Mixed Bed Ion Exchange Adsorber

Abstract

The present invention refers to new species of an ion exchange adsorber which is suitable for the separation of host cell proteins (HCPs), antibody fragments and low molecular weight substances from solutions containing antibodies. The invention especially refers to a process for purifying biological samples by separating biomolecules of interest and impurities, comprising steps of contacting a sample with said chromatography media consisting of fibers, said fibers having imparted thereon functionality enabling ion exchange chromatography and/or hydrophobic interaction.

Claims

1. A process for purifying a biological sample by separating a biomolecule of interest and impurities, comprising the steps of contacting the sample with a chromatography media consisting of fibers, said fibers having imparted thereon functionality enabling ion exchange chromatography and/or hydrophobic interaction and steps of washing either to remove unbound species or to extract said biomolecule of interest.

2. A process according to claim 1 for purifying a sample comprising a biomolecule of interest and impurities, comprising the steps of: a) providing a sample, b) contacting said sample with a first chromatography media comprising fibers, said fibers having imparted thereon functionality enabling ion-exchange chromatography or hydrophobic interaction chromatography, c) washing said first fiber media to remove unbound species, d) washing said first fiber media to extract said biomolecule of interest, e) contacting said biomolecule of interest with a second chromatography media comprising fibers, said fibers having imparted thereon functionality enabling ion-exchange chromatography or hydrophobic interaction chromatography, f) washing said second fiber media to remove unbound species, and g) washing said second fiber media to extract said biomolecule of interest, but with the proviso that differently functionalized fiber media are used in steps b) and e).

3. A process according to claim 2, wherein in step b) said sample is contacted with a first chromatography media comprising fibers, said fibers having imparted functionality enabling anion-exchange chromatography, and wherein in step e) chromatography media are used comprising fibers having functionality enabling hydrophobic interaction chromatography.

4. A process according to claim 2, wherein in step b) said sample is contacted with a first chromatography media comprising fibers, said fibers having imparted functionality enabling cation-exchange chromatography, and wherein in step e) chromatography media are used comprising fibers having functionality enabling hydrophobic interaction chromatography.

5. A process according to claim 2, wherein in step b) chromatography media are used comprising fibers having functionality enabling hydrophobic interaction chromatography, and wherein said ion-exchange chromatography in step e) is anion-exchange chromatography.

6. A process according to claim 2, wherein in step b) chromatography media are used comprising fibers having functionality enabling hydrophobic interaction chromatography, and wherein said ion-exchange chromatography in step e) is cation-exchange chromatography.

7. The process of claim 2, wherein said first chromatography media in step b) is anion-exchange chromatography, and wherein said second chromatography media in step e) is cation-exchange chromatography.

8. The process of claim 2, wherein said first chromatography media in step b) is cation-exchange chromatography, and wherein said second chromatography media in step e) is anion-exchange chromatography.

9. A process for purifying a sample comprising a biomolecule of interest and impurities, comprising the steps: a) providing a sample, b) contacting said sample with a mixture of a first chromatography media comprising fibers and a second chromatography media comprising fibers, said first chromatography media comprising fibers having imparted thereon functionality enabling ion exchange chromatography, said second chromatography media comprising fibers having imparted thereon functionality enabling ion exchange chromatography, c) washing said mixture of chromatography media to remove unbound species, and d) washing said mixture of chromatography media to extract said biomolecule of interest.

10. The process of claim 9, wherein said first chromatography media is anion-exchange chromatography and said second chromatography media is cation-exchange chromatography or vice versa.

11. A process for purifying a sample comprising a biomolecule of interest and impurities, comprising the steps: a) providing a sample, b) contacting said sample with a mixture of a first chromatography media comprising fibers and a second chromatography media comprising fibers, said first chromatography media comprising fibers having imparted thereon functionality enabling hydrophobic interaction chromatography, said second chromatography media comprising fibers having imparted thereon functionality enabling ion exchange chromatography, c) washing said mixture of chromatography media to remove unbound species, and d) washing said mixture of chromatography media to extract said biomolecule of interest.

12. The process of claim 11, wherein said second chromatography media is cation-exchange chromatography or anion-exchange chromatography.

