Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Stablizing an enzyme by forming a mixture – an adduct or a...
Patent
1987-05-07
1990-01-02
Wax, Robert A.
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Stablizing an enzyme by forming a mixture, an adduct or a...
424 858, 424 88, 435176, 435178, 530350, 530380, 530802, 530830, 536 56, 536102, 536112, C12N 996
Patent
active
048913197
DESCRIPTION:
BRIEF SUMMARY
This invention relates to the protection of proteins and other macromolecules against denaturing during drying.
Macromolecular compounds, especially proteins and polypeptide-containing compounds, commonly exist in their naturally occurring hydrated state in the form of complex, three-dimentional folded conformations generally known as tertiary structures. Very frequently, the activity of the compound, whether as an enzyme, antibody, antigen, flavourant, fluorescent, gelling agent etc., is critically dependent on the tertiary structure and is severely reduced or even eliminated if the structure is disturbed, even though the chemical empirical formula of the compound may not have changed. This is a very serious problem when the protein etc is required in a dry state, for storage etc.
In order to combat this problem various solutions have been proposed. Enzymes for dry immunoassay kits have been protected in liposomes. Vaccines cannot easily be stored in the freeze-dried state and so have to be stored in bulky ampoules. Fluorescent proteins such as the phycobiliproteins of use in immunoassay techniques lose their ability to fluoresce if dried and so cannot be used in dry kits. Yet a further example is the loss of antigen-binding capacity of monoclonal antibodies bound to a resin substrate and dried.
There is thus an urgent need for a means of protecting such substances from deactivation on drying. We have now found that this object can be achieved by drying the compound in the presence of trehalose.
Trehalose, .alpha.-D-glucopyranosyl-.alpha.-D-glucopyranoside, is a naturally occurring non-reducing disaccharide which has previously been associated with cell protection. It is known that some organisms, both plant and animal, can resist desiccation to very low levels of body water during drought conditions. These organisms include brine shrimps cysts (Artemia salina), the resurrection plant (Selaginella lepidophylla) and bakers yeast (Saccharomyces cerevisiae). They all share, as a common feature, the presence of large amounts of trehalose in their cells.
A body of work exists on the effects of various carbohydrates including trehalose on the stabilisation of cell membranes during freezing and dehydration. This work shows trehalose to be significantly superior to other carbohydrates in protecting cellular organelles from the deleterious effects of the loss of bound water.
(Crowe, J.H., Crowe L.M. and Mouradian R. (1983) Cryobiology 20, 346,356;
Crowe, J.H., Crowe L.M. and Chapman D. (1984) Archives Biochem. Biophysics 232, 400-497; and
Crowe L.M., Mouradian R, Crowe J.H., Jackson S.A. and Womersley C. (1984). Biochimia & Biophysica Acta 769, 141-150.)
While there is no consensus view as to how trehalose exerts it protective effects on cells, one hypothesis is that it substitutes for the bound water on membrane components of the living organism and prevents denaturation due to loss of bound (structural) water. We have now found that the effect is exhibited not only in living cells, but surprisingly also in macromolecules themselves in a purified, isolated state.
EP140489A of Wako Pure Chemical Industries discloses an immunoactive substance (e.g. antibody) on a carrier, e.g. glass beads, stabilized against drying at ambient temperature by immersing in a solution of a sugar, optionally together with a protein such as bovine serum albumin. A considerable number of sugars are mentioned as being of use (ribose, glucose, fructose, mannose, galactose, maltose, lactose, sucrose, oligosaccharides and polysaccharides); preferred are lactose, sucrose or dextrin solutions. There is no mention, however, of trehalose.
Patent Application GB 2009198(A) (Behringwerk AG) discloses the lyophilisation of meningococcal polysaccharide and trehalose; British Patent Application GB 2126588A (Asahi Kasei KKK) discloses the stabilization of tumor necrosis factor (TNF) to lyophilisation and freezing by including either a non-ionic surfactant or trehalose (or another sugar); and Published Japanese Patent Application J 58074696A (Iatron Labo
REFERENCES:
patent: 4206200 (1980-06-01), Guthohrlein et al.
patent: 4457916 (1984-07-01), Hayashi et al.
patent: 4675310 (1987-06-01), Chapman et al.
patent: 4762857 (1988-08-01), Bollin, Jr. et al.
Lim et al., "A Study on Environmental Tolerance of Yeast," Misaengmul Hakhoe Chi, 1978, 16(3), 103-10. (CA 92: 126872d).
Loomis et al., "Anhydrobiosis in Nematodes," J. Exp. Zool., 1979, 208(3), 355-60. (CA 91: 87840z).
Quadrant Bioresources Limited
Wax Robert A.
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