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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Polyclonal anti-HSA antibodies were immobilised on CIMmic™ HDZ (hydrazide chemistry, 100 μL bed volume), following an optimised protocol. This CIMmic™ α-HSA column was prepared to quantitatively remove human serum albumin (HSA), the most abundant plasma protein in humans, and obtain albumin-free samples which can be further processed.
The CIMmic™ α-HSA column is optimized to selectively bind and remove HSA from plasma; albumins from other sources are bound with low efficiency. HSA depletion is carried out in two steps: (i) a loading step in which the diluted and filtered plasma sample is loaded into the column, HSA is bound and HSA-depleted plasma is collected downstream of the column and (ii) a regeneration step in which the bound HSA is removed and the column is regenerated.

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The increasing demand for messenger RNA (mRNA) as therapeutic product requires larger production scales, and in turn more efficient extraction techniques. Messenger RNA can be produced by in vitro transcription reactions (IVT) or isolated from eukaryotic cells. One of the most convenient techniques for its extraction is the use of oligo deoxythymine (dT) coupled to a solid support. Oligo dT hybridises to the poly-adenylated tail which is present on most eukaryotic mRNAs, or synthetised onto the molecule during IVT. Contaminant impurities, such as proteins, unreacted nucleotides, plasmid DNA, CAP analogues, partial transcripts, dsRNA side products and enzymes lack the poly-A moiety and are not retained on the solid support.
Chromatography using a solid phase consisting of large channels, such as monoliths, allows high flow rates and low shear forces. This can have a positive impact on recovery and productivity in purification of biologics. In addition, chromatography offers a closed system to minimise the risk of cross-contamination or exposure to RNase degradation, and an easily scalable platform.
CIMmultus™ Oligo dT is a chromatography column with Oligo dT ligands covalently bound on its surface. The sample containing poly-adenylated mRNA is loaded onto the column in a high salt concentration buffer. Salt ions screen the electrostatic repulsion between the negatively charged backbones and allow interaction between the Oligo dT and poly-adenylated tail of mRNA. Before product elution, a wash step at reduced salt concentration removes unspecifically bound contaminants. Elution of messenger RNA occurs under mild conditions in low conductivity buffer at neutral pH. In the absence of salt, electrostatic repulsion between the negatively charged backbones of Oligo dT and poly-adenine destabilises the T–A pairs and releases mRNA from the column.

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Pre-activated CIMmic™ Monolithic Columns are used as a basis for preparation of small volume affinity chromatographic columns as well as enzyme reactors. Small bed volume and flexible design makes them a powerful tool for screening purposes and immobilization protocol optimizations. Range of covalently bound ligands is wide and includes diverse set of proteins, peptides, nucleotides and other affinity ligands. The covalent nature of the bond between the ligand and matrix reduces leaching and improves stability and reusability. Reaction conditions must cater to their specific physiochemical nature.


Successful preparation of an affinity column includes a decision on the appropriate matrix chemistry and determination of an optimal immobilization protocol. Presented case study explores the basics of a coupling protocol optimization using covalent immobilization of Recombinant Prokaryotic Lectins (RPL-Gal1) on CIMmic CDI-0.1 and CIMmic ALD-0.1 columns, as an example. Carboxy imidazole (CDI) and aldehyde (ALD) activated CIMmic™ columns are used for covalent immobilization of amine or thiol containing molecules.

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CIMmic™ Monolithic Columns combine the advantages of the CIM® stationary phase with a flexible design and the possibility to operate with syringe. Discs containing the stationary phase can be easily interchanged inside the custom designed housing. Pre-activated chemistries enable immobilisation of numerous ligands and can be used for preparation of affinity chromatographic columns or enzyme reactors. Their small bed volume is particularly suitable for screening purposes and to optimise immobilisation protocols due to economic usage of often expensive ligands.

Carboxy imidazole (CDI) monolithic chromatographic columns are used for covalent immobilisation of proteins, peptides and other amine or thiol containing molecules. The covalent nature of the carbamate bond between the ligand and matrix reduces leaching and improves stability and reusability.

An example described below shows the feasibility of CIMmic™ CDI-0.1 utilisation for covalent immobilisation of recombinant protein A (r-pA). Additionally the example was used for the evaluation of the reproducibility of CIMmic™ CDI columns. The dynamic binding capacity for human polyclonal immunoglobulin (IgG) was used as metric for comparison of the affinity columns.

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2018

Bottom-up proteomic approach based on tandem mass spectrometry (MS/MS) is a method of choice for identification and quantitation of proteins in the complex biological samples. Followed by quantitative analysis of a protein sample, this approach enables the identification of putative biomarkers for early pathology diagnostics and differentiation. The main challenges confronting this analysis are suppression of low-abundance ions and the limited dynamic range of MS/MS.
Affinity depletion of abundant proteins is an important stage in routine sample preparation prior to tandem mass spectrometry (MS/MS) analysis of biological samples. One such protein is Human Serum Albumin (HSA). In this study, polyclonal αHSA antibodies were immobilised onto a chromatography support to use as immunoaffinity-based column (CIMac™ HDZ -αHSA) for HSA depletion from embryo culture media for in vitro fertilization (IVF).

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As an alternative to conventional particle-packed columns for liquid chromatography, CIM® monolithic supports can be used.1-3 They are extremely permeable and allow very efficient mass transport at low back pressures and good separation efficiency at high flow rates. Consequently, the time of chromatographic separation can be shortened. Such characteristics are very valuable in speciation analysis where preservation of the integrity of individual chemical species of a given element is of crucial importance.4,5 CIM® monolithic disks can be placed together in one housing forming so-called conjoint liquid chromatography (CLC), which combines two different chromatographic modes in one step.
For better understanding of anticancer therapy with Pt-based chemotherapeutics, the quality of pharmaceutical formulations must be assured and the behaviour of the drugs studied by separation and detection of the intact drug and its individual biotransformation species in clinical samples at therapeutically relevant levels. To study the interactions of Pt-based chemotherapeutics with serum proteins, two-dimensional chromatographic separation of serum proteins was suggested, combining size-exclusion and CIM DEAE monolithic.6 Since this procedure is time consuming the potential of the use of CLC was investigated.

