Stabilization of proteins in solution

Details Stabilization of proteins in solution Stabilization of proteins in solution

1996-07-01

2000-10-17

Celsa, Bennett

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Peptide containing doai

514 12, 514 21, 435188, 525 541, 530408, 530410, A61K 3843, C12N 996

Patent

active

061332291

DESCRIPTION:

BRIEF SUMMARY
BACKGROUND OF THE INVENTION

This invention relates to stabilization of proteins in solution, particularly but not exclusively to stabilisation of enzymes. Alternative proteins include antibodies, antigens, serum compliment, vaccine components and bioactive peptides.
Use of enzymes in analytical applications has become well known because enzymes afford a number of significant advantages over conventional analytical chemistry. Enzymes confer specificity, sensitivity and operate under mild analytical conditions. A major disadvantage of enzyme based assays is that the enzyme component is often unstable. This may lead to degeneration of the reagent during storage and spurious results. Various methods have been used to increase the stability of enzymes including immobilisation, chemical modification by cross-linking, polymer grafting or substitution reactions, physical entrapment or encapsulation in polymer matrices or membranes and the addition of chemicals or solvents to the enzyme preparation. Enzyme preparations for use in analytical methods are often supplied in a dry stabilized form using a combination of chemical additives to promote stability. WO90/05182 and WO91/14773 disclose stabilization of enzymes on drying by mixing aqueous solutions of the enzyme with soluble polyelectrolytes and cyclic polyols before removal of water from the solution. Such compositions have not been found to afford significant stabilization prior to dehydration.
According to a first aspect of the present invention a protein stabilizer additive comprises two or more of:


SUMMARY OF THE INVENTION
NH.sub.2 ; NR.sup.1 R.sup.2 wherein R.sup.1 and R.sup.2 may be independently: H, C.sub.1 -C.sub.4 alkyl sulfonate, C.sub.1 -C.sub.4 hydroxyalkyl sulfonate; C.sub.1 -C.sub.4 alkyl-NHC(CH.sub.2 OH).sub.3, C.sub.1 -C.sub.4 alkyl, C.sub.1 -C.sub.4 hydroxyalkyl; C.sub.1 -C.sub.4 alkyl carboxylate;


DESCRIPTION OF THE PREFERRED EMBODIMENTS

Component (a) may be referred to as a "tris" compound. Examples of "tris" compounds include: 1,1',1"-tris(hydroxymethyl)ethane; 1,1',1"-tris(hydroxymethyl)propane; tris(hydroxymethyl)aminomethane or salts thereof for example chloride, maleate, phosphate, succinate salts; 1,3-bis[tris(hydroxymethyl)methylamino]propane; bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane; N-[tris(hydroxymethyl)methyl]-2-aminoethane sulphonate; N-[tris(hydroxymethyl)methyl]-3-aminopropane sulphonate; N-[tris(hydroxymethyl)methyl]-3-amino-2-hydroxypropane sulphonate; N-[tris(hydroxymethyl)methyl]-glycine.
The polyelectrolyte may be a cationic or anionic polyelectrolyte. Amphoteric polyelectrolytes may also be employed. 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 pendant from the chain or may be incorporated in it. Examples of cationic polyelectrolytes include: Coplymers of vinyl pyrollidone and quaternary methyl methacrylate e.g., GAFQUAT.RTM. series (755N, 734, HS-100) obtained from ISP; substituted polyacrylamides; polyethyleneimine, polypropyleneimine and substituted derivatives; polyamine homopolymers (GOLCHEM.RTM. CL118); polyamine co-polymers (e.g., condensates of epichlorohydrin and mono or dimethylamine); polydiallyl dimethyl ammonium chloride (polyDADMAC); substituted dextrans; modified guar gum (substituted with hydroxypropytrimonium chloride); substituted proteins (e.g., quaternary groups substituted on soya protein and hydrolysed collagen); polyamino acids (e.g., polylysine); low molecular weight polyamino compounds (e.g., spermine and spermidine). Natural or artificial polymers may be employed. Cationic polyelectrolytes with MW 150 to 5,000,000, preferably 5000 to 500,000, more preferably 5000 to 100,000 may be employed. An amount of 0.01 to 10% is preferred, more preferably 0.1 to 2% w/v, especially 0.05 to 5%.
The anionic polyelectrolyte is preferably a polymer with anionic groups distributed along the molecular chain. The anionic g

REFERENCES:
patent: 4282316 (1981-08-01), Modrovich
patent: 4318818 (1982-03-01), Letton et al.
patent: 5116729 (1992-05-01), Ismail et al.
patent: 5240843 (1993-08-01), Gibson et al.
Chang et al., J. Parent. Sci. and Tech., vol. 42, No. 2S pp. S3-S26, 1988.
Gibson et al., Anal. Chim. Acta, vol. 279 No. 1 (Jul. 1993) pp. 185-92.
Caplus AN 1980: 583645, Friedman, Biochem. Biophys. Acta (1980) 619(3) 650-9.

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