Containers for chromatography media

Abstract

The invention relates to containers or bags for chromatographic media and methods of packing chromatography columns using such containers. The bags may be used for storing and/or transporting chromatographic media and can be inserted directly into the chamber of a chromatography column in readiness for use.

Claims

1. A flexible bag containing chromatographic medium, said flexible bag comprising: an exterior wall of a non-porous material having attached thereto a first and a second liquid distribution element, thereby defining a compartment for chromatographic medium therein; said first liquid distribution element being opposed to said second liquid distribution element; and a medium filling port; wherein the first liquid distribution element and/or the second liquid distribution element are welded to said exterior wall and the first and second liquid distribution elements are each formed from material having pores that are too small to allow chromatographic medium to pass through but are porous to liquids.

2. The flexible bag according to claim 1, wherein said flexible bag has a tubular configuration and additionally comprises an interior wall, wherein the first liquid distribution element is welded to said exterior wall and the second liquid distribution element is welded to said interior wall.

3. The flexible bag according to claim 2, for use in radial chromatography.

4. The flexible bag according to claim 1, wherein the flexible bag has a cylindrical configuration and comprises a first and a second end piece attached to the exterior wall and adjacent to the first and second distribution element respectively, both said end pieces comprising an opening for receipt of an inlet or outlet for carrier liquid.

5. The flexible bag according to claim 4, wherein the first and second end piece are welded to the exterior wall.

6. The flexible bag according to claim 4, wherein the first and second end piece are made from a rigid or semi-rigid material.

7. The flexible bag according to claim 1, wherein the first liquid distribution element and the second liquid distribution element is selected from the group consisting of filter, mesh, net and sinter.

8. The flexible bag according to claim 1, wherein the first liquid distribution element and the first end piece are an integrated unit, and the second liquid distribution element and the second end piece are an integrated unit.

9. The flexible bag according to claim 4, for use in axial chromatography.

10. The flexible bag according to claim 1, wherein said flexible bag additionally comprises a second compartment having an inlet for hydraulic fluid.

11. The flexible bag according to claim 1, wherein the wall is made from a plastic polymeric material.

12. The flexible bag according to claim 4, additionally comprising a semi-rigid housing around the exterior wall to provide rigidity in an axial and/or radial direction.

13. The flexible bag according to claim 1, wherein the chromatographic medium is wet or dry.

14. The flexible bag according to claim 13, wherein said wet or dry chromatographic medium is sterile.

15. The flexible bag according to claim 1, the bag having been sterilised by radiation and/or autoclaving.

16. A method of packing a chromatography column, said method comprising the steps of: a. inserting a flexible bag according to claim 1 into a chamber of a chromatography column; b. closing the column housing; c. forming or compressing a packed bed of chromatographic medium.

17. The method according to claim 16, wherein said flexible bag contains dry chromatographic medium and said packed bed is formed by adding liquid to swell said medium to give a swollen volume Vs in a liquid of substantially 105-120% of the column chamber.

18. The method according to claim 16, wherein the flexible bag contains wet medium, the column housing is rigid and the packed bed is formed or compressed by axial compression of the bag.

19. The method according to claim 18, wherein the column comprises a hydraulic chamber and the packed bed is formed or compressed by filling said chamber with a hydraulic fluid.

20. The method according to claim 19, wherein said hydraulic chamber is a flexible bag.

21. The method according to claim 18, wherein the flexible bag comprises a second compartment and the packed bed is formed or compressed by filling said second compartment with a hydraulic fluid.

22. The method according to claim 18, wherein the column comprises a movable piston or adapter and the packed bed is formed or compressed by axial movement of said piston or adapter.

23. The method according to claim 18, wherein the flexible bag containing wet medium is placed over a rigid core within the column chamber and the packed bed is formed or compressed by radial compression of the flexible bag.

24. The method according to claim 23, wherein the force is exerted by radial compression of the column housing.

25. The method according to claim 24, wherein radial compression of said column housing is effected by tightening radial bands on the outside of the housing.

26. The method according to claim 23, wherein volumetric compression of the wet medium is 5 to 20%.

27. The method of any of claim 16, additionally comprising sterilising the column with radiation.

28. A chromatography column comprising the flexible bag according to claim 1.

29. The chromatography column according to claim 28, wherein the chromatography column is configured for separating or purifying a chemical.

30. The chromatography column according to claim 29, wherein said chemical is a protein.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIGS. 1a and 1b show schematic, sectional views of one embodiment of a welded bag (10) according to the invention that has a semi-ridged housing that can be packed by axial compression.

(2) FIGS. 2a, b and c are schematic sectional plan views of a cartridge containing a bag in accordance to the invention which can be packed by radial compression.

