Chemistry: molecular biology and microbiology – Treatment of micro-organisms or enzymes with electrical or... – Cell membrane or cell surface is target
Reexamination Certificate
2001-12-22
2004-03-02
Park, Hankyel T. (Department: 1648)
Chemistry: molecular biology and microbiology
Treatment of micro-organisms or enzymes with electrical or...
Cell membrane or cell surface is target
C435S285200
Reexamination Certificate
active
06699698
ABSTRACT:
DESCRIPTION
The invention relates to a method and an apparatus for the selective in situ separation and enrichment of substances.
Separation, isolation and disintegration constitute a unit in most separation methods in biology and medicine. Biological material is disintegrated for the purpose of subsequent enrichment, separation or isolation of individual substances or groups of substances or compartments.
The isolation of substances from the most diverse biological materials has been a long-standing practice. Both unambiguous characterization and use in various fields such as pharmacy or medicine, for example, in most cases require the chemically uniform compound, i.e. the pure substance. Within the entire life science sector, therefore, separation methods represent one of the most important foundations for the identification of substances and their use. Said isolation or separation often constitutes a problem, depending on the specific isolation task, for example regarding the purity of the substance to be isolated.
The isolation, separation or disintegration of biological material, for example organisms, tissues, biological cells, organelles, micelles, viruses etc., as a rule constitutes the first step in the analysis or extraction of cell constituents. Such constituents can, for example, be nucleic acids, proteins, metabolites, pigments etc. As the quality of all subsequent steps is determined by the disintegration of the biological material, said disintegration occupies a key position. Novel disintegration methods are therefore of interest for a multiplicity of procedures and have a proportionately large potential for being marketed profitably as a product. Disintegration methods are a prerequisite, in the same way as explained above for the separation methods, for life science fields, for example genomics, proteomics and many others. Methods of disintegrating biological material are not universal, but are geared very specifically to the particular requirements. The known methods—mechanical and nonmechanical disintegration methods—are toxic, expensive, time-consuming and laborious, as well as being limited to specific applications. Moreover, there is a high risk of cross-contamination which has a major impact on the quality of all subsequent process steps, especially in sensitive detection methods such as, for example, the PCR-based nucleic acid detection methods. With the known mechanical methods, moreover, standardization and automation is more difficult, or it is virtually impossible to combine them with separation and isolation methods.
The standard separation and isolation methods include filtration, centrifuging, crystallization, distillation, extraction, electrophoresis, chromatography and magnetism-based methods.
Finally, a distinction is drawn between analytical and preparative methods. Analytical methods are used to detect specific substances in mixtures, while preparative methods are employed for concentrating or extracting larger quantities of as pure a substance as possible.
The use of pulsed electric fields for separation purposes has not been known hitherto.
Instead it is known to use such fields within the context of electroporation (Prausnitz, M. R. et al., in Biophysical Journal 66 (1994), 1522-1530, U.S. Pat. No. 5,019,034, U.S. Pat. No. 5,273,525, U.S. Pat. No. 5,304,120, U.S. Pat. No. 5,389,069, U.S. Pat. No. 5,422,272). With electroporation, from one to at most ten electric pulses (impulse number) are used, as a rule, over the particular treatment time. Depending on the pulse frequency, the treatment duration is at most a matter of seconds, the field strength of the pulses being chosen such that the critical voltage (V
c
, equal to about 1 volt) across the membrane of the cell to be electroporated is exceeded (with spherical objects, the relationship v=3/2 E
0
r applies, where r is the cell radius). Electroporation is generally carried out under mild conditions, i.e. for example at room temperature, it being essential not to exceed or drop below the maximum physiologically acceptable temperature of the respective target organism or target cell (U.S. Pat. No. 5,466,587, U.S. Pat. No. 5,545,130, U.S. Pat. No. 5,547,467, U.S. Pat. No. 5,749,847).
Similar conditions are employed with cell fusion, the emphasis here too being on the choice of the mildest conditions possible, to achieve a high success rate in the fusion aimed for (“Electroporation and Electrofusion” in: Cell Biology, Plenum Press, New York and London (1989), editors: Neumann, E., Sowers, A. E. and Jordan, C. A.).
Finally, to achieve complete disruption of biological cells, pulsed electric fields are likewise used in some cases, the cell constituents being released uncontrolledly. In the process, pulse numbers greater than 20,000 are generally used, the field strength as with electroporation generally being above the critical voltage (V
c
) of 1 volt across the membrane of the cell to be disrupted. The temperatures used for disruption of the biological cells are generally quite high, i.e. they are far above the physiologically suitable temperatures, since the aspect of cell preservation no longer plays a part and, in contrast, the disruption is to be accelerated and completed by employing extreme conditions (U.S. Pat. No. 5,235,905, U.S. Pat. No. 5,447,733, U.S. Pat. No. 5,393,541).
It is hitherto unknown, by means of the conventional methods, for biological material to be sorted, separated and disintegrated and for substances to be released and to be concentrated or isolated directly, or to be purified. Conventional methods, especially separation methods are generally preceded by a time-consuming and expensive sample preparation after cell disruption or work-up, especially sorting and/or disintegration, of the biological material. Thus, the biological material is often first homogenized and lysed, and then is subjected to further processing steps and finally to the separation method. Only in exceptional cases can the homogenate be subjected directly to a separation method. Usually, however, the homogenate is then centrifuged and the supernatant as a crude extract is subjected to a separation method, first requiring adjustment of pH, ionic strength and other parameters.
The object of the invention is therefore to provide a method of separating and disintegrating substances from and/or on biological materials, which renders time-consuming and expensive sample preparation unnecessary and in a single process step leads to the selective in situ release and/or separation of the desired substance(s), a further object of the method being to enable a universal, standardized and consequently automated separation or disintegration of biological material, e.g. organisms, tissues, cells, organelles, micelles, viruses etc., in conjunction with the release of the constituents.
The present invention achieves this object by providing a method for the selective in situ separation of one or more substances from a substance mixture present in a liquid medium by means of a stationary and a mobile phase, wherein the stationary phase is a constituent of a biological material present in the liquid medium and the mobile phase is the liquid medium and wherein the biological material present in the liquid medium is subjected to pulsed electric fields having a field strength of up to 200 V/cm. The substance mixture to be separated can, prior to the separation, disintegration, isolation or enrichment according to the invention, be present in the biological material, or outside it, or both. This means that the substance mixture can be present directly in the liquid medium or enclosed in the biological material in the liquid medium. Preferably, in the process, i.e. during and/or after the treatment with the pulsed, electric fields, one or more desired substances are released from the biological material, are concentrated in the liquid medium and can then be separated from the biological material by means of conventional methods such as e.g. centrifuging or filtration of other, undesirable substances and/or the bi
Bentsian Elkin
Bernhagen Jürgen
Brunner Herwig
Geiger Georg
Vitzthum Frank
Fraunhofer-Gesellschaft Zur Forderung Der Angewandten Forschung
Ostrolenk Faber Gerb & Soffen, LLP
Park Hankyel T.
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