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Method for measuring viscosity of protein solution

a protein solution and viscosity technology, applied in the direction of instruments, indirect flow property measurement, peptides, etc., can solve the problems of affecting the overall development, difficult to reduce the viscosity with absolute certainty, and the inability to perform viscosity evaluation. , to achieve the effect of reducing the viscosity and low the viscosity

Inactive Publication Date: 2017-12-21
CHUGAI PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides methods for measuring the viscosity of protein solutions using small amounts of sample. This is achieved by utilizing small angle X-ray scattering (SAXS) which has been conventionally used in analyzing molecular shapes of proteins in solutions. This allows for accurate measurement of viscosity and the selection of proteins with regulated viscosity, as well as the production of modified proteins with regulated viscosity, decreased viscosity, or low viscosity. The invention can be carried out with a small amount of sample, resulting in efficient use of resources.

Problems solved by technology

Therefore, viscosity is a physical property issue that greatly affects the overall development.
That is, since high viscosity is caused by the properties of the protein molecule itself, it is difficult to reduce viscosity with absolute certainty through formulation design.
However, large quantities of samples are required to obtain actual values for viscosity at high concentrations, and thus it is impractical to perform viscosity evaluation in the early stages of development when candidate molecules are being assessed.
However, only rough correlations have been observed; and particularly with regard to antibodies with strongly working intermolecular interactions, there were problems with the precision of the parameters when considering them as a viscosity prediction system (Non-patent Documents 2 to 4).
However, using this technique to obtain parameters for measuring the viscosity of a protein solution has not been known.

Method used

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  • Method for measuring viscosity of protein solution
  • Method for measuring viscosity of protein solution
  • Method for measuring viscosity of protein solution

Examples

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Effect test

example 1

[Example 1] Correlation Between Particle Size Parameters and Viscosity for Mab1, Mab2. And MAb3

[0092]MAb1: a bispecific antibody that recognizes blood coagulation factor IX and / or activated blood coagulation factor IX, and blood coagulation factor X and / or activated blood coagulation factor X. ACE910 (Q499-z121 / J327-z119 / L404-k), which is a bispecific antibody described in a non-patent document (PLoS One. 2013:8(2):e57479) and a patent document (WO 2012 / 067176) (a bispecific antibody in which the H chain comprising the amino acid sequence of SEQ ID NO: 3 and the L chain of SEQ ID NO: 5 are associated, and the H chain comprising the amino acid sequence of SEQ ID NO: 4 and the L chain of SEQ ID NO: 5 are associated), was prepared according to the description in the aforementioned non-patent document or the patent document. As described in the aforementioned patent document, ACE910 has an activity of substituting for the function of coagulation factor VIII.

[0093]MAb2: an anti-IL-6 rece...

example 2

[Example 2] Correlation of Viscosity with Particle Size Parameters and Molecular Weight Parameters for MAb1, MAb3, and MAb4

[0098]MAb1, MAb3 (both are the same as those described in Example 1), and MAb4 (humanized anti-IL-6 receptor antibody; generic name: tocilizumab) were used to prepare samples in which the antibody concentration is 15 mg / mL in the formulated solutions of Table 2. Microcapillary cells were filled with approximately 10 μL of the samples; and the particle size parameters, which are the apparent particle radius of gyration Rgapp (nm) and the apparent maximum particle diameter Dmaxapp (nm), as well as the scattering intensity I(0) (a.u.) at zero scattering angle as a value proportional to the apparent molecular weight were calculated using SAXSess mc2 (Anton Paar). The results appear to show that the larger these values are, the higher the associativity of the antibodies. In order to more clearly detect the difference in associativity between the samples, measurements...

example 3

[Example 3] Correlation Between Particle Size Parameters and Viscosity for MAb2 and MAb4

[0102]MAb2 and MAb4 (same as in Example 1 and Example 2, respectively) were used to examine whether differences in viscosity between the antibody prior to amino acid sequence modification (Mab4) and the antibody in which a portion of the amino acid sequence has been substituted with other amino acids (Mab2) can be evaluated using the measurement methods of the present invention. The respective antibodies were used to prepare samples in which the antibody concentration is 15 mg / mL in the formulated solutions of Table 3. Microcapillary cells were filled with approximately 10 μL of the samples, and the particle size parameters, which are scattering intensity I(0) at zero scattering angle, apparent particle radius of gyration Rgapp, and apparent maximum particle diameter Dmaxapp(nm), were calculated using SAXSess mc2 (Anton Paar). The results appear to show that the larger these values are, the highe...

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Abstract

The inventors discovered that viscosity of a protein solution can be estimated by measuring the apparent particle size or apparent molecular weight by a small angle X-ray scattering (SAXS) method or X-ray solution scattering method, which enables measurement of small amounts of samples, and then correlating those measurement results with viscosity of the protein solution.

Description

TECHNICAL FIELD[0001]The present invention relates to methods for measuring viscosity of a protein solution, and particularly to methods for measuring viscosity of a protein solution with a small quantity of sample.BACKGROUND ART[0002]Recently, pharmaceutical preparations containing proteins such as antibodies as an active ingredient are being developed into solution preparations for use in subcutaneous administration, in consideration of their convenience and such. Along with this, the protein such as an antibody as an active ingredient in the drug solutions is being used at higher concentrations. When developing a high-concentration protein drug solution, the viscosity of the solution greatly affects manufacturability and usability of the solution. Therefore, viscosity is a physical property issue that greatly affects the overall development. The increase of solution viscosity (high viscosity) accompanying the increase of concentration is considered to be caused by electrostatic a...

Claims

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Application Information

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IPC IPC(8): C07K16/00G01N11/00G01N23/201C07K16/28C07K16/30C07K16/36
CPCC07K16/00C07K16/36C07K16/2866C07K16/303C07K2317/24G01N23/201C07K2317/94C07K2317/31G01N11/00C07K16/18C07K2317/90G01N2223/054G01N2223/612G01N2011/006G01N2011/008G01N33/487G01N33/6803G01N33/6854
Inventor FUKUDA, MASAKAZUHAYASAKA, AKIRAIGAWA, TOMOYUKIMAEDA, ATSUHIKOFUJINO, MACHIKO
Owner CHUGAI PHARMA CO LTD
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