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.
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. Carboxyimidazole (CDI) and aldehyde (ALD) activated CIMmic™ columns are used for covalent immobilization of amine or thiol containing molecules.
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.
Carboxyimidazole (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.
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). 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.
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).
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.
The application describes separation of Ni species by assembling four weak CIM DEAE anion-exchange disks into a monolithic column. The concentrations of the Ni species eluted from the column were quantified by post-column isotope dilution inductively coupled plasma mass spectrometry (ID)-ICP-MS. The Ni binding ligands eluted under the chromatographic peaks were identified off-line by tandem electro spray mass spectrometry (ESI-MS-MS), scanning for negative ions.
The mild chromatographic conditions of the CIM DEAE disks preserved chemical species and enabled separation of negatively charged Ni complexes.4 NH4NO3 was chosen as eluent since it enabled separation of Ni species and is compatible with ICP-MS and mass spectrometry detectors.
Human coronavirus OC43 (HCoV-OC43) is a frequent cause of respiratory tract illness, ranging from common cold to severe disease. The research on coronaviruses and medical application of coronaviral vectors/vaccines requires a quality material of high purity. Unfortunately, virus preparations are highly contaminated with cell debris and purification requires laborious, cost-ineffective procedures.
Here, we report a simple and efficient method for coronavirus concentration and purification by the example of HCoV-OC43. To achieve this, virus chromatography was performed on CIM QA monolithic columns (BIA Separations), with immobilized positively charged quaternary amines. The quality of the obtained virus stock was assessed with SDS Page electrophoresis, followed by Western blot analysis. Finally, infectivity of recovered virus was evaluated by titration.
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.
Influenza vaccines are still predominantly produced in embryonated chicken eggs and the purification processes barely have changed during the years. There is a growing need for fast, efficient and economical vaccine production.
So far, monolithic supports have been used successfully in virus purification and concentration, as well as in the purification of virus-like particles (VLP) propagated in cell cultures.
Therefore, our aim was to prove the applicability of monoliths in purification of influenza virus A propagated in embryonated chicken eggs.
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.
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.
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).
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.
Downstream processing of viruses in virus vaccine or virus vector production accounts for up to 70% of the overall production costs. Immunoaffinity chromatography is a powerful purification technique due to its high specificity but is disadvantageous by the fact that the elution conditions are often detrimental for both the immobilized proteins and target antigens, especially viruses.
This application note describes the mumps virus purification using monolith-based immunoaffinity stationary phase and recently invented native elution of the bound viruses using amino acid solutions under physiological pH.
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.
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.
Determining the concentration of viruses is a crucial step in any production process. The most commonly used methods for virus quantification are either based on the infectivity of the virus (plaque assay, TCID50) determination of their genomic material (qPCR), or protein content (SRID, ELISA) and are very cumbersome and time consuming. HPLC analytical methods represent a fast alternative to these assays since they provide information on the virus content and purity in a matter of minutes. In addition to that, the data obtained is very reproducible and accurate.
For any kind of quantification, a calibration curve obtained with a virus standard is needed. The work presented in this application note shows the excellent performance of the CIMac™ Adeno Analytical Column – a monolith based anion exchange column, designed for fast and reproducible analyses of adenoviruses.
Adeno-Associated Virus (AAV)-based vectors of various serotypes are considered to have high potential in human gene therapy and genetic vaccination applications. During manufacturing of AAV vectors undesired, incomplete particles are co-produced. They lack recombinant viral genomes and consist of empty capsid proteins only. Empty capsids increase the required dose of AAV virus for medical applications and are thought to cause immunological reactions against the vector capsid, leading to unwanted side effects. Removal of empty capsids during manufacturing, as well as the ability to quantify the amount of empty AAV particle content in a formulation is hence a critical requirement for any AAV production process.
Current methods for preparative separation of empty capsids (CsCl or iodixanol gradients) are challenging to scale-up and are not suitable for large-scale production. Furthermore, analytical methods for detection of empty capsids and determination of full to empty particle ratio (electron microscope (EM) assay, total particle assay [ELISA] combined with genome copy titration [qPCR]) are time- and labour consuming, influenced by operator technique or do not provide readily available reagents for different serotypes of AAV.
A new approach for separation of full and empty AAV8 particles by exploiting minor charge differences is presented in this application note. By using linear gradient elution on a CIM QA Disk Monolithic Column, a simple, rapid and reproducible assay for analysis of AAV particles is introduced. The method was successfully applied to AAV8 particles prepared by two different manufacturing processes.
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.