C. Kasper, L. Meringova, R. Freitag, T. Tennikova
Journal of Chromatography A, 798 (1998) 65-72
A fast affinity method for the semi-preparative isolation of recombinant Protein G from E. coli cell lysate is proposed. Rigid, macroporous affinity discs based on a glycidyl methacrylate–co-ethylene dimethacrylate polymer were used as chromatographic supports. The specific ligands (here human immunoglobulin G, hIgG) were immobilized by the one-step reaction between native epoxy groups of the polymer surface and ϵ-amino groups of the IgG molecules. No intermediate spacer was necessary to reach full biological activity of the ligand. The globular affinity ligands are located directly on the pore wall surface and are thereby freely accessible to target molecules (here Protein G) migrating with the mobile phase through the pores. It is shown that the conditions chosen for the hIgG immobilization do not involve an active site of the protein and thus do not bias the formation of the affinity complex. Chromatographically determined constants of dissociation of hIgG–Protein G affinity complexes confirm the high selectivity of this separation method. Two different aspects of the affinity separation are discussed, which differ mostly in terms of scale. In disc chromatography, high volumetric flow velocities are possible because of the small backpressure. Since in addition the mass transfer is more efficient, it becomes possible to achieve very short analysis times. The discs proposed can be used in a single-step enrichment of Protein G from lysates of non-pathogenic E. coli. Gel electrophoresis data are used to demonstrate the high degree of purity achieved for the final product.
D. Josić, H. Schwinn, A. Štrancar, A. Podgornik, M. Barut, Y. P. Lim, M. Vodopivec
Journal of Chromatography A, 803 (1998) 61–71
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 IV, 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.
R. Giovannini, R. Freitag, T. B. Tennikova
Anal. Chem. 1998, 70, 3348-3354
Membrane adsorbers are well established in protein chromatography. The present paper investigated for the first time the behavior of polynucleotides on these stationary phases, taking a 7.2-kb predominantly supercoiled plasmid as example. Gradient and isocratic elution was studied. In contrast to protein high-performance membrane chromatography (HPMC), isocratic elution is possible in DNA chromatography. In the case of gradient elution, much higher salt concentrations can be used in the starting buffer. Under optimized conditions, both approaches led to a splitting of the single plasmid peak into three maximums, which corresponded to the threealbeit isolated bands in the agarose gel. Presumably the three fractions were supercoiled, nicked, and open circular plasmid DNA. Linearization of the plasmid lowered the adsorption energy, and the linearized plasmid eluted earlier than the nonlinearized one. The HPMC experiments were compared to similar ones performed using a conventional packed-bed anion-exchange column (BioScale Q2, 7 × 52 mm, 10-μm porous particles) and a novel monolithic-type anion-exchange column (UNO Q1, 7 × 35 mm). The results and characteristic differences observed in these experiments were interpreted in the light of the newly developed theory of HPMC.
A. Štrancar, M. Barut, A. Podgornik, P. Koselj, H. Schwinn, P. Raspor, D. Josić
Journal of Chromatography A, 760 (1997) 117-123
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.
A. Štrancar, P. Koselj, H. Schwinn, D. Josić
Analytical Chemistry, Vol. 68, No. 19, October 1, 1996
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 α1-antitrypsin and clotting factor IX and the immobilization of enzyme glucose oxidase on an epoxy-activated compact porous disk.
N.I. Dubinina, O.I. Kurenbin, T.B. Tennikova
Journal of Chromatography A, 753 (1996) 217-225
Since the influence of column length on protein resolution in high-performance liquid chromatography (HPLC) is not clear, different viewpoints presented in the literature are analysed in detail. The influence of gradient steepness on the length of the working column part (X0) or the part of a column in which the quasi-steady state is attained was studied. The equation for estimating the X0 value was obtained for the general case of the retention model. It was shown that at steep gradients only a short part of the column is used as the working part on which all separation processes develop. The other part of a column is a ballast where the protein zone migrates in a regime of parallel transfer. These results form a theoretical basis for high-performance membrane chromatography. As was shown experimentally, this method makes it possible to perform protein separation at low gradient times with appropriate resolution, comparable with that of HPLC.
B. G. Belenkii, V. G. Malt'sev
BioFeature, BioTechniques, 288, Vol. 18, No. 2 (1995)
In gradient chromatography for proteins migrating along the chromatographic column, the critical distance X0 has been shown to exist at which the separation of zones is at a maximum and band spreading is at a minimum. With steep gradients and small elution velocity, the column length may be reduced to the level of membrane thickness-about one millimeter. The peculiarities of this novel separation method for proteins, high-performance membrane chromatography (HPMC), are discussed and stepwise elution is shown to be especially effective. HPMC combines the advantages of membrane technology and high-performance liquid chromatography, and avoids their drawbacks.
D. Josić, Y.P. Lim, A. Štrancar, W. Reutter
Journal of Chromatography B, 662 (1994) 217-226
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.
T. B. Tennikova, F. Švec
Journal of Chromatography A, Volume 646, Issue 2, 3 September 1993, Pages 279–288
High-performance membrane chromatography (HPMC) is a very effective chromatographic method in which all the mobile phase flows through the separation medium. The effects of process variables such as concentration of displacement agent, flow-rate and gradient slope on HPMC separations in the ion-exchange, hydrophobic interaction and reversed-phase modes were studied using model protein mixtures. The basic relationships characterizing column HPLC also apply in HPMC. Whereas the efficiency of the HPMC membrane does not depend on flow-rate, the resolution increases with increasing gradient volume. Separations obtained with a continuous linear gradient were used for the design of a stepwise gradient profile which decreases the consumption of both time and mobile phase in separations of proteins. According to calculations, the protein diffusivity enhanced by the convective flow through the membrane is about four orders of magnitude higher than the “free” diffusivity of the protein in the stagnant mobile phase located in the pores of a standard separation medium. This considerably speeds up the process and improves the efficiency of the separation.
D. Josić, J. Rauch, K. Löster, O. Baum, W. Reutter
Journal of Chromatography A, 590 (1992) 59-76
Porous discs made of poly(glycidyl methacrylate) were used for high-performance membrane chromatography (HPMC) of proteins. In model experiments, separations of standard proteins by anion-exchange HPMC using a DEAE disc were carried out. The influences of sample distribution and disc diameter and thickness on separation performance were studied. The separation disc allowed a scaling-up from analytical (diameter 10 mm) to semi-preparative (diameter 50 mm) dimensions. In an application study, separations with anion-exchange and affinity HPMC were carried out using different complex samples such as rat serum and plasma membrane proteins. In all experiments the results on poly(glycidyl methacrylate) discs were comparable to those achieved on adequate high-performance liquid chromatographic (HPLC) columns. However, the separations on HPMC discs could be carried out faster than corresponding separations on HPLC columns. The pressure drop on the discs was low even at high flow-rates. The experiments show that the poly(glycidyl methacrylate) discs used are especially suitable for the isolation of proteins and other biopolymers which occur in a diluted state in complex mixtures.
T. B. Tennikova, B. G. Belenkii, F. Švec
Journal of Liquid Chromatography, 13(1), 63-70 (1990)
Basing on the fact that only short layers of a chromatographic column contribute to the separation in the interaction chromatography, 1 mm thick membranes from macroporous methacrylate polymer provided with functional groups were synthetized and used for protein separation. The chromatograms show that the separation is fully comparable with that experienced on a filled column but the advantage of a membrane is up to two orders of magnitude lower pressure during the process and very high loading reaching up to 40 g/m2. This recommends the high performance membrane chromatography also for large scale preparative separations.