(3) FIG. 3a, b and c are schematic diagrams illustrating the component parts of a column which contains a bag according to the invention which can be packed by radial compression. FIGS. 3d and e illustrate how the component parts are fitted together and operate.

(4) FIGS. 4a, b and c show results from low temperature storage experiments with pH 7.4, 5.0, and 9.0 respectively.

DETAILED DESCRIPTION OF THE DRAWINGS

(5) FIGS. 1a and 1b show schematic, sectional views of one embodiment of a welded bag (10) according to the invention placed in a column (20). The bag (10) consists of a first (13) and a second (14) liquid distribution element welded to a wall (12) of a non-porous material. The distribution elements (13, 14) are bed supports or flits or nets or sinters which act as filters, having pores that are too small to allow chromatographic media to pass through but are porous to liquids. The non porous material or film is made of a plastic polymer, those based upon polyethylene being particularly suitable for welding or moulding. For example, suitable films include American Renolit Infuflex Barrier 9101 (SOLMED? BF 9101; American Ronolit Crop, La Porte, Ind., USA), American Renolit BF-1400 (SOLMED? BF-1400 Film; American Renolit Corp, La Porte, Ind., USA), American Renolit 4301 (SOLMED? Granuflex, American Renolit Crop, La Porte, Ind., USA), Mitos Durapure (Mitos Technologies, Phoenixville, Pa., USA) and MILLIPORE? Pureflex Process Container Film, (Millipore Corp, Billerica, Mass., USA).

(6) The bag also comprises a chromatography medium filling port (16) through which dry or wet media or slurry is added to the bag (10). In the embodiment shown in FIG. 1b, the bag (10) has one compartment (11) for receipt of media (not shown) via port (16). Although not shown in FIGS. 1a or 1b, the bag (10) also comprises a first and second end piece attached to the exterior wall (12) and adjacent to each of the distribution elements (13, 14), respectively. The end pieces are made from a rigid or semi-rigid material that prevents seepage of carrier liquid from the bag (10) and has openings for receipt of an inlet (25) and outlet (26) for carrier liquid. In one embodiment, the opening takes the form of a resilient septum (not shown) which receives the inlet (25) or outlet (26) for carrier liquid. Suitable materials for the production of the end pieces include inert metals and plastics such as polypropylene. Preferably the end piece is welded or moulded to the exterior wall (12).

(7) In one embodiment (not shown) the end piece and the distribution element may be integrated into a single unit; suitable materials may include semi-flexible plastics such as polypropylene. The unit can be prepared by machining or moulding.

(8) The use of rigid or semi-rigid materials in the component parts of the bag allows for the formation or compression of the packed bed. Preferably, the bag and its component parts are fabricated from biologically and chemically inert materials which are approved by regulatory authorities, such as the US Food and Drug Administration, for the manufacture of drugs.

(9) The bag (10) may also comprise a second compartment (17), adjacent to the first compartment (11) of the bag (10) which acts as a hydraulic chamber to compress the first compartment (11) as described below. In other embodiments, the second compartment (17) may be a separate bag adjacent to the first compartment (11) which acts as a hydraulic chamber.

(10) In FIGS. 1a and 1b, the bag (10) is enclosed by the column (20) housing (22) and lid (21), at least one of these components being semi-rigid or rigid. The bag (10) is filled with a dry powder or wet slurry of chromatography media through port 16. The column (20) may be sterilised by gamma irradiation on filling with media prior to use, or alternatively the bag may be sterilised by gamma irradiation or autoclaving on charging with media, and then stored or shipped to a user.

(11) To prepare the media bed for chromatographic separation, a user will insert the the bag (10) into a chamber of a chromatography column (20), close the column housing (22) and lid (21) such that walls of the bag (10) fit tightly within the chamber and, in one embodiment, use the flow packing method to compress the bed (not shown). The packed column could then also be sterilised by gamma radiation prior to use.

(12) In another embodiment, the second compartment or hydraulic chamber (17) can be filled with hydraulic fluid via port (24) to axially compress the first compartment (11) of the bag (10) containing media in order to maintain bed stability.

(13) Alternatively, the first compartment (11) can be compressed mechanically by a piston or adapter present in the column chamber.

(14) Separation of chemical compounds, such as proteins, may now occur by flowing a carrier liquid containing the compounds into the bag 10 (via inlet 25) and through the chromatographic media. the eluant being collected at outlet 26. Alternatively, the carrier flow can he reversed, such that port 26 serves as an inlet and eluant is collected at port 25.

(15) In another embodiment, the user will insert the bag (10) into a chamber of an axial chromatography column (20), close the column housing (22) and lid (21) such that the bag is confined by the rigid housing and arrange the column in a vertical direction to apply liquid to the bag such that a consolidated bed is formed. When forming the consolidated bed by application of flow in downward direction, a gap may be formed above the consolidated bed.

