TARGET MOLECULE CAPTURE FROM CRUDE SOLUTIONS

Abstract

The present invention refers to a method for the separation of peptide aggregates and fragments from solutions containing target peptide.

Claims

1. A method for separating peptide aggregates and fragments from solutions containing the target peptide, comprising the steps of (a) providing the sample containing the target peptide; (b) contacting the sample with a hydrophobic chromatography material for a suitable period of time and adsorbing the peptides, (c) recovering the target peptide by use of different solvent compositions and thereby separating aggregated peptides and peptide fragments from the target peptide.

2. A method of claim 1, wherein the hydrophobic chromatography material, is particulate, and which is made of cross-linked polymer, selected from the group vinylbenzene, styrene, ethylstyrene, poly(ethyl)styrene-divinylbenzene, and poly(ethyl)styrene-divinylbenzene ethyleneglycol-dimethylacrylate resin.

3. A method of claim 1, wherein the particulate hydrophobic chromatography material is a resin, which is composed of a cross-linked polymer consisting of styrene and divinylbenzene in a ratio 98:2 up to 10:90% by weight or of polystyrene, which is cross-linked with copolymer of divinylbenzene and ethylenglycoldi-methacrylate in a ratio of 98:2 up to 10:90% by weight.

4. A method according to claim 1, wherein the particulate hydrophobic chromatographic separation material has mean particle diameters in the range of 10 μm to 600 μm, preferably in the range of 20 μm to 150 μm, most preferably in the range of 20 μm to 63 μm.

5. A method according to claim 1, wherein the particulate hydrophobic chromatographic separation material consists of hydrophobic porous polymer beads having pore sizes in the range of 4-500 nm, preferably in the range of 10-30 nm, most preferred in the range of 13 nm to 25 nm.

6. A method according to claim 1, wherein in step b) an aqueous solution containing the target peptide, having a pH value in the range of 2-11, preferably in a range of 3-8 and a conductivity in the range of 1-150 mS/cm, preferably in the range of 2-50 mS/cm, is contacted with a hydrophobic chromatography material.

7. A method according to claim 1, wherein in step b) the hydrophobic chromatographic material is exposed to 30-100 mg of target peptide per ml of packed bed, preferably to 50-80 mg of target peptide per ml of packed bed at a flow rate in the range of 150-1000 cm/min, preferably in the range of 300-900 cm/min.

8. A method according to claim 1, wherein in step c) the hydrophobic chromatographic material is exposed to an aqueous solution comprising organic solvent in direct or gradient manner, whereby depending on the concentration of the contained organic solvent a partial separation between the aggregated and target peptide is achieved.

9. A method according to claim 1, wherein in step c) a selective desorption and separation of the bound components is achieved using various ratios of organic solvents contained in an aqueous mixture.

10. A method according to claim 1, wherein the separation of peptide aggregates is processed after a target molecule refolding step.

11. A method according to claim 1, wherein after contacting the sample with a hydrophobic chromatography material for a suitable period of time and adsorbing the peptides, the loaded chromatography material is subjected to post peptide refolding solutions for selectively reducing the level of aggregated substances for a suitable period of time.

12. A method according to claim 1, wherein the separation and purification sequence includes a treatment with an ion exchange resin.

13. A method according to claim 1, wherein the separation and purification is carried out in a bind and elute or flow-through mode, whereby the flow velocity is adjusted in the range of 150 cm/min-1000 cm/min, and especially between 300-900 cm/min.

14. A method according to claim 1, wherein in step c) organic solvents selected from the group ethanol, 1-propanol and dipropylenglycol are used to selectively desorb the adsorbed substance from the hydrophobic chromatography material.

15. A method according to claim 1, wherein in step b) the sample is contacted with a hydrophobic chromatography material in form of polymer beads in a liquid chromatography column having a diameter ranging from 1 to 100 cm, preferably in the range of 5 to 50 cm, and where the column is operated at pressures up to 100 bar, and preferably at pressure ranging from 0.2 to 80 bar.

16. A method according to claim 1, wherein the sample is contacted with a hydrophobic chromatography material in form of polymer beads in a liquid chromatography column having a diameter in the range of 10 to 50 cm and where the column is operated at pressures in the range of 0.2 to 80 bar.

17. A method according to claim 1, wherein in step a) a crude insulin solution originating from E. coli expression system is provided.

Description

FIGURE LIST

[0086] FIG. 1: Non-reducing SDS-PAGE of static capture insulin on P00446, LiChroprep® RP-18 and Eshmuno® S. 1 ml of 5 mg/ml insulin @ 50 mM Acetate pH 4 was added to 100 μl equilibrated particles. Adsorption for 30 minutes @ 25° C. Elution for 30 minutes @25° C. with 500 μl 50 mM Acetate, 40 Vol % DPG, pH 4 respectively 50 mM phosphate, 500 mM NaCl, pH 8 for Eshmuno® S. Indicated lanes: M—molecular weight marker; S—start material, SA—supernatant after adsorption, BA—beads after adsorption.

