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In vitro glycoengineering of antibodies

A technology of antibody and glycosylation, applied in peptide preparation methods, chemical instruments and methods, organic chemistry, etc., can solve problems such as changes in the distribution of glycan species

Pending Publication Date: 2019-08-02
F HOFFMANN LA ROCHE & CO AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] However, antibodies produced by current expression systems (e.g., CHO cells) exhibit heterogeneous glycan patterns, resulting in variations in the distribution of different glycan species within different batches of produced antibodies

Method used

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  • In vitro glycoengineering of antibodies
  • In vitro glycoengineering of antibodies
  • In vitro glycoengineering of antibodies

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0334] On-column galactosylation of bulk material

[0335] Regenerate, equilibrate and wash Protein A or Kappa select columns by applying 2 column volumes of Regeneration Buffer 1, 10 column volumes of Equilibration Buffer and 4 column volumes of Wash Buffer 1

[0336] · Apply 2 mg IgG to the column (batch material)

[0337] Wash with 10 column volumes of Wash Buffer 1

[0338] Apply 2 mL of galactosylation reaction solution (containing 0.033 mg / ml GalT) and let 0.8 mL flow through

[0339] ·Incubate at 25°C (2, 7 or 24 hours)

[0340] Wash with 8 column volumes of Wash Buffer 1

[0341] Elute with the corresponding elution buffer (for protein A, 2 column volumes; for Kappa select, 8 column volumes) and adjust the pH using 1M Tris buffer (pH 9.0)

Embodiment 2

[0343] On-column sialylation of IgG1 bulk material (Protein A)

[0344] Regenerate, equilibrate and wash the Protein A column by applying 2 column volumes of Regeneration Buffer 1, 10 column volumes of Equilibration Buffer and 10 column volumes of Wash Buffer 3

[0345] · Apply 2 mg IgG to the column (batch material)

[0346] Apply 2 mL of sialylation reaction solution (3.3 mg / ml CMP-NANA, + / -AP) and let 0.8 mL flow through

[0347] Incubate at 37°C (2, 7, 24 or 48 hours) and 25°C (48 hours), respectively

[0348] Wash with 4 column volumes of Wash Buffer 3

[0349] ·Elute with 2 column volumes of elution buffer protein A (sodium citrate) and adjust the pH using 1M Tris buffer (pH9.0)

[0350] On-column sialylation of IgG1 bulk material (Kappa select)

[0351] Regenerate, equilibrate and wash the kappa Select column by applying 2 column volumes equilibration buffer, 3 column volumes regeneration buffer 2, 4 column volumes equilibration buffer and 2 column volumes wash buff...

Embodiment 3

[0359] Sequential galactosylation and sialylation of cell culture supernatants

[0360] • Regenerate and equilibrate the Protein A or Kappa Select column by applying 2 column volumes of regeneration buffer 1 and 10 column volumes of equilibration buffer.

[0361] Apply 1 mg IgG (in supernatant) to the column

[0362] Wash with 10 column volumes of Equilibration Buffer, then 2 column volumes of Wash Buffer 2 and 6 column volumes of Wash Buffer 1

[0363] Apply 2 mL of galactosylation reaction solution and let 0.8 mL flow through

[0364] ·Incubate at 25°C for approximately 6 to 24 hours (to allow sufficient galactosylation)

[0365] Wash with 8 column volumes of Wash Buffer 1, 10 column volumes of Equilibration Buffer, 2 column volumes of Wash Buffer 2 and 6 column volumes of Wash Buffer 3

[0366] Apply 2 mL of sialylation reaction solution and let 0.8 mL flow through

[0367] · Incubation (eg 25°C for 2, 7 or 24 hours respectively or even longer)

[0368] Wash with 8 col...

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Abstract

Herein is reported a method for producing an antibody comprising the steps of: forming an antibody-antibody light chain affinity ligand complex, wherein the antibody light chain affinity ligand is immobilized on a solid phase, by applying a solution comprising the antibody to the immobilized antibody light chain affinity ligand, and incubating the complex formed in the previous step with one or more enzymes to modify the glycosylation of the antibody, thereby producing the antibody.

Description

[0001] The present invention belongs to the field of antibody engineering. In vitro glycoengineering methods for glycosylation in antibody Fc regions are reported in more detail herein. Background technique [0002] IgG is the most abundant antibody isotype, with IgGl antibodies being the subclass exhibiting the most significant degree and array of effector functions. IgG1 antibodies are the most commonly used antibodies in immunotherapy, where ADCC and CDC are generally considered important. Within the antibody structure, the CH2 domain as well as the IgG hinge region play a major role in Fc-mediated antibody effector functions. Each CH2 domain contains a conserved glycosylation site (numbered according to Kabat's EU Index) at the asparagine residue at approximately position 297, to which the glycan moiety is covalently bound (Wright, A. and Morrison, S.L., TIBTECH 15 (1997) 26-32). In mature IgG molecules, glycans are buried between CH2 domains, affecting the tertiary str...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/00C12P21/08C07K16/00
CPCC12P21/00C07K16/00C07K2317/21C07K2317/24C07K2317/41C07K2317/55C07K1/22C07K2317/52C12P21/005
Inventor R·法尔肯施泰因H·沃尔克S·马利克M·托曼M·弗赖赫尔范罗曼I·格伦特R·多恩M·欣加尔
Owner F HOFFMANN LA ROCHE & CO AG