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2024

Klemen Božič, Ajda Sedlar, Špela Kralj, Urh Černigoj, Aleš Štrancar, Rok Sekirnik

Biotechnology and Bioengineering, 2024, 1–11

High purity of plasmid DNA (pDNA), particularly in supercoiled isoform (SC), is used for various biopharmaceutical applications, such as a transfecting agent for production of gene therapy viral vectors, for pDNA vaccines, or as a precursor for linearized form that serves as a template for mRNA synthesis. In clinical manufacturing, pDNA is commonly extracted from Escherichia coli cells with alkaline lysis followed by anion exchange chromatography or tangential flow filtration as a capture step for pDNA. Both methods remove a high degree of host cell contaminants but are unable to generically discriminate between SC and open-circular (OC) pDNA isoforms, as well as other DNA impurities, such as genomic DNA (gDNA). Hydrophobic interaction chromatography (HIC) is commonly used as polishing purification for pDNA. We developed HIC-based polishing purification methodology that is highly selective for enrichment of SC pDNA. It is generic with respect to plasmid size, scalable, and GMP compatible. The technique uses ammonium sulfate, a kosmotropic salt, at a concentration selective for SC pDNA binding to a butyl monolith column, while OC pDNA and gDNA are removed in flow-through. The approach is validated on multiple adeno-associated virus- and mRNA-encoding plasmids ranging from 3 to 12 kbp. We show good scalability to at least 300 mg of >95% SC pDNA, thus paving the way to increase the quality of genomic medicines that utilize pDNA as a key raw material.

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2023

Ivana Petrović Koshmak, Hana Jug, Katja Vrabec, Ana Mavri, Valentina Novak, Petra Dekleva, Veronika Fujs, Maja Leskovec and Aleš Štrancar

Electrophoresis 2023; 1–11

Adenoviruses are well-known viral vectors that have been previously used in gene therapy and as a vaccine-delivery vehicle for humans and animals. During the COVID-19 pandemic, it gained renewed attention, but at the same time, it raised concerns due to side effects observed with some of the resulting vaccines administered to patients. It has been indicated that these side effects might be attributed to impurities present in the final product. Therefore, constant enhancement of the vaccine purity and further improvement of impurity detection methods are needed. In this work, we showcase an example of industry-relevant adenovirus bioprocess optimization. Our data show the effect of upstream parameters on the bioburden introduced to the downstream process. We provide an example of process optimization using a combination of the PATfix analytical method, ddPCR, infectivity, total DNA, and total protein analyses to optimize cell density, multiplicity of infection, and length of production. Additionally, we provide data illustrating the robustness of the convective interaction media quaternary amine monolithic chromatography step. This anion exchange strategy was shown to remove over 99% of protein and DNA impurities, including those unable to be addressed by tangential flow filtration, while maintaining high adenovirus recoveries.

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Rok Sekirnik, Head Process Development mRNA | pDNA, Sartorius BIA Separations, d.o.o.

mRNA Production and Synthesis, InformaConnect eBook, November 2023

The use of mRNA vaccines in the fight against SARS-CoV-2 during the COVID-19 pandemic, has demonstrated their enormous potential as a highly effective therapeutic modality and has led to a revolution in biomedicine. Since then, mRNA has also shown promise in the treatment of cancer, rare diseases, anaphylaxis, CRISPR-Cas9-based therapy and others. There are currently >100 clinical trials registered with mRNA being the drug substance. The technology to produce mRNA therapeutics can still be considered as being in the early stages of development, as multiple academic and industrial laboratories across the world invest significant funding and talent, with the intention to increase productivity and purity in the production of mRNA drug substance and lipid nanoparticles that would deliver affordable mRNA therapeutics. This article aims to present a technological paradigm of harnessing the potential of chromatography as a purification tool with high selectivity and scalability that are unmatched by other purification methods, as well as analytical technology that can provide timely and accurate information on the quality and quantity of the mRNA product, its precursors and side-products.

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Ana Ferjančič Budihna, Anže Martinčič Celjar, Sergeja Lebar, Andreja Gramc Livk and Aleš Štrancar

Cell & Gene Therapy Insights 2023; 9(9), 1231–1247 | DOI: 10.18609/cgti.2023.161

Rapid advancement of mRNA technology, as a response to the COVID-19 crisis, prompted an increased need for precise analytical methods to support the fast-paced mRNA process development. Accurate and robust analytics are required to support modifications in the mRNA production process, protocols, raw materials, in vitro transcription reaction, purification methods, scale-up, or final formulation processes, to ensure high quality and safety of the final product. This Innovator Insight demonstrates the application of an ion pair reverse phase chromatographic analytical method as a robust analytical tool to determine mRNA fragmentation while also separating in vitro transcription components from the main product. The method’s efficacy is assessed through a comprehensive stability study of a mRNA standard at different temperatures. The chromatographic analytical results are compared to the ones obtained by the capillary gel electrophoresis, a well-established method for the analysis of fragmented mRNA.