[0087] FIG. 2: Elution peak of the captured crude insulin from P00446 polystyrene particles

[0088] FIG. 3: Non-reducing SDS-PAGE of dynamic crude insulin capture on P00446. Indicated lanes: M—Perfect Protein Marker; F—feed (starting material at pH 3.5), FF—flow through, A1-A5—elute fractions

[0089] FIG. 4: Non-reducing SDS-PAGE of dynamic capture raw insulin on P00446 (Column 1.1 ml diameter 10 mm). using ÄKTA FPLC system. 10 CV Equillibration with 25 mM Tris, 100 mM Arginine, pH 7. 30 ml of 1 mg/ml raw insulin were loaded with 1 ml/min flowrate. Elution in 20 CV from 0% till 100% 60 Vol. % DPG. Lanes: M—molecular weight marker; S—start material; FT—flow through.

[0090] FIG. 5: Non-reducing SDS-PAGE of dynamic crude pSCP194 capture on P00446 (Column 1.1 ml diameter 10 mm). Lanes: M—PerfectProtein™ molecular weight marker; F—feed (supernatant after high pressure lysis of cells @ pH 8.0, 300 mM NaCl); collected fractions from P00446 column: flow through, wash, elute).

[0091] FIG. 6: Non-reducing SDS-PAGE of dynamic crude insulin capture on P00446 and PS02 (Column 1.1 ml diameter 10 mm). Lanes: S—start material, FT—flow through fraction, PS02 B12-A8 elute fractions, 30410 A6-A1 elute fractions.

[0092] FIG. 7: Chromatogram of Insulin/aggregates purification with PP00446 at pH 3.5 using a linear gradient and Dipropylene glycol in elution buffer.

[0093] FIG. 8: Non-reducing SDS-PAGE analysis of all fractions during the Insulin/aggregates purification with PP00446 at pH 3.5 using a linear gradient and Dipropylene glycol in elution buffer. Lanes: M—SeeBlue®Plus2 Prestained Standard, F—Feed Insulin starting material@pH 3.5, Load—flowthrough collection during loading, Wash—flow through collection during wash step, elutes—elute fraction A1-A5 from P00446 column. Elution conditions: 50% DPG in 50 mM Glycine, 50 mM Acetic acid pH 3.5 gradient.

[0094] FIG. 9: Chromatogram of Insulin/aggregates purification with PP00446 at pH 3.5 using a linear gradient and Ethanol in elution buffer.

[0095] FIG. 10: Non-reducing SDS-PAGE analysis of all fractions during the Insulin/aggregates purification with PP00446 at pH 3.5 using a linear gradient and Ethanol in elution buffer. Lanes: M—SeeBlue®Plus2 Prestained Standard, F—Feed Insulin starting material@pH 3.5, L—flowthrough collection during loading, W—flow through collection during wash step, elutes—elute fraction E1-E5 from P00446 column. Elution conditions: 50% DPG in 50 mM Glycine, 50 mM Acetic acid pH 3.5 gradient.

[0096] FIG. 11: Chromatogram of Insulin/aggregates purification with PP00446 at pH 8.0 using a linear gradient and Dipropylene glycol in elution buffer.

[0097] FIG. 12: Non-reducing SDS-PAGE analysis of all fractions during the Insulin/aggregates purification with PP00446 at pH 8.0 using a linear gradient and Dipropylene glycol in elution buffer. Lanes: M—SeeBlue®Plus2 Prestained Standard, F—Feed Insulin starting material@pH 8.0, L—flowthrough collection during loading, W—flow through collection during wash step, elutes—elute fraction E1-E5 from P00446 column. Elution conditions: 50% DPG in 50 mM TRIS pH 8.0 gradient.

[0098] FIG. 13: Chromatogram of Insulin/aggregates purification with PP00446 at pH 3.5 using a step gradient and Dipropylene glycol in elution buffer.

[0099] FIG. 14: Non-reducing SDS-PAGE analysis of all fractions during the Insulin/aggregates purification with PP00446 at pH 3.5 using a step gradient and Dipropylene glycol in elution buffer. Lanes: M—SeeBlue®Plus2 Prestained Standard, F—Feed Insulin starting material@pH 3.5, L—flowthrough collection during loading, W—flow through collection during wash step, elutes—elute fraction E1-E7 from P00446 column. Elution conditions: 50% DPG in 50 mM Glycine, 50 mM acetic acid pH 3.5.

[0100] FIG. 15: Chromatogram of Insulin/aggregates purification with PRLP-S at pH 8.0 using a step gradient and Dipropylene glycol in elution buffer.

