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.
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.
- 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.
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.
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.
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.
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.
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.
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.
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.
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
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.
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.
Linearised pDNA is currently the starting point of In-Vitro-Transcription processes to synthesize mRNA. Large scale purification protocols for manufacturing of pDNA used for Gene Therapy applications typically include two chromatography steps. The first step captures both linear, open circular and supercoiled pDNA species. The polishing step enriches supercoiled pDNA, while discarding other isoforms. We describe a single-step-capture strategy to maximize the recovery of pDNA for further linearization.
The increasing demand for messenger RNA (mRNA) as a therapeutic product requires larger production scales and more efficient extraction techniques. In this poster, fast and efficient way to purify poly-adenylated mRNA using affinity chromatography on CIMmultus™ Oligo dT column is presented.
The poly-adenylated tail of mRNA interacts with covalently bound oligo dT ligands in high-salt loading conditions, where electrostatic repulsion between negatively charged backbones of both, mRNA and oligo dT, are reduced and H-bonding in T-A base pair is emphasized. High salt concentration additionally screens out attractive electrostatic interactions between mRNA and other components in the process sample, thus facilitating aggregate reduction in purified product.
Immunoaffinity columns using antibodies as ligands against mammalian proteins could be used for different applications in protein expression control and, if a standard available, for direct protein quantification in complex sample solutions. Additionally, these columns are ideal for polishing step of recombinant proteins, such as mammalian receptor Fc fusion proteins. Most importantly, such columns could extract a significant amount of a single membrane protein from native source, suitable for downstream analyses, such as mass spec analysis of their glycans. Immunoaffinity chromatographic monoliths against RAE-1 GPI anchored glycoprotein were developed (CIMmic HDZ - @RAE-1 column) as a part of Glycomet project with the main goal to analyze the antigen glycoprofile.
Hydrazide-activated (HDZ) columns were proven to be a product of choice for making the most effective immunoaffinity columns. They take advantage of a special hydrazide linkage that binds antibodies through the carbohydrate residues on their Fc regions. This leaves the antigen-binding domains fully accessible to enable the most effective capture of desired target (Figure bellow).
CIMac™ HDZ monoliths make HDZ-immobilized antibody columns even more effective. Because of their large channel size and the efficiency of convective mass transport, they eliminate the long loading residence times that are required for affinity chromatography on porous particle columns. Flow rates of 5–10 column volumes per minute allow complete purifications in a few minutes, even when the source material contains a low concentration of antigen. The same performance is achieved whether a small peptide or a large bio-assemblage like a virus particle or extracellular vesicle is isolated. The combination of HDZ monoliths and the immobilization protocol offers a strong tool for fast antigen isolation from complex biological sample (plasma, lysate, etc.) and consequently sensitive antigen quantification. An example of CIMac™ HDZ application is a purification of fibrinogen from human plasma.
The upstream and downstream monoclonal antibody (mAb) bioprocessing makes them susceptible to physical and chemical modifications. In the biotechnological production process of mAbs, structural variations may arise due to some enzymatic activity. Antibody charge variants have gained considerable attention in the biotechnology industry due to their potential influence on stability and biological activity and cation-exchange chromatography (CEX) is one of the typical approaches for mAb charge variant analyses. We tested several CEX columns under different conditions and the best column for isotype separation was weak cation-exchanging CIMac COOH chromatographic monolith in pH gradient. We have proven a flow independent separation of mAb charge variants and in this way, a resolution comparable to classical CEX particulate-based analytical columns was achieved in only 6 min analysis time.
CIMac™ r-Protein A Analytical Column is short bed, high performance monolithic column . Primarly is intended for fast, efficient, and reproducible qualitative and quantitative analyses of Immunoglobulin G (IgG). It is suitable for use with HPLC and UPLC systems. Quantification of Immunoglobulin G is possible between 0.2 μg and 20 μg. Its small volume and short column length allow operation at high volumetric flow rates ( up to 3mL/min). The information about product quantity and purity is thus generated in just 1 minute! The column has innovative symmetric design for bi-directional flow, also extending column lifetime.
Biological samples often consist of a main component, such as albumin in serum, and many other constituents, present in smaller quantities, but nevertheless of high importance in biological systems. When detection of the low-abundance molecules is needed, the main component could interfere with the analyte, complicating the analysis or even making it impossible. In such cases a possible approach is to remove the interfering main component from the sample before the analysis.
Monolithic columns (CIM®) are a great foundation to build affinity chromatography methods, as they offer fast flow rates and can be modified to accomodate various ligands. We selected two most promising approaches for oriented binding of antibodies to the monolithic support. One approach was to bind antibodies to a protein A (pA) column with consequent crosslinking of the protein complex. The other approach was to chemically activate antibodies and bind them selectively to hydrazide-modified (HDZ) monolith surface.
Interactions between antibodies and their antigens are highly selective and therefore immensely popular for affinity chromatography. Consequently, numereous antibody immobilizations were performed on monolithic supports via different activation chemistries in the last decade. Despite the work already done there was no systematic study, where as many as possible activation chemistries were tested for the immobilization of a model monoclonal antibody with subsequent chromatographic characterization of the affinity support. In this work, various preactivated CIM monolithic columns were used for the immobilization of a model monoclonal IgG.