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Polona Megušar, Rok Miklavčič, Matevž Korenč, Jure Ličen, Tina Vodopivec, Urh Černigoj, Aleš Štrancar, and Rok Sekirnik

Electrophoresis. 2023; 1–11 

Messenger RNA (mRNA) has emerged as a modality with immense therapeutic potential. Recent innovations in production process of mRNA call for procedures to isolate pure mRNA drug substance (DS) with high yield, high capacity, scalability, and compatibility with GMP production systems. Novel RNA modalities, such as circular RNA (circRNA), have further driven the need for non-affinity capture possibilities which are already widely used in the biopharmaceutical industry, for example, in monoclonal antibody processing. The principle that multimodal ion exchange/hydrogen bonding chromatography can be used to separate mRNA from in vitro transcription components has recently been demonstrated. Here, we apply and refine this approach to be suitable for scalable purification of multiple mRNA constructs with sufficient yields, purity, and stability, for use in mRNA production process. Binding capacity of the PrimaS-modified monolithic chromatographic column for mRNA enabled up to 7 mg/mL product isolation in a single chromatographic run, with 98% recovery and room temperature stability of the eGFP mRNA demonstrated for up to 28 days. This approach is independent of construct size or the presence of polyadenylic acid tail and is applicable for capture of a wide variety of RNAs, including mRNA, self-amplifying RNA, circRNA, and with optimization also smaller RNAs such as transfer RNA and others.

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Juan Martínez, Verónica Lampaya, Ana Larraga, Héctor Magallón, and Diego Casabona

Frontiers in Molecular Biosciences, volume 10, 2023.

After the COVID-19 pandemic, messenger RNA (mRNA) has revolutionized traditional vaccine manufacturing. With the increasing number of RNA-based therapeutics, valuable new scientific insights into these molecules have emerged. One fascinating area of study is the formation of double-stranded RNA (dsRNA) during in vitro transcription (IVT) which is considered a significant impurity, as it has been identified as a major trigger in the cellular immune response pathway. Therefore, there is a growing importance placed to develop and optimize purification processes for the removal of this by-product. Traditionally, efforts have primarily focused on mRNA purification after IVT through chromatographic separations, with anion exchange and reverse phase chromatography emerging as effective tools for this purpose. However, to the best of our knowledge, the influence and significance of the quality of the linearized plasmid have not been thoroughly investigated. Plasmids production involves the growth of bacterial cultures, bacterial harvesting and lysis, and multiple filtration steps for plasmid DNA purification. The inherent complexity of these molecules, along with the multitude of purification steps involved in their processing, including the subsequent linearization and the less-developed purification techniques for linearized plasmids, often result in inconsistent batches with limited control over by-products such as dsRNA. This study aims to demonstrate how the purification process employed for linearized plasmids can impact the formation of dsRNA. Several techniques for the purification of linearized plasmids based on both, resin filtration and chromatographic separations, have been studied. As a result of that, we have optimized a chromatographic method for purifying linearized plasmids using monolithic columns with C4 chemistry (butyl chains located in the surface of the particles), which has proven successful for mRNAs of various sizes. This chromatographic separation facilitates the generation of homogeneous linearized plasmids, leading to mRNA batches with lower levels of dsRNA during subsequent IVT processes. This finding reveals that dsRNA formation is influenced not only by RNA polymerase and IVT conditions but also by the quality of the linearized template. The results suggest that plasmid impurities may contribute to the production of dsRNA by providing additional templates that can be transcribed into sequences that anneal with the mRNA molecules. This highlights the importance of considering the quality of plasmid purification in relation to dsRNA generation during transcription. Further investigation is needed to fully understand the mechanisms and implications of plasmid-derived dsRNA. This discovery could shift the focus in mRNA vaccine production, placing more emphasis on the purification of linearized plasmids and potentially saving, in some instances, a purification step for mRNA following IVT.

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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.

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Rok Sekirnik

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%.

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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.

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Maja Leskovec, Andrej Raspor, Veronika Fujs, Andrej Mihevc, Aleš Štrancar

Electrophoresis. 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.

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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.

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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.

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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.

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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.

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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.

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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.

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2022
  • 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.

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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.

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  • 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

Abstract:

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.

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  • 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.

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