Chromatography Media

Abstract

The present invention relates to a novel chromatography media, more closely a novel IMAC (Immobilized Metal Affinity Chromatography) media. The novel chromatography media comprises a pentaligand and provides high dynamic binding capacity as well as high purity of the sample proteins purified on the media of the invention.

Claims

1. A method for purification of a biomolecule on a medium, the method comprising: loading a sample on an immobilized metal affinity chromatography (IMAC) medium comprising a pentadentate ligand coupled to a chromatography bead Q having a diameter of 5 μm to 60 μm, wherein the sample comprises a chelating agent, and the dynamic binding capacity at 10% breakthrough (QB10%) is more than double that of conventional IMAC media.

2. The method of claim 1, wherein the conventional IMAC media comprises a bead having a diameter greater than 60 μm.

3. The method of claim 1, wherein the diameter of the chromatography bead Q is 5 μm to 40 μm.

4. The method of claim 3, wherein the chromatography bead Q exhibits an increased number of repeated bindings (off-on events) in a column compared to conventional IMAC media comprising a bead having a diameter greater than 60 μm.

5. The method of claim 1, wherein the dynamic binding capacity at QB10% is 3 to 6 times greater than that of conventional IMAC media.

6. The method of claim 1, wherein the sample comprises a biomolecule labelled with at least two His-residues.

7. The method of claim 6, wherein the biomolecule is labelled with at least six His-residues.

8. The method of claim 1, wherein the chelating agent comprises ethylenediaminetetraacetic acid (EDTA).

9. The method of claim 1, wherein the immobilized metal affinity chromatography (IMAC) medium is coated with a dextran layer.

10. The method of claim 1, wherein the immobilized metal affinity chromatography (IMAC) medium comprising the pentadentate ligand coupled to the chromatography bead Q has the following formula: ##STR00005## wherein Q is the chromatography bead, S is a spacer, L is an amide linkage, X is COOH, and n=2-3.

11. The method of claim 10, wherein n is 2, and S is a hydrophilic chain of C and O comprising at least 3 atoms.

12. The method of claim 10, wherein n is 2, Q is charged with Ni2+, and the immobilized metal affinity chromatography (IMAC) medium comprising the pentadentate ligand coupled to the chromatography bead Q has the following structure: ##STR00006##

13. The method of claim 12, wherein the spacer (S) is derived from 2-(allyloxy)methyl)oxirane ##STR00007## and the immobilized metal affinity chromatography (IMAC) medium comprising the pentadentate ligand coupled to the chromatography bead Q has the following structure: ##STR00008##

14. The method of claim 1, wherein Q is a porous natural or synthetic polymer.

15. The method of claim 14, wherein Q comprises agarose.

16. The method of claim 1, wherein Q is made of agarose, and the diameter of Q is 30 μm to 40 μm.

17. The method of claim 1, wherein Q is charged with metal ions selected from the group consisting of Cu2+, Ni2+, Zn2+, Co2+, Fe3+, and Ga3+.

18. The method of claim 1, wherein Q comprises magnetic particles.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0027] FIG. 1 Chromatogram showing a test of dynamic binding capacity (QB10%) for MBP-His of commercial HisTrap excel (bold line) versus Excel HP prototype LS018819 (dotted line). The arrows indicate 10% breakthrough during sample application. The absorbance curve at 280 nm shows later breakthrough for the prototype.

[0028] FIG. 2 Diagram of QB10% results for commercial HisTrap excel and Excel HP prototype LS018819. Sample: MBP-His.

[0029] FIG. 3 Chromatogram showing a test of dynamic binding capacity on (QB10%) for GFP-His of commercial HisTrap excel (bold line) and Excel HP prototype LS019382 (dotted line). The arrows indicate 10% breakthrough during sample application. The absorbance curve at 280 nm shows later breakthrough for the prototype, with less loss of target protein.

[0030] FIG. 4. Diagram of QB10% results for commercial HisTrap excel and Excel HP prototype LS019382. Sample: GFP-His.

[0031] FIG. 5 Purification of GFP-His in E coli lysate. Analysis by reduced SDS-PAGE (Amersham WB system). Lane 1: Start sample, Lane 2: eluted peak HisTrap excel, Lane 3: eluted peak Excel HP prototype LS019382.

[0032] FIG. 6 Purification of GFP-His in E coli lysate (eluted fractions). SDS-PAGE at reduced conditions. Lane 1: Reference (IMAC Sepharose High Performance), Lane 2: Epoxy-activated resin prototype LS018835B, Lane 3: Dextran coated resin prototype LS018835A. The separate Lane 4 shows analysis of the pre-peak obtained with the reference.