(16) In another embodiment, the walls of the bag will be displaced to reduce said gap formed above the consolidated bed in order to reduce the hold-up volume of column and/or to mechanically stabilise the consolidated bed.

(17) In use, a liquid sample containing a chemical (such as a protein, peptide, antibody, nucleic acid or mixtures thereof) or a cell (or cellular mixture) is loaded onto the consolidated bed and allowed to equilibrate. The bag is then removed from the column and may be stored at low temperature (e.g. ?20? C.) to minimise chemical or cellular loss or breakdown due to chemical, photochemical or biological instability. Alternatively, the bag may be transported at low temperature (such as, for example, ?20? C.) to another location. Following storage and/or transport at low temperature, the bag may be equilibrated to an operating temperature at or above 4? C., placed in a semi rigid or rigid column and the chemical or cell eluted from the chromatographic medium. Alternatively, the chemical or cell which is loaded on the chromatographic medium may be subjected to processing, such as a chemical or biological reaction, following equilibration and replacement of the bag in the rigid or semi-rigid column at temperatures at or above 4? C. The product of the processing step may then be eluted from the column.

(18) In one embodiment, the axial column of FIG. 1 is designed in a cylindrical shape. In another embodiment, the axial column is designed having a cross-sectional shape deviating from a circle found at the cylindrical shape, such as being of oval or rectangular shape. The shape of the rigid housing confining the column is adapted accordingly. For example, the column could be made to adopt to the shape of a 2D pillow bag or a 3D rectangular bag that has been fitted with suitable distribution systems at inlet and outlet side. In vet another embodiment, the cross section of the column and bag may vary across the axial position in between the end pieces as being rectangular or oval at the end pieces and being circular or oval in the middle of the column, for example.

(19) In another embodiment, the rigid housing is opened and closed at the side of the column and housing instead of having a removable lid as depicted in FIG. 1. In yet another embodiment, the rigid housing consists of two or more wall segments mounted together after having inserted the bag.

(20) FIGS. 2a, b and c are schematic sectional plan views of a bag (210) in accordance to the invention which can be packed by radial compression. The bag (210) is tubular in nature with a hollow core or centre (230). In the embodiment shown, the exterior (212) and interior (219) walls of the bag (210) have welded opposed liquid distribution elements (213, 214) which allow passage of carrier liquid from the exterior to the interior of the bag (or in the reverse direction depending upon the direction of flow). In the embodiment shown, the bag (210) has semi-rigid walls (222) which can exert a radial, compressive force against the walls (212, 219) of the bag (210). The outer or exterior wall (212) of the bag has indents (215) which allow mild compression of the media filled bag (210) in response to the radial force of the semi-rigid walls (222). The bag (210) is stable under such mild compression and may be stored or transported to a user (FIG. 2a).

(21) Bed packing is achieved by applying a radial mechanical compression to the walls (222) of the bag (210) as indicated by the arrows in FIG. 2b. As can be seen in FIG. 2c, the radial forces exerted on the bag (210) closes the indents (215) and is transmitted through the bag, compressing the media within.

(22) In another embodiment mild compression of bed is achieved using the flow packing method described above.

(23) FIGS. 3a, b and c show the component parts of a radial column using a bag 310 according to the invention. FIG. 3a illustrates a cylindrical rigid core (340) with a stand (342) and locking lid (344). The lid 344 has apertures or connectors to accommodate an outlet port (346) for eluant and a media filling port (348). FIG. 3b depicts an outer cylindrical shell (350), typically composed of a semi rigid plastic sheet (351), which can wrapped around the radial flow bag 310 shown in FIG. 3c and used to radially compress it. In the embodiment shown, the length of the sheet (351) is greater than the external circumference of the bag 310 such that there is an overlap 354 to allow for changes in the diameter of the sheet (351) during compression. Other functional designs, such as pleating of the sheet, are also possible to achieve this purpose. The shell (350) has an aperture or connector (352) to accommodate a carrier liquid inlet (325).

(24) FIG. 3c illustrates a bag (310) according to the invention. The bag has a tubular configuration with a hollow core 330. A first liquid distribution element (313) and second liquid distribution element (314) are welded to the exterior (312) and interior (319) walls of the bag (310), respectively. A media inlet port (316) is used to fill the hag with media, typically in the form of a slurry, as indicated by the arrow in FIG. 3c. Carrier liquid enters the bag via inlet 325, flows through the media bed (not shown) and exits via outlet (326, see arrows).

(25) A radial flow column (360) is prepared by inserting the bag (310) over the rigid core (340), wrapping the cylindrical shell (350) around the exterior wall of the bag, and locking the lid (344) into position (FIG. 3d). The bag is filled with media slurry via inlet port 316 and excess media removed via outlet 326 or 325, prior to sealing the inlet port (316). The media in the bag may then be equilibrated with carrier liquid which will enter the column via inlet 325 and exit via port 326. In one embodiment, the bed is compressed by the flow packing method describe above.

