Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Stablizing an enzyme by forming a mixture – an adduct or a...
Patent
1991-06-27
1993-08-31
Lilling, Herbert J.
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Stablizing an enzyme by forming a mixture, an adduct or a...
435 25, 435189, C12N 902, C12N 998
Patent
active
052408434
DESCRIPTION:
BRIEF SUMMARY
This invention relates to stabilisation of proteins, particularly but not exclusively of enzymes in the dry state.
Few enzymes are inherently stable in solution. Many have a tendency to become denatured when held in solution. Various workers have attempted to stabilise enzymes either by adding compounds such as sugars or glycerol to solutions of them or by freeze drying. These methods often cause a loss of activity. Alternative methods of stabilisation have involved drying of enzymes with stabilisers in a presence of a solid support such as cellulose fibre or polyacrylamide. U.S. Pat. No. 4,451,569 disclosed stabilisation of glutathione peroxidase by freezing the enzyme with one of a number of sugars including arabinose, glucose, xylitol and sorbitol. Freeze drying is expensive to operate on a large scale and often results in denaturation.
PCT/GB86/00396 discloses stabilisation of proteins by use of the disaccharide trehalose.
According to a first aspect of the present invention a method of protecting proteins against denaturation on drying comprises mixing an aqueous solution of the protein with a soluble cationic polyelectrolyte and a cyclic polyol, and removing water from the solution.
Stabilisation in accordance with this invention enhances the activity of freshly dried enzymes and other proteins. The stability upon storage is also enhanced.
The proteins may include enzymes, antibodies, antigens, serum complement, vaccine components and bioactive peptides.
Drying of proteins and especially enzymes is important for many applications, for example use in diagnostic or analytical aids such as test strips which may be stored for prolonged periods before use. Transportation of enzymes or other proteins in solution is inconvenient and expensive.
Although freeze drying may be employed, The present invention facilitates use of the vacuum drying and air drying without denaturation. Vacuum drying and air drying milder processes and are much cheaper to operate.
The cyclic polyol may incorporate one or more alicyclic rings and may have at least one side chain. Compounds having 5 to 10 hydroxyl groups may be preferred. Non-reducing polyols are preferred. Di and trisaccharides are particularly efficaceous but other cyclic polyols, for example inositol may also be used. The polyol may be chosen to suit both the enzyme or other protein and also the polyelectrolyte in question. Lactitol, lactose, maltose and sucrose are especially preferred in conjunction with DEAE-dextrin, lactitol having been found to be most suitable for many applications. Sorbitol is suitable for use with cholesterol oxidase, cholesterol esterase and other enzymes. Cellobiose may also be used. The amount of polyol may lie in the preferred range of 1 to 20%, more preferably 2 to 10%, most preferably 5 to 10%.
The cationic polyelectrolyte is preferably a polymer with cationic groups distributed along the molecular chain. The cationic groups, which are preferably quaternary ammonium derived functions, may be disposed in side groups pendent from the chain or may be incorporated in it. Natural or artificial polymers may be employed. Natural polymers such as polysaccharides are preferred since many artificial polymers contain residual traces of the inorganic polymerisation catalyst.
Diethylaminoethyl dextran (DEAE-dextran) and chitosan are preferred although polyethyleneimine is also suitable. Polysaccharides with MW 5000 to 500 000, preferably 5000 to 20 000, more preferably 5000 to 10 000 may be employed. An amount of 0.1 to 10% is preferred, especially 0.5 to 2%.
The pH at which enzymes are dried in accordance with this invention may be important to optimise retention of activity both upon drying and after subsequent storage. The optimum pH for a particular enzyme may be determined by simple experimentation.
Alcohol oxidase has been formed to retain activity between pH 7 and 8, preferably at pH 7.8.
Cholesterol oxidase, dependent on the source, dries best at pH 5 or 9.
Uricase may be dried at pH 9.
Cholesterol esterase dependent on source may be dried
REFERENCES:
patent: 3133001 (1964-05-01), Muset
patent: 3413198 (1968-11-01), Deutsch
patent: 3950133 (1976-04-01), Monte et al.
patent: 3950223 (1976-04-01), Yugari et al.
patent: 4011169 (1977-03-01), Diehl et al.
patent: 4465770 (1984-08-01), Modrovich
patent: 4824938 (1989-04-01), Koyama et al.
patent: 4950596 (1990-08-01), Cheng et al.
patent: 4965203 (1990-10-01), Silbering et al.
patent: 5102788 (1992-04-01), Cole
Wang et al. "Technical Report No. 10" Parenteral Formulations of Proteins & Peptide: Stability & Stabilizers vol. 42 Suppl 1988 S4-S26.
Back et al. "Biochem" vol. 18 No. 23 1979 pp. 5191-5197.
Chemical Abstracts, vol. 106, 1987, Abstract No. 115739X.
Gibson Timothy D.
Woodward John R.
Cranfield Biotechnology Ltd.
Lilling Herbert J.
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