13. A housing comprising a packed bed of fibers; said packed bed having a first layer and a second layer, said first layer comprising fibers having imparted thereon functionality enabling ion-exchange chromatography, and said second layer comprising fibers having imparted thereon functionality enabling hydrophobic interaction chromatography.

14. The housing of claim 13, wherein said ion-exchange chromatography is anion-exchange chromatography or cation-exchange chromatography.

15. A housing comprising a packed bed of fibers; said packed bed having a first layer and a second layer, said first layer comprising fibers having imparted thereon functionality enabling hydrophobic interaction chromatography, and said second layer comprising fibers having imparted thereon functionality enabling ion-exchange chromatography.

16. The housing of claim 15, wherein said ion-exchange chromatography is anion-exchange chromatography or cation-exchange chromatography.

17. A housing comprising a packed bed of fibers; said packed bed having a mixture of a first chromatography media and a second chromatography media, said first chromatography media comprising fibers having imparted thereon functionality enabling hydrophobic interaction chromatography, and said second chromatography media comprising fibers having imparted thereon functionality enabling ion-exchange chromatography.

18. The housing of claim 17, wherein said ion-exchange chromatography is anion-exchange chromatography or cation-exchange chromatography.

19. A process for purifying a sample comprising a biomolecule of interest and impurities, comprising; providing a sample, contacting said sample with a first chromatography media comprising fibers, said first fibers having imparted thereon functionality enabling ion-exchange chromatography and a second chromatography media comprising fibers, said second fibers having imparted thereon functionality enabling ion-exchange chromatography.

20. The process of claim 19, wherein said first chromatography media are cation-exchange fibers and said second chromatography media are anion-exchange fibers.

21. The process of claim 19, wherein said first chromatography media are anion-exchange fibers and said second chromatography media are cation-exchange fibers.

22. The process of claim 19, wherein said first functionality enables purification in a flow-through mode and said second functionality enables purification in a flow-through mode.

23. The process of claim 19, wherein said first functionality enables purification in a bind/elute mode and said second functionality enables purification in a bind/elute mode.

24. The process of claim 19, wherein said first functionality enables purification in a flow-through mode and said second functionality enables purification in a bind/elute mode.

25. A process for purifying a sample comprising a biomolecule of interest and impurities, comprising; providing a sample, contacting said sample with a first chromatography media comprising fibers, said first fibers having imparted thereon functionality enabling ion-exchange chromatography and a second chromatography media comprising fibers, said second fibers having imparted thereon functionality enabling hydrophobic interaction chromatography.

26. The process of claim 25, wherein said first chromatography media are cation-exchange fibers and said second chromatography media are hydrophobic interaction chromatography fibers.

27. The process of claim 25, wherein said first chromatography media are anion-exchange fibers and said second chromatography media are hydrophobic interaction chromatography fibers.

28. The process of claim 25, wherein said first functionality enables purification in a flow-through mode and said second functionality enables purification in a flow-through mode.

29. The process of claim 25, wherein said first functionality enables purification in a bind/elute mode and said second functionality enables purification in a bind/elute mode.

30. The process of claim 25, wherein said first functionality enables purification in a flow-through mode and said second functionality enables purification in a bind/elute mode.

31. The process of claim 19, wherein said first chromatography media and said second chromatography media are arranged in a mixture.

32. The process of claim 19, wherein said first chromatography media and said second chromatography media are arranged in layers.

33. The process of claim 25, wherein said first chromatography media and said second chromatography media are arranged in a mixture.

34. The process of claim 25, wherein said first chromatography media and said second chromatography media are arranged in layers.

Description

FIGURES

[0193] FIG. 1: A standard mAb purification scheme is shown, which employs DSP including a bind-elute chromatography purification step followed by a flow-through polishing step.

[0194] FIG. 2: A schematic flow scheme of the separation process is shown of a mAb purification by tandem chromatography (CEX media and HIC) including the steps: [0195] i. post-protein A mAb load (conductivity 3 mS, pH 5) [0196] ii. high salt mAb elution (conductivity 30-100 mS, pH 5), where the product mAb is eluted from the upper CEX media layer but binds to the lower HIC media layer and [0197] iii. low salt mAb elution (conductivity 3 mS, pH 5), where the product mAb is eluted from the lower HIC media layer by elution with a low ionic strength eluent

[0198] FIG. 3: shows HCP clearance from the mAb04 Protein A feed after flow through purification in the form of bar charts, where the described media from example 5 are used (AEX fiber, CEX fiber, and a blend of both AEX and CEX media).

