Purification and concentration of bacteriophage T4 using monolithic chromatographic supports.

Phages are gaining importance due to their wide usage. In this work strong anion exchange monolithic chromatographic column was used for single step phage purification. Most of the proteins and DNA were removed and recovery of approximately 70% of infective virus was reproducibly achieved. 30 ml of phage sample was purified in around 10 min.

Production of oligoglucuronans using a monolithic enzymatic microreactor.

A glucuronan lyase (EC 4.2.2.14) was immobilized on a monolithic Convective Interaction Media (CIM) disk. The immobilization yield was equal to 29% of the initial activity and 35% of the initial protein amount. Degradations of three glucuronans with various O-acetylation degrees were investigated and compared with degradations using free enzyme. The immobilized glucuronan lyase was inhibited by the O-acetylation degree like the free enzyme. (1)H NMR analyses were used to study the O-acetylation degree of oligoglucuronans and demonstrated that the average degrees of polymerization were inclusive between 4 and 13 after 24h of degradation. This first immobilization of a glucuronan lyase constitutes a new tool to produce oligoglucuronans.

Fast and efficient separation of immunoglobulin M from immunoglobulin G using short monolithic columns.

Certain diagnostic, analytical and preparative applications require the separation of immunoglobulin G (IgG) from immunoglobulin M (IgM). In the present work, different ion-exchange methacrylate monoliths were tested for the separation of IgG and IgM. The strong anion-exchange column had the highest dynamic binding capacity reaching more than 20mg of IgM/ml of support. Additionally, separation of IgM from human serum albumin, a common contaminant in immunoglobulin purification, was achieved on the weak ethylenediamino anion-exchange column, which set the basis for the IgM purification method developed on convective interaction media (CIM) supports. Experiments also confirmed flow independent characteristics of the short monolithic columns.

Pressure drop characteristics of poly(high internal phase emulsion) monoliths.

Today, monoliths are well-accepted chromatographic stationary phases due to several advantageous properties in comparison with conventional chromatographic supports. A number of different types of monoliths have already been described, among them recently a poly(high internal phase emulsion) (PolyHIPE) type of chromatographic monoliths. Due to their particular structure, we investigated the possibility of implementing different mathematical models to predict pressure drop on PolyHIPE monoliths. It was found that the experimental results of pressure drop on PolyHIPE monoliths can best be described by employing the representative unit cell (RUC) model, which was originally derived for the prediction of pressure drop on catalytic foams. Models intended for the description of particulate beds and silica monoliths were not as accurate. The results of this study indicate that the PolyHIPE structure under given experimental condition is, from a hydrodynamic point of view, to some extent similar to foam structures, though any extrapolation of these results may not provide useful predictions of pressure versus flow relations and further experiments are required.

Characterisation of metal-chelate methacrylate monoliths.

Monoliths are attractive stationary phases for purification of large biomolecules like proteins because of their flow-unaffected properties. Isolation of histidine containing proteins to high purity can be efficiently performed using metal-chelate interactions within a single chromatographic step. In this work, we investigated properties of commercial metal-chelate methacrylate monoliths-Convective Interaction Media (CIM). Analytical CIM disk monolithic columns and CIM 8 ml monolithic columns were used for purification of tumor necrosis factor-alpha (TNF-alpha) analog LK-801 and green fluorescence protein with 6 histidine tag (GFP-6His). In both cases, purity over 90% was achieved. Dynamic binding capacity at 10% of breakthrough was around 17-18 mg/ml for LK-801 and around 30 mg/ml for GFP-6His. Adsorption isotherm revealed that the maximal capacity is achieved at protein concentration above 60 microg/ml. Dynamic binding capacity and resolution were found to be flow unaffected.

Methacrylate monolith chromatography as a tool for waterborne virus removal.

