Liquid purification or separation – With means to add treating material – Chromatography
Reexamination Certificate
1999-06-02
2001-11-13
Therkorn, Ernest G. (Department: 1723)
Liquid purification or separation
With means to add treating material
Chromatography
C210S512100, C210S635000, C210S656000, C210S659000
Reexamination Certificate
active
06315900
ABSTRACT:
BACKGROUND OF THE INVENTION
Purification systems using glass or metal tubes which contain a packed column of separation medium, for example, beads or particles, are known. These tubes are known as column boxes. Because the separation medium is compacted within the column boxes, the flow rates are slow and the column boxes have a limited capacity. Therefore, prior art purification technology has focused on increasing the porosity of the separation medium to increase the flow rates and capacity within the column box. The object of these known systems is to purify the largest amount of material within the shortest amount of time while keeping the amount of contaminants low and the product yields high. One problem with the old purification technology is that increasing porosity of the separation medium achieved faster flow rates and capacity, but reduced product yields and quality.
A past method, which does not use a column box, utilized a dynamic (i.e., mechanical forces at the site of filtration) filtration system with a variety of separation medium. This old method and apparatus also failed in part because the sheering forces of the dynamic system damaged the separation medium and the bio-compounds being purified.
Prior methods of affinity separation involving dynamic filtration of cellulose and non-cellulose beads have the disadvantage that the beads degrade due to sheering forces inherent in dynamic filtration. These sheering forces irreversibly damage bead supports and the sheer-sensitive biological molecules grafted or bound to them. Generally, many porous beads will fragment due to the agitation and sheering forces contained in the dynamic affinity separation apparatus. The present invention overcomes the disadvantages of the prior art by utilizing a static affinity separation method which is gentle on beads and biological molecules, but causes intermixing of the target compound to be purified with the sheer-sensitive beads and biological molecules which flow through the static tangential flow filters.
The prior art discloses the use of polystyrene beads in dynamic filtration apparatuses for affinity separation of biological compounds. Certain disadvantages are involved in the use of polystyrene beads, such as high non-specific adsorbing of biological molecules on their hydrophobic surfaces. Further, polystyrene beads have open pores on the surface of the bead which entrap contaminants which will co-purify with wanted products and decrease yield and purity of a target compound. Polystyrene has been found to exhibit a high degree of agglomerate and to adhere to the filters used in dynamic affinity separation methods. This agglomeration of the polystyrene beads allows for debris to become trapped and spoils the filtration affinity system by clogging the filter. A preferred embodiment of the present invention overcomes the disadvantages of the prior art by adding a multitude of linkers to the bead surface to increase bead coating. Bead coating reduces unwanted agglomeration and filter clogging. This multitude of linkers reduces agglomeration and non-specific binding, resulting in increased stability and reduced entrapment of unwanted contaminants, while enabling the attachment of the target compounds desired to be separated.
The prior art discloses the use of cellulose in dynamic filtration apparatuses for affinity separation of biological compounds. The prior art cellulose particles available are highly porous particles which exhibit entrapment of target and contaminants. Moreover, these highly porous prior art cellulose particles can swell from 25% to 400% of their original size in aqueous medium. Additionally, the prior art cellulose particles are highly sensitive to sheering forces, thus resulting in fragmentation in a dynamic filter. The present invention uses non-porous cellulose beads in a static tangential flow system to prevent bead fragmentation.
BRIEF SUMMARY OF THE INVENTION
A preferred embodiment of the present invention provides an improved affinity separation method and system comprising an affinity separation media with low porosity and low non-specific binding, and a fluid containing a target compound to be isolated which is capable of binding onto the affinity separation media in a fluid mixing loop in a static filtration apparatus. The static filtration apparatus comprises an intermixing-chamber containing a filtration medium having upstream and downstream sides, an inlet in fluid communication with the upstream side of the filtration medium, and a filtrate outlet in fluid communication with the downstream side of the filtration medium, wherein a tangential flow is created for intermixing the affinity separation media and target compound in the fluid. The fluid is capable of passing through the filtration medium while the affinity separation media are substantially incapable of passing through the filtration medium. The affinity separation media are separated from the fluid by opening the filtrate outlet so as to allow the fluid to pass through the filtration medium of the static filtration apparatus. The filtrate can be thereby rendered substantially free of the target compound. If the recovery of the target compound is desired and/or if the affinity separation media are to be reused, the affinity separation media are then thoroughly washed, and the target compound is eluted from the affinity separation media, filtered, and ultimately the target compound is recovered from the filtrate and the affinity separation media may be reused.
The preferred invention apparatus and system ideally utilizes small, reconstituted cellulose particles having no pores and low non-specific binding properties. The cellulose particles of the preferred embodiment of the present invention consist of small, substantially spherical bodies with a near complete absence of irregularities, holes, cracks and the like. The cellulose bodies are made from viscose. This improvements results in uncross-linked, high density, spherical cellulose separation support beads without substantial holes, voids or craters on their surface. In certain circumstances, as where ligands are attached to the cellulose particles, chromatographic separation may be optimized when substrate/sorbent interactions take place exclusively on the outside surface of the particle. In such cases, any presence of holes of a size that may accommodate a substrate molecule cannot be tolerated; otherwise diffusion based interferences may adversely effect resolution of pure compounds.
The cellulose beads are essentially non-crystalline. Electron micrograph sections of the particles mounted in an epoxy matrix display a structure whereby the cellulose particles show a dense non-porous outer shell with an approximate thickness of 1,000 to 2,000 angstroms and a more porous interior of the closed-cell type. The shape of the particles is essentially spherical. The cellulose particles are essentially non-swellable and stable in pH range between about 1 and 13.
The current invention relates to a unique and novel method which eliminates the column box and utilizes low porosity. The present method for the affinity separation of bio-compounds uses a static filtration system instead of the column box to achieve flow rates of liters per minute instead of milliliters per minute, which is typical of high porosity separation media. Therefore, a low porosity, low non-specific binding separation medium, generally the lowest being reconstituted cellulose affinity particles, is preferred.
An advantage of the present inventive method is the provision of the exceptionally efficient separation through an affinity separation procedure of a compound from a dilute solution. A preferred embodiment of the present invention provides a means for lessening the number of processing steps required to perform an affinity separation as compared to known affinity separation methods, thereby increasing the overall yield of the separation method.
A further advantage of the preferred embodiment of the invention is that the present inventive method is able to be con
Garner William Darwin
Stipanovic Bozidar
Accurate Polymers
Davis , Wright, Tremaine, LLP
Therkorn Ernest G.
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