[0199] FIG. 4: Modified surface of the winged nylon fiber media with a selection of reactive methacrylic monomers ((hydroxyethyl)methacrylate, poly(propyleneglycol)-monomethacrylate, (phenyl)methacrylate, (n-butyl)methacrylate, (n-hexyl)methacrylate)) in presence of 0.4 M cerium(IV) ammonium nitrate.

TABLES

[0200] Table 1: Characteristics of columns packed with either AEX fibers, CEX fibers or a blend of both AEX and CEX fibers in view of bed depth and column volume, pressure and flowrate, permeability and velocity.

[0201] Table 2: Flow-through purification data for mAb04 using AEX fiber media, CEX fiber media and blended AEX/CEX fiber media columns in view of the characteristics, flow through, loading of mAb on fibers, recovery of mAb, HCP concentration and LRV of HCP.

[0202] The present description enables the person skilled in the art to apply the invention comprehensively. Even without further comments, it is therefore assumed that a person skilled in the art will be able to utilise the above description in the broadest scope.

[0203] If anything is unclear, it goes without saying that the publications and patent literature cited should be consulted. Accordingly, these documents are regarded as part of the disclosure content of the present description.

[0204] For better understanding and in order to illustrate the invention, examples are given below which are within the scope of protection of the present invention. These examples also serve to illustrate possible variants. Owing to the general validity of the inventive principle described, however, the examples are not suitable for reducing the scope of protection of the present application to these alone.

[0205] Furthermore, it goes without saying to the person skilled in the art that, both in the examples given and also in the remainder of the description, the component amounts present in the compositions always only add up to 100% by weight or mol %, based on the composition as a whole, and cannot exceed this, even if higher values could arise from the percent ranges indicated. Unless indicated otherwise, % data are % by weight or mol %, with the exception of ratios, which are shown in volume data, such as, for example, eluents, for the preparation of which solvents in certain volume ratios are used in a mixture.

[0206] The temperatures given in the examples and the description as well as in the claims are always in ° C.

EXAMPLES

Example 1

Graft Polymerization of Un-Modified Nylon Fibers

[0207] 10 g Allasso nylon fibers and water (466 ml) are added into a 500 ml bottle. 14 ml 1M HNO.sub.3 (14.4 mmol) are added to the reaction mixture, followed by the addition of 1.2 ml of a 0.4 M ammonium cerium(IV) nitrate solution in 1M HNO.sub.3 (0,480 mmol). The reaction mixture is agitated for 15 minutes. 3.39 g Glycidyl methacrylate (GMA, 24 mmol) are added. Now the agitated reaction mixture is heated to 35° C. for 1 hour. After cooling down to room temperature, the solids are washed with DI water (3×300 ml) and the damp material is used immediately in the following step.

Example 2

Q-Functionalization of Epoxy-Functionalized Fibers (AEX Fiber Media)

[0208] The damp GMA functionalized fibers from example 1 are added into a 2 L bottle together with water (500 ml) and a solution of 50 wt % trimethylamine (aq.) in methanol (500 ml). The mixture is agitated for 18 hours at room temperature. Then the fiber solids are subsequently washed with a solution of 0.2 M ascorbic acid in 0.5 M sulphuric acid (3×400 ml), DI water (3×400 ml), 1M sodium hydroxide solution (3×400 ml), DI water (3×400 ml) and ethanol (1×400 ml). Subsequently, the material is placed in an oven to dry at 40° C. for 48 hours.

Yield: 11.74 g of a white fibrous solid

Example 3

Graft Polymerization of Un-Modified Nylon Fibers (CEX Fiber Media)

[0209] 10 g Allasso nylon fibers and water (460 ml) are added into a 1000 ml bottle. 29.8 ml 1M HNO.sub.3 solution (30 mmol) are added to the reaction mixture, followed by the addition of a solution 7.46 ml of a 0.4 M ammonium cerium(IV) nitrate solution in 1M HNO.sub.3 (3.00 mmol). The reaction mixture is agitated for 15 minutes. Then 61.5 g 3-sulfopropylmethacrylate potassium salt (3-SPMA, 250 mmol) are added and the resulting agitated reaction mixture is heated to 35° C. for 18 hours. After cooling to room temperature, the fiber solids from each bottle are washed with DI water (3×300 ml), 0.2 M ascorbic acid in 0.5 M sulphuric acid (3×300 ml), DI water (3×300 ml), 1M sodium hydroxide solution (3×300 ml), DI water 3×300 ml) and ethanol (1×300 ml). The prepared material is then placed in an oven to dry at 40° C.

