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2012

In the last few years pharmacology has made a big step towards the new type of drugs, called biological drugs. Popularity and market for biological drugs grew exponentially, so did the need for fast and inexpensive purification. Classic liquid chromatography columns were unable to separate biological compounds in industrial quantities, therefore the scientists were looking for alternatives. One of them are monolithic materials. Monolithic materials, especially methacrylate monoliths, are becoming more and more popular in separation processes due to their fast separations, low pressure drop and mechanical stability.

In the context of preparing new columns and improving existing ones, we need to know every single chemical as well as mechanical property of our monolithic material. Here we present some key data and interesting correlations between mechanical and structural properties of GMA-co-EDMA porous monolith. In the first paragraph we compare nonmodified and DEAE modified monoliths with different average pore size and porosity, regarding to their compression and tension properties. The second paragraph deals with the impact of these parameters on the permeability of the column during separation.

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Commonly, epoxide-based monoliths used as porous supports in affinity chromatography are synthesized from glycidyl methacrylate (GMA) and ethylene glycol dimethacrylate (EDMA) by free radical polymerization.

We prepared an epoxide-based monolith by self polymerization of polyglycidyl ethers where the epoxy groups serve as functional groups for the polymerization reaction as well as for the immobilization of the ligand.

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Monolith technology has been employed in chromatography for a variety of applications using diverse substrates. The development of different column chemistries has led to the Thermo Scientific ProSwift line of monolith columns for analytical protein separation by ion exchange and reversed phase. Separation of biomolecules can be achieved at elevated linear velocities with minimal loss of resolution. Columns are designed to withstand extreme pH cleaning, desired for sterilization. The backbone and functionalization are optimized for high mass loading for small-scale preparative applications, the ideal first dimension separation of crude biological samples. Combined with increased sensitivity of a 1 mm format, detection of proteins of very low copy number in a crude samples is achievable.

We discuss here the ability to produce highly-reproducible columns with excellent stability as well as characteristics required for fast small-scale preparative analysis. HPLC column selection is a challenging task, specifically where the mixture contents is somewhat unknown. Many factors influence the choice of column used; chemistry, robustness, and reproducibility. For quality assurance, columns should be chosen that are reproducible both run to run and batch to batch. To prevent cross-contamination between samples, carryover and sterilization should be considered. For semi-prep, a combination of high mass loading and good resolution enable increased purity of peak fractions. Format and operational flow rate should be considered with respect to multidimensional analysis.

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2010

Analysis of a large number of samples requires chromatographic support that not only enables fast separation and purification of a target biomolecule from a complex matrix but also support an automation of a process. The methacrylate 96-well monolithic plate format enables both. 96-well monolithic plate reduces experimental time because it allows fast and efficient evaluation of parameters for binding and elution conditions. This format is a quicker alternative to several consecutive tests on chromatographic column.

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In recent decades much work has been done on the development and optimisation of chromatographic supports in order to achieve efficient purification of biomolecules.

In the presented study we have investigated hydrodynamic and chromatographic properties of weak anion-exchange grafted monoliths (DEAE). Varying the concentration of the grafted polymer, grafted monoliths with different layer thickness and degree of branching were obtained. This results in a different hydrodynamic and chromatographic behavior of the examined monoliths such as permeability, ionic capacity and dynamic binding capacity (DBC) for the BSA protein. The DBC increases with the grafted layer thickness probably due to higher number of binding sites available for binding of the macromolecules. However, longer chains contribute to the reduction of the pore volume which results in a higher pressure drop. The latter can be additionally increased when biomolecules of interest are bound to the matrix. From this data information about the penetration depth into the grafted layer can be obtained giving an insight into the binding mechanism. Since the flow-unaffected properties were preserved even for large biomolecules, grafted monoliths may become a resin of choice for downstream processing of various macromolecules.

