Method of stabilizing protein

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...

AI Technical Summary

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 existing in the CDR, is substituted with aspartic acid. The TF binding activity of anti-human TF antibody was suggested to decrease significantly due to the deamidation of Asn54 existing in the CDR2 region of the anti-human TF antibody heavy chain (H chain). The amino acid adjacent to Asn54 in the CDR2 region of anti-human TF antibody heavy chain is Gly55. These two amino acids form a primary sequence Asn-Gly that is easily deamidated. Therefore, the possibility to suppress deamidation of Asn54 by substituting this Gly55 with another amino acid was considered. Thus, the present inventors prepared mutants wherein the glycine adjacent to the asparagine was substituted with other amino acids to measure their binding activities. As a result, it was discovered that the substitution of glycine that is located adjacent to asparagine with other amino acids did not reduce the activity, and also suppressed the known instability due to deamidation.

[0046] Generally, an antibody is inactivated by amino acid substitution in the CDR. However, the present inventors revealed that the activity of an antibody is retained even after the substitution of an amino acid adjacent to asparagine in the CDR, and hence the stability of the antibody can be improved. Therefore, according to the present invention, an amino acid adjacent to an asparagine in the CDR is effectively substituted with another amino acid. Glycine is a suitable target as the amino acid adjacent to the asparagine. Particularly, glycine contained in the “Asn-Gly” sequence that is particularly easily deamidated is the most suitable target.

Problems solved by technology

Reduction in protein activity is a very important problem from the medical and pharmaceutical perspectives.

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