Chemistry: electrical and wave energy – Processes and products – Electrophoresis or electro-osmosis processes and electrolyte...
Reexamination Certificate
1998-11-19
2002-04-30
Warden, Jill (Department: 1743)
Chemistry: electrical and wave energy
Processes and products
Electrophoresis or electro-osmosis processes and electrolyte...
C204S455000, C204S601000, C204S605000
Reexamination Certificate
active
06379515
ABSTRACT:
The present invention relates to a process in stages for the selective separation and/or determination of charged organic compounds. In this process, mixtures containing these organic compounds are subjected to conditions of high binding affinity of capillary affinity gel electrophoresis, whereby the capillary tube is filled with a gel based on polymers as the separating agent, to which the specific recognition elements (receptors) for one or several organic compounds are covalently bound. By applying an electric field, the undesired portions of the mixture of substances are separated optionally whilst splitting them open, and then the affinity conditions are changed, so that the bound compounds are eluted and detected, optionally whilst splitting them open. Further objects of the invention are the separating agents which may be filled into the capillary tubes for the capillary affinity gel electrophoresis and their usage for the separation of charged organic compounds.
Column-chromatography processes have acquired great importance and found a wide application for the preparatory separation and purification of, in particular, biologically active compounds. Of the liquid chromatography processes, gel chromatography with polymeric gels as separating agents, or the stationary phase, is particularly widespread. Since, in many cases, this method does not satisfy requirements, improvements are always being proposed. Some time ago, it was proposed that specific receptors, for example oligonucleotides or proteins, should be bound to the polymeric gels, and that owing to their interaction and affinity, they would bind to biological molecules, the molecules would be retained under certain conditions of elution and allow the undesired secondary components to be separated. With the change in elution conditions, the affinity may be influenced so that it is possible to elute the desired, purified compound. This method of affinity chromatography has been described for example by H. W. Jarrett in the Journal of Chromatography, Biomedical Applications, 618 (1993), pages 315 to 31, or by S. Ostrove in Methods of Enzymologie, volume 182 (1990), pages 357 to 379.
Another known method for the preparatory separation is the gel electrophoresis process for separating charged compounds, which is used in particular in the biochemical field for the separation of peptides, antigens, antibodies and oligo- to polynucleotides. Normally, plates or rods of the gel are used with the covalently bound receptors, as described for example in DE-A-2 712 344, by V. Horejsi in Analytical Biochemistry 125, pages 358 to 369 (1982) and in the Journal of Chromatography, 376 (1986), pages 49 to 67, by G. L. Igloi in Biochemistry 27 (1988), pages 3842 to 3849, and by H. Goubran-Botros et al. in the Journal of Chromatography, 597 (1992), pages 357 to 364. This method may also be set up as a column process, see for example FR-A-2 402 716, the description of which discloses the continuous elution and concentration of a the testerone antibody with the assistance of a polymer-bound antigen at a pH of just below the isoelectric point of the antibody.
For analytical separation and determination processes of charged organic compounds, the capillary gel electrophoresis method is more suitable, since on the one hand one can use smaller amounts of samples, this method is considerably more sensitive and high resolutions may be attained. Also, this method is faster to carry out and can even be operated automatically. An example which may be mentioned is the analytical separation of oligonucleotides, as described by A. Paulus et al. in the Journal of Chromatography, 507 (1990), pages 113 to 123.
Proposals have also already been disclosed, which link capillary gel electrophoresis with affinity chromatography. In WO 95/10344, polymer globules with covalently bound receptors are bound chemically to the inside wall of a capillary tube. In this way, in a continuous elution process, the separation output ought to be improved. However, the number of covalently bound receptors is very small and restricted, due to the specific construction, so that the sensitivity is often unsatisfactory. Furthermore, the chemical binding of the separation material to the inside wall of the capillary tube is very complicated and restricts its application.
In the Journal of Chromatography 632 (1993), pages 137 to 142, Y. Baba et al. describe a continuous process for the separation of oligodesoxynucleotides by means of capillary affinity gel electrophoresis. By using polyvinyl adenine as the stationary phase, selectivity ought to be improved. However, this aim cannot be completely attained, since although certain interactions between the analytical substance and the receptor do take place, no fixed bindings arise as when using complementary receptors. Therefore, the selectivity is wholly unsatisfactory for many purposes, especially for separating oligonucleotide mixtures, the continuous elution also making a substantial contribution to this.
In Anal. Chem. 64 (1992), pages 2872 to 2874, S. Birnbaum et al. describe the separation of optical isomers of tryptophan by continuous elution using capillary affinity gel electrophoresis, whereby the chiral selector employed is a BSA gel (BSA=bovine serum albumin) which is crosslinked with glutaraldehyde. However, the separation and detection of analytical mixtures using such gels as separating agents is not possible, since specific targeted bindings to receptors are not possible.
In EP-A-0 671 626, capillary tubes are described for capillary affinity gel electrophoresis, in which receptors are bound to the inside wall of a first part of the capillary tube and the remainder of the capillary tube is filled with a buffer. In the separation process, for example, a mixture of labelled and unlabelled analytical molecules may be bound to the receptors and the unbound portions eluted. Afterwards, the elution conditions are changed (pH change, addition of organic solvent, other buffers) and the labelled and unlabelled analytical molecules are dissolved and then separated in the subsequent gel. In this construction also, due to the specific set-up, the number of covalently bound receptors is too small to obtain separations with high sensitivity. In addition, production of the capillary tubes is complicated and the chemical possibilities of binding to receptors are greatly restricted by the material of the capillary tubes and the functional groups present.
In the Journal of Chromatography A 652 (1993), pages 247 to 252, P. Sun et al. describe the covalent binding of BSA to a high molecular weight dextran (M
r
2,000,000). The immobilised polymer is used as a replaceable chiral separating agent for the enantiomers of leucovorin in capillary affinity gel electrophoresis, whereby a continuous procedure is similarly employed. In this process also, the selectivity and sensitivity leave a lot to be desired; in particular, the selective separation and detection of target analytical substances, as well as other analytical components, with high selectivity and sensitivity, is not possible with the process described.
It has now surprisingly been found that target molecules even in capillary affinity gel electrophoresis may be retained completely by selecting certain conditions, and eluted completely by changing the conditions, when using as separating agent polymer gels with covalently bound receptors complementary to target analytical substances. It was also surprisingly found that the analytical substances and mixtures of analytical substances may be separated and detected (1) with very high selectivity and sensitivity, as well as (2) rapidly and specifically, and (3) other components of the analytical substance may be simultaneously separated and determined, and (4) furthermore separations of this type may be carried out in fully-automatic on-line apparatus, if in the capillary affinity gel electrophoresis (a) a polymer gel is used as the separating agent, the gel containing receptors which are complementary to one or several of the
Muscate-Magnussen Angelika
Natt François
Paulus Aran
Novartis AG
Perkins Coie LLP
Starsiak Jr. John S.
Warden Jill
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