Liquid purification or separation – Processes – Liquid/liquid solvent or colloidal extraction or diffusing...
Reexamination Certificate
2001-03-12
2003-09-16
Therkorn, Ernest G. (Department: 1723)
Liquid purification or separation
Processes
Liquid/liquid solvent or colloidal extraction or diffusing...
C210S656000, C210S198200
Reexamination Certificate
active
06620326
ABSTRACT:
FIELD OF THE INVENTION
EBA is an abbreviation for Expanded Bed Adsorption. This is a technology used within the pharmaceutical and diagnostic industries for the purification of, i.a., proteins and peptides from a vast variety of extracts and raw materials. The present invention relates to various improvements for the known EBA technology.
BACKGROUND OF THE INVENTION
A traditional purification process for a mixture comprising one or several target molecules could be purification on a packed column (i.e. not EBA), however this requires multiple operational steps such as filtration and centrifugation and a number of steps in order to ensure that impurities and particles are removed before the mixture is applied to a suitable packed column. These steps are necessary in order to avoid that the packed column clogs up. In the packed column, a given chromatographic medium is present for binding of the molecule(s) which are the target for the purification. This chromatographic medium can be adapted to various purification purposes.
The main principle in EBA is to keep the chromatographic medium fluidised and thereby also allow particles to pass through the column. By using the EBA technology, it is in many instances possible to avoid the above-mentioned operational steps before application of the raw material to the column. In this manner, time and expenses for these processes are reduced making EBA a valuable technology which is economically recommendable for the purification of a countless number of molecules. It reduces time and expenses for a lot of equipment.
In order to utilise the EBA technology, an EBA column and a suitable chromatographic medium should be used.
A brief presentation of the steps used in the EBA technology will be given in the following.
1. An adequate quantity of chromatographic medium is placed in an EBA column.
2. A flow through the medium from below and up through the medium is initiated. The medium is fluidised.
3. The medium is rinsed in the column and the salt concentration and pH are set.
4. Raw material is applied. The medium binds the target molecule(s).
5. Remaining raw material is rinsed out from the column.
6. The column is packed. The matrix is sedimented.
7. The liquid above the medium is drained.
8. The target molecule is eluted off the medium.
9. Rinsing and regeneration of the chromatographic medium (optionally).
Steps 1-5 and 9 are implemented in expanded bed mode. In step 6, the flow has stopped, in step 7, liquid is drained through the bottom, and in step 8, the flow comes from above and down, i.e. in packed mode.
Before the raw material is applied to the column, it should be ensured that the expansion is stable. This can be done visually or by determining the theoretical bottoms (“Expanded Bed Adsorption” by Pharmacia Biotech, page 14). A double determination must not deviate by more than 20%. During visual inspection, any channels and jet streams are located. If the medium moves in small circles, and local jet streams and channels are not observed, it is considered to be stably expanded. By the term jet streams is understood a clear stream of liquid and thus medium locally in the EBA column. If the jet stream flows upwards at one place, it will typically flow downwards at another. Further information about EBA technology can be found in the book “Expanded Bed Adsorption” by Pharmacia Biotech.
The Chromatographic Medium
In EBA technology, only density controlled particles which can adsorb a given target molecule are used. Density and diameter of the medium determine the extent of the flow to which the particles can be exposed without being flushed out of the column. The particles used are preferably round. The diameter is proportional to the square of the fall velocity. This means that two spheres of equal density will not necessarily fall at the same velocity. The fall velocity is proportional to the difference of density between the liquid and the material of which the sphere is produced to the power of one, demonstrating the great significance of the diameter.
In liquid chromatography at low pressure, many types of chromatographic medium are used. These media may either have a higher density than the surrounding liquid in which case the medium will precipitate, or a lower density than the surrounding liquid in which case the medium will float. One example of these density modified particles and their application in fluidised columns is described in WO 92/00799.
In packed column chromatography, a medium which is not density controlled is employed. In practice, water will be led into the top of the column so that the liquid flows from above and down. The medium will move in the same direction as the liquid flow. At the same time, the quantity of the medium is limited as the medium at a given point will pack so hard that the liquid cannot flow freely. Another factor determining whether the medium will pack down hard or not is the velocity at which liquid is led through the column, i.e. the liquid flow.
DKIEP 0538350 T3 discloses chromatographic adsorption particles having covalently bound thereto at least one active substance for binding of molecules in a liquid chromatographic liquid bed process. These adsorption particles are formed of a porous composite material with pores permitting access for the said molecules to the interior of the composite material. The spheres can be produced having a given density and diameter. The density is controlled by incorporation of one or more inert particles in the chromatographic medium, the number, material and percentage of the inert particles being significant for the ultimate density of the chromatographic medium. In addition, the pore size can be controlled. The density controlled particles can be viewed as inert heavy/light particles coated with a hydrophilic layer, a conglomeration compound such as an agarose layer of different concentration and thus pore size. See
FIG. 1
a
for two examples of the described spheres. Here, two types of products which are marketed today are shown. The Streamline matrix by Pharmacia Biotech, Sweden, and the UpFront Matrix by UpFront Chromatography A/S, Denmark. These have been produced according to the same principle with one or more particles inside a sphere of agarose.
The book “
Expanded Bed Adsorption
” by Pharmacia, Sweden, discloses that the size and density of the individual sphere at a given flow situates the sphere at a specific position in the column. The small and light spheres will move to the upper part of the expanded matrix while large, heavy particles will move towards the lower part. The result is that the particles settle at their ideal position after a suitable period of time. When this has taken place, expansion will be stable.
Columns
DK/EP 0538350 T3 discloses a liquid bed reactor as a down/upflowing liquid fluid bed reactor comprising a vertical reactor container with an inlet, an outlet, a fluidised particle bed of chromatographic adsorbent particles and means for initiating movement which are located near by or in the fluidised particle layer which is closest to the liquid inlet. There is a mixed zone, i.e. a stirring zone, the size of which is determined by the degree of stirring, the liquid flow and the quantity of matrix in the reactor container. Above/below this zone is a non-mixed zone in which a so-called plug flow is achieved. By the term plug flow is understood a movement of the liquid as a band through the container and consequently also through the matrix.
An example of such a reactor container is an UpFront column 20™ which is an upflow reactor developed by UpFront Chromatografy A/S, Copenhagen, Denmark. See
FIG. 1 and 2
for the construction thereof and examples of application in fluidised and packed mode, respectively.
This reactor container is constructed in such a manner that a supporting net with a pore size of 50 &mgr;m is located at the bottom. Below the supporting net is an outlet/inlet which is primarily used as an outlet during elution. A motor axis on which a stirrer is secured extends down the middle of this net. T
Lihme Allan Otto Fog
Oehlenschlæger René
Olsen Brian A.
Zafirakos Elias
Birch & Stewart Kolasch & Birch, LLP
Therkorn Ernest G.
Upfront Chromatography A/S
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