Enteric viruses are commonly present in environmental waters and represent the major cause of waterborne infections and outbreaks. Since traditional wastewater treatments fail to remove enteric viruses in the water purification process, they are released daily into environmental waters. Monolithic supports have enabled chromatography to enter the field of virology. They have been successfully used in virus purification and concentration. In this work quaternary amine (QA) methacrylate monoliths were exploited to remove enteric viruses from wastewater treatment plant effluent. Expectedly, chromatographic processing of such a complex medium was troublesome, even for monoliths, characterized by extremely large pore dimensions. This problem was solved by introducing a pre-step chromatography using hydroxyl (OH) methacrylate monoliths. This way, molecules, that would hinder virus binding to the anion-exchanger monolith, were removed. As a result, the OH pre-column reduced backpressure increase on the subsequent anion-exchanger column, and increased both QA column binding capacity and life time. Wastewater effluent samples were successfully purified from five waterborne enteric viruses (rotavirus, norovirus genogroup I and II, astrovirus, sapovirus), below the detection limit of RT-qPCR. The breakthrough of the rotavirus binding capacity was not reached for concentrations that significantly exceeded those expected in effluent waters. The obtained results confirm that methacrylate monoliths can be a valuable tool for simultaneous removal of different waterborne viruses from contaminated water sources.

Very fast analysis of impurities in immunoglobulin concentrates using conjoint liquid chromatography on short monolithic disks.

Transferrin and albumin are often present in immunoglobulin G (IgG) concentrates and are considered as impurities. Therefore, it is important to determine their concentration in order to obtain a well-characterized biological product. Here, we describe their determination based on conjoint liquid chromatography (CLC). The established method combines two different chromatographic modes in one step: affinity and ion-exchange chromatography (IEC) combined in one column. Therefore, two CIM Protein G and one CIM quaternary amine (QA) monolithic disks were placed in series in one housing forming a CLC monolithic column. Binding conditions were optimized in a way that immunoglobulins were captured on the CIM Protein G disks, while transferrin and albumin were bound on the CIM QA disks. Subsequently, transferrin and albumin were eluted separately by a stepwise gradient with sodium chloride, whereas immunoglobulins were released from the Protein G ligands by applying low pH. A complete separation of all three proteins was achieved in less than 5 min. The method permits the quantification of albumin and transferrin in IgG concentrates and has been successfully validated.

Application of monoliths for downstream processing of clotting factor IX.

In this paper, the application of monolithic columns for downstream processing of different clotting factor IX concentrates is shown. Determination of basic chromatographic conditions as well as investigations on the regeneration of disk- and tube-shaped monolithic columns using human serum albumin as a model protein, were performed. Separation of factor IX and vitronectin, a possible impurity in commercial factor IX concentrates was accomplished using disk-shaped monolithic columns. These same applications were also carried out with identical results on up-scaled tube-shaped monolithic columns. Since these media allow very fast separations, this method can be successfully applied not only to an in-process control of the purification of factor IX but also to other biopolymers from human plasma. Besides, the same application on the up-scaled tube-shaped monolithic column was successfully carried out.

Extraction of Triton X-100 and its determination in virus-inactivated human plasma by the solvent--detergent method.

For inactivation of lipid-enveloped viruses during the production of fresh frozen and lyophilized human plasma, the solvent-detergent method was applied. In this process, the solvent tri-n-butyl phosphate is removed by extraction with castor oil. The removal of the non-ionic detergent Triton X-100 is performed by solid-phase extraction using reversed-phase supports. For this purpose, different polymer- and silica-based supports were tested. The highest capacity for Triton X-100 was achieved with C18 silica gels. These supports can bind more than 0.1 ml of Triton X-100 per ml of support. None of the proteins, e.g., clotting factors, bind to the support and therefore they pass through the column and their biological activity is hardly affected. The determination of detergent during the production process was also studied. The application of special columns allowing direct sample injection was introduced. This is a simple method for the rapid in-process determination of Triton X-100 in human plasma by reversed-phase chromatography under isocratic conditions. Using the method developed here, less than 1.0 ppm of Triton X-100 can be detected in less than 12 min without any sample pretreatment.