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CIMac™ Analytical Columns are high-performance monolithic columns offering all the advantages of a special continuous short polymeric bed and are primarily intended for fast, efficient and reproducible separations of biomolecules like large proteins – antibodies (IgG, IgM), plasmid DNA, phages and viral particles. Their small volume and short column length allow the operation at high volumetric flow rates (from 1 to 30 column volumes/min) thus enabling receiving the information about the product quantity and purity in just a few minutes. These columns are pre-packed in dedicated stainless steel housings and allow user friendly connections to HPLC equipment. The product family offers strong cation exchange, strong and weak anion exchange and specialty analytical column for plasmid DNA. All columns can be effectively used for the in-process and final control of various samples from different purification process steps.

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The biotechnological production of recombinants proteins consists of two main processes, upstream (biosynthesis) and downstream (protein purification) process. During the last decades the upstream process for mammalian cell culture has been improved significantly yielding in high amounts of protein. This development however led to a new challenge : the downstream process became a bottle-neck because of the large amounts of protein per batch in combination with the protein specific behaviors at high concentration.

In protein purification preparative chromatography is synonymous to “column chromatography”, and the favorable statics of a column are out of question for the physical requirements of beaded matrices. However, when approaching larger scales the physical dimensions of chromatography columns turn unfavorable: shallow gel beds of wide diameters. The footprint of such device increases drastically as does the weight, consequently resulting in limitations regarding floor space and floor bearing force.

A suitable chromatographic base matrix that is not obliged to a distinctive column design is a single piece of polymer – a monolith. Leaving the conventional column design, we have constructed a device for a monolith of rectangular shape, with the size of the monolith only limited by total weight (e.g. for handling and / or transportation). Using this design in a modular way, the individual modules can be stacked to make use of the height of a room at a very low footprint. A specific distribution system for feeding the monolith modules has been designed to allow a true linear scale-up from laboratory to large technical scale.

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2007

Membrane based anion exchangers are being used increasingly for purification of monoclonal antibodies. The transition from particle-based anion exchangers is driven partly by the convenience of membranes and partly by the cost saving associated with their disposability, however the feature that makes them functionally superior is more effective mass transport.

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2006

Commercially available CIM® disk monolithic columns are intended for very fast analyzes and laboratory purification. Their shape is a compromise to achieve acceptable resolution and binding capacity what make them suitable for wide range of laboratory applications. Separations of complex protein mixtures can be carried out within just a few seconds because of flow unaffected resolution and, on the other hand, purification can be effectuated with high productivity due to flow-unaffected dynamic binding capacity [1]. However, in many cases in the field of molecular biology, only a limited amount of sample is available. In such a case it is beneficial to work with small columns having high resolution or they can be used as affinity columns or bioreactors saving significant amount of valuable ligand. Having this goal in mind we developed CIM® disks with the volume of 1/10th and 1/100th of original volume. In comparison to conventional CIM® disks, they exhibit higher resolution and lower limit of detection, therefore smaller concentrations of target macromolecules can be detected. The separation ability and the protein capacity were tested on anion and cation exchange 3.4 mL and 34m L mini disk monolithic columns.

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Analysis of a large numbers of samples requires chromatographic supports that not only enable fast separation and purification of a target biomolecules from a complex matrix but are also involved in an automation process. The 96 – microtiter plate format enables both. Although they are routinely used for decade's only recently few reports about the microtiter plates bearing monoliths as a separation media, were reported [1]. Because of advantageous properties such as flow unaffected dynamic binding capacity and resolution 96 - microtiter plates with methacrylate based monolith were prepared. Characterisation of such plate demonstrated that uniform flow rate can be achieved through all wells and no leakage is present. Efficient separation of proteins was achieved within minute. Furthermore CLC (Conjoined Liquid Chromatography) concept [2] originally derived for analytical columns on CIM disk, can easily be extrapolated to microtiter plates. We demonstrated that multidimensional chromatography with 96 – well plate is feasible and can further accelerate screening processes.

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2005

The analysis of molecular interactions is a key part of the drug discovery process, and analytical techniques are available for studying in vitro the ligand/target complex since the early stage of the drug development process.

With regard to the assessment of the activity of chemical libraries, the affinity chromatography on HPLC immobilized-enzyme column (or immobilized enzyme reactors, IMER) is one of most promising methodologies for HTS applications.

Human recombinant acetylcholinesterase (hAChE) represents a well-known target for drug-discovery in Alzheimer’s Disease.

