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US 5,849,296 AGranted
Crosslinked protein crystals
AssigneeVertex Pharmaceuticals Incorporated
InventorsManuel A. Navia, Nancy L. St. Clair
Life of this patent
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AssigneeVertex Pharmaceuticals Incorporated
InventorsManuel A. Navia, Nancy L. St. Clair
Estimating the term…
A protein such as an enzyme or antibody is immobilized by crosslinking crystals of the protein with a multifunctional crosslinking agent. The crosslinked protein crystals may be lyophilized for storage. A preferred protein is an enzyme such as thermolysin, elastase, asparaginase, lysozyme, lipase or urease. Crosslinked enzyme crystals preferably retain at least 91% activity after incubation for three hours in the presence of a concentration of Pronase.TM. that causes the soluble uncrosslinked form of the enzyme to lose at least 94% of its initial activity under the same conditions. A preferred enzyme:Pronase.TM. ratio is 1:40. Enzyme crystals that are crosslinked may be microcrystals having a cross-section of 10.sup.-1 mm or less. Crosslinked enzyme or antibody crystals may be used in an assay, diagnostic kit or biosensor for detecting an analyte, in an extracorporeal device for altering a component of a fluid, in producing a product such as using crosslinked thermolysin crystals to produce aspartame, in separating a substance from a mixture, and in therapy.
A protein crystal crosslinked with a multifunctional crosslinking agent, said crosslinked protein crystal having resistance to exogenous proteolysis, such that said crosslinked protein crystal retains at least 91% of its stability, as measured in terms of degradation, after incubation for three hours in the presence of a concentration of Pronase.TM. that causes the soluble uncrosslinked form of the protein that is crystallized to form said protein crystal that is crosslinked to lose at least 94% of its stability, as measured in terms of degradation, under the same conditions.
An enzyme crystal crosslinked with a multifunctional crosslinking agent, said crosslinked enzyme crystal having resistance to exogenous proteolysis, such that said crosslinked enzyme crystal retains at least 91% of its initial activity after incubation for three hours in the presence of a concentration of Pronase.TM. that causes the soluble uncrosslinked form of the enzyme that is crystallized to form said enzyme crystal that is crosslinked to lose at least 94% of its initial activity under the same conditions, wherein said crystal is in lyophilized form.
A lipase crystal crosslinked with a multifunctional crosslinking agent, said crosslinked lipase crystal having resistance to exogenous proteolysis, such that said crosslinked lipase crystal retains at least 91% of its initial activity after incubation for three hours in the presence of a concentration of Pronase.TM. that causes the soluble uncrosslinked form of the lipase that is crystallized to form said lipase crystal that is crosslinked to lose at least 94% of its initial activity under the same conditions, wherein said lipase is selected from the group consisting of Geotrichum candidum lipase, Candida cylindracea lipase and porcine lipase.
An enzyme-catalyzed method of producing a selected product, comprising the steps of:
Dependencies are inferred from the claim text.