Transfer of gradient chromatographic methods for protein separation to Convective Interaction Media monolithic columns.

Convective Interaction Media (CIM) columns are monolithic columns optimized for the separation of macromolecules. Some of them operate in the axial mode while others operate in the radial mode depending on the column size. In this work we tested the approach suggested by Yamamoto [Biotechnol. Bioeng., 48 (1995) 444] for transfer of gradient methods between columns of different size. A simplified equation for transfer was derived together with a criterion for its application. Separation was evaluated for a standard protein mixture and peroxidase enzymes present in fermentation broth. Salt and pH gradients were applied. Similar resolutions were obtained for each sample on all columns which demonstrates that the proposed approach can be successfully used for method scale-up on this type of column.

Use of compact, porous units with immobilized ligands with high molecular masses in affinity chromatography and enzymatic conversion of substrates with high and low molecular masses.

Different ligands with high molecular masses are immobilized on compact, porous separation units and used for affinity chromatography. In subsequent experiments different enzymes are immobilized and used for converting substrates with low and high molecular masses. Disk or tube with immobilized concanavalin A (ConA) are used as model systems for lectin affinity chromatography. The enzyme glucose oxidase is used as a standard protein to test the ConA units. Subsequently glycoproteins from plasma membranes of rat liver are separated, using units with immobilized ConA. The enzyme dipeptidyl peptidase i.v., which is used as a model protein in the experiments, is enriched about 40-fold in a single step, with a yield of over 90%. The results are only slightly better than those obtained with ConA when it is immobilized on bulk supports. The important improvement lies in the reduction of separation time to only 1 h. Experiments concerning the isolation of monoclonal antibodies against clotting factor VIII (FVIII) are carried out on disks, combining anion-exchange chromatography and protein A affinity chromatography as a model for multidimensional chromatography. Both IgG (bound to the protein A disk) and accompanying proteins (bound to the anion-exchange disk) from mouse ascites fluid are retarded and eluted separately. With the immobilized enzymes invertase and glucose oxidase (GOX) the corresponding substrates with low molecular masses, saccharose and glucose, are converted. It is shown that the amount of immobilized enzyme and the concentration of the substrate are responsible for the extent of the conversion, whereas the flow-rates used in the experiments have no effect at all. The influence of immobilization chemistry was investigated with GOX. Indirect immobilization with ConA as spacer proved to be the best alternative. With trypsin, immobilized on a disk, substrates with high molecular masses are digested in flow-through. For optimal digestion the proteins have to be denatured in the buffer for sodium dodecyl sulfate-polyacrlyamide gel electrophoresis prior to application. In contrast to the conversion of substrates with low molecular masses, flow-rates play an important part in conversion of substrates with high molecular masses. With lower flow-rates a higher degree of digestion is achieved.

Application of compact porous tubes for preparative isolation of clotting factor VIII from human plasma.

Membranes as well as compact porous disks are successfully used for fast analytical separations of biopolymers. So far, technical difficulties have prevented the proper scaling-up of the processes and the use of membranes and compact disks for preparative separations in a large scale. In this paper, the use of a compact porous tube for fast preparative separations of proteins is shown as a possible solution to these problems. The units have yielded good results, in terms of performance and speed of separation. The application of compact porous tubes for the preparative isolation of clotting factor VIII from human plasma shows that this method can even be used for the separation of very sensitive biopolymers. As far as yield and purity of the isolated proteins are concerned, the method was comparable to preparative column chromatography. The period of time required for separation was five times shorter than with corresponding column chromatographic methods. Compact porous disks made of the same support material can also be used for in-process analysis in order to control the separation. The quick response, which is obtained from these units within 5 to 60 s, allows close monitoring of the purification process.

