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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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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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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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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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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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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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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Lab scale production of recombinant human monoclonal antibodies (mAbs) is required for the identification and characterization of lead clones with potential therapeutic value. For this purpose, many mAbs need to be screened. MAbs titers in this type of production scale tend to be quite low (from 0.01 – to 0.1 mg/mL), therefore a substantial amount of material needs to be processed to obtain the right amount of purified mAbs. Speed of processing and the ability to capture mAbs from diluted harvest stock are essential in this type of mAbs purification.


In this application note, a quick purification procedure using a CIM® r-Protein A-80 Tube Monolithic Column that generated up to 100 mg of mAbs with a purity of more than 95 % is described. Elution of mAbs is performed using a two-dimensional gradient (pH 7.2 to 2.5; NaCl 150 to 500 mM), allowing gentle elution of a wide range of mAbs at moderate pH (pH ~4) without any method optimization. Using this procedure, approximately 30 different mAbs were purified, processing up to 5 L of loading material (2 times diluted clarified harvest).

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Immunoaffinity columns were prepared by immobilization of Protein G on CIM® Epoxy Disk, CIM® Epoxy tube (1 mL) and an activated, particle based agarose support. A comparison of productivity was performed by loading centrifuged human plasma and resulted in superior productivity of CIM® monolithic supports.

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A sample of mouse ascites was subjected to separation of proteins and isolation of IgG by means of CLC. Configuration of CLC was chosen to have CIM® QA Disk (Anion Exchange Chromatography) as first and CIM® Protein A Disk (Affinity Chromatography) as second column. The single step operation resulted in baseline separation of proteins and IgG. Elution of proteins was affected by linear salt gradient elution, while elution of IgG was effected by a stepwise pH shift.

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A mixture of IgG, HSA and IgM standard was loaded on CIM® EDA Disk and eluted in linear salt gradient at a flow rate of 4 mL/min (12 CV/min). A complete separation of IgM from IgG and HSA was obtained within 1.5 minute.

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A Hemoglobin A1c reference standard was loaded on CIM® SO3 monolithic column and eluted in a mixed stepwise and linear gradient. HbA1a, HbA1b and HbA0 variants were separated and a complete determination of HbA1c (including equilibration) was obtained within 1.1 minute.

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The demand for monoclonal antibodies is invariably increasing on an annual basis. To satisfy increasing demands, faster and cheaper ways of manufacturing are explored. 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 a quickly developed process, the use of reliable and fast analytical techniques are crucial. Moreover, this analytical technique should than be preferably used also for in-process control during manufacturing stage.


Here we present fast and reliable method for processing and analyzing IgG, IgA ang IgM using CIM® QA Disk Monolithic Column, which thrive upon speed, repeatability and high capacity.

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