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.

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

3. The method according to claim 1, wherein the first enzyme is a galactosyltransferase and the second enzyme is a sialyltransferase.

4. The method according to claim 3, wherein the galactosyltransferase is 4GalT1.

5. The method according to claim 3, wherein the sialyltransferase is ST6.

6. The method according to 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.

7. The method according to claim 6, 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 about 75 mM to about 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 about 750 mM to about 1250 mM sodium chloride (NaCl).

8. A method for producing a glycosylation modified antibody comprising: applying a solution comprising an antibody with a glycosylation at the N-glycosylation site to an antibody affinity ligand bound to a solid phase, enzymatically modifying the glycosylation at the 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 the enzymatic modification to the ligand-bound antibody thereby producing a first modified antibody, recovering the first modified antibody from the antibody affinity ligand, 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 separating the glycosylation modified antibody from the second enzyme in a cation exchange chromatography.

9. The method according to claim 1, wherein the antibody is a bivalent monospecific antibody or a bivalent bispecific antibody or an antibody Fab fragment.

10. The method according to claim 1, wherein the antibody is a chimeric or humanized or human antibody.

11. The method according to claim 8, wherein the antibody is a monoclonal antibody.

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

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

14. A glycosylation modified antibody produced with the method according to claim 1.

15. A pharmaceutical formulation comprising the glycosylation modified antibody according to claim 14 and a pharmaceutically acceptable carrier.

16. The glycosylation modified antibody of claim 14 for use as a medicament.

17. The method according to claim 1 further including 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.

18. The method according to claim 17, wherein the first enzyme is a galactosyltransferase and the second enzyme is a sialyltransferase.

19. The method according to claim 1 further including 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.

20. The method according to 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.

Description

DESCRIPTION OF THE FIGURES

[0273] FIG. 1 SDS-page of the eluted fractions of the S-Sepharose separation as reported herein and according to Example 5.

EXAMPLES

[0274] GalT reaction solution (5 mM MnCl2, 10 mM UDP-Gal, 100 mM MES, 0.05 mg/ml GalT, pH 6.5)
153 mg UDP-Gal (MW=610.27 g/mol)
32 mg MnCl2 (MW=125.84 g/mol)
single use: 460 L GalT (c=5.43 mg/mL; 10 g/2 mg antibody-->10 g in 300 L=0.033 mg/ml)
multiple use: 460 L GalT (c=5.43 mg/mL; 15 g/1 mg AK-->15 g in 300 L=0.05 mg/mL)
in 100 mM MES buffer pH 6.5
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)
50 L ZnCl (100 mM solution: 13.6 mg in 1 mL 50 mM MES)
28 L alkaline phosphatase (AP) (c=20 mg/mL, MW=56,000 g/mol)
single use: 167 mg CMP-NANA (1000 g/2 mg antibody-->1000 g pro 300 L=3.34 mg/mL)
multiple use: 83.5 mg CMP-NANA (500 g/1 mg AK-->500 g pro 300 L=1.67 mg/mL)
single use: 6 mL ST6 (c=5.45 mg/mL, target: 200 g in 300 L (2 mg AK)=0.67 mg/mL)
multiple use: 6 mL ST6 (c=5.45 mg/mL, target: 200 g in 300 L (1 mg AK)=0.67 mg/mL)
in 50 mM MES buffer pH 6.5

Buffers:

[0275] Regeneration buffer 1 (0.1 M phosphoric acid)
Regeneration buffer 2 (3 M Guanidine-HCl)
Equilibration buffer (25 mM Tris, 25 mM NaCl, 5 mM EDTA, pH7.1)
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) Wash buffer 2 (1 M Tris, pH 7.2)
Wash buffer 3 (50 mM MES, pH 6.5): Wash buffer 100 mM MES 1:1 with distilled H2O
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)
Elution buffer protein A (25 mM Na-citrate, pH 2.8)

Example 1

Galactosylation of Bulk Material on Column

[0276] 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 [0277] apply 2 mg of IgG (bulk material) onto the column [0278] wash with 10 column volumes wash buffer 1 [0279] apply 2 mL galactosylation reaction solution (with 0.033 mg/ml GalT), let 0.8 mL flow through [0280] incubate respectively at 25 C. (2, 7 or 24 h) [0281] wash with 8 column volumes wash buffer 1 [0282] 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

Sialylation of IgG1 Bulk Material on Column (Protein A)

