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%.
Kaja Bažec, Mirjam Krašna, Andrej Mihevc, Maja Leskovec, Aleš Štrancar, Mojca Tajnik Sbaizero
Electrophoresis. 2023; 1–10
Adeno-associated virus (AAV) vectors are crucial tools for gene therapy applications. As AAVs are administered in vivo, stringent purity requirements must be met, necessitating the development of various downstream processing strategies in accordance with regulatory guidelines. In this context, we focus on the non-affinity serotype-independent recombinant AAV (rAAV) capture step, which involves the use of Convective Interaction Media (CIM) cation-exchange SO3 monoliths. We analyzed differentially pretreated viral samples obtained from the Sf9 cell line and applied these samples to the capture SO3 chromatography step. We conducted screening experiments using CIM SO3 0.05 mL monolithic 96-well plates with buffers of varying pH, sodium chloride concentrations, and the inclusion of poloxamer 188, aiming to select the optimal binding mobile phase. Dynamic binding capacity was defined for different pretreatments and the optimal conditions were subsequently retested using the industrial purification CIMmultus line. The results demonstrated a high overall vector recovery (51%) and a significant reduction in impurities (99.98% for protein reduction and 99.25% for DNA reduction) using the selected capture step parameters, thereby confirming the successful optimization of the rAAV capture step in the downstream process using monoliths.
Maja Leskovec, Andrej Raspor, Veronika Fujs, Andrej Mihevc, Aleš Štrancar
Electrophores. 2023; 1–9
Preferential exclusion chromatography (PXC) sometimes described as hydrophobic interaction chromatography is a well-known, but not widely used technique for purification of Adeno-associated viruses. It employs high molarity of preferentially excluded cosolvent (salt in our case). The downside of this method is that high molarity of salt can lead to aggregation and precipitation of different compounds from the sample. In the case of viruses that are excreted to medium, the concentration of impurities is much lower compared to cell lysates, and PXC can be used as a first chromatographic, serotype independent step to concentrate and purify adeno-associated virus (AAV). Here, we explored PXC for adherent and suspension harvests using monolithic chromatographic columns (CIMmultus). Suspension extracellular adeno-associated virus, serotype 9 (AAV9) harvest had more impurities compared to adherent harvest, therefore it required higher input regarding method development. Final conditions for suspension harvest included higher molarity of binding salt and using more open channel format of chromatographic column (6 µm channel size). Vector genome analysis with droplet digital polymerase chain reaction (ddPCR) revealed 84% and 97% recovery for suspension and adherent AAV9 harvest, respectively. After PXC capture step, adherent AAV9 was purified by already described ion exchange techniques. Overall process vector genome recovery, from clarified harvest to anion exchange elution fraction, was 54% measured by ddPCR. Residual host cell DNA was measured at 40 ng per 1E13 vector genome, and empty AAV was below 5% in final anion exchange chromatography fraction.
Troy Rogerson, Guoling Xi, Amanda Ampey, Jon Borman, Sally Jaroudi, Dan Pappas, Thomas Linke
Electrophoresis. 2023; 1– 11.
The use of viral vectors for vaccine, gene therapy, and oncolytic virotherapy applications has received increased attention in recent years. Large-scale purification of viral vector-based biotherapeutics still presents a significant technical challenge. Chromatography is the primary tool for the purification of biomolecules in the biotechnology industry; however, the majority of chromatography resins currently available have been designed for the purification of proteins. In contrast, convective interaction media monoliths are chromatographic supports that have been designed and successfully utilized for the purification of large biomolecules, including viruses, viruslike particles, and plasmids. We present a case study on the development of a purification method for recombinant Newcastle disease virus directly from clarified cell culture media using strong anion exchange monolith technology (CIMmultus QA). Resin screening studies showed at least 10 times higher dynamic binding capacity of CIMmultus QA compared to traditional anion exchange chromatography resins. Design of experiments was used to demonstrate a robust operating window for the purification of recombinant virus directly from clarified cell culture without any further pH or conductivity adjustment of the load material. The capture step was successfully scaled up from 1 mL CIMmultus QA columns to the 8 L column scale and achieved a greater than 30-fold reduction in process volume. Compared to the load material, total host cell proteins were reduced by more than 76%, and residual host cell DNA by more than 57% in the elution pool, respectively. Direct loading of clarified cell culture onto a high-capacity monolith stationary phase makes convective flow chromatography an attractive alternative to centrifugation or TFF-based virus purification procedures.