[0101] FIG. 16: Non-reducing SDS-PAGE analysis of all fractions during the Insulin/aggregates purification with PRLP-S at pH 8.0 using a step gradient and Dipropylene glycol in elution buffer. Lanes: M—SeeBlue®Plus2 Prestained Standard, F—Feed Insulin starting material@pH 8.0, L—flowthrough collection during loading, W—flow through collection during wash step, elutes—elute fraction E1-E7 from P00446 column. Elution conditions: 50% DPG in 50 mM TRIS pH 8.0.

EXAMPLES

Base Beads:

Synthesis of Polystyrene Based Material (Such as P353, P374 and P375)

[0102] 25.6 g Polyvinylalcohol and 0.38 g SDS are dissolved in 614.2 g water to form the water phase for the following suspension polymerization. The organic phase is formed by a homogenous solution of 19.94 g ethylvinylbenzene, 75 g divinylbenzene, 41.57 g ethylene glycol dimethacrylate, 90.24 g toluene, 90.24 g 2-ethyl-1-hexanole and 0.96 g AIBN. The organic phase is added to the water phase in a reactor vessel and the two phases are emulsified at 25° C. with a stirrer at 480 rpm to achieve the anticipated particle size distribution. After 60 min 640 g water are added and the reaction mixture is heated up to 72° C. For two hours the temperature is kept at 72° C. and then increased to 82° C. The mixture is polymerized at 82° C. for additional two hours. Following polymerization the suspension is filtered on a filter funnel and the particles are washed with 1.5 liter water of 60° C., followed by 5 liter methanol at 60° C., 5 liter of toluene and 2 liter of methanol at 40° C. The final product is dried in a vacuum oven for 24 hours at 50° C. and 50 mbar. The yield regarding dry mass is quantitative. Depending from the anticipated particle size distribution the final product is classified by sieving according to procedures which are state of the art.

Example 1

[0103] In this experiment, different materials are evaluated for their ability to adsorb recombinant peptide-insulin. For the following example Polystyrene (PS) particles P00446 are used for capture of pure insulin (A11382IM, life technologies) in comparison to the common used LiChroprep® RP-18 (113900, Merck Millipore) beads and one cation exchange material (e.g. Eshmuno® S, 120078, Merck Millipore). 100 μl particles are washed with 1 ml of 50 mM Acetate pH 4. Afterwards 1 ml of a 5 mg/ml insulin solution in 50 mM Acetate pH 4 was adsorbed for 30 minutes at 25° C. After centrifugation the supernatant is removed (5 μl were mixed with 5 μl gel loading buffer, NP0007, life technologies). Particles are washed with 1 ml 50 mM Acetate pH 4 and split in 2 tubes. After centrifugation the supernatant is discarded. For beads after adsorption one part of the particles are resuspended in 500 μl gel loading buffer. The other part is eluted with 500 μl of 50 mM Acetate, 40 Vol % DPG, pH 4 respectively 50 mM phosphate, 500 mM NaCl, pH 8 for Eshmuno® S for 30 minutes at 25° C. After centrifugation the supernatant is removed (5 μl were mixed with 5 μl gel loading buffer). Particles are washed with 500 μl 50 mM Acetate pH 4. After centrifugation the supernatant is discarded. For beads after elution the particles are resuspended in 500 μl gel loading buffer. After heating all samples for 10 minutes at 99° C. 10 μl of supernatant/eluate respectively 5 μl of bead-samples are loaded on 4-12% NuPAGE® Novex® Bis-Tris Gel (NP0336, life technologies). Gel is run for 25 minutes at 200 V constant. After washing with pure water it is stained with SimplyBlue™ SafeStain (LC6065, life technologies) and destained with pure water.

[0104] As shown in FIG. 1 the polystyrene beads P00446 adsorb insulin nearly complete which corresponds to a static binding capacity of 50 mg protein per ml particle-suspension. 40 Vol % Dipropylene glycol is a useful desorption solution. The new technology of PS particles is comparable to the common used cation exchange process. In comparison LiChroprep® RP-18 (as an alternative reverse phase material) does not adsorb insulin under the given conditions.

Example 2

[0105] In this experiment, different materials are evaluated for their ability to adsorb crude insulin A. For the following example Polystyrene (PS) particles P00446 are packed in a 10 mm diameter 12 mm long column using 20% ethanol 150 mM NaCl solution. The packed column is equilibrated using 50 mM Glycine/50 mM Acetic acid buffer pH 3.5 for at least 20 column volumes at 1 ml/min. The crude insulin solution A is adjusted to pH 3.5 (insulin concentration˜1.7 g/L). 60 ml of obtained solution is directly loaded on the equilibrated column at 1 ml/min and the flow through fraction collected in a separate flask. After loading, column is washed with 10CV using equilibration solution. The elution of the captured crude insulin is performed using a gradient elution from 0-100% of 50% ethanol in 50 mM Glycine/50 mM Acetic acid buffer pH 3.5 in 30 CV at 1 ml/min (FIG. 2). The fractions are collected and subjected to non-reducing SDS-PAGE analysis (FIG. 3).

