2005
D. JOSIĆ, MK. BROWN, F. HUANG, H. CALLANAN, M. RUCEVIC, A. NICOLETTI, J. CLIFTON, DC. HIXSON. Use of selective extraction and fast chromatographic separation combined with electrophoretic methods for mapping of membrane proteins. Electrophoresis, 2005, 26, 2809-2822.
A model system for selective solubilization and fast separation of proteins from the rat liver membrane fraction and purified rat liver plasma membranes for their further proteomic analysis is presented. For selective solubilization, high-pH solutions and a concentrated urea solution, combined with different detergents, are used. After extraction, proteins are separated by anion-exchange chromatography or a combination of anion- and cation-exchange chromatography with convective interaction monolithic supports. This separation method enables fast and effective prefractionation of membrane proteins based on their hydrophobicity and charge prior to one-dimensional (1-D) and 2-D electrophoresis and mass spectrometry. By use of this sample preparation method, the less-abundant proteins can be detected and identified.
A. PODGORNIK, A. ŠTRANCAR. Convective Interaction Media(R) (CIM) - Short layer monolithic chromatographic stationary phases. Biotechnol Annu Rev., 2005, 11, 281-333.
Modern downstream processing requires fast and highly effective methods to obtain large quantities of highly pure substances. Commonly applied method for this purpose is chromatography. However, its main drawback is its throughput since purification, especially of large molecules, requires long process time. To overcome this problem several new stationary phases were introduced, among which short layer monoliths show superior properties for many applications. The purpose of this review is to give an overview about short methacrylate monolithic columns commercialised under the trademark Convective Interaction Media(R) (CIM). Their unique properties are described from different perspectives, explaining reasons for their application on various areas. Approaches to prepare large volume methacrylate monolithic column are discussed and optimal solutions are given. Different examples of CIM monolithic column implementation are summarised in the last part of the article to give the reader an idea about their advantages.
A. PODGORNIK, J. VIDIČ, J. JANČAR, N. LENDERO, V. FRANKOVIČ, A. ŠTRANCAR. Noninvasive Methods for Characterization of Large-Volume Monolithic Chromatographic Columns. Chem Eng Technol, 2005, 28, 1435-41.
Chromatographic monoliths exhibit several properties like flow-unaffected resolution and dynamic binding capacity, low pressure drop and high capacity for extremely large molecules, which are advantageous to the purification of large biomolecules such as proteins, DNA or even viruses. Recently, large-volume monolithic columns were introduced enabling the monoliths to be incorporated into industrial downstream processes. Due to the monolithic structure, however, conventional methods for resin characterization cannot be applied to monoliths, which limit their usage under cGMP conditions. In this article, a methodology of how to overcome this problem and to determine the most important parameters is presented. Several fast and noninvasive methods for monolith characterization are introduced. Their usage enables accurate determination of monolith pore size distribution and specific surface area, uniformity of the monolith as well as type and density of the active groups before or during monolith usage.
M. BARUT, A. PODGORNIK, P. BRNE, A. ŠTRANCAR. Convective Interaction Media Short Monolithic Columns: Enabling Chromatographic Supports for the Separation and Purification of Large Biomolecules. J Sep Sci, 2005.
New therapeutics that are being developed rely more and more on large and complex biomacromolecules like proteins, DNA and viral particles. Manufacturing processes are being redesigned and optimized both upstream and downstream to cope with the ever-increasing demand for the above target molecules. In downstream processing, liquid chromatography still represents the most powerful technique for achieving high yield and high purities of these molecules. In most cases, however, the separation technology relies on conventional particle based technology, which has been optimized for the purification of smaller molecules. New technologies are, therefore, needed in order to push the downstream processing ahead and into the direction, which will provide robust, productive and easy to implement methods for the production of novel therapeutics. New technologies include the renaissance of membranes, various improvements of existing technologies, but also the introduction of a novel concept – the continuous bed or monolithic stationary phases. Among different introduced products, Convective Interaction Media (CIM) Short Monolithic Columns (SMC) that are based on methacrylate monoliths exhibit some interesting features that make them attractive for these tasks. SMC can be initially used for fast method development on the laboratory scale and subsequently efficiently transferred to preparative and even more importantly to industrial scale. A brief historical overview of methacrylate monoliths is presented, followed by a short presentation of theoretical considerations that had led to the development of short monolithic columns. The design of these columns, as well as their scale-up to large units, together with the methods for transferring gradient separations from one scale to another are addressed. Non-invasive methods that have been developed for the physical characterization of various batches of SMC, which fulfill the regulatory requirements for cGMP production, are discussed. The applications of SMC for the separation and purification of large biomolecules, which demonstrate the full potential of this novel technology for an efficient downstream processing of biomolecules are also presented.