(26) In another embodiment, the outer shell (350) is radially compressed to form a packed bed of chromatography media in the bag. Radial compression can be effected in a number of ways, such as pulling the overlap 354 to constrict and compress the hag against the rigid core (FIG. 3e). Other means for compression are possible, such as clamping or tightening radial bands. Once the outer shell 350 has been compressed it is secured in position to stabilise the packed bed of media. (for example, by use of tie bands 370). The column is now ready to be used for chromatographic separation of chemicals, such as proteins and/or peptides. The sample containing the chemical(s) to be separated or purified are dissolved in carrier liquid and applied to the column. The carrier liquid enters the bag via inlet 325 and first distribution element 313, partitions over the media. bed (not shown) where separation occurs and passes through the second element 314 to exit the column at outlet port 326. It will he understood, that the column may be run in the reverse direction by reversing the direction of flow.

Low Temperature Storage Experiments

(27) The experiments detailed below are described fully in Applicant's co-pending U.S. patent application 61/370,878 entitled Stabilization and Storage Media and Method, filed on 5 Aug. 2010. Where national law permits, the content is hereby incorporated by reference in its entirety herein as if it had been specifically incorporated.

(28) It is generally known that the storage of proteins (e.g. antibodies) in solution leads to aggregation over time. By freezing the protein solutions in a carefully designed buffer environment at slow and controlled rates of freezing aggregation can be reduced, but the aggregate content will inevitably be higher after storage. Experiments were then performed using an alternative storage approach by using different gel media to bind or encapsulate the target proteins.

Storage of Human Polyclonal IgG with a Low Initial Dimer Content in Solution or on Gel Media

(29) The monomer fraction of human polyclonal IgG (1% dimer) was first concentrated to 121.5 AU (A280 nm) using VIVASPIN? 6 ultrafiltration units and filtered with 0.2 .mu.m syringe filters. Various experimental capture storage hydrogel media, and control (noncapture SEC) media (see Materials above) were dispensed into 96 well filter plates (MULTITRAP? plates). The MULTITRAP? protocol was followed using the concentrated monomer fraction of human polyclonal IgG as antibody sample. In parallel antibody samples were mixed with the various buffers used for the different gel media for subsequent storage in solution at ?20? C. 6.7 .mu.l antibody solution was mixed with 13.3 .m.u.l of each buffer respectively.

(30) Buffers used for samples and equilibration/washing of gel media were typical adsorption capture buffers for the different media used: pH 5 IEX=20 Na-Acetate, 0.02% (w/v) Na-azide, pH 5.0 pH 9 IEX=20 mM Na-Glycine, 0.02% (w/v) Na-azide, pH 9.0 PBS-10 mM Na-Phosphate, 2.7 mM KCl, 0.14M NaCl, 0.02% (iv) Na-azide, pH 7.4 pH 5 HIC=25 mM Na-Acetate, 0.5 mM EDTA, 0.75M (NH4)2SO4, pH 5.0

(31) The various chromatography gel media were stored in the 96-well MULTITRAP? plate. The MULTITRAP? plates were stored in the fridge for 4 weeks at 4-8? C. (the protocol incubation time), whereas the antibody samples in solution were stored at ?20? C. for 4 weeks including 6 freeze thaw cycles. All samples were run in triplicate except for three of the anion exchange gel media, CAPTO? M Q, Q SEPHAROSE? FE and Q SEPHORASE? XL, that were run as single samples. After incubation or storage for 4 weeks, either the non-bound antibodies (flow-through) were collected (gel filtration SEC media SEPHADEX? G-50 and SUPERDEX? 200) or the bound and then eluted antibodies (all media except gel filtration media). Elution and strip buffers used were: elution buffer: 3.3.times.PBS=30 m Na-Phosphate, 8.1 mM KCl, 0.42 M NaCl,. pH 7.4 (for those samples of which buffers used for samples and equilibration/washing of gel media were pH 5 IEX and pH 9 IEX); elution buffer: 0.1 M Na-Glycine pH 2.9 (for those samples of which buffers used for samples and equilibration/washing of gel media were PBS); elution and strip buffer: 20 mM Tris-HCl pH 7.5 (for those samples of which buffers used for samples and equilibration/washing of gel media were pH 5 HIC); strip buffer: 10 mM NaOH, 1M NaCl for those samples of which buffers used for samples and equilibration/washing of gel media were (pH 5 IEX and pH 5 HIC); strip buffer: 0.5M Acetic acid (for those samples of which buffers used for samples and equilibration/washing of gel media were PBS and pH 9 IEX) Elution was performed in two steps, first with elution buffers and secondly with strip buffers. The latter are typically in bioprocessing used to remove any residual protein not eluted by the elution buffersand to prepare column for possible additional use. Both elution fractions for each sample were analyzed respectively. All samples, stored in solution, on SEC control media or on binding media, were analyzed by SEC. The results are shown in Tables 1A-1D.

