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2023

Immobilized enzyme reactors (IMERs) stand as innovative biotechnological constructs, seamlessly merging the catalytic proficiency of enzymes with the advantages of solid support matrices. Immobilized enzymes offer notable benefits such as improved stability, the potential to operate within a continuous system over extended durations, reusability of the enzyme, as well as reduced production costs and product purification steps. The aim of this study was to prepare a functional IMER on monolithic support for efficient pDNA linearization, that could be used in in vitro transcription reaction for messenger ribonucleic acid (mRNA) production.

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Extracellular vesicles (EVs) are a diverse group of particles secreted by all living cells. Numerous different therapeutic applications of both native and engineered EVs are currently in different stages of clinical development. Nevertheless, considerable challenges are still present in the manufacturing, purification and analytics of EVs. Many factors can influence the final product, therefore an all-inclusive approach to development of the bioprocess is needed. Cell culture parameters and production platform selection might alter the number and composition of EVs. Furthermore, raw materials used in upstream production, such as media and supplements, can greatly impact the chromatographic purification. In this study, we evaluated EV production in different HEK293-derived cell lines. Separation on a strong anion exchange column CIMmultus®-EV was used to assess the abundance of different EV populations. Multi-detector PATfix® SEC analytics coupled with antibody labeling was then used to analyze chromatographic fractions. Furthermore, the analytical methods and performance in downstream processing were applied in the optimization of the upstream process.

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Cells release extracellular vesicles (EVs) of different sizes and intracellular origin. Due to their heterogenicity, the isolation of the target EV population from a mixture of supernatant-derived particles can be challenging. Anion exchange chromatography (AEX) exploits the negative charge on EV surface molecules for binding to the positively charged solid phase. CIMmultus® EV, an AEX chromatography monolith column, can separate EVs in subpopulations based on charge and offers insight into the heterogenicity of particles. Besides the availability of preparative tools for separation, combining multiple orthogonal and complementary characterization tools is crucial for defining the EV product of interest. In this work, we used a multiple-detector PATfix® system for the analysis of CIMmultus EV-fractionated samples. Samples were analyzed for the presence of EV-related tetraspanins using the fluorescence detector. PATfix MALS 3609 detector was used for the analysis of particle-containing samples and calculation of particle sizes.

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The cost of mRNA production is driven by IVT reagents, particularly the co-transcriptional capping reagents. Optimization of mRNA yield is therefore crucial for lowering the cost of mRNA production. To monitor the IVT reaction over time, we implemented a rapid at-line HPLC monitoring of consumption of NTPs and production of mRNA, with a sub-3 min read-out. Use of CIMac PrimaS analytical column allowed us to determine and adjust key IVT components that influence the kinetics of mRNA production and are critical for optimization of continuous addition of reagents, i.e. fed-batch IVT.

Fed-batch reactions can also be performed by continuous feeding, requiring automated control system. We used Ambr® 250 bioreactor platform, demonstrating for the first time its potential for mRNA production. First we designed a fed-batch IVT reaction in a thermal shaker, sampled and analyzed at-line by CIMac PrimaS analytics. Based on NTP consumption kinetics, the Ambr® 250 protocol was then designed to feed a defined mixture of NTP-Mg 2+ continuously.

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New development in the modern biotechnology increased the need for plasmid DNA (pDNA) with sizes above 10 kbp (large pDNA), but their chromatographic purification is often challenging due to low process yields and column clogging. There are 
indirect proofs that open circular (OC) pDNA isoform is the main troublemaker due to its physical entrapment within the narrow channels of chromatographic media. Increasing the channel size of chromatographic support should decrease the negative 
impact and improve the chromatographic performance. The aim of the study was to use novel Convective Interaction Media® (CIM®) monolith chromatographic columns with large, 6 µm channels, for analytical and preparative separation of pDNA. The effect of supercoiled (SC), OC and linear (LIN) pDNA isoforms on chromatographic performance was thoroughly evaluated.