[0283] 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 [0284] apply 2 mg of IgG (bulk material) onto the column [0285] 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 [0286] incubate respectively at 37 C. (2, 7, 24 or 48 hours) and 25 C. (48 h) [0287] wash with 4 column volumes wash buffer 3 [0288] 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) [0289] 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 [0290] apply 2 mg of IgG (bulk material) onto the column [0291] wash with 3 column volumes wash buffer 3 [0292] apply 2 mL sialylation reaction solution (3.3 mg/ml CMP-NANA, +/AP), let 0.8 mL flow through [0293] incubate respectively at 37 C. (2, 7, and 24 h) and at 25 C. (24 h) [0294] wash with 3 column volumes wash buffer 3 [0295] elute with 8 column volumes of Elution buffer Kappa select and use 1 M Tris buffer (pH 9.0) for pH adjustment

Example 3

Sequential Galactosylation and Sialylation of Cell Culture Supernatant

[0296] regenerate and equilibrate protein A respectively Kappa select columns by applying 2 column volumes regeneration buffer 1, 10 column volumes equilibration buffer [0297] apply 1 mg of IgG (in supernatant) onto the column [0298] wash with 10 column volumes equilibration buffer, then 2 column volumes wash buffer 2 and 6 column volumes wash buffer 1 [0299] apply 2 mL galactosylation reaction solution, let 0.8 mL flow through [0300] incubate at 25 C. for about 6 to 24 h (to allow for sufficient galactosylation) [0301] 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 [0302] apply 2 mL sialylation reaction solution, let 0.8 mL flow through [0303] incubate (e.g. 25 C. respectively for 2, 7 or 24 h or even longer) [0304] wash with 8 column volumes wash buffer 1 [0305] 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

Sequential Galactosylation and Sialylation of Bulk Material

[0306] 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 [0307] apply 1 mg of IgG (bulk material) onto the column [0308] wash with 10 column volumes wash buffer 1 [0309] apply 2 mL galactosylation reaction solution, let 0.8 mL flow through [0310] incubate at 25 C. for about 6 to 24 h (to allow for sufficient galactosylation) [0311] 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 [0312] apply 2 mL sialylation reaction solution, let 0.8 mL flow through [0313] incubate (e.g. 25 C. respectively for 2, 7 or 24 h or even longer) [0314] wash with 8 column volumes wash buffer 1 [0315] 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

In Solution Galactosylation and Sialylation and Enzyme Recovery

[0316] incubation of 25 mg antibody, 2.5 mg galactosyltransferase and 2.5 mg sialyltransferase in 10 mL 50 mM MES buffer pH 6.4 [0317] after reaction application of reaction solution to an S-Sepharose column (0.510 cm) equilibrated with 50 mM MES pH 6.4 [0318] washing with 5 column volumes of 50 mM MES pH 6.4 to remove unbound material [0319] washing of column with 20 column volumes 40 mM Tris buffer pH 7.4 and thereby eluting the galactosyltransferase [0320] re-equilibrated with 50 mM MES pH 6.4 [0321] eluting the antibody with a buffered solution comprising 30 mM MES pH 5.6 and 95 mM NaCl (40 column volumes) [0322] eluting the sialyltransferase with a linear gradient over 10 column volumes to 50 mM MES pH 6.4 and 1 M NaCl [0323] fractions containing the target enzymes or the humanized antibody were pooled and concentrated using ultrafiltration devices (Amicon Ultra-15, 10 kDa)

Example 6

Enzyme Re-Use Testing

[0324] galactosyltransferase: incubation of 500 g antibody in 78.5 L reaction buffer (100 mM MES, 10 mM UDP-Gal, 5 mM MnCl2, pH 6.5) with 2.5 g galactosyltransferase at 37 C. for a defined time period, e.g. 6.5 h or 24 h [0325] sialyltransferase: incubation of 500 g antibody in 61.8 l water, 250 g CMP-NANA dissolved in water, 50 g sialyltransferase, 200 nM alkaline phosphatase, 0.1 mM ZnCl2 at 37 C. for a defined time period, e.g. 6.5 or 24 hours [0326] analysis of the galactosylation by qTOF-ESMS: denaturation and reduction of the sample (approx. 250 g antibody, 4 M guanidinium, TCEP); buffer exchange to 20% acetonitrile with 1% formic acid; ESMS analytics