Application of semi-industrial monolithic columns for downstream processing of clotting factor IX.

It has been shown in a previous study that monolithic columns can be used for downstream processing of different concentrates of clotting factor IX [K. Branovic et al., J. Chromatogr. A 903 (2000) 21]. This paper demonstrates that such supports are useful tools also at an early stage of the purification process of factor IX from human plasma. Starting with the eluate after solid-phase extraction with DEAE-Sephadex, the use of monolithic columns has allowed much better purification than that achieved with conventional anion-exchange supports. The period of time required for separation is also much reduced. In up-scaling experiments, separations are carried out with 8, 80 and 500 ml columns. A volume of 1830 ml of DEAE-Sephadex eluate, containing a total of 27.6 g of protein and 48500 IU of factor IX is applied to the 500 ml monolithic column. This corresponds to a separation on a pilot scale. The results of this separation after up-scaling are comparable to those obtained with the 8 ml column on a laboratory scale.

Large-scale methacrylate monolithic columns: design and properties.

Monoliths represent a special class of chromatographic supports. In contrast to other stationary phases, they consist of a single piece of highly porous material through which a sample is mainly transported by convection. As a consequence, monoliths enable fast separations and exhibit flow-unaffected properties, which make them attractive for purification of macromolecules like proteins or DNA. In this work, methacrylate-based monolithic columns with the bed volume up to 8000 ml are characterized. They perform high-resolution separations of several hundreds of grams of proteins per hour by utilizing liter per minute flow rates. They are incompressible under these operating conditions and resistant to strong alkaline conditions.

Application of Convective Interaction Media (CIM) disk monolithic columns for fast separation and monitoring of organic acids.

The separation of organic acids on the anion-exchange monolithic support, commercially available as Convective Interaction Media (CIM), is presented in this study. It is demonstrated that citric, isocitric, pyruvic, fumaric, malic, and alpha-ketoglutaric acid can be successfully separated using a CIM monolithic column of suitable user-adjustable length. The effect of the mobile phase composition on the separation is investigated. CIM monolithic columns of adjustable length from 3 to 18 mm are compared regarding the resolution and the back pressure. It is shown that the CIM monolithic column of 12 mm in length enables a good separation of all six organic acids within 3 min and exhibits a linear dependence of back pressure versus flow rate. The resolution and the dynamic binding capacity are found to be flow-unaffected. A filtrated sample of bioprocess supernatant is analyzed without previous pretreatment, which indicates the possibility of online monitoring of small molecules during the bioprocess using CIM monolithic columns.

Development and characterization of methacrylate-based hydrazide monoliths for oriented immobilization of antibodies.

Convective interaction media (CIM; BIA Separations) monoliths are attractive stationary phases for use in affinity chromatography because they enable fast affinity binding, which is a consequence of convectively enhanced mass transport. This work focuses on the development of novel CIM hydrazide (HZ) monoliths for the oriented immobilization of antibodies. Adipic acid dihydrazide (AADH) was covalently bound to CIM epoxy monoliths to gain hydrazide groups on the monolith surface. Two different antibodies were afterwards immobilized to hydrazide functionalized monolithic columns and prepared columns were tested for their selectivity. One column was further tested for the dynamic binding capacity.

Miniaturized monolithic disks for immunoadsorption of cardiac biomarkers from serum.

