On May 12th, the biaseparations.com website will be retired and migrated to sartorius.com. Learn more about our combined offering today!
2022

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.

Attachments

Full view

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.

Attachments

Full view

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.

Attachments

Full view

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.

Attachments

Full view

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.

Attachments

Full view

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.

Attachments

Full view

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.

Attachments

Full view

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

Attachments

Full view

Optimizing processing steps in sc pDNA isolation is critical for obtaining good process yields as well as high product purity. HPLC 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 HPLC 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.

Attachments

Full view

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.

Attachments

Full view

2020

HPLC with convective chromatography media (e.g.monolith) offers a rapid analytical method to characterise complex mixtures. Transcription reaction used for production of mRNA represents such a mixture, with components varying in size, chemical and physical properties. A new analytical HPLC approach (PATfix) presented here uses CIMacPrimaS to separate IVT components such as triphosphate-nucleotides (NTPs), enzymes, DNA template and RNA in a very short gradient.

Attachments

Full view

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.

Attachments

Full view

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.

Attachments

Full view

2005

The rapidly growing interest in the area of proteomics induces intensive efforts to find robust, automated and sensitive high-throughput analytical tools. In this context, the concept of solid-phase digestion (ex. trypsin immobilization on a solid support[1]) has received great attention in the last years. Trypsin (EC 3.4.21.4) has been covalently immobilized on different monolithic supports and resulting bioreactors used as immobilized enzyme reactors (IMERs) for on-line digestion, peptide separation and peptide mapping. Bioreactors efficiencies were evaluated with different recombinant proteins after on-line digestion. The technique used for the separation and identification of peptides was high-performance liquid chromatography coupled with electrospray ionisation tandem mass spectrometry (LC-ESI-MS/MS).

Attachments

Full view

2004

The availability of sufficient quantities of quality DNA is always a crucial point in DNA based methods, i.e. for PCR, DNA sequencing, Southern blotting, and microarrays [1]. The same is true for the PCR-based methods for detection of genetically modified food [2]. During the production chain foods passes several physical, biological, and chemical processes, which all negatively influences on the quantity of available DNA. The phenomenon is especially expressive when high temperature treatment is performed at low pH [3]. The existing methods for DNA isolation from food cannot always fulfill the expectations of quantity and quality of isolated DNA. Furthermore they usually include 100 mg of sample and are difficult to scale-up [4]. Four major chromatographic modes are used for the separation of DNA: size-exclusion, anion-exchange, ion-pair reversephased, and slalom chromatography. Of these, anion-exchange chromatography combined with micropellicular packing is described as the most prominent technique so far [1].
Anion-exchange CIM® (Convective Interaction Media) monolithic columns allow fast and flow unaffected separation of several biomolecules, including nucleic acids [5].

Attachments

Full view

2003

The only four drugs approved for the clinical treatment of Alzheirner’s Disease (tacrine. rivastigmine, donepezil and galantamine) are acetylcholinesterase inhibitors which act by maintaining high levels of acetylcholine at the muscarinic and nicotinic receptors in the central nervous system. Human acetylcholinesterase (HuAChE) represents a widely studied target enzyme and it is still object of research for the development of new drugs as enzyme inhibitors.

In a previous paper we reported the immobilisation of AChE on a silica based chromatographic column (50 x 4.6 mm 1.0.) The yeld of immobilization and the stability of the AChE-IMER were considered satisfactory, but some problems arose. The length of the IMER and the large amount of enzyme covalently bound to the chromatographic support resulted in catalysis product long elution times and some inhibitors aspecific matrix absorption with delayed enzyme activity recovery. In order to avoid these complications and considering the high rate of AChE enzymatic reaction. we decided to reduce the dimension of the solid support for immobilization, hence the amount of immobilized enzyme, by selecting a monolithic matrix disk (12 x 3 mm I.D.).

CIM® (Convective Interaction Media) monolithic supports (Biaseparations. Lubiana) represent a novel generation of stationary phases used for liquid chromatography, bioconversions, and solid phase synthesis. As opposed to individual particles packed into chromatographic columns, CIM® supports are cast as continuous homogeneous phases and provide high rates of mass transfer at lower back pressure.

In the present work a CIMK disk with immobilised human recombinant acetylcholinesterase (HuAChE-ClM® Disk) was developed. The activity of immohilised enzyme, the long term stability and reproducibility were tested. HuAChE-CIM® disk was applied as an immobilised enzyme micro-reactor (micro-IMER) in on-line HPLC system for inhibitory potency determination of known AChE inhibitors.

Full view

Traces of DNA in RNA samples represent impurities that could affect results of mRNA quantification and cDNA synthesis. In most cases, the DNA impurities in RNA samples are removed using enzyme deoxyribonuclease (DNase), which specifically breaks down DNA. In order to avoid the addition of DNase into the analyzing sample, the use of immobilized DNase on solid support is recommended. Because of the DNA size, very few supports available on the market enable efficient interaction between immobilized enzyme and DNA.

In recent years a new group of supports named monoliths was introduced. Because of enhanced exchange between mobile and stationary phase separation and bioconversion processes are significantly accelerated. Therefore also the efficiency of DNA removal using immobilised enzyme might be competitive to the degradation with free enzyme.

Attachments

Full view

The availability of sufficient quantities of quality DNA is always a crucial point in DNA based methods, i.e. for PCR, DNA sequencing, Southern blotting, and microarrays [1]. The same is true for the PCR-based methods for detection of genetically modified food [2]. During the production chain foods passes several physical, biological, and chemical processes, which all negatively influences on the quantity of available DNA. The phenomenon is especially expressive when high temperature treatment is performed at low pH [3].

The existing methods for DNA isolation from food cannot always fulfill the expectations of quantity and quality of isolated DNA. Furthermore they usually include 100 mg of sample and are difficult to scale-up [4]. Four major chromatographic modes are used for the separation of DNA: size-exclusion, anion-exchange, ion-pair reversephased, and slalom chromatography. Of these, anion-exchange chromatography combined with micropellicular packing is described as the most prominent technique so far [1].

Attachments

Full view

The availability of sufficient quantities of quality DNA is always a crucial point in DNA-based methods, i.e. for PCR, DNA sequencing, Southern blotting, and microarrays [1]. The same is true for the PCR-based methods of GMO detection in food [2]. During the production chain foods passes several physical, biological, and chemical processes, which all negatively influences on the quantity of available DNA. The phenomenon is especially expressive when high temperature treatment is performed at low pH [3].

The existing methods, for DNA isolation from food, cannot always fulfill the expectations of quantity and quality of isolated DNA. Furthermore they usually include 100 mg of sample and are difficult to scale-up [4]. Four major chromatographic modes are used for the separation of DNA: size-exclusion, anionexchange, ion-pair reverse-phased, and slalom chromatography. Of these, anionexchange chromatography combined with micropellicular packing is described as the most prominent technique so far [1].

Attachments

Full view

2000

Strains of the anaerobic bacterial genus are thought to play an important role in fiber degradation. sp. Mz5 was previously isolated from the rumen of a black and white Friesian cow and its xylanolytic activity was proved to be at least 1,65 times higher than the activities of all of the compared well known xylan-degrading rumen bacterial species and strains (1). High xylanolytic activity was the reason for partial isolation of its xylanases in order to study their special characteristics and possible biotechnological applications later.

Attachments

Full view