[0106] As shown in FIG. 3 the polystyrene beads P00446 adsorb insulin nearly complete which correspond to a binding capacity of >80 mg protein per ml packed bed. 50 Vol % ethanol is a useful desorption solution (e.g. recovery 101.33%), enabling to use polystyrene particles for aggregate and target molecule separation, that is not noticed using ion exchangers for the crude insulin capture (data not shown).

Example 3

[0107] In this experiment, particulate material, consisting of poly(ethy)styrene (m) crosslinked with divinylbenzene copolymers (PS-DVB), is evaluated for its ability to adsorb crude insulin B. For the following example Polystyrene (PS) particles P00446 are packed in a 10 mm diameter 12 mm long column using 20% ethanol 150 mM NaCl solution. The packed column is equilibrated using 50 mM TRIS, 100 mM Arginine buffer pH 7.0 for at least 20 column volumes at 1 ml/min. The crude insulin solution B originating from E. coli expression system is adjusted to pH 3.5 and 30 ml of obtained solution is directly loaded on the equilibrated column at 1 ml/min and the flow through fraction collected in a separate flask. After loading, column is washed with 10CV using equilibration solution. The elution of the captured crude insulin is performed using a gradient elution from 0-100% of 60% dipropylenglycol in 50 mM Glycine/50 mM Acetic acid buffer pH 3.5 in 20 CV at 1 ml/min. The fractions are collected and subjected to non-reducing SDS-PAGE analysis (FIG. 4).

[0108] As shown in FIG. 4 the polystyrene beads P00446 adsorb insulin (5% purity) from the crude solution and dipropylenglycol solution can be applied to separate insulin fragments from aggregates and target achieving >40% purity.

Example 4

[0109] In this experiment, particulate materials consisting of poly(ethyl)styrene (m), crosslinked with divinylbenzene copolymers (PS-DVB) are evaluated for their ability to adsorb crude protein pSCP194. For the following example Polystyrene (PS) particles P00446 are packed in a 10 mm diameter 12 mm long column using 20% ethanol 150 mM NaCl solution. The packed column is equilibrated using 50 mM TRIS, 100 mM Arginine buffer pH 7.0 for at least 20 column volumes at 1 ml/min. The crude E. coli lysate containing protein pSCP194 is adjusted to pH 7.0 and 20 ml of obtained solution is directly loaded on the equilibrated column at 1 ml/min and the flow through fraction collected in a separate flask. After loading, column is washed with 10CV using equilibration solution. The elution of the captured crude insulin is performed using a gradient elution from 0-100% of 60% dipropylenglycol in 50 mM Glycine/50 mM Acetic acid buffer pH 3.5 in 20 CV at 1 ml/min. The fractions are collected and subjected to non-reducing SDS-PAGE analysis (FIG. 5).

[0110] As shown in FIG. 5 the polystyrene beads P00446 adsorb pSCP194 protein from the crude e. coli lysate solution and dipropylenglycol solution can be applied to elute the captured target achieving >80% purity.

Example 5

[0111] In this experiment, particulate material consisting of poly(ethyl)styrene (m), crosslinked with divinylbenzene copolymers (PS-DVB) and material consisting of ethylene glycol dimethylacrylate (PS-DVB-EGDMA) copolymers in various ratios is evaluated for its ability adsorb insulin in presence of urea.

For the following example Polystyrene (PS) particles P00446 are packed in a 10 mm diameter 12 mm long column using 20% ethanol 150 mM NaCl solution. The packed column is equilibrated using 50 mM Glycine/50 mM acetic acid buffer pH 3.5 for at least 20 column volumes at 1 ml/min. The crude insulin solution containing aggregated insulin after incubation in 8M urea solution is diluted to 2M urea concentration using pure water adjusted to pH 3.5. 50 ml of obtained solution is directly loaded on the equilibrated column at 1 ml/min and the flow through fraction collected in a separate flask. After loading, the column is washed with 10CV using equilibration solution. The elution of the captured crude insulin is performed using a gradient elution from 0-100% of 60% dipropylenglycol in 50 mM Glycine/50 mM Acetic acid buffer pH 3.5 in 20 CV at 1 ml/min. The fractions were collected and subjected to non-reducing SDS-PAGE analysis (FIG. 6).

[0112] As shown in FIG. 6 the polystyrene beads P00446 and PS02 adsorb insulin from the crude 2M urea containing solution and dipropylenglycol solution can be applied to elute the captured target achieving >90% purity (fractions A3-A4 and A11-A12).

