Method for in vitro glycoengineering of antibodies

Abstract

Herein is reported a method for the enzymatic production of an antibody with a modified glycosylation in the Fc-region comprising the steps of incubating the antibody light chain affinity ligand-bound monoclonal antibody with a glycosylation in the Fc-region with a first enzyme for a time sufficient and under conditions suitable to modify the glycosylation of the Fc-region, recovering the antibody from the antibody light chain affinity ligand, incubating the recovered antibody in solution with a second enzyme for a time sufficient and under conditions suitable to modify the glycosylation of the Fc-region to a defined form, separating the antibody with the modified glycosylation in the Fc-region from the second enzyme in a cation exchange chromatography, and thereby producing the antibody with a modified glycosylation in the Fc-region.

Claims

1. A method for the enzymatic production of a glycosylation modified antibody comprising a) incubating a first antibody affinity ligand-bound monoclonal antibody with a glycosylation at an N-glycosylation site with a first enzyme and a first activated sugar residue for a time sufficient and under conditions suitable to modify the glycosylation at the N-glycosylation site thereby producing a first modified antibody, (b) recovering the first modified antibody from the antibody affinity ligand, (c) incubating the recovered first modified antibody in solution with a second enzyme and a second activated sugar residue for a time sufficient and under conditions suitable to modify the glycosylation at the N-glycosylation site of the first modified antibody thereby producing the glycosylation modified antibody, and (d) separating the glycosylation modified antibody from the second enzyme in a cation exchange chromatography, wherein the first enzyme is a galactosyltransferase and the second enzyme is a sialyltransferase.

2. The method of claim 1, wherein the cation exchange chromatography material has a matrix of cross-linked agarose with sulfopropyl cation exchange groups.

3. The method of claim 1, wherein the galactosyltransferase is β4GalT1.

4. The method of claim 1, wherein the sialyltransferase is ST6.

5. The method of claim 1, wherein the step of separating comprising, i) applying a solution comprising the first enzyme and second enzyme and the glycosylation modified antibody to a cation exchange chromatography material, ii) washing the cation exchange chromatography material with a wash solution to remove unbound compounds from the cation exchange chromatography material but without eluting the glycosylation modified antibody, iii) applying a first solution to the cation exchange chromatography material and thereby eluting the first enzyme from the cation exchange chromatography material, iv) applying a second solution to the cation exchange chromatography material and thereby eluting the glycosylation modified antibody from the cation exchange chromatography material, and v) applying a linear gradient to the cation exchange chromatography material and thereby eluting the second enzyme from the cation exchange chromatography material.

6. The method of claim 5, wherein the solution of step i) is a 2-(N-morpholino)ethanesulfonic acid (MES) buffered solution with a pH value from pH 5.0 to pH 6.5, the wash solution of step ii) is a 2-(N-morpholino)ethanesulfonic acid (MES) buffered solution with a pH value from pH 5.0 to pH 6.5, the first solution of step iii) is a tris(hydroxymethyl)aminomethane (TRIS) buffered solution with a pH value from pH 6.6 to pH 8.0, the second solution of step iv) is a 2-(N-morpholino)ethanesulfonic acid (MES) buffered solution with a pH value from pH 5.0 to pH 6.5 comprising 75 mM to 125 mM sodium chloride (NaCl), the linear gradient is from the solution of step iv) to a 2-(N-morpholino)ethanesulfonic acid (MES) buffered solution with a pH value from pH 5.0 to pH 6.5 comprising 750 mM to 1250 mM sodium chloride (NaCl).

7. A method for producing a glycosylation modified antibody comprising: applying a solution comprising an antibody with a glycosylation at an N-glycosylation site to an antibody affinity ligand bound to a solid phase, enzymatically modifying a glycosylation at an N-glycosylation site of the antibody by applying a solution comprising a first glycosylation modifying enzyme and a first activated sugar residue for a time sufficient and under conditions suitable for enzymatic modification to the ligand-bound antibody thereby producing a first modified antibody, recovering the first modified antibody from the antibody affinity ligand, and incubating the recovered first modified antibody in solution with a second enzyme and a second activated sugar residue for a time sufficient and under conditions, wherein the first enzyme is a galactosyltransferase and the second enzyme is a sialyltransferase.

