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.

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.

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.

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.

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.

Screening for peptide affinity ligands on CIM monoliths.

Screening of peptide ligands for affinity chromatography usually involves incubation with the target protein in a batch system. In an additional step, peptides with fast binding kinetics have to be selected in respect to satisfactory performance under flow conditions on a support ensuring optimal three-dimensional presentation of the peptide. We have developed a rapid screening system based on peptide synthesis and screening on CIM((R)) disks. The disk size was minimized to fit into microplates usually applied for solid-phase extraction. In combination with a vacuum manifold, semi-automated peptide synthesis and screening for binding to a target protein under simulated chromatography conditions are possible. Various analytical methods can be applied for parallel and automated determination of the quantity, integrity, or activity of the target protein in the flow through or bound to the affinity support. This system also allows parallel screening for suitable chromatographic conditions like running buffer, washing, and elution conditions.

Effect of experimental error on the efficiency of different optimization methods for bioprocess media optimization.

Four optimization methods (Simplex, Rosenbrock, iterative factorial experimental design (IFED) and genetic algorithms) for the optimization of the biotechnological media composition under conditions where the measured quantities are subjected to the experimental error were compared. The computer simulations were performed on some of the selected two- to six- parameter biotechnological models. The optimization process was modified in such a way that the experimental error was considered. The results show that the optimization efficiency increases when this new termination criteria is implemented. In addition, the method efficiency becomes independent of the experimental error. In general, Simplex and Rosenbrock methods need fewer experiments and their distribution of necessary experiments is narrower than for IFED and genetic algorithms. The increase of model parameters that need to be optimized results in a decrease in the method efficiency and in an increase of the average number of required experiments. The results were further verified on the cultivation of Saccharomyces cerevisiae and were found to be in good agreement with the results obtained from the computer simulations.

Isolation and characterization of Mn(III) tartrate from Phanerochaete chrysosporium culture broth.

High initial Mn(II) concentration results in accumulation of a Mn(III) tartrate complex in the growth medium of Phanerochaete chrysosporium. Since Mn(III) is the major oxidant in ligninolysis by manganese peroxidase, the role of accumulated complex should not be neglected when degradation experiments by a crude culture filtrate are performed. To study the Mn(III) complex oxidative potential it was isolated by absorption to polyamide followed by desorption with an alkaline methanol solution. High performance liquid chromatography analysis and atomic absorption spectroscopy confirmed that the isolate was Mn(III) tartrate. Oxidation of 2,2'-azino-bis(3-ethylbenz-thiazoline-6-sulfonate) was used for testing the temperature and pH stability of the isolate that also intensively oxidized 2,6-dimethoxyphenol. In comparison with the non-isolated complex in the culture filtrate, the isolate showed increased temperature and pH stability. The oxidative potential of the isolated Mn(III) tartrate was additionally tested by decolorization of the synthetic dye Indigo carmine.

The effect of agitation and nitrogen concentration on lignin peroxidase (LiP) isoform composition during fermentation of Phanerochaete chrysosporium.

Convective Interaction Media (CIM) monolithic columns were applied for the HPLC monitoring of Phanerochaete chrysosporium lignin peroxidase (LiP) isoforms during cultivation. The influence of the agitation mode (circular, elliptic) and rate (130 and 200 rpm), as well as the initial nitrogen concentration (1.6-6 mM) in the growth medium was investigated. Identical rotation rate but different agitation modes resulted in different LiP activities and isoenzyme compositions. On the other hand, at different agitation types and rates, similar LiP activities were obtained at different isoenzyme compositions. Although LiP H2 and LiP H6/H7 were predominant isoenzymes obtained at various cultivation conditions, relative isoenzyme amounts differ considerably when initial nitrogen concentration was changed between 1.6 and 5 mM.

Involvement of intracellular Ca2+ in the regulation of bovine leukemia virus expression.

The calcium ionophore A23187, which was used to increase the intracellular calcium concentration ([Ca2+]i), was analyzed for effects on bovine leukemia virus (BLV) expression in two BLV infected cell lines. To clarify the role of intracellular free calcium in this response, [Ca2+]i was measured during ionophore treatment with the fluorescent calcium indicator Fura-2. Elevation of intracellular calcium under these conditions caused an enhancement of BLV gp51 and p24 synthesis as well as an activation of the BLV long terminal repeat (LTR) in a dose-dependent manner. Furthermore, it was observed that elevated levels of intracellular calcium following A23187 stimulation lead to activation of NF-kappaB. Based on inhibitor studies, we hypothesize that the effect of A23187 on BLV expression appears to be mediated by PKC.

