Recombinant baculovirus is extensively used for expression of recombinant proteins in insect cells. The appeal of baculovirus systems lies in their high level expression within an eukaryotic system, providing target proteins with appropriate posttranslational modifications. Recent approaches as vector in human gene therapy applications indicate a new dedication for baculovirus.


In any field of operation the increasing demand of highly pure baculovirus requests efficient, robust and scaleable purification strategies. Traditional techniques such as ultracentrifugation and tangential flow filtration are efficient in terms of virus concentration, but suffer from low yield and clearly lack robustness and scalability. In this application sheet we introduce a CIM monolith based purification process for infective baculovirus.

The protocol provides high recovery of active virus, efficient removal of host cell impurities, ease of use and straight forward scale up.

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Orthoreoviruses are dsRNA, non-enveloped viruses that can cause severe enteric and respiratory infections in humans and other animals. It is speculated that these viruses might be an important zoonotic pathogen. As such, orthoreoviruses can cause infections of undetermined etiology which are difficult to resolve. Next-generation sequencing (NGS) is a new technology which enables gathering a huge amount of genomic information from a sample in a short period of time. NGS is being increasingly applied in animal screenings for pathogen discovery and has a great potential in clinical microbiological diagnostics. However, the preparation of high-quality and high-quantity nucleic acid samples is a major concern for efficient application of the method.


CIM QA® disk in combination with NGS was used for discovering a novel reovirus in stool samples of a child with gastroenteritis infection of undetermined etiology. Two different starting samples were compared: clarified stool suspension and supernatant from cell culture inoculated with clarified stool suspension.

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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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60 mL of non-concentrated periplasmic fraction from Prevotella bryantii cultivation medium were subjected to two-step purification on CIM® DEAE 8 mL tube. Both purification steps were performed under similar chromatographic conditions, i.e. same column, buffer systems and flow rate (7 mL/min). Gradient slopes varied to achieve isolation of the desired fraction. The elution fraction of interest of purification No1 was subjected to a second purification on the same column. By altering the linear gradient pure 66 kDa-Endoxylanase for production of antibodies was isolated.

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50 mL of crude enzyme solution were loaded on CIM® DEAE 8 mL tube and eluted by a linear gradient at a flow rate of 1.2 mL/min. Analysis of the elution fraction resulted in 86.1% recovery and 4 times concentration of the sample, having a purity of >95%.

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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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Plasmids in the size range of 21 to 93 kbp were harvested from E.coli, desalted and concentrated by 2-propanol precipitation, and subjected to chromatography on CIM® DEAE Disk. Elution was effected by a combination of linear and stepwise gradient at a flow rate of 3 CV/min. Analysis of eluted supercoiled pDNA indicated that 21 and 39 kbp plasmids remained intact while larger plasmids (62 and 93 kbp) partially degraded during the purification step.  

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pDNA isoform separation was performed on CIM® C4 1 mL tube in linear descending salt gradient mode. Crude bacterial lysate containing pDNA was incubated with 4 M ammonium sulfate for 90 minutes at room temperature. Prior loading on CIM® C4 1 mL tube the solution was filtered. Elution and isoform separation was achieved by descending ammonium sulfate gradient and resulted in an sc:oc ratio of 99:1.  

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Bacteriophage T4 was harvested from E.coli suspension. The clarified and 0.22µm filtered solution was subjected to single step separation on CIM® QA Disk. An optimized stepwise elution gradient resulted in 78% recovery of infective bacteriophage in the elution fraction, while efficient separation of phages from DNA and proteins was obtained.

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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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A supernatant from Phanerochaete chrysosporium cultivation was loaded on CIM® QA Disk, and elution was effected by a linear gradient at a flow rate of 3 mL/min (9 CV/min). Baseline separation of isoenzymes H2, H6/H7, H8 and H10 was achieved in less than 3 minutes.

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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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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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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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Diluted samples of live attenuated measles and mumps virus were each loaded on CIM® DEAE Disk. Concentrated eluates of viral RNA were subjected to molecular detection by PCR. It was demonstrated that enrichment of viral RNA on a CIM® DEAE Disk prior PCR is feasible and successful.

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Adenovirus vectors have proven as useful tool for gene therapy, vaccine therapy and basic biology studies. The increasing importance of the recombinant adenoviruses pushes the limits of research in the field of adenovirus purification methods. There is a global focus on large scale production of adenovirus vectors, providing high titres combined with fast, effective and reliable purification methods.


Because of the physico-chemical properties adenovirus vectors possess, they can effectively be purified using ion-exchange chromatography. Here we present a simple and rapid method for adenovirus vectors purification using ion-exchange CIM ®QA chromatographic supports (Figure 1). CIM® monolithic supports are a new generation of chromatographic supports able to meet the GMP and GLP requirements in the field of virus purification.

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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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