?
- Are there analytical chromatography solutions to improve my process?
- How to control dsRNA contamination in drug substance?
This paper is an overview of the use of chromatography in the complete production of mRNA, from plasmid to pure mRNA, including analytical HPLC.
Rok Sekirnik and Tomas Kostelec
BioProcess International's special report, December 2021
Rapid response to global pandemics requires the manufacture of billions of vaccine doses within months. This short timeline must allow for design and testing of active ingredients, development of production and purification processes, clinical evaluations, regulatory filings, and manufacturing. Existing purification methods often have been adopted from laboratory-scale techniques to allow rapid implementation, and those have provided adequate product quality. But future mRNA development will require optimized production and purification processes.
Chromatography has been a workhorse of biomanufacturing for decades, including for monoclonal antibodies, plasmid DNA, viruses, and other modalities — as well as for supporting analytics. As an emerging therapeutic modality, mRNA production requires the development of new methodologies to suit its peculiar physicochemical profile: large, charged, and relatively unstable. Due to requirements for high purity, these methodologies will be based in large part on chromatography.
This article describes the versatility of chromatography when applied to mRNA production, starting with the purification of the key raw material (plasmid DNA) to final polishing of mRNA drug substance.
- How does chromatography affect RNA stability?
- Can HPLC be used for RNA stability assessment?
- Is precipitation a good approach for small-scale RNA purification?
- How do HPLC, bioanalyser, AGE, and other methods compare?
Thermal and freeze-thaw stability of a model RNA construct purified by various methods is evaluated by a range of analytical methods. Results indicate a stark contrast in stability, and discuss applicability of analytical methods for stability assessment.
Matevž Korenč, Nina Mencin, Jasmina Puc, Janja Skok, Kristina Šprinzar Nemec, Anže Martinčič Celjar, Pete Gagnon, Aleš Štrancar, Rok Sekirnik
Cell & Gene Therapy Insights 2021; 7(9), 1207–1216
One of the major challenges of mRNA based vaccines has been their requirement for distribution and storage at extremely low temperatures, indicating that exposure of mRNA to suboptimal physico-chemical conditions can result in degradation and loss of potency; it is unclear whether this is due to instability of mRNA drug substance, or LNP-encapsulated mRNA, or both. In this study we compare the stability of model mRNA drug substance (eGFP, 995 nt) prepared by affinity chromatography with the stability of mRNA purified by precipitation. We show that both purification methods lead to highly pure mRNA drug substance, however, mRNA purified by chromatography remains stable for 28 days at 37°C, whereas mRNA purified by precipitation is subject to significant degradation under the same storage conditions. We conclude that chromatography eliminates elements and/or conditions with adverse impact on the quality of mRNA to a greater extent than precipitation method and that choosing appropriate purification strategy is crucial not only to achieve target purity but also to obtain a stable product with retained integrity.
Tomas Kostelec, Rok Sekirnik, Anže Martinčič Celjar, Kristina Šprinzar Nemec, Andreja Gramc Livk, Pete Gagnon, Aleš Štrancar
BioProcess International, June 2021
COVID-19 has focused a spotlight on the ability of mRNA technology to accelerate vaccine development and approval. That same technology can hasten development and approval of other therapeutic classes, including cancer immunotherapy, protein replacement, and gene therapy. Fulfilling those opportunities imposes significant challenges on process developers and manufacturers to improve existing processes. Scale-up to produce millions of doses (tens of kilograms) compounds those challenges. Furthermore, every step of the journey requires high-performance analytical methods, to ensure patient safety and maximize productivity.
