Anionic exchange-hydrophobic mixed mode

Abstract

Solid supports and ligands are provided for purification of biomolecules by mixed-mode anion exchange-hydrophobic chromatography. Compositions can have the formula Support-(X)N(R1, R2)-R3-L-Ar, or a salt thereof, wherein: Support is a chromatographic solid support; X is a spacer or absent; R1 and R2 are each selected from hydrogen and an alkyl comprising 1-6 carbons; R3 is an alkyl comprising 1-6 carbons or a cyclo alkyl comprising 1-6 carbons; L is NR4, O, or S; wherein R4 is hydrogen or an alkyl comprising 1-6 carbons; and Ar is an aryl. Methods are also provided for using solid supports and ligands to purify biomolecules such as monomeric antibodies.

Claims

1. A chromatographic solid support linked to a ligand having the formula: Support-(X)N.sup.+(R.sup.1, R.sup.2)R.sup.3-LAr, wherein the positively-charged nitrogen is in salt form, and wherein X is a spacer or absent; R.sup.1 and R.sup.2 are each selected from hydrogen and an alkyl comprising 1-6 carbons; R.sup.3 is an alkyl comprising 1-6 carbons or a cyclo alkyl comprising 1-6 carbons; L is NR.sup.4, O, or S; wherein R.sup.4 is hydrogen or an alkyl comprising 1-6 carbons; and Ar is an aryl.

2. The chromatographic solid support of claim 1, wherein R.sub.1 and R.sub.2 are hydrogen.

3. The chromatographic solid support of claim 1, wherein R.sup.3 is an alkyl comprising 1-4 carbons.

4. The chromatographic solid support of claim 1, wherein R.sup.3 is an alkyl comprising 2 carbons.

5. The chromatographic solid support of claim 1, wherein the aryl is a phenyl.

6. The chromatographic solid support of claim 1, wherein the aryl is a substituted phenyl.

7. The chromatographic solid support of claim 6, wherein the substituted phenyl is an alkyl-substituted phenyl.

8. The chromatographic solid support of claim 1, wherein R.sub.1 and/or R.sub.2 are an alkyl comprising 1-6 carbons and Ar is an alkyl-substituted phenyl.

9. The chromatographic solid support of claim 1, wherein the ligand is N-phenylethylenediamine or a salt thereof.

10. The chromatographic solid support of claim 1, wherein the ligand is 2-phenoxyethylamine or a salt thereof.

11. The chromatographic solid support of claim 1, wherein X is absent.

12. The chromatographic solid support of claim 1, wherein X is a spacer.

Description

EXAMPLE

Example 1

(1) Generation of Matrix Comprising N-phenylethylendiamine

(2) UNOsphere Diol (20 mL), a copolymer of 3-allyloxy-1,2-propanediol and vinyl pyrrolidinone, crosslinked with N,N-methylenebisacrylamide and with a diol density of 200-300 mol/mL, was used in the form of spherical beads. The beads were suspended in 20 mL of either 0.1 M sodium acetate or water. Sodium periodate was added to a concentration within the range of 50 to 100 mM, and the resulting mixture was incubated at room temperature (approximately 70 F. (21 C.)) for 3-24 hours. The reaction resulted in conversion of the diol groups to aldehyde groups in the range of 150-250 mol/mL. The resulting aldehyde-functionalized resin was transferred to a 20 mL column where it was washed with 100 mL of water.

(3) Twenty milliliters of UNOsphere aldehyde resin was then suspended in 20 ml of 0.20 M sodium phosphate containing 0.6 g of N-phenylethylenediamine at pH 7.0. After this mixture was incubated (shaking, 200 rpm) at room temperature for 15 minutes, 200 mg NaBH.sub.3CN was then added and the reaction was allowed to continue for 3-20 hours. The N-phenylethylenediamine concentration in the reaction was determined to be in the range of 25-200 mM. At the end of the reaction, resin was transferred to a 20 ml column, washed with 3 CV of water followed by 1-2 CV of 0.1N HCl, and then washed with 5 CV water. The N-phenylethylenediamine ligand density was in the range of 25-100 mol/ml.