(32) TABLE-US-00001 TABLE 1A Dimer content in eluted fractions from human polyclonal IgG stored on gel media at 4-8? C. or frozen in solution at ?20? C. for 4 weeks in pH 5 IEX buffer (20 mM Na-Acetate, 0.02% (w/v) Na-azide, pH 5.0). Gel media % dimer replicate comment CAPTO? MMC 1.72 1 CAPTO? MMC 0.84 1 CAPTO? MMC 0.75 3 CAPTO? S 2.95 1 CAPTO? S 2.53 2 CAPTO? S 1.68 3 SP SEPHAROSE? FF 2.68 1 SP SEPHAROSE? FF 2.21 2 SP SEPHAROSE? FF 2.58 3 SP XL 2.44 1 SP XL 1.84 1 SP XL 1.72 3 Storage in solution (?20? C.) % dimer replicate comment pH 5 IEX 3.41 1 pH 5 IEX 3.23 2 pH 5 IEX 3.92 3

(33) All samples stored on these gel media contained less antibody dimer content compared with storing the samples in solution at ?20.degree. C. The start material contained 1.0% dimer and two of the replicates using CAPTO? MMC as storing gel media contained less dimer than this initial dimer content level.

(34) TABLE-US-00002 TABLE 1B Dimer content in eluted fractions from human polyclonal IgG stored on gel media at 4-8? C. or frozen in solution at ?20? C. for 4 weeks in PBS buffer (10 mM Na-Phosphate, 2.7 mM KCl, 0.14M NaCl, 0.02% Na azide, pH 7.4). Gel media % dimer replicate comment nProtein A 0.31 1 SEPHAROS? 4 FF nProtein A 0.30 2 * <0.3, difficult to integrate peaks SEPHAROSE? 4 FF nProtein A 0.30 3 * <0.3, difficult to integrate peaks SEPHAROSE? 4 FF MABSELECT? 0.52 1 MABSELECT? 0.50 2 * <0.5, difficult to integrate peaks MABSELECT? 0.50 3 * <0.5, difficult to integrate peaks SUPERDEX? 200 4.25 1 SUPERDEX? 200 4.54 2 SUPERDEX? 200 4.14 3 SEPHADEX? G-50 4.93 1 SEPHADEX? G-50 5.18 SEPHADEX? G-50 4.94 3 Storage in solution (?20? C.) % dimer replicate comment PBS, pH 7.4 3.24 1 PBS, pH 7.4 3.27 2 PBS, pH 7.4 3.00 3

(35) Samples stored on the affinity gel media contained less antibody dimer compared with storing the samples in solution at ?20? C. However, storage on noncapture SEC media resulted in higher levels of dimer. The start material contained 1.0% dimer and using nProtein A or MABSELECT? as storing gel media resulted in less dimer content than the initial dimer content.

(36) TABLE-US-00003 TABLE 1C Dimer content in eluted fractions from human polyclonal IgG stored on gel media at 4-8? C. or frozen in solution at ?20? C. for 4 weeks in pH 5 HIC buffer (25 mM Na-Acetate, 0.5 mM EDTA, 0.75M (NH4)2SO4, pH 5.0). Gel media % dimer replicate comment Butyl SEPHAROSE? HP 1.31 1 Butyl SEPHAROSE? HP 1.05 2 Butyl SEPHAROSE? HP 1.25 3 CAPTO? Phenyl high sub. 0.10 1 CAPTO? Phenyl high sub. 0,10 2 * <0.1 CAPTO? Phenyl high sub. 0.00 3 * <0.1 pH HIC 2% poor binding of sample at 0.75M AmSO pH HIC 2% poor binding of sample at 0.75M AmSO pH HIC 2% poor binding of sample at 0.75M AmSO pH HIC 4% AMBN poor binding of sample at 0.75M AmSO pH HIC 4% AMBN poor binding of sample at 0.75M AmSO pH HIC 4% AMBN poor binding of sample at 0.75M AmSO SEPHAROSE? HP 16% poor binding of sample at 0.75M AMBN AmSO SEPHAROSE? HP 16% poor binding of sample at 0.75M AMBN AmSO SEPHAROSE? HP 16% poor binding of sample at 0.75M AMBN AmSO CAPTO? Phenyl low sub. 1.15 1 CAPTO? Phenyl low sub. 0.87 2 CAPTO? Phenyl low sub. 1.26 3 SEPHAROSE? HP 16% poor binding of sample at 0.75M AMBN AmSO SEPHAROSE? HP 16% poor binding of sample at 0.75M AMBN AmSO SEPHAROSE? HP 16% poor binding of sample at 0.75M AMBN AmSO Storage in solution (?20? C.) % dimer Replicate comment pH 5HIC 1.55 1 pH 5HIC 1.74 2 pH 5HIC 1.69 3

(37) The overall recoveries from the HIC gel media were poor, probably due to poor binding of the antibodies due to using too low a conductivity (salt concentration) in the adsorption buffer. To promote binding to the HIC gel media a higher concentration of (NH4) 2SO4 was needed. The results that were obtained showed that binding to the gel media gives less dimer during storage compared with storage in solution and for CAPTO? Phenyl high substitution (higher ligand concentration) media the dimer content was less than 1.0% (level in the start material).