Example 6

[0113] In this experiment, particulate material consisting of poly(ethyl)styrene (m), crosslinked with divinylbenzene copolymers (PS-DVB) and ethylene glycol dimethylacrylate (PS-DVB-EGDMA) copolymers in various ratios, is evaluated for its ability to purifying Insulin from its aggregates at pH 3.5 using a linear elution gradient and Dipropylene glycol organic solvent in aqueous buffer. For the following example 1 ml Polystyrene (PS) particles PP00446 are packed in a 10 mm diameter 6.2 long column using grinding beads for lengthening the column. For column packing 20% Ethanol 150 mM NaCl is prepared. The packed column is equilibrated using 50 mM Glycine/50 mM Acetic acid pH 3.5 (equilibration buffer) for 20 column volumes at 1 ml/min. The Insulin/aggregates solution is adjusted to pH 3.5 by adding acetic acid (insulin concentration: ˜1.5 mg/ml). 92 ml of prepared solution is loaded on the equilibrated column at 1 ml/min while the flow through is collected in a flask. After accomplished loading the column is rinsed with equilibration buffer for 15 column volumes at 1 ml/min to wash out unbound Insulin molecules. The flow through during the wash out step is collected in a second flask. Subsequently the elution is initialized using a linear gradient from 0-100% of 50% Dipropylene glycol in 50 mM Glycine/50 mM Acetic acid (elution buffer) in 40 column volumes at 1 ml/min. The eluates are fractionated in 10 ml per fraction. FIG. 7 depicts the chromatogram recorded during the Insulin/aggregates purification. The collected fractions are subjected to non-reducing SDS-PAGE analysis (FIG. 8).

[0114] As shown in FIG. 8 PP00446 resin is overloaded by Insulin/aggregates solution. The measured dynamic binding capacity of PP00446 reached >80 mg protein per ml packed bed. It becomes apparent that 50 Vol % Diproylene glycol is not only a suitable desorption solution (e.g. recovery: 115%), but also enable the separation of Insulin from its aggregates during linear gradient elution.

Example 7

[0115] In this experiment, particulate materials consisting of poly(ethyl)styrene (m) crosslinked with divinylbenzene copolymers (PS-DVB) and ethylene glycol dimethylacrylate (PS-DVB-EGDMA) copolymers in various ratios is evaluated for its ability to purifying Insulin from its aggregates at pH 3.5 using a linear elution gradient and ethanol. For the following example 1 ml Polystyrene (PS) particles PP00446 are packed in a 10 mm diameter 6.2 long column using grinding beads for lengthening the column. For column packing 20% Ethanol 150 mM NaCl is prepared. The packed column is equilibrated using 50 mM Glycine/50 mM Acetic acid pH 3.5 (equilibration buffer) for 20 column volumes at 1 ml/min. The Insulin/aggregates solution is adjusted to pH 3.5 by adding acetic acid (insulin concentration: ˜2.0 mg/ml). 50 ml of prepared solution is loaded on the equilibrated column at 1 ml/min while the flow through is collected in a flask. After accomplished loading the column is rinsed with equilibration buffer for 15 column volumes at 1 ml/min to wash out unbound Insulin molecules. The flow through during the wash out step is collected in a second flask. Subsequently the elution was initialized using a linear gradient from 0-100% of 50% Ethanol in 50 mM Glycine/50 mM Acetic acid (elution buffer) in 40 column volumes at 1 ml/min. The elutes were fractionated in 10 ml per fraction. FIG. 3 depicts the chromatogram recorded during the Insulin/aggregates purification. The collected fractions are subjected to non-reducing SDS-PAGE analysis (FIG. 9).

[0116] As shown in FIG. 10 PP00446 resin is overloaded by Insulin/aggregates solution. The measured dynamic binding capacity (DBC) of PP00446 reached >60 mg protein per ml packed bed. The results expose that 50 Vol % Ethanol can be used as desorption solution (recovery: 96%). In contrast to the elution under comparable conditions with Dipropylene glycol, the purification from this example results in lower DBC as well as in deteriorated recovery.

Example 8

[0117] In this experiment, particulate materials consisting of poly(ethyl)styrene (m) crosslinked with divinylbenzene (PS-DVB) and ethylene glycol dimethylacrylate (PS-DVB-EGDMA) copolymers in various ratios is evaluated for its ability to separate insulin from its aggregates at pH 8.0 using a linear elution gradient and dipropylen glycol.