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Affinity chromatography is a key method for protein purification. Its main advantage is in the high specificity which enables purification of a single protein from complex biological mixtures. For practical use the specific ligand should be immobilised on insoluble matrix. As a matrix, standard chromatographic supports are commonly used. They are normally in form of small (some m in diameter) particles containing pores to provide high specific surface resulting in high binding capacity. The pores are normally closed on one side, thus the liquid inside them is stagnant and the molecules are transported to the active site by diffusion. Since the diffusion coefficients for macromolecules, such as proteins, are very low, diffusion determines the overall process dynamics. As a consequence, separation or purification of the proteins takes normally 0.5 to 1h even on analytical scale.

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2004

Tissue plasminogen activator (t-PA) is serine protease which converts plasminogen into plas-min dissolving the major component of blood clots, fibrin. So, it can be extremely useful in clinical practice to help curing of heart attack victims. The most available way protein producing is genetic engineering where separation and purification of goal protein are one of the important steps in protein producing process.

Recently developed High performance monolithic disk chromatography, HPMDC, seems to be a very attractive way for study quantitative affinity parameters of recombinant proteins with different ligands as well as for protein separations and purifications. High process speed prevents the denatura-tion due to temperature and solvents influence. The better mass transfer mechanism (convection rather than diffusion) allows to consider only the biospecific reaction as time limiting.

It is known that plasminogen, which is the natural substratum for t-PA, can be successfully used as affinity ligand to separate t-PA from cellular media. However, the use of synthetic ligands for affinity chromatography is more preferable due to their higher stability and lower total cost.

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2003

Traces of DNA in RNA samples represent impurities that could affect results of mRNA quantification and cDNA synthesis. In most cases, the DNA impurities in RNA samples are removed using enzyme deoxyribonuclease (DNase), which specifically breaks down DNA. In order to avoid the addition of DNase into the analyzing sample, the use of immobilized DNase on solid support is recommended. Because of the DNA size, very few supports available on the market enable efficient interaction between immobilized enzyme and DNA.

In recent years a new group of supports named monoliths was introduced. Because of enhanced exchange between mobile and stationary phase separation and bioconversion processes are significantly accelerated. Therefore also the efficiency of DNA removal using immobilised enzyme might be competitive to the degradation with free enzyme.

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The only four drugs approved for the clinical treatment of Alzheimer’s Disease (tacrine, rivastigmine, donepezil and galantamine) are acetylcholinesterase inhibitors which act by maintaining high levels of acetylcholine at the muscarinic and nicotinic receptors in the central nervous system. Human acetyicholinesterase (HuAChE) represents a widely studied target enzyme and it is still object of research for the development of new drugs as enzyme inhibitors.

In a previous paper il] we reported the immobilisation of AChE on a silica based chromatographic column (50 x 4.6 mm I.D.) The yield of immobilization and the stability of the AChE—IMEN were considered satisfactory, hut some problems arose. The length of the IMER and the large amount of enzyme covalently bound to the chromatographic support resulted in catalysis product long elution times and some inhibitors aspecific matrix absorption with delayed enzyme activity recovery. In order to avoid these complications and considering the high rate of AChE enzymatic reaction, we decided to reduce the dimension of the solid support for immobilization, hence the amount of immobilized enzyme, by selecting a monolithic matrix disk (12 x 3 min I.D.).

CIMa (Convective Interaction Media) monolithic supports (Bia Separations, Ljubljana) represent a novel generation of stationary phases used for liquid chromatography, bioconversions, and solid phase synthesis. As opposed to individual particles packed into chromatographic columns, CIM supports are cast as continuous homogeneous phases and provide high rates of mass transfer at lower back pressure.

In the present work a CIM® disk with immobilised human recombinant acetylcholinesterase (HuAChECIM€ Disk) was developed. The activity of immobilised enzyme, the long term stability and reproducibility were tested. HuAChECIM disk was applied as an immobilised enzyme micro-reactor (micro-IMER) in on-line HPLC system for inhibitory potency determination of known AChE inhibitors.