(38) TABLE-US-00004 TABLE 1D Dimer content in eluted fractions from human polyclonal IgG stored on gel media at 4-8.degree. C. or frozen in solution at ?20? C. for 4 weeks in pH 9 IEX buffer (20 mM Na-Glycine, 0.02% (w/v) Na-azide, pH 9.0). Gel media % dimer replicate comment CAPTO? Adhere 0.69 1 CAPTO? Adhere 0.95 2 CAPTO? Adhere 0.75 3 CAPTO? Q 2.27 1 CAPTO? Q no sample CAPTO? Q no sample Q SEPHAROSE? FF 3.84 1 Q SEPHAROSE? FF no sample Q SEPHAROSE? FF no sample Q XL 3.31 1 Q XL no sample Q XL no sample Storage in solution (?20? C.) % dimer Replicate comment pH 9IEX 3.39 1 pH 9IEX 3.35 2 pH 9IEX 3.58 3

(39) Samples stored on CAPTO? Adhere gel media contained less antibody dimer content compared with storing the samples in solution at ?20.degree. C. The dimer content was less than 1.0% as found in the start material. Storage on the other gel media resulted in about the same levels of dimeric proteins as storage in solution.

(40) Storage of human polyclonal IgG with a high initial dimer content in solution or on gel media

(41) Polyclonal human IgG (GAMMANORM? 165 mg/ml) was diluted to 30 mg/ml with following equilibration/wash buffers: 20 mM Na-Acetate, 0.02% (w/v) Na-azide, pH 5.0 20 mM Na-Glycine, 0.02% (w/v) Na-azide, pH 9.0 10 mM Na-Phosphate, 2.7 mM KCl, 0.14M NaCl, 0.02% (w/v) Na-azide pH 7.4 50 ruM Na-Acetate, 1 mM EDTA, 1.5M (NH4) 2SO4, pH 5.0

(42) Some protein was precipitated upon mixing the antibody solution with the fourth buffer solution containing 1.5M (NH4) 2504. The absorbance of these start materials were measured (on clarified solutions):

(43) TABLE-US-00005 Sample buffer A280 cm.sup.?1 20 mM Na Acetate, 0.02% (w/v) Na azide, pH 5.0 40.6 20 mM Na Glycine, 0.02% (w/v) Na azide, pH 9.0 40.5 10 mM Na Phosphate, 2.7 mM KCl, 0.14M NaCl, 0.02% 40.8 500 mM Na Acetate, 1 mM EDTA, 1.5M (NH.sub.4).sub.2SO.sub.4, pH 28.5 5.0

(44) SPINTRAP? columns were filled with 40 .mu.l of following gel media (i.e. 200 .mu.l 20% gel slurry) and were equilibrated according to the SPINTRAP? protocol: CAPTO? MMC mixed mode media, 20 mM Na-Acetate, 0.02% (w/v) Na-azide, pH 5.0 CAPTO? S cation exchange media, 20 mM Na-Acetate, 0.02% (w/v) Na-azide, pH 5.0 CAPTO? Adhere mixed mode media, 20 mM Na-Glycine, 0.02% (WO Na-azide, pH 9.0 CAPTO? Q anion exchange media, 20 mM Na-Glycine, 0.02% (w/v) Na-azide, pH 9.0 MABSELECT?, affinity media, 10 mM Na-Phosphate, 2.7 mM KO, 0.14M NaCl, 0.02% (w/v) Na-azide, pH 7.4 SEPHADEX? G-50, control SEC media, 10 mM Na-Phosphate, 2.7 mM KCl, 0.14M NaCk, 0.02% (w/v) Na-azide, pH 7.4 CAPTO? Phe hs, phenyl ligand containing HIC media, 50 mM Na-Acetate, 1 mM EDTA, 1.5M (NH.sub.4).sub.2SO.sub.4, pH 5.0 pH HIC 6%, pH responsive polymer based HIC media, 50 mM Na-Acetate, 1 mM EDTA, 1.5M (NH.sub.4).sub.2SO.sub.4, pH 5.0 pH HIC 16%, pH responsive polymer based HIC media, 50 mM Na-Acetate, 1 mM EDTA, 1.5M (NR.sub.4).sub.2SO.sub.4, pH 5.0

(45) After equilibration, 40 ?l of sample was added to each column (matching buffers). Three SPINTRAP? columns of each gel media containing bound antibodies were stored at room temperature (+20? C.), in fridge (+4-8? C.) and in the freezer (?20? C.) respectively. In parallel, aliquots of antibodies in solution containing the various buffers were also stored at the same temperatures as the SPINTRAP? columns.

