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2022

Robust and precise chromatographic analytical methods are key for efficient development of mRNA production process. Three different analytical methods, which utilize three different column chemistries, are embedded in a ready-to-use PATfix™ mRNA analytical platform to support mRNA process development and product quantification and characterization.

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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 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.

Due to an increasing productivity of IVT reaction, finding conditions that increase binding capacity of Oligo dT has been an intense focus of development. Multi-parallel approaches, such as screening in multi-well plate format, can significantly cut the development time by screening multiple conditions at once. 96-well plates can then be scaled-up to preparative scale, such as CIMmultus™ product line operated by chromatographic skids.

Continue to products for screening

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CIMac™ pDNA Analytical Column is powerful tool for pDNA quantification for in-process control or in a QC laboratory. The column can separate pDNA isoforms from each-other and from RNA impurities. Monitoring of pDNA production leads to a controlled and robust process, and can result in consistent high quality of the final product.

Optimised methods are a key component of a well-functioning analytical system, sometimes requiring time-consuming method development and steep learning curves. The following two methods described in this quick start guide can provide a starting point for pDNA purity and isoform analysis.

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2021

Optimized analytical methods are key components of a well-functioning analytical system, while method development usually comes with a time-consuming learning curve and optimization.

PATfix pDNA analytics platform, designed for in-process control of linear pDNA production, enables monitoring of pDNA linearization progression, as shown in Figure 1. Fully optimized and validated analytical methods, as well as guidelines for buffer and sample preparation come as part of the PATfix system, allowing users to focus on their specific application. In addition, the PATfix pDNA analytical package includes a pDNA calibration standard, which enables accurate quantification of the pDNA species of interest.

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2020

Miniaturised immobilised enzymatic reactors can be used for small scale digestion of proteins. There is need for such devices; small scale devices are used either for processing of analytical sample quantities, or as proof of concept before protein digestion at larger scale. This application note compares the performance of a flow through miniaturised immobilised enzymatic reactor (μIMER) with in-solution batch digestion of simple proteins and complex matrices. Automation of peptide analysis by coupled LC-MS is explored as an option to increase throughput. In the cases evaluated, the miniaturised immobilised enzymatic reactor offered comparative results to overnight in-solution digestion, within less than 10 minutes.


Pre-activated CIMmic™ monolithic columns with 100 μL bed volume were immobilised with trypsin from bovine pancreas. This small format allows coupling to HPLC for on-line protein digestion, as well as syringe (manual) operation of the IMER. Pre-treated samples (denatured, alkylated and ultra-filtered) are injected into the column, and the eluate (tryptic digests) are subjected to LC-ESI-MS-MS analysis for protein identification and post-translational modification (PTM) determination.

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2019

A purification of synthetic oligonucleotides by using CIM™ monolith was evaluated. In this case study, the CIM™ anion exchange column had the capability to resolve oligonucleotides with small difference in comparative chain length.

A crude reaction mixture of synthetic oligonucleotide was loaded onto the CIM™ anion exchange column. Sample elution was achieved by salt concentration gradient. In comparison with conventional media, CIM™ monolith indicated higher resolution for major impurities.

Advantages of the characteristic properties of the CIM™ monolith were evaluated based on the high throughput purification of oligonucleotides under the identified gradient separation conditions. Over 99 % HPLC purity for the target oligoDNA was achieved by one-step purification from the crude reaction mixture.

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Pre-activated CIMmic™ monolithic columns are cost efficient tools for screening of immobilisation conditions and small scale proof-of-concept testing of custom affinity columns and enzymatic reactors. Each column is assembled from a dedicated housing and discs containing a chromatography medium. With a bed volume of 100 μL, sample requirements are minimal, while inserting multiple discs in the housing adapts the column volume to application requirements. Different surface modifications of the discs enable immobilisation of a wide variety of ligands.

The increasing demand for messenger RNA (mRNA) as a therapeutic product requires larger production scales, and in turn more efficient extraction techniques. One of the most convenient techniques for its extraction is the use of oligo deoxythymidine (dT) coupled to a solid support [1]. Oligo dT hybridises to the poly-adenylated tail which is present on most eukaryotic mRNAs, or synthetised onto the molecule during IVT, while other contaminant impurities (proteins, unreacted nucleotides, plasmid DNA, CAP analogues, partial transcripts, dsRNA side products and enzymes) lack the poly-A moiety and do not adhere to the solid support.

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2018

Coupling trypsin enzyme onto chromatographic supports provides a platform to reuse the enzyme and automate the hydrolysis process. A monolithic chromatographic support, such as Convective Interaction Media (CIM®), enables mass transfer of molecules within its channels exclusively by convective flow. This results in enzymatic conversion which is not limited by diffusion, making CIM® monoliths ideal for the preparation of immobilised monolith enzymatic reactors (IMERs). Sartorius BIA Separations offers CIMac™ trypsin IMER with a bed volume of 0.1 mL as analytical platform for mass spectrometry (MS)-based proteomics. Larger volume IMERs (up to 80 mL) are available for industrial scale production of protein hydrolysates. The following example describes the enzymatic production of β-Lactoglobulin (β-Lg) hydrolysates using monoliths with 2 μm (N1) or 6 μm (N2) average channel diameter.