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2001

We have developed a screening procedure for peptide ligands for affinity chromatography on the same monolithic support. CIM® monolithic columns used conventionally for analytical and preparative separation of proteins and polynucleotides were minimized to fit into 96 well solid phase extraction plates. Peptide synthesis and screening were performed on the same format using a vacuum manifold for liquid throughput.

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The development of new chromatographic supports with the aim to improve their chromatographic, hydrodynamic and mechanical properties is continually going on.

CIM Convective Interaction Media® monolithic columns represent a new chapter in every mode of the chromatography. Monolithic columns consist of a single piece of a highly porous polymer with a bimodal pore size distribution, forming flow-through channels [1]. Since all of the mobile phase flows through the pores, molecules to be separated are transported to the active sites by convection [2]. Therefore, the entire analysis can be completed in a very short time.

In this work, the performance of novel semi-preparative CIM® RP-SDVB disk monolithic column for separating proteins and peptides has been investigated. Since the column length in the case of gradient separations commonly used for large molecules, does not play a significant role, CIM® RP-SDVB disk monolithic column are extremely short, typically of only 3 mm. The effect of decreasing column length on the resolution under the conditions of a linear gradient has been presented.

Finally, a 1 minute purification of oligodeoxynucleotide from the synthetic mixture has been performed.

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Gene therapy which is becoming more and more important in human health care requires the purification of high molecular mass compounds, so called nanoparticles (e. g. viruses and plasmids). The method of choice to ensure proper purity would be chromatography.

Most of the chromatographic supports available on the market at the moment can not follow the requests for such work due to low binding capacity for large molecules, limitation with regards to the time of the separation process and requests for CIP (cleaning in place) and SIP (sanitation in place).

Monolithic supports represent a new generation of chromatographic supports. In contrast to conventional particle supports, where the void volume between individual porous particles is unavoidable, these supports consist of a single monolith highly interconnected with larger and smaller open flow-through channels. Due to the structure, molecules to be separated are transported to the active sites on the stationary phase by convection, resulting in very short separation times. This is especially true for large molecules.

In this work we will present the use of monolithic supports for the separation of different nanoparticles on analytical and preparative scales. It will be shown that monolithic supports can overcome the limitations of particle-based supports for the analytics and isolation of big molecules and represent a major step towards the safe and efficient purification or production of nanoparticles.

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CIM® (Convective Interaction Media) supports represent the fourth generation of chromatographic supports monoliths. These glycidyl methacrylate based monolithic columns are commercially available under the name CIM®. In contrast to conventional porous particles the morphology of the CIM® supports is characterised by a single monolithic unit that contains pores, opened on both sides. These pores are highly interconnected forming a flow-through network. Thus the whole mobile phase is forced to run through these open pores, therefore, the mass transfer between stationary and mobile phase is based on convection rather than on diffusion. This transport mechanism enables very fast separations and purifications of components as well as flow-unaffected resolution and dynamic binding capacity. The latter is especially important for the purification of molecules on preparative level where the productivity is essential. One of the key features of monolithic units is their pore size distribution that should enable low backpressure at high throughputs together with high specific surface area, needed for high binding capacity. In this work the effect of different parameters on binding capacity of CIM® monolithic columns is presented.

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There is a rapidly increasing development of new materials in the field of chromatographic supports stimulated by the need to achieve fast and reliable separation of different solutes.

The generally used chromatographic supports are based on beads packed in the columns. Although these stationary phases have been continuously improved over the last decades, there are still some limitations present. The absence of flow within the pore matrix of particles, slow diffusional mass transfer of solutes, in some cases high back pressure and laborious handling represent the major hindrances.

Development of monolithic materials is a chronicle of efforts to overcome problems of packed particles. Monolithic separation media, made in one piece, contain only flow-through pores, which significantly augment the mass transfer based on convection. This enables use of high mobile phase velocities along with low back pressures and therefore fast separations without decrease of resolution.

This report presents the preparation of glycidylmethacrylatestearylmethacryate- ethylenedimethacrylate and styrene-divinylbenzene monoliths. The porous structure of the support obtained will be discussed. Finally, a few examples of separation of proteins and small molecules with monolithic CIM C-18 and CIM SDVB disks in reversed-phase chromatography will be displayed.

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