Patent Application
20050208637
This is a 371 of PCT/EP 2003/006564 filed 21 Jun. 2003 and claims priority of DE 102 33 542.7 filed 23 Jul. 2002.
Spherical carriers in the form of gels containing affinity ligands have been employed for a long time in many areas of biotechnology for purification and separation of many different types of biomolecules. An example of such an application is the use of affinity ligands based on an agarose gel, commercially available in matrices such as aqueous suspensions or in lyophilized form. A persistent problem with such matrices containing affinity ligands is that it is difficult if not impossible to dry the matrices once they have become swollen in an aqueous medium because the small gel spheres are irreversibly damaged during drying. Preservation and transport of such gels thus presents a considerable logistical problem.
From EP 0 787 523 A1 it is known that ligands of a carrier material can be coupled for separation of substances having an affinity for the ligands. The function of the ligands is to bind a single target substance or even an entire class of substances which specifically adsorb to the ligands. It is further known from DE 196 17 775 A1 that membrane adsorbers or membranes can carry ligands which are capable of interacting with at least one substance in a liquid phase. The transport of the liquid phase through the membrane occurs in this case convectively due to a pressure differential. A chief drawback of such known separations of biomolecules is that the presence of water moisture in the carrier or membrane involves the risk of microbial attack, thereby requiring the addition of preservatives. But at the same time, the carriers or membranes tend to dry out, which drying must be suppressed with complicated procedures in order to prevent loss of the bioactivity of the ligands.
A primary object of the present invention is therefore to provide membranes for separation of biomolecules from a fluid by means of affinity ligands that permits elimination of complicated and costly wet storage of the membranes.
The foregoing object is achieved by the present invention, which allows dry storage of the membrane with the affinity ligand, yet retains the activity of the affinity ligand.
Because the membrane can be stored practically without a significant loss of activity, storage and transportation costs can be significantly reduced and the separation of biomolecules is simplified.
There are essentially three aspects to the invention: (1) a microporous membrane infused with an affinity ligand capable of interacting with at least one type of biomolecule in a fluid; (2) a filtration module for the separation of biomolecules from a fluid, comprising a housing and at least one membrane of the type noted in (1); and (3) a method for the separation of biomolecules from a fluid by one or more membranes of the type noted in (1) or by a filtration module of the type noted in (2).
Surprisingly, it has been found that membranes charged with affinity ligands such as proteins can be stored dry for a relatively long period of time without a loss of activity. A particularly suitable class of microporous membranes of this type is that which is commercially available from Sartorius AG of Göttingen, Germany under the trade name of Sartobind®. The term “dry” as used herein should be understood as relating to membranes or membrane bodies whose pore volume is substantially filled with air. This does not exclude those cases where the inner surface of the pores is covered with a highly volatile organic substance.
Suitable membranes are polymeric microporous membranes such as cellulose acetate (CA), cellulose nitrate (CN), polyamide, polyether sulfone (PES), polypropylene (PP) and polyvinylidine fluoride (PVDF). The diameter of the pores for such membranes should be from 0.01 to 15 μm, preferably from 0.2 to 5 μm. The thickness of such membranes is from 100 to 500 μm, preferably from 200 to 300 μm. Such membranes are preferably chemically activated, so that the affinity ligands can be chemically coupled thereto. However, physical binding of the affinity ligands to the membranes is also possible. In a preferred embodiment, the membrane of the invention is impregnated with glycerine, which aids in preventing damage to the membrane's microporous structure during the drying process.
Adsorptive affinity ligands are well known to persons skilled in the art, and include the following:
In another preferred embodiment of the invention, selective separation of different biomolecules can be achieved by using a plurality of membranes having affinity ligands coupled thereto. Moreover, the types of membranes can be adjusted in a relatively simple manner, depending on the relevant separation problem. The membranes can be arranged in a housing in multiple layers and can also be arranged serially in single housing chambers or in different housings.
