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Method for detecting multispecific antibody light chain mispairing

a multi-specific antibody and light chain mispairing technology, applied in the field of multi-specific antibody light chain mispairing detection, can solve the problem of hampered light chain mispairing in a complete multi-specific antibody

Inactive Publication Date: 2017-04-27
F HOFFMANN LA ROCHE & CO AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The patent text describes a method of creating a humanized antibody by combining the specificity and affinity of a non-human antibody with human antibody sequences. This helps to reduce the immunogenicity of the antibody and maintain its effectiveness. The text also describes a way to alter the CH3 domains of a multispecific antibody using a technology called "knob-into-holes." This helps to increase the stability and yield of the antibody. Overall, the patent text provides technical methods for creating humanized antibodies with improved function and effectiveness.

Problems solved by technology

It has been found that for the determination of light chain mispairing in a multispecific antibody limited proteolysis of the (multispecific) antibody has to be performed prior to MS analysis as the determination of light chain mispairing in a complete multispecific antibody is hampered by isobaric mass interference.

Method used

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  • Method for detecting multispecific antibody light chain mispairing
  • Method for detecting multispecific antibody light chain mispairing
  • Method for detecting multispecific antibody light chain mispairing

Examples

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specific embodiments

[0314]1. Use of a limited digestion with a proteolytic enzyme of a multispecific antibody for the analysis of the multispecific antibody's light chain pairing.[0315]2. Use of a limited digestion with a proteolytic enzyme of a multispecific antibody for the determination of light chain mispairing in the multispecific antibody.[0316]3. Use of a limited digestion with a proteolytic enzyme of a multispecific antibody produced by a recombinant mammalian cell for the selection of a multispecific antibody producing mammalian cell.[0317]4. The use according to any one of embodiments 1 to 3, wherein the proteolytic enzyme is selected from the group consisting of Lys-C, Asp-N, Arg-C, Glu-C and chymotrypsin.[0318]5. The use according to embodiment 4, wherein the proteolytic enzyme is Lys-C.[0319]6. The use according to any one of embodiments 1 to 5, wherein the incubating is for 35 to 45 minutes.[0320]7. The use according to embodiment 6, wherein the incubating is for about 40 minutes.[0321]8....

example 1

[0394]Method for the Determination of Light Chain Mispairing of a Multispecific Antibody after Limited Proteolytic Digestion

[0395]The expected primary structures were analyzed by electrospray ionization mass spectrometry (ESI-MS) of the limited LysC digested CrossMabs. Advantageously the antibody has been deglycosylated in advance.

[0396]The VH / VL CrossMabs was deglycosylated with N-Glycosidase F in a phosphate or Tris or histidine buffer at 37° C. for up to 17 h at a protein concentration of 1 mg / mL and an antibody: enzyme ratio of 100:1. The limited Lys-C (Roche Diagnostics GmbH, Mannheim, Germany) digestions was performed with 100 μg deglycosylated VH / VL CrossMabs in a Tris buffer pH 8 at 37° C. for 40 min.

[0397]Prior to mass spectrometry the samples were desalted via HPLC on a Sephadex G25 column (GE Healthcare).

[0398]The total mass was determined via ESI-MS on a maXis 4G UHR-QTOF MS system (Bruker Daltonik) equipped with a TriVersa NanoMate source (Advion).

example 2

Method for the Determination of Light Chain Mispairing of a Tetravalent Bispecific Antibody

[0399]The tetravalent bispecific antibody was deglycosylated with N-Glycosidase F in a phosphate or Tris or histidine buffer at 37° C. for up to 17 h at a protein concentration of 1 mg / ml and an antibody:enzyme ratio of 100:1. The limited Lys-C(Roche Diagnostics GmbH, Mannheim, Germany) digestions was performed with 100 μg deglycosylated tetravalent bispecific antibody in a 100 mM citrate buffer pH 3.7 at 37° C. for 16 hours at a protein concentration of approx. 0.9 mg / mL and an antibody:enzyme ratio of 50:1.

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Abstract

Use of a limited digestion with a proteolytic enzyme of a multispecific antibody for the analysis of the multispecific antibody's light chain pairing.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application is a continuation of International Patent Application No. PCT / EP2015 / 057164, having an international filing date of Apr. 1, 2015, the entire contents of which are incorporated herein by reference, and which claims benefit under 35 U.S.C. §119 to European Patent Application No. 14163165.5, filed on Apr. 2, 2014 and European Patent Application No. 15155361.7, filed on Feb. 17, 2015.BACKGROUND OF THE INVENTION[0002]Herein is reported a method for the determination of light chain mispairing in a multispecific antibody preparation based on limited proteolytic digestion and ESI-MS analysis. The method can be used for quality control or cell line selection.[0003]In 1995 Yamaguchi et al. (J. Immunol. Meth. 181 (1995) 259-267) reported a method for proteolytic fragmentation with high specificity of mouse immunoglobulin G using lysyl endopeptidase, clostripain, metalloendopeptidase and V8 protease, whereby under the reaction condit...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): G01N33/68C12Q1/37
CPCG01N33/6848C12Q1/37G01N33/6857G01N33/6854
Inventor MOLHOJ, MICHAELKOENIG, MAXIMILIANELARRAILLET, VINCENTMONTAN, KATJAGRUENWALDER, BIANCA
Owner F HOFFMANN LA ROCHE & CO AG
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