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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

Monoclonal antibody (mAb) charge variants have gained considerable attention in the biotechnology industry, due to their potential influence on stability and biological activity of the active pharmaceutical. Cation-exchange chromatography (CEX) is historically one of the typical approaches for mAb purification and characterization, especially charge variant analysis. We have proven the flow independent separation of mAb charge variants using CIMac COOH monolithic column in a pH gradient. In this way, a resolution comparable to classical CEX particulate-based analytical columns was achieved in only 6 min analysis time. Therefore a CIMac COOH column is a perfect choice for fast Process Analytical Control (PAT) of purified mAb samples.

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PEGylation involves the formation of a stable covalent bond between activated poly (ethylene glycol) polymers and polypeptidic drugs and molecules. This process causes a change in protein hydrophobicity and results in variance between the obtained conjugates. Despite this, hydrophobic interaction chromatography (HIC) is used less frequently for separation of PEGylation reaction products than other techniques. Separation of PEGylated conjugates of Ribonuclease A (RNase A) via HIC on monolithic supports was analysed in this work. The protein was PEGylated in the N-terminal amino group with 20 kDa methoxy poly (ethylene glycol) propionaldehyde.

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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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Transferrin (Tf) is a glycoprotein that transports iron to cells and has two N-glycosylation sites in humans – at asparagine 432 and asparagine 630. Carbohydrate-deficient Tf, which lacks one or both N-glycans, is the most common marker for congenital disorders of glycosylation.1 Altered Tf glycosylation has also been reported in hepatocellular carcinoma2 and chronic alcohol consumption.3,4 High-throughput Tf purification and glycan characterisation methods are under extensive development in order to facilitate screening of glycosylation patterns for population, genetic and clinical studies.

This application note describes the development of an immunoaffinity purification method on a CIMac™ analytical column with immobilised anti-transferrin antibodies (@Tf) and the successful transfer of the method to the monolithic 96-well plate (CIM® @Tf-0.2 monolithic 96-well plate). The affinity purification method has been used for Tf isolation from human blood plasma followed by ultra-performance liquid chromatography (UPLC) analysis of Tf N-glycosylation.

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CIMac™ r-Protein A Analytical Column is a short bed, high performance monolithic column. It is intended for fast, efficient, and reproducible qualitative and quantitative analyses of Immunoglobulin G (IgG) and suitable for use with HPLC and UPLC systems. Quantification of IgG 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 3 mL/min). The information about product quantity and purity is thus generated in just 1 minute! The column has an innovative symmetric design for bi-directional flow contributing to longer lifetime.

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The CIMacTM antibody immobilization platform enables an elegant immobilization of antibodies, which can be used as immunosorbents in specific diagnostic applications as well as in downstream processes. In this work we show the dependence of the coupling strategy on CIM monolith with the chromatographic efficiency of final immunoaffinity adsorbent. Different activation chemistries  were tested for the immobilization of two model monoclonal antibodies (mAbs) with subsequent chromatographic characterization of the affinity support.

Column used for this application note were CIMac CDI, AE, EDA, HDZ, rpA.

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2015

The CIMac™ antibody immobilisation platform enables an elegant immobilisation of antibodies, which can be used as immunoaffinity adsorbents in specific diagnostic applications. Immobilization of antibodies to solid chromatographic supports is avery elegant solution for preparation of reusable immunoaffinity assays that can lower the price but also increase the accuracy and lower the limit of detection of biological assays for diagnostic purposes. Therefore, it is important that reusing a column for multiple sample analysis does not affect chromatographic/analysis performance. In this application note, an example of reusability of the hydrazide-based CIMac™ column ( HDZ ) with immobilised monoclonal antifibrinogen for consecutive purification of fibrinogen from human plasma is shown.

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In diagnostic applications, fibrinogen is measured with clotting activity assay variations, together with other blood clotting factors. In research and preparative applications, larger quantities of anti-coagulants, such as EDTA and citrate, or coagulants such as Ca2+ are present in the samples, preventing successful and/or accurate fibrinogen concentration measurements. In such cases an alternative measurement method is needed.
The CIMac™ antibody immobilisation platform provides an elegant solution for immobilisation of antibodies, which are used as immunoaffinity adsorbents. It has been shown that several analytical runs can be performed on a single column. This simplicity is also combined with a fast method, where a chromatographic analysis can be completed in 4 min. Used column was hydrazide ( HDZ )-based CIMac™ column.

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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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The demand for human normal immunoglobulin is invariably increasing on an annual basis. To satisfy increasing demands, different manufacturing processes are used to isolate immunoglobulins from human plasma. A quest for alternative paths in manufacturing not only requires development of most economical manufacturing process, but also rapid method development and development of good analytics for monitoring of manufacturing. For an efficient development of the purification methods as well as for in-process control during manufacturing stage, the uses of reliable and fast analytical techniques are crucial.
Fast and reliable fingerprint-based method for characterization of immunoglobulin G (IgG) prepared from Cohn I+II+III paste in two chromatographic steps is presented here. The fingerprint method bases on partial separation of proteins in linear gradient on CIMac™ QA 0.1 mL column. Partial separation of proteins does not allow simple quantitative analysis of the samples, however a very accurate qualitative information about the composition of the sample being analyzed can be obtained in less than 5 minutes.

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