8. The method of claim 1, wherein the antibody is a bivalent monospecific antibody or a bivalent bispecific antibody or an antibody Fab fragment.

9. The method of claim 1, wherein the antibody is a chimeric or humanized or human antibody.

10. The method of claim 7, wherein the antibody is a monoclonal antibody.

11. The method of claim 1, wherein the antibody is an antibody of the human IgG1 or IgG4 subclass.

12. The method of claim 1, wherein the N-glycosylation site is an Fc-region N-glycosylation site at asparagine residue 297 (numbering according to Kabat) or is a Fab region N-glycosylation site.

13. The method of claim 1 further comprising the steps of: recovering the first enzyme used in incubating step a) thereby producing a recycled first enzyme; recovering the second enzyme used in incubating step c) thereby producing a recycled second enzyme; and incubating a second antibody that has a glycosylation at an N-glycosylation site with at least one of the recycled first enzyme or the recycled second enzyme for a time sufficient and under conditions suitable to modify the glycosylation at the N-glycosylation site of the second antibody.

14. The method of claim 1 further comprising the steps of: recovering the first enzyme used in incubating step a) thereby producing a recycled first enzyme; recovering the second enzyme used in incubating step c) thereby producing a recycled second enzyme; and repeating incubating step a) with the recycled first enzyme, and repeating incubating step c) with the recycled second enzyme.

15. The method of claim 1, wherein the step of separating comprising, i) applying a solution comprising the second enzyme and the glycosylation modified antibody to a cation exchange chromatography material, ii) applying an eluting solution to the cation exchange chromatography material and thereby eluting the glycosylation modified antibody from the cation exchange chromatography material, and iii) applying a linear gradient to the cation exchange chromatography material and thereby eluting the second enzyme from the cation exchange chromatography material.

16. The method of claim 1, wherein the antibody affinity ligand is a light chain affinity ligand.

17. The method of claim 1, wherein the antibody affinity ligand is an Fc region affinity ligand.

18. A method for the enzymatic production of an antibody with a modified glycosylation at an N-glycosylation site, comprising a) incubating the antibody light chain affinity ligand-bound monoclonal antibody with a glycosylation at an N-glycosylation site with a first enzyme and a first activated sugar residue for a time sufficient and under conditions suitable to modify the glycosylation at the N-glycosylation site thereby producing a first modified antibody, b) recovering the first enzyme and producing thereby a recycled first enzyme, c) recovering the first modified antibody from the antibody light chain affinity ligand, d) incubating the recovered first modified antibody in solution with a second enzyme and a second activated sugar residue for a time sufficient and under conditions suitable to modify the glycosylation at the N-glycosylation site of the first modified antibody thereby producing the glycosylation modified antibody; e) separating the glycosylation modified antibody from the second enzyme in a cation exchange chromatography, optionally recovering the second enzyme and producing thereby a recycled second enzyme, and f) repeating step a) with the first recycled enzyme of step b) at least once and repeating step d) with the second recycled enzyme of step e) at least once, wherein the first enzyme is a galactosyltransferase and the second enzyme is a sialyltransferase.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1 SDS-page of the eluted fractions of the S-Sepharose separation as reported herein and according to Example 5.

EXAMPLES

(2) GalT reaction solution (5 mM MnCl2, 10 mM UDP-Gal, 100 mM MES, 0.05 mg/ml GalT, pH 6.5)

(3) 153 mg UDP-Gal (MW=610.27 g/mol)

(4) 32 mg MnCl2 (MW=125.84 g/mol)

(5) single use: 460 μL GalT (c=5.43 mg/mL; 10 μg/2 mg antibody.fwdarw.10 μg in 300 μL=0.033 mg/ml)

(6) multiple use: 460 μL GalT (c=5.43 mg/mL; 15 μg/1 mg AK.fwdarw.15 μg in 300 μL=0.05 mg/mL)

(7) in 100 mM MES buffer pH 6.5

(8) ST6 reaction solution (0.1 mM ZnCl2, 200 nM AP, 50 mM MES, 1.7 mg/ml CMP-NANA, 0.7 mg/ml ST6, pH 6.5)