Yield: 11.38 g of a white fibrous solid

Example 4

Blended Ion-Exchange Media Column Packing

[0210] 0.35 g of a slurry of the described fiber media (see Table 1) in 25 mM Tris pH 8 is added into a 6.6 mm ID Omnifit column. The fiber media is compressed to a bed depth of 3.0 cm (1.03 ml column volume, 0.35 g/ml fiber packing density). Fiber bed permeability is assessed by flowing 25 mM Tris pH 8 buffer through the column at a flow rate of 2.0 ml/min and measuring the column pressure drop by means of an electric pressure transducer. Fiber bed permeability values are also provided in Table 1.

TABLE-US-00001 TABLE 1 Characteristics of columns packed with either AEX fibers, CEX fibers or a blend of both AEX and CEX fibers Pressure, Permeability Bed depth, [PSI] [mDarcy] Media type, [cm] flowrate velocity Column type amount [g] CV [ml] [ml/min] [cm/h] AEX column AEX fibers, 3.0 cm, 23.5 PSI 185 mDa, example 2 1.03 ml  2.0 ml/min 350 cm/h 0.35 g CEX column CEX fibers, 3.0 cm, 20.0 PSI 269 mDa, example 3, 1.03 ml  2.5 ml/min 440 cm/h 0.35 g AEX and CEX AEX fibers, 3.0 cm, 28.0 PSI 144 mDa blended column example 2, 1.03 ml  1.9 ml/min 330 cm/h 0.18 g CEX fibers, example 3, 0.18 g

Example 5

[0211] Comparison of HCP Removal from mAb04 Protein a Elution with AEX Fiber, CEX Fiber, and a Blend of Both AEX and CEX Media

[0212] A cell culture of mAb04 was clarified and then captured at a concentration of 7.2. mg/ml using Protein A column chromatography. The pH of the mAb04 Protein A elution was then adjusted to pH 5 with Tris base for storage and then filtered through a Stericup-GP 0.22 μm Millipore ExpressPLUS membrane (1 L, catalogue number: SCGPU02RE, Millipore Corp. Billerica, Mass., 01821, USA). The pH of the solution was adjusted to pH 7.0 with Tris base just prior to use. The resulting solution was then filtered through a Stericup-GP 0.22 mm Millipore Express PLUS membrane (1 L, catalogue number SCGPU02RE, Millipore Corp. Billerica, Mass., 01821, USA).

[0213] Three columns containing functionalized fibers were prepared as described in example 4. The first 1 ml column consisted of AEX fibers functionalized with quaternary ammonium ligands from example 2 (lot ID # JA7654-163B). The second 1 ml column consisted of CEX fibers functionalized with sulfonate ligands from example 3 (lot ID# JA7654-131). The third 1 ml column consisted of a blend of equal quantities of AEX fibers functionalized with quaternary ammonium ligands from example 2 (JA7654-163B) and CEX fibers functionalized with sulfonate ligands from example 3 (JA7654-131), see example 4. The three columns are equilibrated with a buffer solution (25 mM Tris at pH 7).

[0214] 120 ml of a Protein A elution pool is passed through each column at a flow rate of 0.33 ml/min giving a residence time of 3 min in each fiber packed column. Three 40 ml fractions are collected from each column. Pooled samples representing the elution pool composition after 80 ml and 120 ml, which have passed through the column, are submitted for analysis. The solutions are analyzed for host cell protein (HCP) and IgG concentration. HCP analysis is performed using a commercially available ELISA kit from Cygnus Technologies, Southport, N.C., USA, catalogue number F550, following kit manufacturer's protocol. IgG concentration is measured using an Agilent HPLC system equipped with a Poros® A Protein A analytical column. Results are summarized in Table 3 and FIG. 3.