M.
PETERKA, D. GLOVER, P. KRAMBERGER, M. BANJAC, A. PODGORNIK, M. BARUT, A.
ŠTRANCAR.
Short Monolithic Columns —An enabling technology for the purification of
proteins, DNA, and viruses.
BioProcessing Journal. March/April 2005, 1-6.
The last 30 years have seen rapid and dramatic developments in recombinant DNA technology and the related biological sciences. Therapeutic molecules for human use must be pure and free of contaminants. Consequently, purification or downstream processing is one of the most important and most expensive steps in the production process. Although, different techniques can be used for the purification of large molecules and particles, liquid chromatography is the method of choice because it increases productivity and provides the high level of purity required for human use. In principle, liquid chromatography is a slow and stressful process for biomolecule purification during which undesirable modifications can occur. The use of a fast process under mild conditions can overcome these problems. Methacrylate monoliths represent the key to the biochromatographic purification and downstream processing of biomolecules such as proteins, DNA, and viruses.
DJ.
JOSIĆ.
Foreword (EDITORIAL).
J. Chromatogr. A, 2005, 1065,
1.
A. JUNGBAUER.
Chromatographic
media for bioseparation.
J. Chromatogr. A, 2005, 1065, 3-12.
Bioseparation processes are dominated by chromatographic steps. Even primary recovery is sometimes accomplished by chromatographic separation, using a fluidized bed instead of a fixed bed. In this review, the action principles, features of chromatography media regarding physical and chemical properties will be described. An attempt will be made to establish categories of different media. Characteristics for bioseparation are the large pores and particle sizes. To achieve sufficient capacity for ultralarge molecules, such as plasmids or nanoparticles, such as viruses monoliths are the media of choice. In these media, the mass transport is accomplished by convection, and thus, the low diffusivity can be overcome. Common to all modern chromatography media is the fast operation. There are examples where a residence time of less then 3 min, is sufficient to reach the full potential of the adsorbent.
T.B. TENNIKOVA, J. REUSCH.
Short
monolithic beds: history and introduction to the field.
J. Chromatogr.
A, 2005, 1065, 13-17.
The history of the development of short monolithic beds is described.
G.A. PLATONOVA, T.B. TENNIKOVA.
Affinity
processes realized on high-flow-through methacrylate-based macroporous
monoliths.
J. Chromatogr. A, 2005, 1065, 19-28.
The technology for preparation of rigid macroporous polymers suggested in the late 1980s has become a powerful instrument for the development of a novel scientific and practical field. At present, monolithic stationary phases are widely used in the processes of bioseparation (chromatography), bioconversion (enzyme reactors) as well as in other processes based on interphase mass distribution (for example, solid phase peptide and oligonucleotide synthesis). Bioaffinity modes of suggested dynamic methods are very promising for their use in different analytical processes (immunological, ecological, medical and other types of analytical monitoring), preparative isolation of blood proteins such as myoglobin, hemoglobin, immunoglobulins, etc. and also recombinant products directly from cell supernatants or lysates. For the first time, it has been shown that bioaffinity pairing with participation of immobilized on carefully designed rigid supports is very fast and the whole process of affinity separation can be realized within second time scale. The principle of bioaffinity recognition is generaly at the construction of biological reactors (for example, enzyme reactors). Improved kinetics of biocatalized reactions is explained by a minimal influence on the surface of the used sorbent. Very perspective field is the use of discussed monoliths for solid phase chemical synthesis of fragments of biological macromolecules (peptides and oligonucleotides). Several examples of these applications will be presented and discussed.
N. LENDERO, J. VIDIČ, P.
BRNE, A. PODGORNIK, A. ŠTRANCAR.
Simple method for determining the amount
of ion-exchange groups on chromatographic supports.
J. Chromatogr.
A, 2005, 1065, 29-38.