Artaches A. Kazarian, Wesley Barnhart, Iain D.G. Campuzano, Jeremy Cabrera, Theodore Fitch, Jason Long, Kelvin Sham, Bin Wu, Justin K. Murray
Journal of Chromatography A,Volume 1634, 2020
The current study investigates a method for purification of the G-quadruplex secondary structure, naturally formed by a guanine-rich 21-mer oligonucleotide strand using a monolithic convective interaction media quaternary amine (CIM-QA) column under ion-exchange conditions. The monolithic support was initially evaluated on a preparative scale against a highly efficient TSKgel SuperQ-5PW ion-exchange support designed for oligonucleotide purification. The CIM analogue demonstrated clear advantages over the particle based support on the basis of rapid separation times, while also affording high purity of the G-quadruplex. Various parameters were investigated including the type of mobile phase anion, cation, pH and injection load to induce and control quadruplex formation, as well as enhance chromatographic separation and final purity. Potassium afforded the most prominent quadruplex formation, yet sodium allowed for the highest resolution and purity to be achieved with a 30 mg injection on an 8 ml CIM-QA monolithic column. This method was applied to purify in excess of 300 mg of the quadruplex, with excellent retention time precision of under 1% RSD. Native mass spectrometry was utilized to confirm the identity of the intact G-quadruplex under non denaturing conditions, while ion-pairing reversed-phase methods confirmed the presence of the single stranded oligonucleotide in high purity (92%) under denaturing conditions.
The key advantage of the purification method enables isolation of the G-quadruplex in its native state on a milli-gram scale, allowing structural characterization to further our knowledge of its role and function. The G-quadruplex can also be subsequently denaturated at elevated temperature causing single strand formation if additional reactions are to be pursued, such as annealing to form a duplex, and evaluation in in vitro or in vivo studies.
P. Gagnon, B. Goričar, Š. Peršič, U. Černigoj, A. Štrancar
Cell & Gene Therapy Insights 2020; 6(7), 1035–1046
One of the barriers to development of industrial purification platforms for large mRNA has been an inadequate selection of high-performing capture-purification tools. Hybridization-affinity uses a polythymidine (Oligo dT) ligand to base-pair with the polyadenine tail of mRNA. It can be used for capture but it cannot discriminate dsRNA (double-stranded) from ssRNA (single-stranded) and it supports only brief cleaning with 100 mM sodium hydroxide. Traditional anion exchangers elute only mRNA smaller than about 500 bases unless the columns are heated to 50–70°C. Hydrophobic interaction chromatography (HIC) and reverse phase chromatography (RPC) separate ssRNA from dsRNA and short transcripts, but their sensitivity to fouling by proteins and aggregates makes them better suited for polishing than for capture. Better capture options are needed to meet the needs of large clinical trials, scale-up, and manufacture of vaccines. Beyond that, a new spectrum of gene therapy treatments await. This article introduces two new capture options that both eliminate dsRNA, DNA, and proteins in a wash step, then provide high-resolution polishing of ssRNA in an elution gradient at ambient temperature. One represents a new class of anion exchangers. The other exploits hydrogen bonding. Both support prolonged exposure to 1 M sodium hydroxide. Easy transition to either HIC or RPC provides high-resolution orthogonal polishing.
E. S. Sinitsyna, J. G. Walter, E. G. Vlakh, F. Stahl, C. Kasper, T. B. Tennikova
Talanta 93 (2012) 139-146
Macroporous monoliths with different surface functionalization (reactive groups) were utilized as platforms for DNA analysis in microarray format. The slides based on a copolymer glycidyl methacrylate-co- ethylene dimethacrylate (GMA-EDMA) have been chosen as well known and thoroughly studied standard. In particular, this material has been used at optimization of DNA microanalytical procedure.
The concentration and pH of spotting solution, immobilization temperature and time, blocking agent and coupling reaction duration were selected as varied parameters. The efficiency of analysis performed on 3-D monolithic platforms was compared to that established for commercially available glass slides. As a practical example, a diagnostic test for detection of CFTR gene mutation was carried out. Additionally, the part of presented work was devoted to preparation of aptamer-based test-system that allowed successful and highly sensitive detection both of DNA and protein.
G. A. Platonova, T. B. Tennikova
Journal of Chromatography A, 1065 (2005) 75–81(2005) 75–81
High-performance monolithic disk affinity chromatography was applied to the investigation of formation of complexes between (1) complementary polyriboadenylic and polyribouridylic acids, e.g. poly(A) and poly(U), respectively, (2) poly(A) and synthetic polycation poly(allylamine), pAA. Polyriboadenylic acid and poly(allylamine) were immobilized on macroporous disks (CIM disks). Quantitative parameters of affinity interactions between macromolecules were established using frontal analysis at different flow rates.