DETAILED DESCRIPTION OF THE INVENTION

[0033] One of the main difficulties in IMAC purification is the challenge of obtaining both high purity and high capacity. High purity is often sacrified at the expense of high capacity and vice versa. There is a number of available IMAC resins, for different samples and different purposes. For example, Ni Sepharose High Performance (GE Healthcare Bio-Sciences AB) has high capacity while TALON Superflow (Clontech) has lower capacity but results in higher purity in comparison. Ni Sepharose excel is (GE Healthcare Bio-Sciences AB) a pentadentate resin which can be used for all types of samples (also metal stripping samples), results in high purity but has low capacity with loss of target protein during sample application.

[0034] A universal IMAC resin which combines all the benefits, providing high final purity, high capacity and the possibility to purify all types of samples would be very desirable.

[0035] The invention will now be described more closely in association to some non-limiting Examples and the accompanying drawings.

Experimental

Materials and Methods

IMAC Prototypes

[0036] 1. Excel HP prototypes [0037] LS018819 Excel ligand coupled to Sepharose High Performance, allyl content 170 μmole/ml [0038] LS019382 Excel ligand coupled to Sepharose High Performance, allyl content 189 μmole/ml [0039] Reference column: HiTrap excel, 1 ml, GE Healthcare

2. Dextran Coated Prototypes

[0040] LS018835A Dextran coated IMAC Sepharose High Performance [0041] Reference column: LS018835B NaOH treated epoxy activated IMAC Sepharose High Performance

[0042] The prototype resins were packed in 1 ml HiTrap columns according to the HiTrap packing method (GE Healthcare Bio-Sciences AB). A slurry concentration of 50-60% was used for packing of the HiTrap columns.

Test of Breakthrough, Purity and Resolution

[0043] Dynamic binding capacity (DBC) was tested by loading purified histidine-tagged maltose binding protein (MBP-His) and green fluorescent protein (GFP-His) to the column. Absorbance was registered and the capacity at 10% breakthrough (QB10%) of the sample absorbance was calculated.

[0044] Purity and resolution was tested by gradient purifications of GFP-His in E coli lysate. The histidine tagged protein was eluted by imidazole buffer and fractions were collected. Reduced SDS-PAGE was used for purity analysis.

Samples for Test of Dynamic Binding Capacity

[0045] Histidine(6)-tagged Green Flourescent Protein (GFP-His) in 17% glycerol, 20 mM sodium phosphate, 500 mM NaCl, pH 7.4. Concentration 2.5 mg/ml.

[0046] Histidine(6)-tagged Maltose Binding Protein (MBP-His) in 20 mM sodium phosphate, 500 mM NaCl, pH 7.4. Concentration 1.4 mg/ml.

Samples for Test of Final Purity and Resolution

[0047] Histidine(6)-tagged Green Flourescent Protein (GFP-His) in E coli, 20 mM sodium phosphate, 500 mM NaCl, pH 7.4. Concentration ˜3 mg/ml.

[0048] The samples were centrifuged (20 000 g for 10 minutes) and the supernatants were 0.45 μm filtrated when injected to the column.

Buffers

[0049] Binding buffer, A: 20 mM sodium phosphate, 500 mM NaCl, pH 7.4

[0050] Elution buffer, B: 500 mM imidazole in binding buffer

Chromatography Methods

[0051]

TABLE-US-00001 Test: Dynamic binding capacity. Excel HP prototype. Chromatography system: AKTA avant A25. Column Flow volumes rate Step (CV) % B (ml/min) Comments Equilibration 5 0 1 Sample 38 ml or 0 1 38 ml GFP-His or 70 ml application 70 ml MBP-His Wash 5 0 1 Elution 8 100 1 Re- 5 0 1 equilibration

TABLE-US-00002 Test: Purity and resolution. Excel HP Prototype. Chromatography system: AKTAavant A25. Column Flow volumes rate Step (CV) % B (ml/min) Equilibration 5 0 1 Sample 2 ml 0 1 His-tagged protein in E coli application lysate. Load: ~6 mg his-tagged protein. Wash 5 0 1 Elution 20 0-100 1 Gradient elution Re- 4 0 1 equilibration

TABLE-US-00003 Test: Purity and resolution. Dextran coated prototype. Chromatography system: AKTAavant A25. Column Flow volumes rate Step (CV) % B (ml/min) Equilibration 5 4 1 Sample 2 ml 4 1 His-tagged protein in E coli application lysate. Load: ~6 mg his-tagged protein. Wash 5 4 1 Elution 20 4-100 1 Gradient elution Re- 4 4 1 equilibration

[0052] SDS-PAGE under reduced conditions was performed using Amersham WB system. The samples were first buffer exchanged using Amersham WB Minitrap kit.

