Glycerol-free formulations for reverse transcriptases

Patent metadata

Field	Value
Publication number	US-12018302-B2
Application number	US-202217678451-A
Country	US
Kind code	B2
Filing date	Feb 23, 2022
Priority date	Aug 8, 2017
Publication date	Jun 25, 2024
Grant date	Jun 25, 2024

Abstract

Glycerol-free enzyme formulations are described. In some embodiments, a glycerol-free enzyme formulation is stabilized by high salt concentration. The glycerol free enzyme formulation may comprise a reverse transcriptase enzyme.

First claim

What is claimed is:

1. A stabilized enzyme formulation comprising:
a. an enzyme and
b. a glycerol-free buffer having high ionic strength comprising salt(s) providing Na+ and/or K+ ions;

wherein the high ionic strength is at least 0.3 M ionic strength and further wherein the melting temperature of the enzyme increases as compared to the same enzyme in a buffer of 50 mM Hepes pH 7.0, 6.7 mM NaCl, 0.1 mM DTT, 0.27% sucrose, and 0.007 mM EDTA, wherein the enzyme is a MMLV reverse transcriptase.

2. The stabilized enzyme formulation of claim 1 , wherein the high ionic strength is at least 0.5 M ionic strength.

3. The stabilized enzyme formulation of claim 1 , wherein the melting temperature increases by 0.5° C. to 5° C.

4. The stabilized enzyme formulation of claim 1 , wherein the formulation further comprises sorbitol.

5. The stabilized enzyme formulation of claim 4 , wherein the formulation comprises 20% sorbitol.

6. The stabilized enzyme formulation of claim 1 , wherein the stability is maintained after at least 20 freeze-thaw cycles.

7. The stabilized enzyme formulation of claim 1 , wherein the formulation is stable for at least 5 days at 25° C.

8. The stabilized enzyme formulation of claim 1 , wherein the formulation is stable for at least 4 months at −20° C.

9. The stabilized enzyme formulation of claim 1 , wherein the glycerol-free buffer comprises no more than 1% glycerol.

10. The stabilized enzyme formulation of claim 1 , wherein the glycerol-free buffer comprises no more than 0.5% glycerol.

11. The stabilized enzyme formulation of claim 1 , wherein the glycerol-free buffer comprises no more than 0.1% glycerol.

12. A method of storing a stabilized enzyme formulation comprising providing the stabilized enzyme formulation of claim 1 and storing the enzyme formulation for at least 5 days at 25° C. and/or at least 4 months at −20° C., wherein the stabilized enzyme formulation retains at least 70% activity after storage.

Assignees

• Thermo Fisher Scientific Baltics Uab

Inventors

• Siurkus Juozas
• Kavaliauskas Darius
• Matulis Daumantas
• Baranauskiene Lina

Classifications

• C12Y207/07049

  RNA-directed DNA polymerase (2.7.7.49), i.e. telomerase or reverse-transcriptase · CPC title

• C12N15/1096 Primary

  cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR · CPC title

• C12N9/1276

  RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase · CPC title

• C12N9/96 Primary

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