(46) 60 ?l of matching buffer was added to all SPINTRAP? columns after an incubation time of 24-26 days. The flow-through (non-binding fraction) was collected and the absorbance (A280 nm) was measured.

(47) The various gel media was washed, then eluted with 400 ?l of following elution buffers: CAPTO? MMC, 20 mM Na-Phosphate, 1M NaCl, pH 7.0 CAPTO? S, 20 mM Na-Phosphate, 1M NaCl, pH 7.0 CAPTO? Adhere, 0.5M Acetic acid CAPTO?Q, 20 mM Na-Phosphate, 1M NaCl, pH 7.0 MABSELECT?, 0.5M Acetic acid SEPHADEX?G-50, not eluted, flow-through fraction collected CAPTO? Phe hs, 20 mM Na-Phosphate, 1M NaCl, pH 7.0 pH HIC 6%, 20 mM Na-Phosphate, 1M NaCl, pH 7.0 pH HIC 16%, 20 mM Na-Phosphate, 1M NaCl, pH 7.0

(48) The absorbance (A280 nm) was measured on the eluted fractions. All samples from storage in solution and flow-through/elution fractions from the various gel media were analyzed by SEC. The antibody recovery and the dimer content are summarized in Table's 2A-4D.

(49) TABLE-US-00006 TABLE 2A Antibody recovery and dimer content in eluted fractions after storage of human polyclonal IgG on gel media or in solution. Buffer for storage: 20 mM Na-Acetate, 0.02% (w/v) Na-azide, pH 5.0. Initial dimer content directly after dilution was 16.9%. Storage on % dimer after Total protein Monomer IgG gel media Temp. ? C. 3.5 weeks recovery % recovery % CAPTO? 20 8.0 24 27 MMC 20mM 5 11.1 71 82 Acetate pH ?20 10.7 76 87 5.0 CAPTO? S 20 6.4 87 98 20 mM 5 8.5 93 108 Acetate pH ?20 8.3 88 101 5.0 In solution % dimer after Total protein Monomer IgG storage Temp. ? C. 3.5 weeks recovery % recovery % 20 mM 20 11.6 Acetate pH 5 13.8 5.0 ?10 13.0

(50) 1.0 Storage on CAPTO?S results in much reduced dimer content and the antibody recovery is high. CAPTO? MMC gives slightly lower dimer content after storage compared with storage in solution with good recoveries at lower temperatures.

(51) TABLE-US-00007 TABLE 2B Antibody recovery and dimer content in eluted fractions after storage of human polyclonal IgG on gel media or in solution. Buffer for storage: 20 mM Na-Glycine. 0.02% (w/v) Na-azide, pH 9.0. Initial dialer content directly after dilution was 24.4%. Storage on % dimer after Total protein Monomer IgG gel media Temp. ? C. 3.5 weeks recovery % recovery % CAPTO? 20 40 88 111 Adhere 20 mM 5 1.5 93 122 Glycine pH ?20 2.0 93 109 9 CAMPO? 20 74 72 91 20 mM 5 9.0 57 74 Glycine pH ?20 7.5 66 77 9 In solution % dimer after Total protein Monomer IgG storage Temp. ? C. 3.5 weeks recovery % recovery % 20 mM 20 20.9 Glycine pH 5 23.5 9 ?20 14.5

(52) Storage on CAPTO? Adhere gives much improved results compared with storage in solution. The recoveries are very high with possible conversion of dimers into monomers as evidenced by the higher than 100% recovery of monomer IgG. Results from storage on CAPTO? Q show less dimer content compared with storage in solution, but the recoveries were less than 80%.

(53) TABLE-US-00008 TABLE 2C Human polyclonal IgG antibody recovery and dimer content in eluted fractions after storage. Buffer for storage: 10 mM Na-Phosphate, 2.7 mM KCl, 0.14M NaCl, 0.02% (w/v) Na- azide, pH 7.4. Initial dimer content directly after dilution as 24.6% Storage on % dimer after Total protein Monomer IgG gel media Temp. ? C. 3.5 weeks recovery % recovery % MABSELE CT? 20 1.7 88 108 PBS, pH 5 2.2 89 115 7.4 ?20 2.1 90 116 SEPHADE X? 20 14.4 98 120 PBS, pH 5 17.0 73 94 7.4 ?20 14.4 97 125 In solution % dimer after Total protein Monomer IgG storage Temp. ? C. 3.5 weeks recovery % recovery % PBS, pH 20 18.3 7.4 5 22.4 ?20 22.3

(54) Storage on MABSELECT? gives much improved results compared with storage in solution. The recoveries are very high with possible conversion of dimers into monomers evidenced by the higher than 100% recovery of monomer IgG. Results from storage on SEPHADEX? G-50 show slightly less dimer content compared with storage in solution but have the highest protein recovery of all gel media perhaps due to the non-binding properties.