Experiment 1: Synthesis of the Excel HP Prototype

[0053] In this experiment the pentaligand described in EP 2164591B1 was coupled to Sepharose High Performance (GE Healthcare Bio-Sciences AB) (bead size diameter 34 μm). This bead has a smaller bead size which increases surface area for coupling compared with resins with larger bead size. The smaller bead size should also result in an increased number of repeated bindings (off-on events) in the column. This might be beneficial to decrease the leakage of target protein during sample application. The slightly larger pore size of High Performance resin compared to conventional IMAC media might also increase accessibility for the target protein.

Step 1: Allylation

[0054] 120 ml Sepharose HP resin was washed with water on a glass filter (p3, 6 GV) and the water sucked. The 120 g sucked resin was then transferred into a jacketed reactor along with 7,5 ml distilled water. Stirring was started and 12 ml 50% NaOH was added to the slurry. The slurry was stirred for 30 minutes and then heated to 47° C. and then 60 ml AGE was added. After ca 18 hours the stirring was stopped and the slurry transferred to a glass filter. The slurry was then washed with water (1 GV ×3), the EtOH (1 GV ×3) and then with water (1 GV ×6).

[0055] Allyltitration (using titration) Allyl content: ˜170 μmol/ml for LS018819.

[0056] Allyltitration (using titration): Allyl content: ˜189 μmol/ml for LS019382.

Step 2: Bromination

[0057] The 100 g/ml dry sucked allylated gel was transferred into a reaction reactor followed by adding 300 ml water and 4.6 g Sodium acetate trihydrate with stirring for 5 minutes. To the reaction mixture about 5 ml Bromine was added until the colour of the gel became strongly dark yellow and the reaction was left for 5 minutes with stirring at r. t. To the reactions mixture about 7.8 g sodium formate was added and the reaction was left with stirring for 15 minutes until the yellow colour disappeared. The gel was washed with (10×1 GV) water on glass filter (P3).

Step 3: Amination Step

[0058] The 100 g brominated gel from step 2 was transferred to a reaction reactor and 150 ml ammonia solution was added and the reaction mixture was left over night at 45° C. The gel was washed with 10×1 GV on glass filter (P3).

Step 4: EDTA Ligand Coupling Step

[0059] The 100 g aminated gel from step 3 was washed with 6×1 GV Acetone and transferred into the reaction reactor and 100 ml Acetone was added. To the reaction mixture 2.9 g DIPEA was added and the reaction was left for 5 minutes with stirring. 5.3 g EDTA was added to the reaction mixture and the mixture was left overnight at 24-28° C. The gel was washed with 3×1 GV Acetone followed by 3×1 GV water. The sucked gel was transferred in to the reactor and 1 GV 2M NaOH was added to hydrolyse the access of unreacted EDTA. The gel was washed on glass filter (P3) with 6×1GV.

[0060] The gel was finally nickel loaded with 0.1 M nickel sulphate.

##STR00002##

Dynamic Binding Capacity

[0061] The dynamic binding capacity, DBC, was tested using two different purified histidine-tagged proteins (MBP-His and GFP-His) and was calculated at 10% breakthrough, QB10%. The loss of the weak-binding MBP-His started almost immediately from commercial HisTrap excel while a delay was detected for the Excel HP prototype LS018819 (FIG. 1). The calculated QB10% was about 5 mg LS018819 MBP-His/ml resin for HisTrap excel and about 30 mg MBP-His/ml resin for the prototype (FIG. 2). Thus, the QB10% was about 6 times better for the prototype.

[0062] A later breakthrough can be expected for the strong-binding GFP-His compared with the weak-binding MBP-His. The obtained QB10% for HisTrap excel was ˜30 mg GFP-His/ ml resin. The Excel HP prototype LS019382 showed further improvement in performance. The absorbance was very low (0 mAU) with no loss of target protein until the end of sample application (FIG. 3). The calculated QB10% was ˜90 mg GFP-His/ ml resin (FIG. 4).

Purity

[0063] High capacity for histidine-tagged proteins may also result in high capacity for impurities containing one or several histidines. The final purity was investigated by adding a sample of GFP-His in E coli lysate to the columns. Low load was used in order to leave free coordination sites left for the impurities to bind. The sample was applied without any imidazole added, and eluted by an imidazole gradient. The eluted peaks were analyzed by reduced SDS-PAGE (FIG. 5). The reason for two major bands in the lanes 1-3 of FIG. 5 can probably be explained by a known truncation of GFP-His (still having the histidine-tag left). The final purity was equal for the two resins.

[0064] Thus, the results show that equal purity was obtained despite the higher capacity of the Excel HP prototype. This could be explained by the fact that the excel ligand is a pentadentate with only one coordination site left for binding to the protein. The six histidine-tag may be beneficial with improved chances to bind to the only coordination site compared with single histidines distributed along the impurity proteins. The results show that both high capacity and high purity was obtained using the Excel HP prototype.