Immunoadsorbers based on 2.0 x 6.0 mm i.d., epoxy-bearing, methacrylate-based monolithic disks were developed in order to target myoglobin and N-terminal pro-natriuretic peptide (NT-proBNP), two biomarkers involved in cardiovascular disease. In both cases, antibodies were successfully coupled to the polymeric disk material. The developed immunoadsorbers permitted the selective isolation of myoglobin and NT-proBNP from human serum. Myoglobin was successfully isolated and detected from serum samples at concentrations down to 250 fmol microL(-1). However, the affinity of the antibodies was not sufficient for the analysis of low-concentration clinical samples. Frontal analysis of anti-NT-proBNP disks revealed the ability of the immunoadsorber to bind up to 250 pmol NT-proBNP, which is more than sufficient for the analysis of clinical samples. Anti-NT-proBNP disks showed good stability over more than 18 months and excellent batch-to-batch reproducibility. Moreover, anti-NT-proBNP disks permitted the isolation of NT-proBNP at concentrations down to 750 amol microL(-1) in serum, corresponding to concentrations of strongly diseased patients. Using reversed-phase trapping columns, the detection of NT-proBNP eluted from immunoadsorbers by mass spectrometry was achieved for concentrations down to 7.8 fmol microL(-1).

Purification of factor VIII and von Willebrand factor from human plasma by anion-exchange chromatography.

Factor VIII (anti-hemophilia A factor) is isolated from human plasma. Purification is carried out by a combination of precipitation and chromatographic procedures. After precipitation, the first step in virus inactivation is achieved through the effect of a non-ionic detergent such as Tween 80, and a solvent, e.g. tri-n-butylphosphate (TnBP). By subsequent anion-exchange chromatography, a highly enriched product is isolated, consisting of a complex formed by factor VIII and von Willebrand factor (FVIII-vWF). This treatment also removes the virus-inactivating reagents to quantities in the low ppm range. The second step in virus inactivation is aimed specifically at the non-enveloped viruses and consists of pasteurization at temperatures higher than 60 degrees C for 10 h. Through the addition of stabilizers, between 80% and 90% of the initial activity of FVIII is preserved during the modified pasteurisation. Along with the possibly denatured proteins the stabilizers, such as sugars, amino acids and bivalent cations, are subsequently removed by ion-exchange chromatography. The two-fold virus inactivation, by solvent/detergent treatment and subsequent pasteurisation, allows the destruction of both lipid-enveloped and non-enveloped viruses. During the procedure FVIII is stabilized through the high content of vWF. The complex consisting of FVIII and vWF can be dissociated by adding calcium ions. Subsequently both glycoproteins from this complex are separated from one another by further anion-exchange chromatography.

Application of high-performance membrane chromatography for separation of annexins from the plasma membranes of liver and isolation of monospecific polyclonal antibodies.

The separation of annexins, calcium-binding plasma membrane-associated proteins from rat liver and Morris hepatoma 7777 by high-performance membrane chromatography (HPMC) is described. The annexins with low molecular masses, CBP 33 and CBP 35, and the annexin with a high molecular mass, CBP 65/67, can be separated within 10 min from one another by anion-exchange HPMC under non-denaturing conditions. The separation devices used consist of compact, porous disks (QuickDisk) on the one hand and of bundled membranes made of cellulose fibers (MemSep) on the other. Both have been found to be equally well suited for this separation. The annexins obtained in this way are subsequently bound to epoxy-activated porons disks and used for the separation of monospecific polyclonal antibodies against the annexin CBP 65/67.

Application of compact porous disks for fast separations of biopolymers and in-process control in biotechnology.

Production and downstream processing in biotechnology requires fast and accurate control of each step in the process. Improved techniques which can be validated are required in order to meet these demands. For these purposes, chromatographic units containing compact porous disks for fast separation of biopolymers were developed and investigated with regard to their performance and speed. The problems that have, in the past, arisen from the use of wide and flat separation units, such as membranes and disks, have chiefly been those of sample distribution and large void volumes before and behind the unit. Improvements in the construction of the cartridge have led to better performance of the compact porous disks and faster separation. Using these disks, three calibration standard proteins could be separated within less than 1 min by an anion-exchange, cation-exchange, and hydrophobic interaction mode. Such units can be used for in-process control in production and downstream processing of biopolymers, as was shown in experiments involving the purification of α(1)-antitrypsin and clotting factor IX and the immobilization of enzyme glucose oxidase on an epoxy-activated compact porous disk.