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2017

Sample displacement chromatography exploits the different relative binding affinities of components in a sample mixture to achieve accummulation of a desired substance on the column before elution. In pharmaceutical applications, requirements for purity and efficacy of plasmid DNA (pDNA) as a therapeutic product are stringent. The separation of linear, supercoiled (sc) and open-circular (oc) pDNA isoforms has already been established on CIM® butyl (C4 HLD) monolithic columns at preprative scale. This process requires high concentration of ammonium sulphate for loading which increases the overall production requirements. Competing adsorption in sample displacement chromatography utilises the binding capacity of the chromatographic resin more efficiently and increases productivity of the chromatographic step.
This application note investigates three monolithic chromatographic supports with different hydrophobicities regarding their applicability for sample displacement of pDNA. CIMac™ C4 HLD (butyl, high ligand density) as a commercial product and pyridine and histamine as custom immobilised columns are compared.

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2016

Plasmid DNA (pDNA) as a pharmaceutical product has stringent requirements of purity and efficacy and often one or more chromatographic steps are used in the downstream process. High ligand density butyl-modified chromatographic monolith (CIMmultus™ C4 HLD, part of CIMmultus™ HiP² Plasmid Process Pack™ 1-1, product number 100.0011-2) is currently used in a polishing step of a pDNA purification process (1), is mainly used for separation of supercoiled (sc) pDNA separation from open circular (oc) and linear pDNA isoforms as well as for removal of remaining gDNA and RNA.
This application note presents a comparison of two different polishing processes employing monoliths, namely bind-elute (BE) and the more recently described (2) sample displacement purification (SDP).

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2014

DNA immunization can potentially induce both, humoral and cellular immune responses, and thus comprises an attractive approach for the development of an effective vaccine against HCV. The pIDKE2 plasmid is the main component of the CIGB's candidate vaccine against Hepatitis C virus (HVC), which is being used in HCV chronically-infected individuals during clinical trials phase 1 and 2.


In order to satisfy the high demanding plasmids consumption for clinical trials, the downstream process was improved to reach the quantities need it for clinical trials.

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2013

Process Analytical Technology (PAT) is of crucial importance in the process of IgM manufacturing, especially in its optimization where fast and reliable analytical methods capable of quantitation of the corresponding recombinant IgM concentration levels in the upstream processes are required.


Convective Interaction Media CIM® strong anion exchange monolithic columns have a great advantage in comparison to particle related methods due to their separation capability based on the convective flow mechanism that proved to be particularly efficient in the separation of large IgM molecules.

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2011

Filamentous phage M13 is a rod shaped non-lytic bacterial virus. M13 genetic material is used for many recombinant DNA processes, and the virus has also been studied for its uses in nanostructures and nanotechnology. The phage has been intensively studied for purposes of phage display and as a delivery vehicle for gene therapy. Phage display was first demonstrated with M13 bacteriophages and the filamentous phage remains a workhorse for this technology. Because of its typical size and rod shape it is considered as a challenging for purification. With large and highly interconnected pores monolithic chromatographic supports are also bridging that problem.


The ability to improve the purification process of M13 and other phages can have a significant impact on the market. By using phages for gene therapy, there will be a decrease in manufacturing time and production costs while enhancing the gene insertion. For phage display, a quicker method for phage purification will allow this powerful tool, which shortens the new drug discovery path and illuminates the basic interactions between different proteins, to be used with higher frequency.

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2010

Bacteriophages are used in a broad range of applications, including phage therapy and phage display. With the growing problem of antibiotic resistance leading to untreatable bacterial infections, they are becoming very interesting as antimicrobial agents, not only in medicine, but also in veterinary medicine, food industry and agriculture. Phages intended for use as antimicrobial agents, especially those for human use, need to be purified of contaminants.


Here we present efficient single step purification method for a Staphylococcus aureus phage VDX-10 from bacterial lysate on a CIM® QA Disk Monolithic Column (Figure 1). The described method can be used also on a larger scale using a CIM® QA-8 mL Tube Monolithic Column (Figure 2).

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Bacteriophages, viruses that infect bacteria, are being used as antibacterial agents, in phage display screening, as gene therapy delivery systems, and for bacteria typing. To use phages in these applications, they must be free of all impurities. A purification and concentration process was recently developed using an ion exchange monolithic column [1]. One of the key challenges faced in phage purification is the monitoring of genomic DNA (gDNA) released to the growth medium which can interfere with the various applications of phages. CIMac™ DEAE Analytical Columns can be used to monitor the fermentation process, evaluate the amount of degraded gDNA to determine the optimal fermentation endpoint and then to efficiently purify the phage particles.

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As the demand for plasmid DNA (pDNA) based gene therapy and vaccines increases, large scale, cost effective, and reproducible pDNA production will be required. The key to success is a real time in-process control method that ensures a high percentage of supercoiled pDNA in the final product. CIMac™ pDNA Analytical Column allows the monitoring of degradation products (open circular and linear pDNA), the removal of impurities (RNA), and ensures that each production step is yielding the amount of supercoiled pDNA anticipated.

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2008

A mixture of 8mer, 10mer, 12mer, 14mer, 15mer and 16mer Oligodeoxynucleotides was loaded on CIM® DEAE Disk and eluted in linear gradient mode at a flow rate of 6 mL/min (17 CV/min). Separation of all nucleotides could be accomplished within 60 seconds.

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