Method of stabilizing protein

Inactive Publication Date: 2005-08-04
CHUGAI PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
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Benefits of technology

[0006] Conventionally, methods to suppress deamidation by altering amino acids in proteins is a useful technique to improve the value, quality and such of pharmaceuticals. Such methods increase the option in the formulation of pharmaceutical preparations, and thus facilitate application of the proteins to various drug forms and administration routes. Therefore, the purpose of the present invention is to provide a method to suppress deamidation of asparagine without influencing the activity of proteins, particularly antibodies.
[0007] The present inventors diligently conducted research focusing on anti-human TF antibody, which use as a pharmaceutical is expected in the art. The antibody was used as an example of a protein for developing a method to suppress deamidation of asparagine without affecting the protein activity. First, a mutated anti-human TF antibody was expressed as a recombinant wherein asparagine that may be deamidated yet e

Problems solved by technology

Reduction in protein activity is a very important pro

Method used

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Examples

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example 1

Measurement of Binding and Neutralizing Activities of Anti-Human TF Antibody Asn54 Substitution Mutant with TF

[0098] Humanized antibody against human tissue factor (TF) described in WO 99 / 51743 is expected to suppress thrombus formation without suppressing the extrinsic blood coagulation reaction through the inhibition of the TF mediated Factor X activation in the intrinsic blood coagulation reaction. This anti-human TF antibody contains humanized heavy chain version i (SEQ ID NO: 25, FIG. 1) and humanized light chain version b2 (SEQ ID NO: 26, FIG. 1). The antibody comprises a few asparagine residues that may be deamidated: such as Asn51 and Asn54 in CDR2 of the heavy chain variable region, and Asn28 in FR1 of the heavy chain variable region. Particularly, Asn54 is contained in an Asn-Gly sequence, and thus is considered to be easily deamidated.

[0099] Pharmaceutical formulation of the antibody has not been established. Under destabilizing conditions of the antibody, the antibody ...

example 2

Measurement of TF Binding and Neutralizing Activities of Gly55 Substitution Mutant of Anti-Human TF Antibody

[0133] The anti-human TF antibody described in WO 99 / 51743 contains the humanized heavy chain version i (SEQ ID NO: 25, FIG. 1) and the humanized light chain version b2 (SEQ ID NO: 26, FIG. 1). Based on its amino acid sequence, mutants were prepared wherein the Gly55 in the heavy chain CDR2 that is considered as an important amino acid in the construction of the loop of CDR2 had been substituted with 19 other amino acids. Then, the binding activity of each mutant with TF was measured. Furthermore, the neutralizing activity and deamidation was observed for the mutants wherein Gly55 had been substituted with Ile, Leu, Phe, Glu and Lys.

[0134] The amino acid sequence of the antibody based on the sequence described by Kabat et al. (Kabat E. A., Wu T. T., Perry H. M., Gottesman K. S. and Foeller C., “Sequences of proteins of immunological interest. 5th ed.”, US Dept. Health and Hu...

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Abstract

The present inventors revealed that deamidation of an antibody can be suppressed without influencing its activity by substituting a glycine that is located adjacent to an asparagine with another amino acid.

Description

TECHNICAL FIELD [0001] The present invention relates to a method for improving protein stability. Specifically, the present invention relates to a method for stabilizing proteins comprising the step of substituting the amino acid that is located adjacent to the amino acid being deamidated in a protein with another amino acid. BACKGROUND ART [0002] Gradual deamidation of amino acids, such as asparagine, in proteins over time is mentioned as a cause of the reduction in protein stability. When proteins, particularly antibodies, are used as pharmaceutical agents for various diseases, they are required to be stable over a long period. However, the activity of antibody decreases with time. The cause for reduction in activity varies in antibodies, and deamidation of amino acids, such as asparagine, comprised in the antibody is also mentioned as one of the causes. [0003] Therefore, proteins can be stabilized by suppressing deamidation of asparagines. Thus, research on suppressing deamidatio...

Claims

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Application Information

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IPC IPC(8): A61K39/395C07K1/107C07K14/705C07K16/00C07K16/36C12N15/09C12P21/08
CPCC07K1/107C07K14/70503C07K2317/41C07K16/36C07K16/00A61P43/00A61P7/02
Inventor SUGO, IZUMITOMONOU, KIKUO
Owner CHUGAI PHARMA CO LTD
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