The objective of this study was to develop a fast, simple, non-destructive, non-toxic and low-priced method for determining the amount of ionic groups on resins, since the conventional titration method fails to give proper results on methacrylate monoliths. After the column had been pre-saturated with a high concentration buffer solution, a low concentration buffer solution of the same pH value was pumped through the column. Measuring pH and absorbance, the profiles with a shape of typical break-through curve were obtained. It was shown that the time of the pH transient, which appeared under such conditions, could be used as a measure of the total ionic capacity of ion-exchange monolithic columns. The effect of the column length, linear velocity and varying concentrations of buffer solutions on the time of the pH transient was examined. The method was shown to be suitable for determining the amount of ionic groups on both anion and cation monolithic columns. In addition, it could also be applied to particle bed columns. The time of the pH transient and the protein dynamic binding capacity were also compared and it was concluded that for a given monolith the protein capacity can be derived from the data obtained by the new method.
Y-P LIM, DJ. JOSIĆ, H.
CALLANAN, J. BROWN, D. C. HIXSON.
Affinity purification and enzymatic
cleavage of inter-alpha inhibitor proteins using antibody and elastase immobilized
on CIM monolithic disks.
J. Chromatogr. A, 2005, 1065, 39-43.
Epoxy-activated monolithic CIM disks seem to be excellent supports for immobilization of protein ligands. The potential use of enzymes, immobilized on monolithic disks for rapid preparative cleavage proteins in solution was investigated. Digestion of complex plasma proteins was demonstrated by using inter-alpha inhibitors with elastase, immobilized on epoxy-activated CIM disks. Recently, a monoclonal antibody against human inter-alpha inhibitor proteins (MAb 69.31) was developed. MAb 69.31 blocks the inhibitory activity of inter-alpha inhibitor proteins to serine proteases. These results suggest that the epitope defined by this antibody is located within or proximal to the active site of the inhibitor molecule. This antibody, immobilized on monolithic disk, was used for very rapid isolation of inter-alpha proteins. The isolated complex protein was used for enzymatic digestion and isolation of cleavage products, especially from inter-alpha inhibitor light chain to elucidate precisely the target sequence for MAb 69.31 by N-terminal amino acid sequencing. Bovine pancreatic elastase immobilized on monolithic disk cleaves inter-alpha inhibitor protein complex into small fragments which are still reactive with MAb 69.31. One of these proteolytic fragments was isolated and partially sequenced. It could be shown that this sequence is located at the beginning of two proteinase inhibitor domains of the inter-alpha inhibitor light chain (bikunin). Elastase immobilized on monolithic disk offers a simple and rapid method for preparative isolation of protease cleavage fragments. The immobilized enzyme is stable and still active after repeated runs. A partial or complete digestion can be achieved by varying the flow rate.
S. YAMAMOTO, A. KITA.
Theoretical
background of short chromatographic layers: Optimization of gradient elution
in short columns.
J. Chromatogr. A, 2005, 1065, 45-50.
Although linear salt gradient elution ion-exchange chromatography (IEC) of proteins is commonly carried out with relatively short columns, it is still not clear how the column length affects the separation performance and the economics of the process. The separation performance can be adjusted by changing a combination of the column length, the gradient slope and the flow velocity. The same resolution can be obtained with a given column length with different combinations of the gradient slope and the flow velocity. This results in different separation time and elution volume at the same resolution. Based on our previous model, a method for determining the separation time and the elution volume relationship for the same resolution (iso-resolution curve) was developed. The effect of the column length and the mass transfer rate on the iso-resolution curve was examined. A long column and/or high mass transfer rate results in lesser elution volume. The resolution data with porous bead packed columns and monolithic columns were in good agreement with the calculated iso-resolution curves. Although the elution volume can be reduced with increasing column length, the pressure drop limits govern the optimum conditions.
J. VIDIČ, A. PODGORNIK,
A. ŠTRANCAR.
Effect of the glass surface modification on the strength
of methacrylate monolith attachment.
J. Chromatogr. A, 2005, 1065,
51-58.
