Liquid purification or separation – With means to add treating material – Chromatography
Reexamination Certificate
1999-03-04
2001-01-23
Therkorn, Ernest G. (Department: 1723)
Liquid purification or separation
With means to add treating material
Chromatography
C210S657000, C422S070000, C422S105000, C436S178000
Reexamination Certificate
active
06177009
ABSTRACT:
The invention concerns an apparatus for treating biomolecules, in particular for isolating nucleic acids having a separation column which has a top-end inlet and a bottom-end outlet and in which a separation device is arranged, and having a collection vessel for collecting the liquid emerging from the outlet.
The detection of viral nucleic acids is becoming increasingly important in many areas of molecular medicine and diagnostics. Improved therapeutic strategies make it necessary to track viral titer (e.g. for HIV) down to very low values. Blood samples and blood donations must be examined with regard to their viral load, the result being to create a very large sample volume. In order to reduce both complexity and sample numbers, samples are combined so that, for example, ten samples can be examined together. This makes it necessary, inter alia, to develop apparatuses and methods with which larger sample volumes can be processed but with which even small viral loads can nevertheless be detected. A similar problem arises in the isolation of DNA quantities from biological fluids, for example urine, body fluids, etc.
Further possible applications are the specific binding and isolation of proteins or peptides which occur in low concentrations in biological matrices, e.g. serum, and must be detected.
EP 0 616 638 B1 and DE 38 43 610 A1 disclose apparatuses which comprise a separation column and a collection vessel. The separation column has a top-end inlet for admitting the sample, and a bottom-end outlet which projects into the collection vessel. A separation device, for example in the form of single- or multi-stage filters, is located in the lower region of the separation column. The separation device is configured so that the biomolecules to be recovered are bound adsorptively to the separation column. If constituents of, for example, cell material are to be recovered, this is usually preceded by several digestion and purification steps. After isolation, the biomolecules are detached from the separation device using an eluate, and collected in a separate collection vessel. They are then available for further examination.
With the known apparatuses, relatively large elution volumes are necessary in order to release the isolated biomolecules. The concentration of the biomolecules in the eluate is correspondingly low. Since only comparatively small volumes can be used in the subsequent detection methods, the analysis of small quantities of biomolecules, for example nucleic acids, presents considerable difficulties.
Attempts have therefore been made to perform a concentration operation by way of ultracentrifuging or ultrafiltration. These methods have not, however, proven particularly successful, since they are poorly reproducible and moreover require a considerable outlay in terms of equipment and tine.
It is the object of the invention to configure an apparatus of the kind cited initially in such a way that without a separate concentration step, a substantially smaller elution volume is sufficient even for large sample volumes. At the same time, cross-contamination is to be reliably prevented.
According to the present invention, this object is achieved in that the ratio between the internal volume of the separation column above the separation device and the surface area of the separation device is at least 10 ml/cm
2
, preferably at least 30 ml/cm
2
; and that a collection vessel is present into which the separation column and the collection vessel joined thereto can be inserted. This can be an ordinary centrifuging vessel having a top-end opening and a shoulder for the receptacle in a centrifuge. It has been found that with this separation column geometry, substantially smaller elution volumes are needed in order to release the purified biomolecules out of the separation device. The volume ratio between sample and eluate is at least 8 and can reach 100. The result is thus a concentration of the biomolecules in the eluate, which eliminates the need for previous or subsequent concentration steps even when the biomolecule concentration in the sample is low, and makes subsequent analysis much easier and more reliable. The external vessel offers a high level of protection against cross-contamination, and serves moreover as a collection vessel when the biomolecules from the sample are being bound to the separation device, and during subsequent washing steps. Comparatively large volumes occur in this context, which can be received by the external vessel. Because of the geometry according to the present invention and the resulting small quantity of eluate, a comparatively small collection vessel can then be used for elution of the purified biomolecules.
In a development of the invention, provision is made for the surface area of the separation device to be less than 0.4 cm
2
, preferably less than 0.3 cm
2
, for an internal volume of at least 15 ml, preferably 20 ml.
The geometry according to the present invention can, in principle, also be attained with cylindrical separation columns. In order for the separation column to be compact, however, it is more advantageous for the internal cross section of the separation column to increase from the separation device toward the inlet, for example constituting a fimnel segment. Cylindrical segments can respectively adjoin the funnel segment at the top and/or bottom end. The separation device should be supported on a shoulder which causes the cross section of the outlet to taper.
In a particularly preferred embodiment, the apparatus has an external vessel into which the separation column and the collection vessel joined thereto can be inserted. This can be an ordinary centrifuging vessel having a top-end opening and a shoulder for the receptacle in a centrifuge. The external vessel offers a high level of protection against cross-contamination, and serves moreover as a collection vessel when the biomolecules from the sample are being bound to the separation device, and during subsequent washing steps. Comparatively large volumes occur in this context, which can be received by the external vessel. Because of the geometry according to the present invention and the resulting small quantity of eluate, a comparatively small collection vessel can then be used for elution of the purified biomolecules. Preferably the external vessel is dimensioned so that the separation column and the collection vessel joined thereto fit substantially completely into the external vessel. The external vessel should have a length which corresponds to the length of the separation column and of the collection vessel joined thereto, and both are held immovably in the external vessel so that changes in position do not occur during centrifuging.
The external vessel should advantageously have a cap in order to enclose the separation column and collection vessel completely and thus form a barrier against cross-contamination. In order to prevent independent movement of the separation column, it is further advantageous if the separation column and the collection vessel joined thereto can be clamped between the cap and the base of the external vessel. The same purpose is served if the separation column has an annular flange, overlapping the upper rim of the external vessel, which can be clamped between the cap and external vessel and thus functions as a seal.
Provision is further made, according to the invention, for the collection vessel to be joinable in airtight fashion to the separation column in order to prevent any leakage of the collected substance. The collection vessel can be configured so as to be slid onto the outlet of the separation column; an adapter can also be interposed for this purpose.
It is understood that the collection vessel is to be closable by way of a cover so that the eluate containing the biomolecules can be safely transported and stored. For this purpose, the cover can be joined in known fashion to the collection vessel by a tape.
REFERENCES:
patent: 4238196 (1980-12-01), Acuff
patent: 4243534 (1981-01-01), Bulbenko
patent: 4270921 (1981-06-01), Graas
Sieber Manfred
Zeidler Robert
Liniak, Berenato, Longacre & White LLC
Macherey Nagel GmbH & Co.
Therkorn Ernest G.
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