Špela Kralj, Špela Meta Kodermac, Ines Bergoč, Tomas Kostelec, Aleš Podgornik, Aleš Štrancar, Urh Černigoj
Electrophoresis. 2023; 1– 13
Increased need for plasmid DNA (pDNA) with sizes above 10 kbp (large pDNA) in gene therapy and vaccination brings the need for its large-scale production with high purity. Chromatographic purification of large pDNA is often challenging due to low process yields and column clogging, especially using anion-exchanging columns. The goal of our investigation was to evaluate the mass balance and pDNA isoform composition at column outlet for plasmids of different sizes in combination with weak anion exchange (AEX) monolith columns of varying channel size (2, 3 and 6 µm channel size). We have proven that open circular pDNA (OC pDNA) isoform is an important driver of reduced chromatographic performance in AEX chromatography. The main reason for the behaviour is the entrapment of OC pDNA in chromatographic supports with smaller channel sizes. Entrapment of individual isoforms was characterised for porous beads and convective monolithic columns. Convective entrapment of OC pDNA isoform was confirmed on both types of stationary phases. Porous beads in addition showed a reduced recovery of supercoiled pDNA (on an 11.6 kbp plasmid) caused by diffusional entrapment within the porous structure. Use of convective AEX monoliths or membranes with channel diameter >3.5 µm has been shown to increase yields and prevent irreversible pressure build-up and column clogging during purification of plasmids at least up to 16 kbp in size.
Nejc Pavlin, Urh Černigoj, Mojca Bavčar, Tjaša Plesničar, Jan Mavri, Martin Zidar, Matevž Bone, Urška Kralj Savič, Tadej Sever, and Aleš Štrancar
Electrophoresis. 2023; 1– 11
High-performance liquid chromatography (HPLC)-based analytical assays are used to effectively monitor purity and quantity of plasmid DNA (pDNA) throughout the purification process. However, the phenomenon of physical entrapment of open circular (OC) isoforms pDNA inside narrow channels of chromatographic support decreases its accuracy and precision and the effect increases with pDNA size. The purpose of the study was to develop a chromatographic method for accurate analytical separation between isoforms of <16 kbp pDNA using weak anion exchanging monolithic column with large (6 µm) convective channels. Purified samples of 4.7 and 15.4 kbp large pDNA with known isoform composition were prepared and their isoforms separated in ascending salt gradient. Both OC and supercoiled (SC) isoforms were baseline separated at a flow rate below 0.5 mL min−1 in a guanidinium chloride (GdnCl) gradient with a ≥95% OC pDNA elution recovery. However, these chromatographic conditions increased 2 times the peak width for linear (LIN) pDNA isoform compared to the results using monoliths with 1.4 µm channel size. If other chaotropic agents, such as urea or thiocyanate (SCN), were added to Gdn ions, the elution volume for LIN isoform decreased. Optimization of combined GdnCl/GdnSCN gradient for pDNA elution resulted in a simple and robust chromatographic method, where OC–LIN and LIN–SC pDNA (up to 15 kbp size) were separated with resolution above 1.0 and above 2.0, respectively. The accessibility and general acceptance of anion exchange chromatography for pDNA analytics give the newly developed method a great potential for in-process control monitoring of pDNA production processes.
Irena Trbojević-Akmačić, Frano Vučković,Tea Pribić, Marija Vilaj, Urh Černigoj, Jana Vidič, Jelena Šimunović, Agnieszka Kępka, Ivana Kolčić, Lucija Klarić, Mislav Novokmet, Maja Pučić-Baković, Erdmann Rapp, Aleš Štrancar, Ozren Polašek, James F. Wilson and Gordan Lauc
Communications Biology volume 6, Article number: 312 (2023)
Human plasma transferrin (Tf) N-glycosylation has been mostly studied as a marker for congenital disorders of glycosylation, alcohol abuse, and hepatocellular carcinoma. However, inter-individual variability of Tf N-glycosylation is not known, mainly due to technical limitations of Tf isolation in large-scale studies. Here, we present a highly specific robust high-throughput approach for Tf purification from human blood plasma and detailed characterization of Tf N-glycosylation on the level of released glycans by ultra-high-performance liquid chromatography based on hydrophilic interactions and fluorescence detection (HILIC-UHPLC-FLD), exoglycosidase sequencing, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). We perform a large-scale comparative study of Tf and immunoglobulin G (IgG) N-glycosylation analysis in two human populations and demonstrate that Tf N-glycosylation is associated with age and sex, along with multiple biochemical and physiological traits. Observed association patterns differ compared to the IgG N-glycome corroborating tissue-specific N-glycosylation and specific N-glycans’ role in their distinct physiological functions.
Lucija Rebula, Andrej Raspor, Mojca Bavčar, Aleš Štrancar and Maja Leskovec
Journal of Chromatography B, Volume 1217, 15 February 2023
Bacteriophages represent immense potential as therapeutic agents. Many of the most compelling applications of bacteriophages involve human therapy, some pertinent to gene therapy, others involving antibiotic replacement. Phages themselves are considered safe for humans. However, phage lysates may contain many kinds of harmful by-products, especially endotoxins of gram-negative bacteria and protein toxins produced by many pathogenic bacterial species. In bacteriophage research and therapy, most applications ask for highly purified phage suspensions, as such it is crucial to reduce proteins, endotoxins, DNA and other contaminants.