(9) 50 μL ZnCl (100 mM solution: 13.6 mg in 1 mL 50 mM MES)

(10) 28 μL alkaline phosphatase (AP) (c=20 mg/mL, MW=56,000 g/mol)

(11) single use: 167 mg CMP-NANA (1000 μg/2 mg antibody.fwdarw.1000 μg pro 300 μL=3.34 mg/mL)

(12) multiple use: 83.5 mg CMP-NANA (500 μg/1 mg AK.fwdarw.500 μg pro 300 μL=1.67 mg/mL)

(13) single use: 6 mL ST6 (c=5.45 mg/mL, target: 200 μg in 300 μL (2 mg AK)=0.67 mg/mL)

(14) multiple use: 6 mL ST6 (c=5.45 mg/mL, target: 200 μg in 300 μL (1 mg AK)=0.67 mg/mL)

(15) in 50 mM MES buffer pH 6.5

(16) Buffers:

(17) Regeneration buffer 1 (0.1 M phosphoric acid)

(18) Regeneration buffer 2 (3 M Guanidine-HCl)

(19) Equilibration buffer (25 mM Tris, 25 mM NaCl, 5 mM EDTA, pH7.1)

(20) Wash buffer 1 (100 mM MES, pH 6.5): 21.3 mg MES in 1000 mL H2O, pH 6.5 (adjusted with 50% (w/v) NaOH)

(21) Wash buffer 2 (1 M Tris, pH 7.2)

(22) Wash buffer 3 (50 mM MES, pH 6.5): Wash buffer 100 mM MES 1:1 with distilled H2O

(23) Elution buffer Kappa select (0.1 M glycine, pH 2.7): 750 mg glycine in 100 mL H2O, pH 2.7 (adjusted with 25% (w/v) HCl)

(24) Elution buffer protein A (25 mM Na-citrate, pH 2.8)

Example 1

(25) Galactosylation of Bulk Material on Column

(26) regenerate, equilibrate and wash protein A respectively Kappa select columns by applying 2 column volumes regeneration buffer 1, 10 column volumes equilibration buffer and 4 column volumes wash buffer 1 apply 2 mg of IgG (bulk material) onto the column wash with 10 column volumes wash buffer 1 apply 2 mL galactosylation reaction solution (with 0.033 mg/ml GalT), let 0.8 mL flow through incubate respectively at 25° C. (2, 7 or 24 h) wash with 8 column volumes wash buffer 1 elute with the respective elution buffer (2 column volumes for Protein A; 8 column volumes for kappa select) and use 1 M Tris buffer (pH 9.0) for pH adjustment

Example 2

(27) Sialylation of IgG1 Bulk Material on Column (Protein A)

(28) regenerate, equilibrate and wash protein A resp. kappa select columns by applying 2 column volumes regeneration buffer 1, 10 column volumes equilibration buffer and 10 column volumes wash buffer 3 apply 2 mg of IgG (bulk material) onto the column apply 2 mL sialylation reaction solution (3.3 mg/ml instead of 1.7 mg/ml CMP-NANA, +/−AP), let 0.8 mL flow through incubate respectively at 37° C. (2, 7, 24 or 48 hours) and 25° C. (48 h) wash with 4 column volumes wash buffer 3 elute with 2 column volumes of Elution buffer (sodium citrate) and use 1 M Tris buffer (pH 9.0) for pH adjustment
Sialylation of IgG1 bulk material on column (Kappa select) regenerate, equilibrate and wash Protein A resp. kappa Select columns by applying 2 column volumes equilibration buffer, 3 column volumes regeneration buffer 2, 4 column volumes equilibration buffer and 2 column volumes wash buffer 3 apply 2 mg of IgG (bulk material) onto the column wash with 3 column volumes wash buffer 3 apply 2 mL sialylation reaction solution (3.3 mg/ml CMP-NANA, +/−AP), let 0.8 mL flow through incubate respectively at 37° C. (2, 7, and 24 h) and at 25° C. (24 h) wash with 3 column volumes wash buffer 3 elute with 8 column volumes of Elution buffer Kappa select and use 1 M Tris buffer (pH 9.0) for pH adjustment