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mRNA has been at the forefront of both scientific and general public interests from the start of the COVID-19 pandemic. However, there are still limited options available for rapid characterization of mRNA containing samples. For precise characterization of an mRNA sample, first the presence and concentration of mRNA molecules in the sample needs to be identified. In the second step, any contaminants in the sample coming from the IVT reaction need to be identified and quantified. All major components of the IVT reaction; nucleotides, capping reagent, enzymes and DNA template may be present in the mRNA sample. In addition, impurities such as shorter, incomplete RNA fragments, and in particular, dsRNA may also be present. Contaminants may also come from the mRNA in vitro instability, caused by spontaneous hydrolyzation of the mRNA backbone. These issues can be mitigated using appropriate analytical tools throughout the mRNA production and purification steps.

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2022
  • How to increase the binding capacity of Oligo dT18?
  • Can a design of experiment approach be used to optimise Oligo dT binding?
  • Is the monolith available in a high throughput format for liquid handlers?
  • Is it possible to use a 96-well plate Oligo dT device?
Buffer conditions (salt, additives) influence mRNA binding on Oligo dT. Three contributing factors were identified and tested: NaCl, MgCl2 and Gu-HCl, the latter leading to a capacity of >6 mg/mL.

Abstract:

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 affording the possibility of affinity purification using oligo-deoxythymidinic acid (Oligo dT) probes covalently coupled to a solid support. Poly-adenylated mRNA forms a stable hybrid with Oligo dT under high-salt conditions which is destabilized when the salt is removed, allowing mRNA to be released. Typical dynamic binding capacity (DBC) of CIMmultus Oligo dT for mRNA is 2-4 mg/mL; ever higher IVT productivity will require higher binding capacities. Screening experiments to elucidate factors affecting CIMmultus Oligo dT binding capacity for mRNA were performed in CIM® 96-well Oligo dT format. A simplified model identified NaCl, guanidine hydrochloride (Gu-HCl) and MgCl2 concentration as the key factors contributing to DBC. Buffer chemistry, buffer pH, salt type and mRNA concentration had little or no effect on DBC.

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Sample displacement chromatography (SDC) is a chromatographic technique that utilizes differences in relative binding affinities of components in a sample mixture under chromatographic conditions. Here, we use SDC approach with CIM® C4 HLD monoliths under hydrophobic interaction chromatography (HIC) conditions to separate plasmid DNA (pDNA) isoforms under overloading conditions, where supercoiled (SC) isoform acts as a displacer of open circular (OC) or linear isoform. High purity of SC isoform is beneficial for use of plasmids as vaccines, transfecting agents for production of gene therapy viral vectors, or as starting material for linearization prior to IVT reaction in production of mRNA vaccines.

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The cost of mRNA production is driven by IVT reagents, particularly the capping reagent. Optimization of mRNA yield is therefore crucial for lowering the cost of mRNA production. In order to monitor IVT reaction over time, we implemented a rapid at-line HPLC monitoring of consumption of NTPs with concomitant production of mRNA, with a sub-3 min read-out. Use of CIMac PrimaS analytical column allowed us to determine and adjust key IVT components that influence the kinetics of mRNA production and are critical for optimization of continuous addition of reagents, i.e. fed-batch IVT.

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CIM® PrimaS column family combines multimodal anion exchange/hydrogen bonding properties, binding molecules with predominantly negative charge. It is used as capture method for purification of mRNA from IVT (in-vitro transcription) reaction mixture with high binding capacity. High salt wash is used to elute the plasmid and other IVT components from the column without affecting binding of ssRNA.

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Microvolume spectrophotometers are commonly used as quick and easy method to measure concentration and purity of nucleic acids. DSP process for purification of mRNA includes unit operations with salt concentrations up to 2.75 M (HIC) or up to 1.25 M (Oligo dT) during load and low salt concentrations during elution.

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mRNA has been at the forefront of both scientific and general public interests from the start of the COVID-19 pandemic. The demand for the mRNA product has been incredible for the last couple of years. However, there are still limited options available for a rapid mRNA quantification and characterization. In this work, mRNA analytics using a CIMac Oligo dT column is presented. mRNA is a specialized group of RNAs that carries the blueprints for building proteins from the cell’s DNA in the nucleus to the ribosomes in the cytoplasm. One of the features of mRNA molecules is a polyadenylated (poly(A)) tail on the 3’ end, that can be up to 250 nucleotides long. This feature enables mRNA to bind to the Oligo dT column. HPLC Oligo dT analytics provide a solution for fast and reproducible quantification of mRNA throughout all the process steps of mRNA production and purification. The presented method was validated using mFix4, an uncapped mRNA analog produced in-house, 3969 nt long molecule with a poly(A )tail length of 95 nucleotides.