The influence of glass surface modification in order to determine strength of the monolith attachment was studied. Modification consists of pre-treatment of the glass with chemicals or boiling in deionized water, silanization and drying has been investigated on different types of glass. Amount of silane groups was determined by measurement of the contact angle between the glass surface and water drop. The highest values were found for soda–lime glass. Strength of the monolith attachment was established by pumping ethanol through the monolithic capillaries and measuring the pressure drop at which monolith was dislodged. Surprisingly, it was found that the critical part of the glass surface modification procedure is glass pre-treatment. Good results were obtained with glass boiled in water for 2.5 h or more.
I. MIHELIČ, D. NEMEC, A.
PODGORNIK, T. KOLOINI.
Pressure drop in CIM disk monolithic columns.
J.
Chromatogr. A, 2005, 1065, 59-67.
Pressure drop analysis in commercial CIM disk monolithic columns is presented. Experimental measurements of pressure drop are compared to hydrodynamic models usually employed for prediction of pressure drop in packed beds, e.g. free surface model and capillary model applying hydraulic radius concept. However, the comparison between pressure drop in monolith and adequate packed bed give unexpected results. Pressure drop in a CIM disk monolithic column is approximately 50% lower than in an adequate packed bed of spheres having the same hydraulic radius as CIM disk monolith; meaning they both have the same porosity and the same specific surface area. This phenomenon seems to be a consequence of the monolithic porous structure which is quite different in terms of the pore size distribution and parallel pore nonuniformity compared to the one in conventional packed beds. The number of self-similar levels for the CIM monoliths was estimated to be between 1.03 and 2.75.
P. KRAJNC, N. LEBER, D.
ŠTEFANEC, S. KONTREC, A. PODGORNIK.
Preparation and characterisation of
poly(high internal phase emulsion) methacrylate monoliths and their application
as separation media.
J. Chromatogr. A, 2005, 1065, 69-73.
Poly(glycidyl methacrylate-co-ethyleneglycol dimethacrylate) monolithic supports were prepared by radical polymerisation of the continuous phase of water in oil high internal phase emulsions. Morphology of monolithic materials was studied by scanning electron microscopy and mercury intrusion porosimetry. The ratio of phase volume and the degree of crosslinking influenced the void size and pore size distribution of resulting polymers. Void sizes between 1 and 10 µm were observed and average pore sizes around 100 nm. Polymers with 60, 75, 80 and 90% pore volume were prepared and even samples with highest pore volume showed good mechanical stability. They were modified to bear weak-anion exchange groups and tested on the separation of standard protein mixture containing myoglobin, conalbumine and trypsin inhibitor. Good separation was obtained in a very short time similar to the separation obtained by commercial methacrylate monoliths. However, higher dispersion was observed. Bovine serum albumin dynamic binding capacity for monolith with 90% porosity was close to 9 mg/ml.
G.A. PLATONOVA, T.B. TENNIKOVA.
Chromatographic
investigation of macromolecular affinity interactions.
J. Chromatogr.
A, 2005, 1065, 75-81.
High-performance monolithic disk affinity chromatography was applied to the investigation of formation of complexes between (1) complementary polyriboadenylic and polyribouridylic acids, e.g. poly(A) and poly(U), respectively, (2) poly(A) and synthetic polycation poly(allylamine), pAA. Polyriboadenylic acid and poly(allylamine) were immobilized on macroporous disks (CIM disks). Quantitative parameters of affinity interactions between macromolecules were established using frontal analysis at different flow rates.
M. BENČINA, K. BENČINA,
A. ŠTRANCAR, A. PODGORNIK.
Immobilization of deoxyribonuclease via epoxy
groups of methacrylate monoliths: Use of deoxyribonuclease bioreactor in
reverse transcription-polymerase chain reaction.
J. Chromatogr. A,
2005, 1065, 83-91.
A deoxyribonuclease bioreactor was prepared by immobilization of deoxyribonuclease I through epoxy groups inherently present on poly (glycidyl methacrylate-co-ethylene dimethacrylate) monoliths. Columns with various levels of DNase activity were prepared varying immobilization temperature, pH, time and method. The apparent Michaelis–Menten constant, Km(app), and turnover number, k3(app), for immobilized DNase determined by on-line frontal analysis method were, respectively, 0.28 g of DNA l-1 and 16 dA260nm min-1 mg-1 of immobilized DNase. The highest activity of immobilized DNase was detected at 1 mM calcium ions concentration and mirrored properties of free enzyme; however, reaction temperature in the range from 25 to 37 °C has no significant effect on activity of immobilized DNase in contrary to free enzyme. The CIM DNase bioreactor was used for elimination of DNA contaminants in RNA samples prior to reverse transcription followed by PCR.