[0118] For the following example 1 ml Polystyrene (PS) particles PP00446 are packed in a 10 mm diameter 6.2 long column using grinding beads for lengthening the column. For column packing 20% Ethanol 150 mM NaCl is prepared. The packed column is equilibrated using 50 mM TRIS pH 8.0 (equilibration buffer) for 20 column volumes at 1 ml/min. The Insulin/aggregates solution is adjusted to pH 8.0 by adding 1M TRIS. Furthermore the conductivity of the Insulin/aggregates solution is set to ˜20 mS/cm by 1M NaCl solution (insulin concentration: ˜0.6 mg/ml). 75 ml of prepared solution was loaded on the equilibrated column at 1 ml/min while the flow through is collected in a flask. After accomplished loading the column is rinsed with equilibration buffer for 15 column volumes at 1 ml/min to wash out unbound Insulin molecules. The flow through during the wash out step is collected in a second flask. Subsequently the elution is initialized using a linear gradient from 0-100% of 50% Dipropylene glycol in 50 mM TRIS pH 8.0 (elution buffer) in 40 column volumes at 1 ml/min. The elutes are fractionated in 10 ml per fraction. FIG. 5 depicts the chromatogram recorded during the Insulin/aggregates purification. The collected fractions are subjected to non-reducing SDS-PAGE analysis (FIG. 11).

[0119] As shown in FIG. 12 PP00446 resin adsorbs insulin and its aggregates nearly completely and reaches a dynamic binding capacity of >70 mg protein per ml packed bed. Moreover by the application of a linear gradient the elution results in purifying Insulin from aggregates (E2, E3), whereas most aggregates are desorbed at an elution buffer concentration >80%.

Example 9

[0120] In this experiment, particulate materials consisting of poly(ethyl)styrene (m) crosslinked with divinylbenzene (PS-DVB) and ethylene glycol dimethylacrylate (PS-DVB-EGDMA) copolymers in various ratios is evaluated for its ability to separate insulin from its aggregates at pH 8.0 using a step elution and dipropylen glycol.

[0121] For the following example 1 ml Polystyrene (PS) particles PP00446 are packed in a 10 mm diameter 6.2 long column using grinding beads for lengthening the column. For column packing 20% Ethanol 150 mM NaCl is prepared. The packed column is equilibrated using 50 mM Glycine/50 mM Acetic acid pH 3.5 (equilibration buffer) for 20 column volumes at 1 ml/min. The Insulin/aggregates solution is adjusted to pH 3.5 by adding acetic acid (insulin concentration: ˜1.8 mg/ml). 50 ml of prepared solution is loaded on the equilibrated column at 1 ml/min while the flow through was collected in a flask. After accomplished loading the column is rinsed with equilibration buffer for 15 column volumes at 1 ml/min to wash out unbound Insulin. The flow through during the wash out step is collected in a second flask. Subsequently the elution is initialized using a step gradient (70% for 40 column volumes, 100% for 20 column volumes) using 50% Dipropylene glycol in 50 mM Glycine/50 mM Acetic acid as Elution buffer at 1 ml/min. The elutes are fractionated in 10 ml per fraction. FIG. 7 depicts the chromatogram recorded during the Insulin/aggregates purification. The collected fractions are subjected to non-reducing SDS-PAGE analysis (FIG. 13).

[0122] As shown in FIG. 14 PP00446 resin bound Insulin and Insulin aggregates (dynamic binding capacity: >70 mg/ml). In this experiment the recovery achieved 96%. Moreover it is proven by the SDS-PAGE analysis that most Insulin aggregates are desorbed from the resin when the elution buffer concentration was higher than 70%. This property enables Insulin aggregate separation by applying an utilizable step gradient.

Example 10

[0123] In this experiment, particulate materials consisting of poly(ethyl)styrene (m) crosslinked with divinylbenzene (PS-DVB) copolymers is evaluated for its ability to remove aggregates from insulin at pH 8.0 using a step elution and dipropylen glycol.

[0124] For the following example 1 ml Polystyrene (PS) particles PRLP-S are packed in a 10 mm diameter 6.2 long column using grinding beads for lengthening the column. For column packing 20% Ethanol 150 mM NaCl is prepared. The packed column is equilibrated using 50 mM TRIS pH 8.0 (equilibration buffer) for 20 column volumes at 1 ml/min. The Insulin/aggregates solution is adjusted to pH 8.0 by adding 1M TRIS. Furthermore the conductivity of the Insulin/aggregates solution is set to ˜20 mS/cm by 1M NaCl solution (insulin concentration: ˜1.6 mg/ml). 50 ml of prepared solution is loaded on the equilibrated column at 1 ml/min while the flow through is collected in a flask. After accomplished loading the column is rinsed with equilibration buffer for 15 column volumes at 1 ml/min to wash out unbound Insulin. The flow through during the wash out step is collected in a second flask. Subsequently the elution is initialized using a step gradient (70% for 40 column volumes, 100% for 20 column volumes) using 50% Dipropylene glycol in 50 mM TRIS pH 8.0 as elution buffer at 1 ml/min. The elutes are fractionated in 10 ml per fraction. FIG. 9 depicts the chromatogram recorded during the Insulin/aggregates purification. The collected fractions are subjected to non-reducing SDS-PAGE analysis (FIG. 15).

