Liquid purification or separation – Processes – Liquid/liquid solvent or colloidal extraction or diffusing...
Reexamination Certificate
2001-04-19
2003-02-25
Fortuna, Ana (Department: 1723)
Liquid purification or separation
Processes
Liquid/liquid solvent or colloidal extraction or diffusing...
C210S650000, C210S502100, C210S198200, C210S634000, C435S299200, C435S007100
Reexamination Certificate
active
06524482
ABSTRACT:
BACKGROUND OF THE INVENTION
Ions such as calcium, magnesium and iron play a vital role in many important biological functions, including cell growth, enzymatic activity, regulatory function and signal transduction. Understanding how these ions control in vitro functions requires a corresponding control of the concentrations of the ions in physiological buffers or culture media.
The simplest way to control the concentration of a particular ion species is to remove it completely from the buffer or media, and then add it back to the desired concentration. In practice, however, this is very difficult as ions exist naturally everywhere. The difficulty of removing Ca
2+
from a solution, for instance, was first recognized by Sidney Ringer in 1883 when he was forced to retract a paper written the year before which stated that calcium was not required for a frog's heart to beat.
One way of removing such ions from solution is by dialysis, but this method is generally slow and cumbersome. Another method of removing such ions is by titration with EDTA. In fact, EDTA in particular is very widely used and forms complexes with most metal ions. However, EDTA is generally a non-specific chelating or complexing agent. Thus, EDTA will bind to the majority of the various divalent metal ions, and as a result, may remove ions other than those targeted. In addition, EDTA titration often requires an excess of the chelating or complexing agent for the complete removal of a targeted ion species from the solution. As such, the presence of these extra surplus chelating or complexing agents in the biological solution may have unanticipated or undesired biological side effects.
SUMMARY OF THE INVENTION
The present invention provides methods for removing species of ions from solutions used in biological applications, thereby allowing controlled study of the biological effect of one or more ions. The methods described herein are particularly effective for preferentially removing calcium ions from biological solutions that contain protein, which is surprising since proteins are known to bind calcium ions, thus, preventing their removal.
In general, a biological solution from which one or more ion species is to be removed is contacted with an ion binding ligand having an affinity for the desired ion wherein the ligand is bound to a substrate such as a particulate solid support or membrane. After the ions have been bound by the ligand-substrate composition, the composition having the ions bound to it (or the solution having a depleted ion content) is removed, leaving the biological solution with a reduced or depleted concentration of the targeted ion. The biological solution can then be compared to a control biological solution, or alternatively, ions can be added back into the depleted biological solution at known concentrations. Under either scenario, the biological function of one or more ions in a biological solution may be studied.
The ligand-substrate composition for use with the present method may be represented by Formula 1, as follows:
S—B—L Formula 1
wherein S is a substrate; L is any ion-binding ligand having an affinity for one or more ion species and having a functional group for attachment to S; and B is the covalent linkage mechanism which attaches S to L.
In a preferred embodiment, the composition for use with the present method will comprise one or more different ion binding ligand(s) (L) that is ion species specific. It is also preferred that the substrate (S) comprises a filtration-type media commonly used in the liquid purification arts, wherein the substrate, such as organic beads, membranes or composite membranes, and is derivatized to have a hydrophilic surface and polar functional groups, to which the ligand may be covalently bound. While the above mentioned substrate materials and forms are preferred other substrates may be used provided functionality is present.
The methods of the invention can be used to filter out ion species from biological solutions, particularly those which contain protein. Such ion species include, but are not limited to, Ca
2+
and Mg
2+
together over other ions, or in some circumstances, Ca
2+
may be preferentially removed over Mg
2+
. These ions can be removed from solutions such as, but not limited to, serum; blood; biological buffers, e.g., Tris(hydroxymethyl)aminomethane; nucleic acid amplification buffers; tissue culture media, e.g., Dulbecco's Modified Eagle Medium (DMEM) (both serum free and serum supplemented); and MS (Murashige and Skoog) medium.
The invention may be carried out by preferably using a filtration or separation device containing the ion binding ligand bound to the substrate. A biological solution from which the ions are to be removed can be caused to flow through this device containing the ion binding ligand bound to the substrate, so that the ions are trapped by the ligand, and the biological solution that is recovered is essentially free of the ion species. This step may be repeated with the same ligand bound substrate or with a different ligand bound substrate in order to increase the biological solution purity, i.e., further deplete the ion species from the biological solution.
If the ligand is bound to solid supports, such as acrylate or glass beads, the beads can be packed into a column through which the biological solution containing the ions would be passed. The ions would be bound by the ligand on the surface of the beads and remain in the column, allowing the biological solution, now with the ion concentration greatly reduced, to pass out of the column. Alternatively, the ion binding ligands can be bound to a filtration membrane through which is passed the biological solution containing the ions that are to be removed. As with the column, the ions will bind to the ligand attached to the membrane. Thus, the solution that is recovered should have a greatly reduced ion concentration.
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Bruening Ronald L.
DiLeo Anthony J.
Jiang Tongbo
Krakowiak Krzysztof E.
Fortuna Ana
IBC Advanced Technologies, Inc.
Thorpe North & Western
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