[0215] The results of the experiment show that combining the mixed bed column containing the AEX and CEX fiber gave greater HCP removal with a log removal value (LRV) of approximately 1.6 LRV. This greatly exceeds the approximately 0.9 LRV of HCP observed for the column containing only the AEX fibers and the approximately 0.6 LRV of HCP observed for the column that contained only the CEX fibers. The greater amount of HCP removed by the column with a blend of both fibers is likely due to the fact that it has two different ligands which are able to adsorb HCP with different characteristics. The AEX functionalized fibers are able to bind to impurities that have negatively charged regions on their surfaces, which is typical for proteins with lower isoelectric points. The CEX functionalized fibers are able to bind impurities that have positively charged regions on their surface, which is typical of proteins with higher isoelectric points.

TABLE-US-00002 TABLE 2 Flow-through purification data for mAb04 using AEX fiber media, CEX fiber media and blended AEX/CEX fiber media columns Loading Flow of mAb through on fibers mAb mAb HCP HCP LRV of train [kg/l] [g/l] recovery [ng/ml] [ppm] HCP untrea-ted — 7.20 — 1876 261 — AEX fibers 0.54 7.15 99% 223 31 0.92 AEX fibers 0.82 7.16 99% 242 34 0.89 CEX fibers 0.54 6.67 93% 476 71 0.57 CEX fibers 0.82 6.82 95% 494 72 0.56 blend of 0.54 6.97 97% 38 5 1.68 AEX and CEX fibers blend of 0.82 7.03 98% 47 7 1.59 AEX and CEX fibers
FIG. 3: shows HCP clearance from the mAb04 Protein A feed after flow through purification using the described media from example 5.

Example 6

[0216] Fiber Media with Hydrophobic Interaction Chromatography Ligand

[0217] The hydrophobic interaction chromatography media described in the text above can be prepared by using the fiber surface modification procedures described in examples 1 and 3 and a methacrylate monomer or other polymerizable functionality selected from a group comprising [0218] methyl methacrylate, [0219] (hydroxyethylmethacrylate, [0220] poly(propyleneglycol)monomethacrylate, [0221] (phenyl)methacrylate, [0222] ((n-butyl)methacrylate, [0223] (n-hexyl)methacrylate).

[0224] The ceric ion redox polymerization procedure described in examples 1 and 3 can be used to directly modify the surface of the Allasso nylon fiber media with reactive methacrylic monomers disclosed in this example. (see FIG. 4) After grafting polymerization and suitable washing procedures (also described in examples 1 and 3), the fiber media now displays an appropriate hydrophobic ligand functionality for hydrophobic interaction chromatography (HIC). The media is now ready to be loaded into a chromatography column or other device for the tandem chromatography application described in the text above.

FIG. 4: Selection of HIC ligands suitable for fiber media of the present invention and process for attachment to the surface of the Allasso nylon fiber media.
i: Allasso fiber media surface modification using a methacrylate monomer selected from the group comprising:
(hydroxyethyl)methacrylate, poly(propyleneglycol)monoethacrylate,
(phenyl)methacrylate, (n-butyl)methacrylate, (n-hexyl)methacrylate), Allasso nylon fiber, 0.4 M cerium(IV) ammonium nitrate, nitric acid, water 35° C., 18 hrs according to the surface modification procedure employed in examples 1 and 3.

Example 7

[0225] Fiber Media Modified with Poly(Hydroxyethylmethacrylate) Ligand for Hydrophobic Interaction Chromatography

[0226] Hydroxyethylmethacrylate (HEMA, 1.69 g, 13 mmol) and water (232.5 ml) are added into a 500 ml bottle. Then 5.00 g of Allasso nylon fibers (Winged Fiber™), are added to this solution. 1M HNO.sub.3 solution (7.21 ml, 7.2 mmol) are added to this reaction mixture, followed by the addition of a 0.4 M solution of ammonium cerium(IV) nitrate in 1 M HNO.sub.3 (0,601 ml, 0,240 mmol). The reaction mixture is heated to 35° C. for 1 hour. After cooling to room temperature, the solids are washed with a solution of 0.2 M ascorbic acid in 0.5 M sulphuric acid (3×100 ml), DI water (3×100 ml) 1 M sodium hydroxide solution (3×100 ml) DI water (3×100 ml) and ethanol (1×100 ml). The material is placed in an oven to dry at 40° C. for 12 hours.

Yield: 5.58 g as a white fibrous solid