[0125] As shown in FIG. 16 PRLP-S resin bound Insulin with a measured dynamic binding capacity >60 mg protein per ml packed bed. Furthermore the SDS-PAGE analysis outlines that solely Insulin monomers are desorbed from the resin with a elution buffer concentration of 70% (E1-E4). It is proven in FIG. 10 that Insulin aggregates are desorbed through the increase of the elution buffer to 100% (E5). The usage of a step elution gradient for Insulin aggregates separation streamlines the Insulin purification process.

Application of Polystyrene Particles for the Capture of Insulin

[0126] For the following example Polystyrene (PS) particles P00446 are used for capture of pure insulin (A11382IM, life technologies) in comparison to the common used LiChroprep® RP-18 (113900, Merck Millipore) beads and one cation exchange material (e.g. Eshmuno® S, 120078, Merck Millipore).

[0127] 100 μl particles are washed with 1 ml of 50 mM Acetate pH 4. Afterwards 1 ml of a 5 mg/ml insulin solution in 50 mM Acetate pH 4 is adsorbed for 30 minutes at 25° C. After centrifugation the supernatant is removed (5 μl are mixed with 5 μl gel loading buffer, NP0007, life technologies). Particles are washed with 1 ml 50 mM Acetate pH 4 and split in 2 tubes. After centrifugation the supernatant is discarded. For beads after adsorption one part of the particles are resuspended in 500 μl gel loading buffer. The other part is eluted with 500 μl of 50 mM Acetate, 40 Vol % DPG, pH 4 respectively 50 mM phosphate, 500 mM NaCl, pH 8 for Eshmuno® S for 30 minutes at 25° C. After centrifugation the supernatant is removed (5 μl were mixed with 5 μl gel loading buffer). Particles are washed with 500 μl 50 mM Acetate pH 4. After centrifugation the supernatant is discarded. For beads after elution the particles are resuspended in 500 μl gel loading buffer. After heating all samples for 10 minutes at 99° C. 10 μl of supernatant/eluate respectively 5 μl of bead-samples are loaded on 4-12% NuPAGE® Novex® Bis-Tris Gel (NP0336, life technologies). Gel is run for 25 minutes at 200 V constant. After washing with pure water it is stained with SimplyBlue™ SafeStain (LC6065, life technologies) and destained with pure water. FIG. 1 shows a non-reducing SDS-PAGE of static capture insulin on P00446, LiChroprep® RP-18 and Eshmuno® S.

Conditions:

[0128] A mixture of 1 ml of 5 mg/ml insulin and 50 mM Acetate pH 4 is added to 100 μl equilibrated particles. Adsorption is carried out for 30 minutes at a temperature of 25° C. Subsequently elution is done for 30 minutes at a temperature of 25° C. with 500 μl 50 mM Acetate and 40 Vol % DPG, pH 4 respectively 50 mM phosphate, 500 mM NaCl, pH 8 for [0129] Eshmuno® S. [0130] Lanes: M . . . MW-Marker [0131] S . . . Start material [0132] SA . . . supernatant after adsorption [0133] BA . . . beads after adsorption [0134] E . . . Eluate [0135] BE . . . beads after elution

[0136] As shown in

FIG. 1 the polystyrene beads P00446 adsorb insulin nearly complete which correspond to a static binding capacity of 50 mg protein per ml particle-suspension. 40 Vol % Dipropylene glycol is a useful desorption solution. The new technology of PS particles is comparable to the common used cation exchange process.

[0137] In comparison LiChroprep® RP-18 (as an alternative reverse phase material) does not adsorb insulin under the given conditions.

Application of Polystyrene Particles for the Capture of Crude Insulin A

[0138] For the following example Polystyrene (PS) particles P00446 are packed in a 10 mm diameter 12 mm long column using 20% ethanol 150 mM NaCl solution.

[0139] The packed column is equilibrated using 50 mM Glycine/50 mM Acetic acid buffer pH 3.5 for at least 20 column volumes at 1 ml/min. The crude insulin solution A is adjusted to pH 3.5 (insulin concentration-1.7 g/L). 60 ml of obtained solution is directly loaded on the equilibrated column at 1 ml/min and the flow through fraction is collected in a separate flask. After loading, the column is washed with 10CV using equilibration solution. The elution of the captured crude insulin is performed using a gradient elution from 0-100% of 50% ethanol in 50 mM Glycine/50 mM Acetic acid buffer pH 3.5 in 30 CV at 1 ml/min (FIG. 2). The fractions are collected and subjected to non-reducing SDS-PAGE analysis (FIG. 3).

FIG. 2 shows the elution peak of the captured crude insulin from P00446 polystyrene particles.
FIG. 3 shows a non-reducing SDS-PAGE of dynamic crude insulin capture on P00446.

