Manufacture of Active Highly Phosphorylated Human Lysosomal Sulfatase Enzymes and Uses Thereof
AssigneeBiomarin Pharmaceutical Inc.
InventorsErno Pungor, Charles Hague, Zhi Chen, Melita Dvorak-Ewell, Michel Claude Vellard, Vish Koppaka
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Abstract
This invention provides compositions of active highly phosphorylated lysosomal sulfatase enzymes, their pharmaceutical compositions, methods of producing and purifying such lysosomal sulfatase enzymes and compositions and their use in the diagnosis, prophylaxis, or treatment of diseases and conditions, including particularly lysosomal storage diseases that are caused by, or associated with, a deficiency in the lysosomal sulfatase enzyme.
Claims42
A recombinant human lysosomal sulfatase enzyme or biologically active fragment, mutant, variant or derivative thereof produced in a END3 complementation group CHO cell or derivative thereof, wherein said END3 complementation group CHO cell or derivative thereof expresses recombinant human sulfatase modifying factor 1 (SUMF1), and wherein said recombinant lysosomal sulfatase enzyme or biologically active fragment, mutant, variant or derivative thereof has a high level of activation, and a high level of phosphorylation.
A recombinant human N-acetylgalactosamine-6-sulfatase (GALNS) enzyme or biologically active fragment, mutant, variant or derivative thereof produced by a END3 complementation group CHO cell or derivative thereof, wherein said END3 complementation group CHO cell or derivative thereof expresses recombinant human sulfatase modifying factor 1 (SUMF1), and wherein said recombinant human GALNS has a high level of activation, and a high level of phosphorylation.
A method for producing an active highly phosphorylated recombinant human lysosomal sulfatase enzyme or biologically active fragment, mutant, variant or derivative thereof, comprising the steps of:
- (a) culturing a Chinese Hamster Ovary (CHO)-derived END3 complementation group cell or derivative thereof;
- (b) preparing a first mammalian expression vector capable of expressing said active highly phosphorylated recombinant human lysosomal sulfatase enzyme or biologically active fragment, mutant, variant or derivative thereof in said END3 complementation group cell or derivative thereof;
- (c) preparing a second mammalian expression vector capable of expressing recombinant human sulfatase modifying factor 1 (SUMF1) or biologically active fragment, mutant, variant or derivative thereof in said END3 complementation group cell or derivative thereof;
- (d) transfecting said END3 complementation group cell or derivative thereof with said first and second expression vectors;
- (e) selecting and cloning of a transfectant of said END3 complementation group cell or derivative thereof that expresses said active highly phosphorylated recombinant human lysosomal sulfatase enzyme or biologically active fragment, mutant, variant or derivative thereof;
- and
- (f) optimizing a cell culture process method for manufacturing said highly phosphorylated recombinant human lysosomal sulfatase enzyme or biologically active fragment, mutant, variant or derivative thereof,
- wherein the recombinant human lysosomal sulfatase enzyme is selected from the group consisting of arylsulfatase A (ARSA), arylsulfatase B (ARSB), iduronate-2-sulfatase (IDS), sulfamidase/heparin-N-sulfatase (SGSH), N-acetylglucosamine-sulfatase (G6S) and N-acetylgalactosamine-6-sulfatase (GALNS).
A method for producing an active highly phosphorylated recombinant human N-acetylgalactosamine-6-sulfatase (GALNS) enzyme or biologically active fragment, mutant, variant or derivative thereof or variant thereof, comprising the steps of:
- (a) culturing a Chinese Hamster Ovary (CHO)-derived END3 complementation group cell or derivative thereof;
- (b) preparing a first mammalian expression vector capable of expressing said active highly phosphorylated recombinant human GALNS or biologically active fragment, mutant, variant or derivative thereof suitable for said END3 complementation group cell or derivative thereof;
- (c) preparing a second mammalian expression vector capable of expressing recombinant human sulfatase modifying factor 1 (SUMF1) or biologically active fragment, mutant, variant or derivative thereof suitable for said END3 complementation group cell or derivative thereof;
- (d) transfecting said END3 complementation group cell or derivative thereof with said first and second expression vectors;
- (e) selecting and cloning of a transfectant of said END3 complementation group cell or derivative thereof that expresses said active highly phosphorylated recombinant human GALNS or biologically active fragment, mutant, variant or derivative thereof;
- and
- (f) optimizing a cell culture process method for manufacturing said active highly phosphorylated recombinant human GALNS or biologically active fragment, mutant, variant or derivative thereof.