J. URTHALER, R. SCHLEGL,
A. PODGORNIK, A. STRANCAR, A. JUNGBAUER, R. NECINA.
Application of monoliths
for plasmid DNA purification: Development and transfer to production.
J.
Chromatogr. A, 2005, 1065, 93-106.
The demand of high-purity plasmid DNA (pDNA) for gene-therapy and genetic vaccination is still increasing. For the large scale production of pharmaceutical grade plasmids generic and economic purification processes are needed. Most of the current processes for pDNA production use at least one chromatography step, which always constitutes as the key-step in the purification sequence. Monolithic chromatographic supports are an alternative to conventional supports due to their excellent mass transfer properties and their high binding capacity for pDNA. Anion-exchange chromatography is the most popular chromatography method for plasmid separation, since polynucleotides are negatively charged independent of the buffer conditions. For the implementation of a monolith-based anion exchange step into a pDNA purification process detailed screening experiments were performed. These studies included supports, ligand-types and ligand-densities and optimization of resolution and productivity. For this purpose model plasmids with a size of 4.3 and 6.9 kilo base pairs (kbp) were used. It could be shown, that up-scaling to the production scale using 800 ml CIM Convective Interaction Media radial flow monoliths is possible under low pressure conditions. CIM DEAE was successfully implemented as intermediate step of the cGMP pDNA manufacturing process. Starting from 200 l fermentation aliquots pilot scale purification runs were performed in order to prove scale-up and to predict further up-scaling to 8 l tube monolithic columns. The analytical results obtained from these runs confirmed suitability for pharmaceutical applications.
S. JERMAN, A. PODGORNIK,
K. CANKAR, N. ČADEŽ, M. SKRT, J. ŽEL, P. RASPOR.
Detection of processed
genetically modified food using CIM monolithic columns for DNA isolation.
J.
Chromatogr. A, 2005, 1065, 107-113.
The availability of sufficient quantities of DNA of adequate quality is crucial in polymerase chain reaction (PCR)-based methods for genetically modified food detection. In this work, the suitability of anion-exchange CIM (Convective Interaction Media; BIA Separations, Ljubljana, Slovenia) monolithic columns for isolation of DNA from food was studied. Maize and its derivates corn meal and thermally pre-treated corn meal were chosen as model food. Two commercially available CIM disk columns were tested: DEAE (diethylaminoethyl) and QA (quaternary amine). Preliminary separations were performed with standard solution of salmon DNA at different pH values and different NaCl concentrations in mobile phase. DEAE groups and pH 8 were chosen for further isolations of DNA from a complex matrix—food extract. The quality and quantity of isolated DNA were tested on agarose gel electrophoresis, with UV-scanning spectrophotometry, and by amplification with real-time PCR. DNA isolated in this way was of suitable quality for further PCR analyses. The described method is also applicable for DNA isolation from processed foods with decreased DNA content. Furthermore, it is more effective and less time-consuming in comparison with the existing proposed methods for isolation of DNA from plant-derived foods.
D. FORCIC, K. BRANOVIC-CAKANIC,
J. IVANCIC, R. JUG, M. BARUT, A. STRANCAR.
Purification of genomic DNA
by short monolithic columns.
J. Chromatogr. A, 2005, 1065, 115-120.
The isolation and purification of nucleic acids is essential for many procedures in molecular biology. After showing that bacterial and eukaryotic genomic DNA can be specifically bound to the CIM DEAE monolithic column, this characteristic was exploited in development of a simple and fast chromatographic procedure for isolation and purification of genomic DNA from cell lysates that does not include the usage of toxic organic solutions. The purity and the quality of the isolate as well as the duration of the procedure was similar to other chromatographic methods used today for isolation of genomic DNA, but the initial sample volume was not restricted.
I. VOVK, B. SIMONOVSKA,
M. BENČINA.
Separation of pectin methylesterase isoenzymes from tomato
fruits using short monolithic columns.
J. Chromatogr. A, 2005,
1065, 121-128.