In this article we present an efficient two-step chromatographic purification method for P. aeruginosa bacteriophage PP-01, using Convective Interaction Media (CIM®) monoliths, that is cGMP compliant and easy to scale-up for most stringent production of the therapeutic phage. First chromatographic step on CIMmultus OH resulted in 100% bacteriophage recovery with a reduction of 98 % protein and more than 99 % DNA content. Polishing was conducted using three different column options, CIMmultus with QA, H-Bond and PrimaS ligands. For PP-01 bacteriophage all three different options worked, but multimodal ligands H-Bond and PrimaS outperformed traditional QA in endotoxin removal (7 log step reduction). Additionally, an HPLC analytical method was developed to estimate phage concentration and impurity profile in different in-process samples. The HPLC method shows good correlation with drop assay titration, provides useful insights and can be run very fast with just 20 min per sample analysis.
Hana Jug, Natalija Hosta, Mojca Tajnik Sbaizero, Aleš Štrancar, Daniela Brodesser, Bianca Tisch, Theresa Heger, Markus Wolschek, Joachim Seipelt and Manfred Reiter
BioProcess International, 9 February 2023
Newcastle disease is an extremely infectious condition among domesticated poultry and other avian species. Its high morbidity and mortality rates among infected birds give the disease significant economic importance. Thus, many commercially available vaccines based on live or inactivated virions are used globally to protect against Newcastle disease infection.
The causative agent is Newcastle disease virus (NDV), which belongs to the Paramyxoviridae family. NDV is a single-stranded, negative sense, enveloped RNA virus of avian origin that is highly attenuated in humans and other primates because of strong host-range restriction. Attenuated NDV has been evaluated as a vector for vaccines against SARS-CoV-2, Ebola, H5N1 influenza, West Nile, and simian immunodeficiency viruses. Oncolytic NDV vectors also hold much promise for immunotherapies against various cancers.
Whether for vaccines or viral-vector therapies, NDV particles must meet certain criteria for yield, purity, and concentration. Previously, we have described a highly effective process for purification of influenza virus produced in Vero cells. Based on CIMmultus SO3 (sulfonate) monolith columns, the process yielded excellent recovery and impurity removal and enabled good manufacturing practice (GMP) scale-up to produce clinical-grade material. Here, we describe our adaptation of the process to NDV purification and thus demonstrate the broad applicability of SO3 monolith technology.
- 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.
Rok Žigon, Mojca Tajnik Sbaizero, Ivana Petrović Koshmak, Veronika Fujs, Maja Leskovec & Aleš Štrancar
Cell & Gene Therapy Insights 2022; 8(10), 1315–1328
Manufacture and purification of recombinant adeno-associated viruses (rAAV) require development and optimization of processes to ensure the best possible quality of the final rAAV product. To do so, different strategies in upstream can be used to achieve the highest possible viral titer and lowest amount of impurities, both of which further influence downstream. Second challenge involves removal of cell debris where different pre-treatments can be utilized. In the next step, optimized capture of rAAV on a cation-exchange chromatography should be developed to remove impurities and achieve a high recovery of rAAV. In the end, several chromatographic options are available to remove empty and defected capsids, so only functional viruses can be isolated. Here, the process of manufacturing and purification of rAAV has been designed using monolithic columns to achieve this important goal of preparing rAAV for the use in gene therapy.
- 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.
Katarina Markovič, Maja Cemazar, Gregor Sersa, Radmila Milačič and Janez Sčančar
Journal of Analytical Atomic Spectrometry
Ceruloplasmin (Cp) is the major copper-carrying (Cu) protein in human plasma. Due to copper's important physiological functions and its role in various diseases, there is a need to quantify the concentration bound to Cp and the exchangeable form of Cu. In the present work, conjoint liquid chromatography (CLC) on short-bed convective interaction media (CIM) monolithic disks was used to separate the Cu bound to low molecular mass (LMM) species, and the Cu bound to Cp and albumin (HSA) in human serum. Two immunoaffinity CIMmic albumin depletion (α-HSA) disks and one CIMmic weak anion-exchange diethylaminoethyl (DEAE) disk were assembled in a single housing, forming a CLC monolithic column. By applying isocratic elution with a 50 mmol L−1 MOPS buffer (pH 7.4) in the first 3 min, followed by gradient elution with 1 mol L−1 NH4Cl (pH 7.4) in the next 9 min, HSA was retained by the α-HSA disk, allowing subsequent separation of the LMM-Cu from the Cu bound to the Cp on the DEAE disk. Further elutio with 0.5 mol L−1 acetic acid in the next 4 min rinsed the HSA from the α-HSA disk. The separated Cu species were quantified by post column isotope dilution inductively coupled plasma mass spectrometry (ID-ICP-MS), while the elution profile of the proteins was followed by UV detection at 278 nm. Quantitative column recoveries were obtained. Good repeatability of the measurement was achieved for Cu-Cp (±1%), while for Cu-HSA and Cu-LMM species the repeatability of the measurements was slightly worse, due to the much lower Cu concentrations (±6% and ±9%, respectively). The developed method required only 20 μL of a 15-times diluted sample. Low limits of detection for the Cu-Cp, Cu-HSA and Cu-LMM species (6.1, 5.3 and 3.3 ng mL−1 Cu, respectively) were obtained. The technique was successfully applied in the determination of Cu-Cp, Cu-HSA and a fraction that most probably corresponds to the Cu-LMM species in the human serum of healthy individuals, kidney transplant patients and cancer patients.
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.