High load enzyme immobilization by crosslinking

Patent metadata

Field	Value
Publication number	US-10400233-B2
Application number	US-201615232915-A
Country	US
Kind code	B2
Filing date	Aug 10, 2016
Priority date	Mar 31, 2014
Publication date	Sep 3, 2019
Grant date	Sep 3, 2019

Abstract

Methods of cross-linking polypeptide molecules are provided, where such methods include combining a cross-linking agent and polypeptide molecules in a solution under conditions suitable for a cross-linking reaction to occur. Also provided are preparations of cross-linked polypeptide molecules, where the polypeptide molecules are cross-linked by essentially unbranched cross-linking groups of at least 40 contiguous atoms. Further provided are test chemistry matrices and methods of making the same, where the matrices include a redox cofactor, an agent capable of eliciting a change in at least one measurable property of an indicator reagent in the presence of redox equivalents, an indicator reagent, and a preparation of cross-linked polypeptide molecules as described herein. Test elements and methods of using the same to diagnose diseases such as diabetes also are provided, where the test elements include a test chemistry matrix as described herein.

First claim

The invention claimed is:

1. A method of cross-linking polypeptide molecules, the method comprising the step of:
combining a cross-linking agent and polypeptide molecules in a solution under conditions suitable for a cross-linking reaction to occur, wherein the cross-linking agent comprises a contiguous chain of at least 40 atoms, wherein the polypeptide molecules are glucose dehydrogenase, and wherein at least 80% of the polypeptide molecules are glucose dehydrogenase.

2. The Method of claim 1 , wherein at least 90% of the polypeptide molecules are glucose dehydrogenase.

3. The method of claim 1 , wherein the cross-linking agent is an unbranched molecule chemically activated at both ends.

4. The method of claim 1 , wherein the cross-linking agent is bis-activated polyethylene glycol.

5. The method of claim 1 , wherein the cross-linking agent has a molecular mass selected from the group consisting of 0.6 kDa to 20 kDa, 0.9 kDa to 10 kDa, 1 kDa to 5 kDa, and 3 kDa.

6. The method of claim 1 , wherein the polypeptide molecules are present in the solution at an initial concentration selected from the group consisting of 0.1 mM to 5 mM, 0.3 mM to 4 mM, and 1.5 mM to 2.5 mM.

7. The method of claim 1 , wherein the molar ratio of cross-linking agent:polypeptide molecules is selected from the group consisting of 0.2:5, 0.5:4, 0.8:3, and 1:2.

8. A preparation comprising cross-linked polypeptide molecules obtained by the method of claim 1 .

9. Cross-linked polypeptide molecules, wherein the cross-linked polypeptide molecules are cross-linked by unbranched cross-linking groups comprising at least 40 contiguous atoms, wherein the polypeptide molecules are glucose dehydrogenase.

10. A test chemistry matrix comprising:
a redox cofactor;
an agent capable of eliciting a change in at least one measurable property of an indicator reagent in the presence of redox equivalents;
an indicator reagent; and
the preparation of cross-linked polypeptide molecules obtained by the method of claim 1 .

11. A diagnostic test element for determining an analyte concentration in a body fluid sample comprising the test chemistry matrix of claim 10 applied on a surface of a carrier.

12. A method of producing a test chemistry matrix, the method comprising the step of:
admixing a redox cofactor, an agent capable of eliciting a change in at least one measurable property of an indicator reagent in the presence of redox equivalents, an indicator reagent, and the preparation of cross-linked enzyme molecules of claim 8 to produce the test chemistry matrix therefrom.

13. The method of claim 12 , wherein two or more of the redox cofactor, the agent capable of eliciting a change in at least one measurable property of an indicator reagent, and the indicator agent are one compound.

Assignees

• Roche Diabetes Care Inc

Inventors

• Horn Carina
• Gawenda Susanne

Classifications

• C12Q1/001

  Enzyme electrodes · CPC title

• C12N9/96

  Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates · CPC title

• G01N2333/904

  acting on CHOH groups as donors, e.g. glucose oxidase, lactate dehydrogenase (1.1) · CPC title

• C12N11/06 Primary

  attached to the carrier via a bridging agent · CPC title

• C12Y101/01047

  Glucose 1-dehydrogenase (1.1.1.47) · CPC title