[0065] In comparison with the current Ni Sepharose excel product the prototype resulted in 3-6 times higher dynamic capacity with significantly lower loss of target protein during sample application. The reason for the increased capacity might be due to the increased surface of Sepharose High Performance (bead size 34 μm) in comparison with Sepharose Fast Flow (bead size 90 μm) and other effects like accessibility due to larger pore size and increased numbers of repeated binding in the column

Experiment 2: Synthesis of the Dextran Coated Prototype

[0066] The purpose of dextran coating was to prevent multipoint attachment of impurities containing one or several histidines, while maintaining the binding of histidine tagged proteins. (New dextran-coated immobilized metal ion affinity chromatography matrices for prevention of undesired multipoint adsorptions, Journal of Chromatography A, 915 (2001) 97-106.) The tetradentate IMAC Sepharose High Performance (GE Healthcare Bio-Sciences AB) was used in this case but the results should also be applicable for pentadentate resins.

[0067] To evaluate the dextran effect two prototypes were made. One with Dextran coupled to LS 018835A it and one control prototype LS018835B which was only treated with NaOH to hydrolyse the epoxy groups.

Step 1: Epoxy Activation

[0068] Approximately 100 ml slurry of the gel (IMAC Sepharose High Performance) was washed with water (5×1 GV) on a glass filter. The gel was then sucked dry and 50 g was weighed into a 250 ml three-necked flask for epoxy activation. To the flask was then added 12 ml water and stirring and heating to 28° C. was started. During stirring 8 ml of 50% NaOH was added and the slurry was then stirred at 28° C. for about 10 minutes after which Epichlorohydrine (12.5 ml) was added and then left with stirring for 3.5 hours. The gel was then washed with water (6×1 GV) on a glass filter.

[0069] Epoxy titration (60 minutes titration, method 018 BL5-3) gave an epoxide content of around 16 μmol/ml for the epoxide activated gel that was used in the couplings.

Step 2: Dextran Coupling Step Prototype LS018835A

[0070] 8 g Dextran TF (10% Dx TF) was dissolved in a Duran flask with 35.2 ml water during rotation stirring for approximately 3 hours. 40 g of drained epoxyactivated gel from above was then added to the flask and the slurry was then heated to 40° C. and rotation stirred for 60 minutes. To the flask was then added 4.8 ml 50% NaOH and 0.1 g NaBH4 and then left with stirring by rotation at 40° C. overnight. The gel was washed with water (10×1 GV).

Step 3: NaOH Treatment of Epoxy Activated Gel Prototype LS018835B

[0071] 10 g of drained epoxyactivated gel from above was added to a 50 ml Falcon tube along with 8.8 ml dest water and shaken to a homogenous slurry. To the tube was then added 1.2 ml 50% NaOH and 0.05 g NaBH4. The tube was then put on a shaking table and heated to 40° C. and left shaking overnight.

[0072] After approximately 18.5 hours the reactions were stopped and the slurries washed with water (approximately 10×2 GV) on a glass filter (p3). The resins were finally nickel loaded with 0.1 M nickel sulfate.

[0073] Scheme 2: General Reaction Scheme of epichlorohydrine activation of IMAC Sepharose High Performance followed by dextran coupling.

1. Epoxyactivation

[0074] ##STR00003##

2. Dextran Coupling

[0075] ##STR00004##

Dry Weight Analysis

[0076] The dry weight of the prototypes was measured using standard method (120° C. drying temperature).

TABLE-US-00004 Dry weight Dry weight increase Prototype (mg/ml) (mg/ml) IMAC Seph HP 79 — LS018835A 84.1 5.1 LS018835B 79.7 0.7

[0077] As can be seen in the table approximately 5 mg/ml of dextran has been coupled to the media. A small increase in dry weight can also be seen for the NaOH treated B prototype.

Purity and Dynamic Binding Capacity

[0078] As described above a dextran layer of ˜10% was added to epoxy-activated IMAC Sepharose High Performance The sample was GFP-His in E coli lysate and elution was performed using an imidazole-gradient. According to the chromatograms a pre-peak with absorbance at 280 nm was detected for the reference but not the dextran coated prototype LS018835A (not shown). The pre-peak lacked absorbance at 490 nm (specific for GFP-His) which indicated a content of contaminants The eluted samples were analyzed by reduced SDS-PAGE (FIG. 6). The results show higher purity for the dextran coated resin but also the epoxy-activated resin LS018835B had higher purity in comparison with the reference. Thus, both prototypes had clearly better purity properties than the reference.