Example 3

(29) Sequential Galactosylation and Sialylation of Cell Culture Supernatant

(30) regenerate and equilibrate protein A respectively Kappa select columns by applying 2 column volumes regeneration buffer 1, 10 column volumes equilibration buffer apply 1 mg of IgG (in supernatant) onto the column wash with 10 column volumes equilibration buffer, then 2 column volumes wash buffer 2 and 6 column volumes wash buffer 1 apply 2 mL galactosylation reaction solution, let 0.8 mL flow through incubate at 25° C. for about 6 to 24 h (to allow for sufficient galactosylation) wash with 8 column volumes wash buffer 1, 10 column volumes equilibration buffer, 2 column volumes wash buffer 2 and 6 column volumes wash buffer 3 apply 2 mL sialylation reaction solution, let 0.8 mL flow through incubate (e.g. 25° C. respectively for 2, 7 or 24 h or even longer) wash with 8 column volumes wash buffer 1 elute with the respective elution buffer (2 column volumes for Protein A; 8 column volumes for Kappa select) and use 1 M Tris buffer (pH 9.0) for pH adjustment

Example 4

(31) Sequential Galactosylation and Sialylation of Bulk Material

(32) regenerate, equilibrate and wash protein A respectively Kappa Select columns by applying 2 column volumes regeneration buffer 1, 10 column volumes equilibration buffer and 4 column volumes wash buffer 1 apply 1 mg of IgG (bulk material) onto the column wash with 10 column volumes wash buffer 1 apply 2 mL galactosylation reaction solution, let 0.8 mL flow through incubate at 25° C. for about 6 to 24 h (to allow for sufficient galactosylation) wash with 8 column volumes wash buffer 1, 10 column volumes equilibration buffer, 2 column volumes wash buffer 2 and 6 column volumes wash buffer 3 apply 2 mL sialylation reaction solution, let 0.8 mL flow through incubate (e.g. 25° C. respectively for 2, 7 or 24 h or even longer) wash with 8 column volumes wash buffer 1 elute with the respective elution buffer (2 column volumes for protein A; 8 column volumes for Kappa select) and use 1 M Tris buffer (pH 9.0) for pH adjustment

Example 5

(33) In Solution Galactosylation and Sialylation and Enzyme Recovery

(34) incubation of 25 mg antibody, 2.5 mg galactosyltransferase and 2.5 mg sialyltransferase in 10 mL 50 mM MES buffer pH 6.4 after reaction application of reaction solution to an S-Sepharose column (0.5×10 cm) equilibrated with 50 mM MES pH 6.4 washing with 5 column volumes of 50 mM MES pH 6.4 to remove unbound material washing of column with 20 column volumes 40 mM Tris buffer pH 7.4 and thereby eluting the galactosyltransferase re-equilibrated with 50 mM MES pH 6.4 eluting the antibody with a buffered solution comprising 30 mM MES pH 5.6 and 95 mM NaCl (40 column volumes) eluting the sialyltransferase with a linear gradient over 10 column volumes to 50 mM MES pH 6.4 and 1 M NaCl fractions containing the target enzymes or the humanized antibody were pooled and concentrated using ultrafiltration devices (Amicon Ultra-15, 10 kDa)

Example 6

(35) Enzyme Re-Use Testing

(36) galactosyltransferase: incubation of 500 μg antibody in 78.5 μL reaction buffer (100 mM MES, 10 mM UDP-Gal, 5 mM MnCl2, pH 6.5) with 2.5 μg galactosyltransferase at 37° C. for a defined time period, e.g. 6.5 h or 24 h sialyltransferase: incubation of 500 μg antibody in 61.8 μl water, 250 μg CMP-NANA dissolved in water, 50 μg sialyltransferase, 200 nM alkaline phosphatase, 0.1 mM ZnCl2 at 37° C. for a defined time period, e.g. 6.5 or 24 hours analysis of the galactosylation by qTOF-ESMS: denaturation and reduction of the sample (approx. 250 μg antibody, 4 M guanidinium, TCEP); buffer exchange to 20% acetonitrile with 1% formic acid; ESMS analytics