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.

Construction of large-volume monolithic columns.

Monolithic supports have become the subject of extensive study in the past years. Despite their advantageous features and many successful chromatographic applications in the analytical scale, only a very few examples of larger volume monoliths were described. In the case of GMA-EDMA monoliths, this can be attributed to the fact that due to the exothermic polymerization a pronounced temperature increase inside the monolith significantly affects the structure. The temperature increase depends on the thickness of the monolith, and consequently, there is an upper limit that allows the preparation of a unit with a uniform structure. In the present work, we have analyzed a heat release during the polymerization and have derived a mathematical model for the prediction of the maximal thickness of the monolithic annulus having a uniform structure. On the basis of the calculations, two annuluses of different diameters were polymerized and merged into a single monolithic unit with a volume of 80 mL. In addition, a special housing was designed to provide a uniform flow distribution in the radial direction over the entire monolith bed. It was shown that such a monolithic column exhibits flow-independent separation efficiency and dynamic binding capacity up to flow rates higher than 100 mL/min. The separation and loading times are in the range of a few minutes. The pressure drop on the column is linearly dependent on the flow rate and does not exceed 2.5 MPa at a flow rate of 250 ml/min.

A method of fast separation of lignin peroxidases using convective interaction media disks.

The HPLC separation of lignin peroxidase isoenzymes using Convective Interaction Media disks containing quaternary amine and diethylaminoethyl ion-exchange active groups is proposed. In contrast to standard HPLC procedures the separation can be performed within a few minutes without considerably affecting the separation resolution. The method is reproducible and gives a linear response of integrated peak area to protein concentration for all measured isoenzymes. The separation resolution is retained unchanged by applying crude culture filtrate instead of a sample previously frozen and dialyzed. The optimized method might therefore be used for on-line monitoring of lignin peroxidase isoenzyme composition during fermentation. On the other hand, the proposed method is comparable in time to the original method of lignin peroxidase activity measurement (proposed by Tien and Kirk), providing additionally the isoenzyme composition.

High-performance membrane chromatography of small molecules.

High-performance membrane chromatography (HPMC) proved to be a very efficient method for fast protein separations. Recently, it was shown to be applicable also for the isocratic chromatography of plasmid DNA conformations. However, no study about the separation of small molecules has been performed until now. In this work, we investigated the possibility of gradient and isocratic HPMC of small molecules with Convective Interaction Media disks of different chemistries and tried to explain the mechanism that enables their separation. We demonstrated that it is possible to achieve efficient separations of oligonucleotides and peptides in the ion-exchange mode as well as the separation of small hydrophobic molecules in the reversed-phase mode. It was shown that similar peak resolution can be provided in both gradient and isocratic modes.

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.

Nonflocculent versus total biomass ratio as a criterion for starting the biomass separation process

We introduce the ratio of nonflocculent versus total biomass as a criterion for starting cell separation from the medium. This criterion can be applied for the automation of the process regardless of the process dynamics. Its minimum indicates the optimum period of time for the start of the separation process with regard not only to nonflocculent cell concentration, but also medium attributes. In contrast to the concentration of nonflocculent cells, which has two minima, first at the beginning of the process and another broader one in the period during which maximum flocculation is present, the ratio has a single minimum and can therefore be implemented as a criterion for cell separation. To calculate the ratio value, in addition to an on-line method for nonflocculent biomass measurement described elsewhere, an on-line method for the total biomass of flocculent yeast is proposed. It is based on the absorbency measurement of the cell biomass, previously deflocculated by EDTA. Therefore, it can be applied in bioprocesses with transparent media and yeast that can be deflocculated by EDTA. Copyright 1998 John Wiley & Sons, Inc.

On-line measurement and analysis of yeast flocculation.

The ability of yeast to flocculate is important in different separation processes, especially in the beer industry. Because of the regulation purposes, there is a need for online monitoring. With the presented measuring set-up, consisting of a peristaltic pump, a photometer, and a computer, it is possible to determine the onset of flocculation as well as to follow flocculation intensity and the concentration of nonflocculated cells. It was found that for the yeast strain Saccharomyces cerevisiae ZIM 198 the decrease of nonflocculated cells (after flocculation has occurred) during the exponential growth can be described by an exponential equation for the first-order process, whereas the increase of free cells due to dispersion of the flocs during the stationary phase follows the form of the growth curve. It was also demonstrated that the absorbency profiles of yeast sedimentation can be described by the second-order equation suggested by Stradford and Keenan for the decrease of cell concentration during sedimentation. (c) 1997 John Wiley & Sons, Inc.

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.