One of the main forms of tomato pectin methylesterase (PME; EC 3.1.1.11) that is applicable to the food industry was isolated from fresh tomato fruit. The extraction of the PME isoenzymes involved washing the fresh tomato flesh with water in order to remove sugars and than solubilizing the enzymes with a diluted HCl solution at pH 1.6. The extract was then neutralized to pH 7.4 using buffer solution. After filtration, the solution was directly fractioned using Convective Interaction Media (CIM®) short monolithic disk column bearing sulfonyl (SO3) groups and using a linear gradient from 0 to 700 mM NaCl. The injection volume was 3 ml and the diameter of the column was 12 mm and length 3 mm. The isolated fractions were monitored for protein content and PME activity. The fraction with the targeted enzyme, which showed NaCl independent activity, was further purified and concentrated by ultrafiltration and finally purified by a second semi-preparative cation-exchange chromatography step using a CIM carboxymethyl (CM) disk monolithic column consisting of two disks and applying a step gradient. From 1 kg of fresh tomato fruits, 7.5 mg of purified PME with molecular mass estimated to be 26 000 by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) was obtained. A fraction with mixed PME and polygalacturonase activity was also obtained. Compared to the published procedures for the isolation and purification of PME from plant materials, this new procedure is much faster and more efficient. The potential application of CIM disk short monolithic columns in the analysis and semi-preparative extraction and isolation of the PME isoenzyme is presented.
K. ISOBE, Y. KAWAKAMI.
Application
of two types of CIM tube column for purification of microbial enzymes.
J.
Chromatogr. A, 2005, 1065, 129-134.
Chromatography conditions for two types of convection interaction media (CIM) tube monolithic column, DEAE-8 and C4-8, were investigated using three enzymes from different microorganisms. The enzymes were adsorbed on a CIM DEAE-8 tube column under the same conditions as conventional DEAE columns. The CIM C4-8 tube column required a high concentration of ammonium sulfate compared to the conventional C4 column for adsorbing the enzymes. The separation of enzymes on the CIM tube column chromatography was not affected at flow rates between 0.15 and 1.25 volumes of the column per min. Both columns were successfully applied to the purification of enzymes from crude enzyme solution. Thus, both CIM tube monolithic columns proved useful in greatly reducing the purification time, and could be used at any stage of enzyme purification.
M. BARTOLINI, V. CAVRINI,
V. ANDRISANO.
Choosing the right chromatographic support in making a new
acetylcholinesterase-micro-immobilised enzyme reactor for drug discovery.
J.
Chromatogr. A, 2005, 1065, 135-144.
The aim of the present study was to optimize the preparation of an immobilized acetylcholinesterase (AChE)-based micro-immobilized enzyme reactor (IMER) for inhibition studies. For this purpose two polymeric monolithic disks (CIM, 3 mm × 12 mm i.d.) with different reactive groups (epoxy and ethylendiamino) and a packed silica column (3 mm × 5 mm i.d.; Glutaraldehyde-P, 40 µm) were selected as solid chromatographic supports. All these reactors were characterized in terms of rate of immobilization, stability, conditioning time for HPLC analyses, optimum mobile phase and peak shape, aspecific interactions and costs. Advantages and disadvantages were defined for each system. Immobilization through Schiff base linkage gave more stable reactors without any significant change in the enzyme behaviour; monolithic matrices showed very short conditioning time and fast recovery of the enzymatic activity that could represent very important features in high throughput analysis and satisfactory reproducibility of immobilization yield. Unpacked silica material allowed off-line low costs studies for the optimization of the immobilization step.
D. FORCIC, KB. CAKANIC, J. IVANCIC, R. JUG, M. BARUT, A. ŠTRANCAR, R. MAZURAN.
Chromatographic detection of residual cellular DNA on short monolithic
columns. Anal Biochem., 2005, 336(2), 273-8.
Analysis of crude samples from biotechnological processes is often required to demonstrate that residual host cell impurities are reduced or eliminated during purification. Current knowledge suggests that a continuous-cell-line DNA can be considered a cellular contaminant rather than a significant risk factor requiring removal to extremely low levels. Anion-exchange chromatography is one of the most important methods used in the downstream processing and analysis of different biomolecules. In this article, an application using Convective Interaction Media monolithic columns to improve the detection of residual cellular DNA is described.