(55) TABLE-US-00009 TABLE 2D Human polyclonal IgG antibody recovery and dimer content in eluted fractions after storage. Buffer for storage: 50 mM Na- Acetate, 1 mM EDTA, 1.5M (NH.sub.4).sub.2SO.sub.4, pH 5.0. Initial dimer content directly after dilution was not measured due to precipitation. Storage on Temp. % dimer after Total protein Monomer IgG gel media ? C. 3.5 weeks recovery % recovery % CAPTO? 20 6.5 31 Phe hs 5 5.7 49 ?20 7.7 57 pH HIC 20 5.9 70 16% 5 6.9 79 ?20 9.0 68 pH HIC 20 5.7 68 6% 5 7.4 80 ?20 8.2 82

(56) For HIC gel media, there was no starting material for comparison due to target precipitation. The absorbance (A280 nm) of the start material was 28.5 AU compared with over 40 AU for the other start materials. The protein recovery after clarification was good for pH HIC 6% and pH 16% gel media. Compared with other gel media, the dimer content after storage was in the low range, showing a positive effect on aggregation stabilization or reduction.

(57) The results for all storage conditions are shown as graphs in FIGS. 4A-C. Storage on CAPTO? Adhere or on MABSELECT? shows very low levels of dimers or aggregates with high recoveries of monomer IgG after storage, reflecting apparent ability of the media to reduce dimer levels during storage. Storage on CAPTO? Adhere or on MABSELECT? shows very low levels of dimers or aggregates with high recoveries of monomer IgG after storage, reflecting apparent ability of the media to reduce dimer levels during storage.

(58) The initial dimer content directly after dilution of GAMMANORM? (165 mg/ml) was between 16.9-24.6% depending on buffer used. After storage for 3.5 weeks at different temperatures, the dimer content was slightly lower in the various solutions.

(59) The buffer composition, pH, salt content and type seem to affect the equilibrium faster and to a higher degree than compared with the protein concentration. Storage of GAMMANORM? in a low salt buffer at pH 5.0 results in much lower dirtier content compared with storage at pH 7.4 or pH 9.0, both directly after dilution and after storage for 3.5 weeks.

(60) The effect of temperature is less obvious.

(61) The best storage condition for GAMMANORM? in solution seems to be storage in 20 mM Na-Acetate, 0.02% Na-azide 5.0 at +20? C. according to these experiments.

(62) However, the most pronounced effect on dimer content was observed when storing antibodies on binding media. Especially on CAPTO?. Adhere and MABSELECT?, although storage on these gel media was done at pH 9.0 and pH 7.4 respectively. The effect was so great, that when calculating the recovery of monomeric IgG, the result was higher than 100% for samples stored on CAPTO? Adhere and MABSELECT? The total protein recovery was never higher than 98% though (SEPHADEX? G-50. non-binding media).

(63) Thus, storing on binding media may have several benefits including: 1. Stabilization of IgG against aggregation (slow down kinetics) 2. Removal of IgG dimer (polishing) 3. Promote monomer formation (reversal of monomer/dimer equilibrium)

(64) Storing a start material with low initial dimer content on gel media would benefit from stabilization and polishing. Starting from high initial dimer content would also benefit from reducing the amount of dimers and increase the percent of monomers.

(65) Results obtained from non-binding media show no or little reduction of dimer content. In the first experiment starting with low initial dimer content, the storage on non-binding gel media (SUPERDEX?200 and SEPHADEX?G-50) resulted in slightly more dimers compared with storing in solution. Starting with high initial dimer content, the storage on SEPHADEX?G-50 resulted in slightly less dimers compared with storage in solution. The overall dimer content was however always higher compared with storing on any capture gel media.

(66) The skilled person will understand that flexible bags in accordance to the invention may be employed for the preparation of separation or reaction units that rely on introducing a particulate material into a column chamber and bringing the material into contact with a fluid as either a packed, expanded, consolidated or fluidized bed for achieving the separation or reaction process.

(67) Whilst the present invention has been described in accordance with various aspects and preferred embodiments, it is to be understood that the scope of the invention is not considered to be limited solely thereto and that it is the Applicant's intention that all variants and equivalents thereof also fall within the scope of the appended claims.