Example 2

(4) Use of Matrix Comprising N-phenylethylendiamine: pH 8.5-4.5 Gradient

(5) The resin with the N-phenylethylenediamine ligand (generated as described above) was packed into a 7 mm (i.d.)5.5 cm column and equilibrated with 20 mM Tris-HCl buffer containing 300 mM NaCl, pH 8.5. 500 l of 6.0 mg/ml solution of a monoclonal IgG antibody containing 5-10% aggregated antibodies, was applied to the column at a flow rate of 2 ml/minute. The antibody was eluted in a 10 ml gradient for equilibration buffer to elution buffer of 20 mM sodium acetate containing 150 mM NaCl at pH 4.5, followed with 30 ml isocratic elution with elution buffer. The collected antibody elution fractions were analyzed by size exclusion high performance liquid chromatography (HPLC-SEC) to determine the content of aggregated antibody in the elution fractions. No antibody aggregates were detected in the antibody elution fractions.

(6) A similar experiment was performed using a Capto Adhere (ligand: N-benzyl-N-methyl ethanolamine) column. Aggregated antibody species were found in all fractions derived from the Capto Adhere column.

Example 3

(7) Use of Matrix Comprising N-phenylethylendiamine: pH 7.0-4.5 Gradient

(8) A similar experiment to the one described above was conducted using an equilibration buffer at pH 7.0 with 20 mM sodium phosphate buffer containing 300 mM NaCl. No aggregated antibody was detected in the monoclonal antibody fractions.

(9) A similar experiment was performed using a Capto Adhere (ligand: N-benzyl-N-methyl ethanolamine) column. Aggregated antibody species were found in fractions derived from the Capto Adhere column.

Example 4

(10) Generation of Matrix Comprising 2-phenoxyethylamine

(11) A matrix comprising the ligand 2-phenoxyethylamine was generated using reaction conditions as provided in Example 1, replacing N-phenylethylenediamine with 2-phenoxyethylamine. 2-phenoxyethylamine ligand density was 49 mol/ml.

Example 5

(12) Use of Matrix Comprising 2-phenoxyethylamine: pH 7.0-4.5

(13) A matrix comprising the ligand 2-phenoxyethylamine was used to purify antibodies similar to the method described in Example 2.

(14) The resin with the 2-phenoxyethylamine ligand was packed into a 7 mm (i.d.)5.5 cm column and equilibrated with 20 mM sodium phosphate buffer containing 300 mM NaCl, pH 7.0. 500 l of 6.0 mg/ml solution of a monoclonal IgG antibody containing 5-10% aggregated antibodies, was applied to the column at a flow rate of 2 ml/minute. The antibody was eluted in a 10 ml gradient for equilibration buffer to elution buffer of 20 mM sodium acetate containing 150 mM NaCl at pH 4.5, followed with 30 ml isocratic elution with elution buffer. The collected antibody elution fractions were analyzed by size exclusion high performance liquid chromatography (HPLC-SEC) to determine the content of aggregated antibody in the elution fractions. No antibody aggregates were detected in the antibody elution fractions.

Example 6

(15) Use of Matrix Comprising 2-phenoxyethylamine: pH 4.5 Flow-Through Mode

(16) A matrix comprising the ligand 2-phenoxyethyl amine was used to purify antibodies similar to the method described in Example 2, except that the antibody sample was applied to the column using an equilibration buffer at pH 4.5.

(17) The resin with the 2-phenoxyethylamine ligand was packed into a 7 mm (i.d.)5.5 cm column and equilibrated with 20 mM sodium acetate containing 75 mM NaCl at pH 4.5. 750 l of 2.0 mg/ml solution of a monoclonal IgG antibody containing 15-20% aggregated antibodies, was applied to the column at a flow rate of 2 ml/minute. The antibody flowed-through the column. The column was washed in a 10 ml gradient for equilibration buffer to elution buffer of 20 mM sodium acetate at pH 4.5, followed with 10 ml isocratic elution with elution buffer. The collected antibody in the flow-through fractions was analyzed by size exclusion high performance liquid chromatography (HPLC-SEC) to determine the content of aggregated antibody. No antibody aggregates were detected in the antibody flow-through fractions.

(18) In the claims appended hereto, the term a or an is intended to mean one or more The term comprise and variations thereof such as comprises and comprising, when preceding the recitation of a step or an element, are intended to mean that the addition of further steps or elements is optional and not excluded. All patents, patent applications, and other published reference materials cited in this specification are hereby incorporated herein by reference in their entirety.