Stabilizing compositions for immobilized biomolecules

• US 9,797,895 B2
• Filed: 03/31/2010
• Issued: 10/24/2017
• Est. Priority Date: 03/31/2009
• Status: Active Grant

• Current Assignee: Leukocare AG
• Original Assignee: Leukocare AG
• Inventors: Margraf, Stefan, Breuer, Anja, Scholz, Martin, Altrichter, Jens
• Primary Examiner(s): LAM, ANN Y
• Application Number:
  US13/262,542
• Publication Number:
  US 20120107829A1
• Time in Patent Office: 2,764 Days
• Field of Search: None
• CPC Class Codes: G01N 33/54393 Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
• CPC Class Codes: G06N 20/00 Machine learning
• CPC Class Codes: G16C 20/30 Prediction of properties of chemical compounds, compositions or mixtures
• CPC Class Codes: Y02A 90/10 Information and communication technologies [ICT] supporting adaptation to climate change, e.g. for weather forecasting or climate simulation

Abstract

The present invention relates to the use of a composition comprising (a) at least three different amino acids, (b) at least two different amino acids and a saponin or (c) at least one dipeptide or tripeptide for stabilizing biomolecules immobilized on a solid carrier. The invention furthermore relates to a method for producing stabilized biomolecules, comprising embedding the biomolecules in the composition according to the invention and a method of producing a solid carrier having biomolecules attached thereto. The invention furthermore relates to a solid carrier producible or produced by the method of the invention and a method of diagnosing a disease using the carrier of the invention.

Claims

1. A method for producing stabilized biomolecules, comprising

(a) reversibly attaching the biomolecules to a solid carrier;

and

(b) embedding the biomolecules in a composition selected from;

(i) a composition comprising at least three different amino acids, or

(ii) a composition comprising at least two different amino acids and a saponin,

wherein the composition of (i) does not contain dipeptides or tripeptides.

2. The method of claim 1, wherein stabilizing biomolecules includes stabilizing the structure and/or activity of biomolecules, enhancing the shelf-life of biomolecules and/or protecting biomolecules against stress-mediated damage.

3. The method of claim 1, wherein the composition comprising at least 3 different amino acids comprises at least 4 or at least 5 different amino acids.

4. The method of claim 3, wherein the composition comprising at least 5 different amino acids comprises at least one amino acid of each group of,

(a) an amino acid with non polar, aliphatic R groups;

(b) an amino acid with polar, uncharged R groups;

(c) an amino acid with positively charged R groups;

(d) an amino acid with negatively charged R groups; and

(e) an amino acid with aromatic R groups.

5. The method of claim 3, wherein the composition comprising at least 5 different amino acids comprises amino acids selected from

(a) alanine, glutamate, lysine, threonine and tryptophan;

(b) aspartate, arginine, phenylalanine, serine and valine;

(c) proline, serine, asparagine, aspartate, threonine, and phenylalanine;

(d) tyrosine, isoleucine, leucine, threonine, and valine;

or

(e) arginine, glycine, histidine, alanine, glutamate, lysine, and tryptophan.

6. The method of claim 1, wherein the composition comprising at least three different amino acids, or the composition comprising at least two different amino acids and a saponin, comprises less than 1% by dry weight cysteine.

7. The method of claim 1, wherein the composition comprising at least three different amino acids, or the composition comprising at least two different amino acids and a saponin, further comprises less than 1% Tween.

8. The method of claim 1, wherein the saponin is glycyrrhizic acid or a derivative thereof.

10. The method according to claim 1 or 9, further comprising sterilizing the solid carrier after step (b).

11. The method according to claim 10 wherein the sterilization of the carrier is effected by ethylene oxide, beta radiation, gamma radiation, X-ray, heat inactivation, autoclaving or plasma sterilization.

12. The method of claim 1, wherein the biomolecules are reversibly attached to the solid carrier via a cleavable linker, and wherein the biomolecule can be released from the solid carrier by using a means for cleaving the linker.

13. The method of claim 12, wherein the cleavable linker is selected from the group consisting of ethylene glycol bis(succinimidylsuccinate), bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone, dithiobis(succinimidylpropionate), disuccinimidyl tartarate, succinimidyl 2-([4,4′

-azipentanamido]ethyl)-1,3′

-dithiproprionate, and sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido)-ethyl-1,3′

-proprionate.

14. The method according to claim 1 or 9 further comprising

(c) subjecting the solid carrier to drying.

9. A method of producing a solid carrier having biomolecules attached thereto, comprising the steps of

(a) reversibly attaching the biomolecules to the solid carrier;

and

(b) incubating the carrier of step (a) in a composition selected from;

(i) a composition comprising at least three different amino acids, or

(ii) a composition comprising at least two different amino acids and a saponin,

wherein the composition of (i) does not contain dipeptides or tripeptides.

15. A solid carrier produced by the method of claim 9.

16. The solid carrier of claim 15, wherein the biomolecules are proteins, peptides, nucleic acids, carbohydrates, lipids, fatty acids, polyalcohols and combinations or modifications thereof, wherein the proteins preferably are antibodies, enzymes, receptors, cytokines, hormones, membrane proteins, growth factors, albumins, globulins, transport proteins or blood coagulation factors.

17. The solid carrier of claim 15, wherein the biomolecules specifically bind to a marker protein indicative for a disease, a non-cellular pathogen, a cell or a toxin.

18. A method of preparing a medical device comprising the solid carrier of claim 15, wherein the medical device is selected from the group consisting of an implant, a tubing, a catheter, a stent, a tubing, a wound dressing and a medical device used in extracorporeal circulation.

19. A method for diagnosing a disease comprising the steps of:

(a) contacting a sample obtained from a patient with a solid carrier according to claim 16 under suitable conditions to allow specific binding of the biomolecules attached to the carrier to said marker protein indicative for a disease, said non-cellular pathogen, said cell or said toxin; and

(b) detecting whether said marker protein indicative for the disease, said non-cellular pathogen, said cell or said toxin has been bound to the biomolecules.

20. A method for producing stabilized biomolecules, comprising

(a) adsorbing biomolecules directly to the surface of a solid carrier, and

(b) covering the biomolecules in a composition selected from:

(i) a composition comprising at least three different amino acids, or

(ii) a composition comprising at least two different amino acids and a saponin,

wherein the composition of (i) does not contain dipeptides or tripeptides,

so that the biomolecules are partially or completely covered by the composition comprising at least three different amino acids, or the composition comprising at least two different amino acids and a saponin, wherein the covered biomolecules can be released from the carrier by adding a liquid to solubilize/dissolve the covered biomolecules.

21. The method of claim 20, further comprising:

sterilizing the covered biomolecules; and

releasing the sterilized covered biomolecules from the carrier.