[0140] As shown in FIG. 3 the polystyrene beads P00446 adsorb insulin nearly complete which correspond to a binding capacity of >80 mg protein per ml packed bed. 50 Vol % ethanol is a useful desorption solution (e.g. recovery 101.33%), enabling to use polystyrene particles for aggregate and target molecule separation, that is not noticed using ion exchangers for the crude insulin capture (data not shown).

Application of Polystyrene Particles for the Capture of Crude Insulin B

[0141] For the following example Polystyrene (PS) particles P00446 are packed in a 10 mm diameter 12 mm long column using 20% ethanol 150 mM NaCl solution.

[0142] The packed column is equilibrated using 50 mM TRIS, 100 mM Arginine buffer pH 7.0 for at least 20 column volumes at 1 ml/min. The crude insulin solution B originating from E. coli expression system is adjusted to pH 3.5 and 30 ml of obtained solution is directly loaded on the equilibrated column at 1 ml/min and the flow through fraction collected in a separate flask. After loading, column is washed with 10CV using equilibration solution. The elution of the captured crude insulin is performed using a gradient elution from 0-100% of 60% dipropylenglycol in 50 mM Glycine/50 mM Acetic acid buffer pH 3.5 in 20 CV at 1 ml/min. The fractions are collected and subjected to non-reducing SDS-PAGE analysis (FIG. 4).

FIG. 4 shows a non-reducing SDS-PAGE of dynamic capture raw insulin on P00446 (Column 1.1 ml diameter 10 mm) using ÄKTA FPLC system. 10 CV Equillibration with 25 mM Tris, 100 mM Arginine, pH 7. 30 ml of 1 mg/ml raw insulin are loaded with 1 ml/min flowrate. Elution in 20 CV from 0% till 100% 60 Vol. % DPG. [0143] Lanes: M . . . MW-Marker [0144] S . . . Start material [0145] FT . . . Flow Through

[0146] As shown in FIG. 4 the polystyrene beads P00446 adsorb insulin (5% purity) from the crude solution and dipropylenglycol solution can be applied to separate insulin fragments from aggregates and target achieving >40% purity.

Application of Polystyrene Particles for the Capture of Crude Protein pSCP194

[0147] For the following example Polystyrene (PS) particles P00446 are packed in a 10 mm diameter 12 mm long column using 20% ethanol 150 mM NaCl solution.

[0148] The packed column is equilibrated using 50 mM TRIS, 100 mM Arginine buffer pH 7.0 for at least 20 column volumes at 1 ml/min. The crude E. coli lysate containing protein pSCP194 is adjusted to pH 7.0 and 20 ml of obtained solution is directly loaded on the equilibrated column at 1 ml/min and the flow through fraction collected in a separate flask. After loading, the column is washed with 10CV using equilibration solution. The elution of the captured crude insulin is performed using a gradient elution from 0-100% of 60% dipropylenglycol in 50 mM Glycine/50 mM Acetic acid buffer pH 3.5 in 20 CV at 1 ml/min. The fractions are collected and subjected to non-reducing SDS-PAGE analysis (FIG. 5).

FIG. 5 shows a non-reducing SDS-PAGE of dynamic crude pSCP194 capture on P00446 (Column 1.1 ml diameter 10 mm).

[0149] As shown in FIG. 5 the polystyrene beads P00446 adsorb pSCP194 protein from the crude e. coli lysate solution and dipropylenglycol solution can be applied to elute the captured target achieving >80% purity.

Application of Polystyrene Particles for the Capture of Crude Insulin in Urea

[0150] For the following example Polystyrene (PS) particles P00446 are packed in a 10 mm diameter 12 mm long column using 20% ethanol 150 mM NaCl solution.

[0151] The packed column is equilibrated using 50 mM Glycine/50 mM acetic acid buffer pH 3.5 for at least 20 column volumes at 1 ml/min. The crude insulin solution containing aggregated insulin after incubation in 8M urea solution is diluted to 2M urea concentration using pure water adjusted to pH 3.5. 50 ml of obtained solution was directly loaded on the equilibrated column at 1 ml/min and the flow through fraction collected in a separate flask. After loading, column is washed with 10CV using equilibration solution. The elution of the captured crude insulin is performed using a gradient elution from 0-100% of 60% dipropylenglycol in 50 mM Glycine/50 mM Acetic acid buffer pH 3.5 in 20 CV at 1 ml/min. The fractions are collected and subjected to non-reducing SDS-PAGE analysis (FIG. 6). FIG. 6: shows a non-reducing SDS-PAGE of dynamic crude insulin capture on P00446 and PS02 (Column 1.1 ml diameter 10 mm).

[0152] As shown in FIG. 6 the polystyrene beads P00446 and PS02 adsorb insulin from the crude 2M urea containing solution and dipropylenglycol solution can be applied to elute the captured target achieving >90% purity (fractions A3-A4 and A11-A12).