Another aspect of the invention is to provide an efficient and cost-effective method for the membrane separation of biomolecules from a fluid, which is possible without the need for complicated wet storage and transport of the membranes used. The method comprises the following steps:
To minimize the risk of microbial attack when water is used as the washing medium, the separation membrane is preferably dried to a water activity of about 40%. As used herein, the term “water activity” means the equilibrium partial pressure of water in the membrane relative to pure water at the same temperature. In step (b) above a strongly volatile organic substance of one or more components that are miscible with the washing medium may be added as an impregnation medium which remains in the membrane during the drying stage. A film can be also formed on the surface of the pores or the membrane can be formed in a swollen state.
Referring to the drawings, wherein like numerals refer to the same elements, there is shown in
Although membrane body 4 can be provided as a single layer arranged in housing 2 as shown in
It is also possible to arrange membranes 3″/membrane bodies 4″ in several layers in one housing 2″, as shown in
A phosphate buffered saline (PBS) solution having a pH of 7.3±0.2 was prepared as described by J. Sambrook et al. in “Molecular Cloning—A Laboratory Manual,” Book 3, Appendix b. 12 (2d ed. 1989) by combining the following components in the concentrations noted in an aqueous solution.
Three 25 mm disks of a regenerated cellulose microporous membrane functionalized with aldehyde groups (Sartobind® Aldehyde Membrane Code 19306) were reacted with Protein A which contains primary amine groups (Repligen Company, Designation rPrA, Lot No. 011038) to form a Protein A affinity ligand coupled via aldehyde/amine chemical links to the membranes. The protein was dissolved in 10 mg/mL of the PBS solution. The three membrane disks were placed in a Petri dish with 2 mL of the Protein A/PBS solution and agitated for three hours at ambient temperature to form reversible Schiff bases from the aldehyde/amine links. The so-formed Schiff bases were reduced by the addition of 10 mg/mL sodium cyanoborohydrate. After the reaction took place, the membranes were removed and transferred to a fresh Petri dish. In order to reduce the remaining aldehyde groups, 5 mL of a solution of sodium borohydride in PBS with a final concentration of 1% was added to the membranes and the same were agitated for another 15 minutes. The membranes were then washed sequentially with the following solutions: PBS; 0.1 M glycine, adjusted to pH 2.7 with HCl; 1 mM HCl in water; 1 mM NaOH in water; and 1 mM NaCl in 0.01 M potassium phosphate, pH 7.0. The membranes were then dried at ambient temperature with an air current for 3 hours and stored at 4° C. while air was substantially excluded.
The membranes were removed from storage after the storage times noted in the table below and tested with respect to their binding capacity for human immunoglobulin of the type IgGlund IgG2. Filtration modules for the tests were made by incorporating three of the membranes described above into a syringe adaptor unit with a diameter of 25 mm and equipped with a disposable syringe (both from Sartorius AG).
Processed human plasma from a local blood bank was diluted with PBS to a ratio of 1:40 and this solution was first filtered through a 0.2 μm membrane. The syringe was filled with 10 mL of the pre-filtered solution and gravity filtration with the Protein A-coupled membranes was carried out. Following this filtration, washing was conducted with 10 mL of PBS and the bound IgG was eluted with 10 mL of 0.1 M glycine, pH 2.7. The absorption of the elution solution was determined at 280 nm with a spectrum photometer and a manually adjustable calibration apparatus using bovine serum albumin (BSA) as a control to determine the protein-binding capacity. The IgG binding capacity of the functionalized membranes for Protein A as a function of time for these tests are shown in the table below. All values were median values obtained with at least 2 measurements.
The terms and expressions which have been employed in the foregoing specification are used therein as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding equivalents of the features shown and described or portions thereof, it being recognized that the scope of the invention is defined and limited only by the claims which follow.
| Number | Date | Country | Kind |
|---|---|---|---|
| 102 33 542.7 | Jul 2002 | DE | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP03/06564 | 6/21/2003 | WO | 11/30/2004 |