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Messenger RNA (mRNA) is becoming a major contributor in the fields of gene therapy and vaccines, including those developed in response to the COVID-19 pandemic. Convective Interaction Media® (CIM®) Styrene divinylbenzene (SDVB) monolithic columns are promising for high resolution purification and separation of mRNA, enabling large-scale production of this molecule. This study demonstrates the ability to prepare homogeneous SDVB monoliths with desired chromatographic properties and economical analytics over the whole size range.

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Endotoxins are robust and persistent impurity, which are native to majority of phage substrates. Two anion exchangers, CIMmultus PrimaS and H-Bond, were tested for their capacity for endotoxin removal in comparison to well known strong anion exchanger, CIMmultus QA. 

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Agarose gel electrophoresis (AGE) analysis is an important method for monitoring of plasmid DNA (pDNA) quality, with ability to separate pDNA isoforms (sc, oc, lin, multimer). Plasmid linearization can be monitored for purposes of producing starting material for mRNA production. Electrophoretic conditions and, more importantly, matrix used for sample dilution before gel loading can affect analytical results. We have observed that purified linear pDNA shows an additional band in AGE analysis of the sample in water medium, which can lead to misinterpretation of results. 

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Extracellular vesicles (EV) are lipid bound products secreted by cells. Among them, exosomes have great potential for clinical applications. Animal and human-derived components used in cell culture, such as fetal bovine serum (FBS), naturally contain exosomes that can cross-contaminate the desired product. In order to study exosomes derived from cells of interest, multiple producers have come up with exosome-depleted FBS (EV (-) FBS) generated using different approaches. In this work we evaluated commercially available EV (-) FBS supplements for residual exosome content and tested their performance in upstream exosome production process. The analysis was performed with PATfix high pressure liquid chromatography system using PATfix size exclusion (SEC) analytical method.

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2021

The recently demonstrated efficacy of mRNA-based Covid-19 vaccines has shown promise of this therapeutic format, but also highlighted the need for higher efficiency of mRNA production to meet enormous needs for global vaccine supply.

Typical mRNA production process involves three key steps: 1) plasmid DNA (pDNA) production in supercoiled (sc) isoform, linearization and purification, 2) in-vitro transcription (IVT) reaction and 3) mRNA purification.

Here we present a chromatographic toolbox and mRNA IVT synthesis for integrated mRNA production from pDNA to mRNA purification, including in-process analytics. This high yield process reduces the overall number of purification steps required, improves recoveries, results in extra low protein impurity and allows for very efficient dsRNA removal.

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The IVT reaction is one of the most expensive steps in mRNA production process and its optimization to reach high mRNA yield is of key importance Standard mRNA quantification techniques like absorbance and fluorescence based assays are time consuming and cannot be performed at line as the IVT reaction progresses In addition, other reaction components like nucleotides and pDNA interfere in the analytical results and reduce the method’s accuracy A new approach shown here uses CIMac PrimaS™ analytical HPLC column to separate and quantify several key IVT components with a very short run time, enabling fast “at line” tracking

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Optimizing processing steps in sc pDNA isolation is critical for obtaining good process yields as well as high product purity. PATfix platform with convective chromatography media (e.g. monolith) offers a rapid analytical method to characterize complex biomolecular mixtures and gives immediate feedback during process development. E coli lysis represents such a challenging step, where multiple critical quality attributes need to be identified and critical processing parameters optimized. This approach leads to better yields and product purity, allowing for simplified downstream steps. A new PATfix analytical platform presented here uses CIMac pDNA column, to separate and characterize plasmid from impurities, allowing for easy optimization of key parameters such as RNA removal.

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In mRNA production process, downstream purification of in vitro transcription (IVT) reaction often relies on precipitation methods which cannot provide resolution, recovery, or reproducibility to consistently produce a safe and effective product with good process economics. mRNA is a large biomolecule (mass of 1000 nt is ~ 150 kDa and >100 nm in diameter) for which porous particle chromatography lacks the ability to support high capacity and throughput to achieve good process economics. Convective flow chromatography media (e.g. monoliths) is an optimal platform for purification. A fully scalable chromatographic purification process is presented for a posttranscriptionally capped in vitro transcribedmRNA.

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