Treatment of alpha-galactosidase a deficiency
AssigneeTranskaryotic Therapies, Inc., a Delaware corporation
InventorsRichard F. Selden, Marianne Borowski, Carol M. Kinoshita, Douglas A. Treco, Melanie D. Williams, Thomas J. Schuetz, Peter F. Daniel
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Abstract
The invention provides highly purified α-Gal A, and various methods for purifying it; α-Gal A preparations with altered charge and methods for making those preparations; α-Gal A preparations that have an extended circulating half-life in a mammalian host, and methods for making same; and methods and dosages for administering an α-Gal A preparation to a subject.
Claims45
A composition comprising a human α-Gal A preparation, purified to at least 98% homogeneity, as measured by SDS-PAGE or reverse phase HPLC.
A composition comprising a human α-Gal A preparation, having a specific activity of at least 2.0×106 units/mg protein.
A method for producing an α-Gal A preparation comprising various α-Gal A glycoforms and purified to at least 98% homogeneity, comprising
- separating the α-Gal A glycoforms from other components on a hydrophobic interaction resin, wherein the α-Gal A preparation is purified to at least 98% homogeneity and wherein the purification does not include a lectin chromatography step.
A method for producing an α-Gal A preparation comprising various α-Gal A glycoforms, purified to at least 98% homogeneity, comprising:
- (a) binding the α-Gal A glycoforms to a cation exchange resin at acidic pH in an equilibration buffer,
- (b) washing the resin with the equilibration buffer to elute the unbound material, and
- (c) eluting the α-Gal A glycoforms using an elution solution selected from the group consisting of a salt solution of 10-100 mM, a buffered solution of pH 4-5, and a combination thereof,
- wherein the α-Gal A preparation is purified to at least 98% homogeneity.
A method for producing an α-Gal A preparation comprising various α-Gal A glycoforms, purified to at least 98% homogeneity, comprising
- separating the α-Gal A glycoforms in a sample from the other components in the sample using a purification procedure comprising a step selected from the group consisting of chromatofocusing chromatography, metal chelate affinity chromatography and immunoaffinity chromatogrphy, wherein the α-Gal A is purified to at least 98% homogeneity.
A human glycosylated α-Gal A preparation, wherein at least 35% of the oligosaceharides are charged.
A human glycosylated α-Gal A preparation, wherein the preparation includes multiple glycoforms, comprising at least 20% complex glycans with 2-4 sialic acid residues.
A human glycosylated α-Gal A preparation, wherein the oligosaccharide charge, as measured by the Z number, is greater than 100.
A human glycosylated α-Gal A preparation, wherein the preparation includes multiple glycoforms, said glycoforms being at least on average between 25-50% phosphorylated.
A human glycosylated α-Gal A preparation, wherein the preparation includes multiple glycoforms, and, wherein between 50-75% of the total glycans are sialylated.
A method for producing a glycosylated α-Gal A preparation having an increased oligosaccharide charge, comprising:
- (a) introducing a polynucleotide which on expression codes for GlcNAc transferase III (GnT-III) into an α-Gal A producing-cell or introducing a regulatory sequence by homologous recombination that regulates expression of an endogenous GnT-III gene;
- (b) culturing the α-Gal A production cell under culture conditions which results in expression of α-Gal A and GnT-III;
- and
- (c) isolating the α-Gal A preparation, wherein the α-Gal A preparation has induced oligosaccharide charge as compared to α-Gal A from an α-Gal A producing-cell that lacks the polynucleotide in step (a).
A method for producing a glycosylated α-Gal A preparation with increased oligosaecharide charge, comprising:
- (a) introducing a polynucleotide which on expression codes for sialyl transferase into an α-Gal A producing-cell or introducing a regulatory sequence by homologous recombination that regulates expression of an endogenous sialyl transferase;
- (b) culturing the α-Gal A production cell under culture conditions which results in expression of α-Gal A and sialyl transferase;
- and
- (c) isolating the α-Gal A preparation, wherein the α-Gal A preparation has increased oligosaccharide charge as compared to α-Gal A from an α-Gal A producing-cell that lacks the polynucleotide in step (a).
A method for producing a glycosylated α-Gal A preparation with increased sialylation, comprising contacting an α-Gal A production cell with a culture medium having an ammonium concentration below 10 nM.
A method for producing a glycosylated α-Gal A preparation having increased phosphorylation, comprising:
- (a) introducing a polynucleotide which on expression codes for phosphoryl transferase into an α-Gal A producing-cell or introducing a regulatory sequence by homologous, recombination that regulates expression of an endogenous phosphoryl transferase;
- ((b) culturing the α-Gal A production cell under culture conditions which result in expression of α-Gal A and phosphoryl transferase;
- and
- (c) isolating the α-Gal A, wherein the isolated α-Gal A has increased phosphorylation as compared to the α-Gal A produced in a cell without the polynucleotide.
A human glycosylated α-Gal A preparation with an extended circulating half-life when administered to a patient, wherein the preparation includes multiple glycoforms, comprising at least 20% complex glycans with 2-4 sialic acid residues.
A human glycosylated α-Gal A preparation with an extended circulating half-life when administered to a patient, wherein the preparation includes multiple glycoforms, said glycoforms being at least on average between 25-51% phosphorylated.
A human glycosylated α-Gal A preparation with an extended circulating half-life when administerd to a patient, wherein the preparation includes multiple glycoforms, and, wherein between 50-75% of the total glycans are sialylated.
A method for producing a glycosylated α-Gal A preparation with a reduced number of sialic acid and terminal galactose residues on the oligosaccharide chains, comprising:
- (a) contacting α-Gal A with neumminidase (sialidase) to remove sialic acid residues, leaving the terminal galactose moieties exposed;
- and
- (b) contacting the desialylated α-Gal A of step (a) with β-galactosidase to remove terminal galactose residues,
- such that the desialylated, degalactosylated α-Gal A product of step (b) has a reduced number of terminal sialic acid or galactose residues on the oligosaccharide chains compared to α-Gal A from uncontacted α-Gal A.
A method for producing a glycosylated α-Gal A with a reduced number of terminal galactose residues on the oligosaccharide chains, comprising:
- contacting α-Gal A with β-galactosidase to remove terminal galactose residues, such that the product has a reduced number of terminal galactose residues on the oligosaccharide chains compared to α-Gal A from uncontacted α-Gal A.
A formulation comprising an α-Gal A preparation that is substantially free of proteins other than α-Gal A.
A formulation comprising an α-Gal A preparation that is substantially free of albumin.
A method for administering an α-Gal A preparation to a subject, comprising administering a dose of between 0.05-5.0 mg of the α-Gal A preparation weekly or biweekly.
A method for delivering α-Gal A preparation to a subject, comprising subcutaneously administering a dose ranging between 0.01-10 mg of the α-Gal A preparation per kg body weight biweekly or weekly.
A method of treating a patient with Fabry disease, comprising administering an α-Gal A preparation to the patient in a dose of between 0.05-5.0 mg of the α-Gal A preparation per kg body weight weekly or biweekly.
A method for treating a patient with Fabry disease comprising subcutaneously administering an α-Gal A preparation to the patient in a dose ranging between 0.01-10 mg of the α-Gal A preparation per kg body weight biweekly or weekly.
A method of treating a patient with a typical variant of Fabry disease, comprising administering to the patient an α-Gal A preparation at a dose of between 0.05-5.0 mg of the α-Gal A preparation per kg body weight weekly or biweekly.
Dependencies are inferred from the claim text.