A recombinant human N-acetylgalactosamine-6-sulfatase (GALNS) enzyme or biologically active mutant, variant or derivative thereof useful for treating a subject suffering from a lysosomal storage disease that is caused by or associated with a deficiency in said GALNS, wherein said GALNS enzyme or biologically active mutant, variant or derivative thereof:
- (a) has a purity of at least about 90% as determined by Coomassie Blue staining when subjected to SDS-PAGE under non-reducing conditions;
- (b) has at least about 90% conversion of the cysteine residue at position 53 to Cα-formylglycine (FGly);
- and
- (c) is N-linked glycosylated at the asparagine residues at positions 178 and 397, wherein at least about 50% of the oligomannose chains attached to the asparagine residue at position 178 are bis-phosphorylated.
A method of treating a subject suffering from a deficiency of a lysosomal sulfatase enzyme comprising administering to the subject in need of said lysosomal sulfatase enzyme a therapeutically effective amount of said lysosomal sulfatase enzyme, wherein said lysosomal sulfatase enzyme is a recombinant human lysosomal sulfatase enzyme or biologically active fragment, mutant, variant or derivative thereof produced by a CHO-derived END3 complementation group cell or a derivative thereof.
A method of treating a lysosomal storage disease that is associated with a deficiency in one or more lysosomal sulfatase enzymes, comprising administering to a subject having a deficiency in said one or more lysosomal sulfatase enzymes a therapeutically effective amount of recombinant human N-acetylgalactosamine-6-sulfatase (GALNS) enzyme or biologically active fragment, mutant, variant or derivative thereof produced by a CHO-derived END3 complementation group cell or a derivative thereof.
A method of treating a subject suffering from Mucopolysaccharidosis type IVa (MPS IVa) or Morquio A syndrome, or Multiple Sulfatase Deficiency (MSD), comprising administering to the subject a therapeutically effective amount of a purified, recombinant human N-acetylgalactosamine-6-sulfatase (GALNS) enzyme or biologically active mutant, variant or derivative thereof, wherein said GALNS or biologically active mutant, variant or derivative thereof:
- (a) has a purity of at least about 90% as determined by Coomassie Blue staining when subjected to SDS-PAGE under non-reducing conditions;
- (b) has at least about 90% conversion of the cysteine residue at position 53 to Cα-formylglycine (FGly);
- and
- (c) is N-linked glycosylated at the asparagine residues at positions 178 and 397, wherein at least about 50% of the oligomannose chains attached to the asparagine residue at position 178 are bis-phosphorylated.
An END3 complementation group CHO cell line derived from G71 or a derivative thereof, wherein the END3 complementation group CHO cell line comprises:
- (a) an expression vector for recombinant human sulfatase modifying factor 1 (SUMF1);
- and
- (b) an expression vector for a recombinant human lysosomal sulfatase enzyme, wherein the recombinant human lysosomal sulfatase enzyme is selected from the group consisting of arylsulfatase A (ARSA), arylsulfatase B (ARSB), iduronate-2-sulfatase (IDS), sulfamidase/heparin-N-sulfatase (SGSH), N-acetylglucosamine-sulfatase (G6S) and N-acetylgalactosamine-6-sulfatase (GALNS).
A method for measuring the activity of a recombinant human lysosomal enzyme to degrade natural substrates, comprising:
- (a) culturing an isolated human cell deficient in the lysosomal enzyme under conditions in which natural substrates for the lysosomal enzyme accumulate;
- (b) contacting the cell with the lysosomal enzyme;
- (c) lysing the cell;
- (d) adding to the cell lysate an enzyme that (i) is specific for natural substrates and (ii) cleaves small oligosaccharides from the natural substrates;
- (e) labeling the small oligosaccharides with a detectable moiety;
- (f) optionally separating the labeled small oligosaccharides;
- (g) detecting the labeled small oligosaccharides;
- and
- (h) determining the activity of the lysosomal enzyme to degrade natural substrates by comparing (i) the amount of labeled small oligosaccharides from cells contacted with the lysosomal enzyme with (ii) the amount of labeled small oligosaccharides from cells not contacted with the lysosomal enzyme,
- wherein a reduction in (h)(i) as compared to (h)(ii) indicates the activity of the lysosomal enzyme to degrade natural substrates.
Dependencies are inferred from the claim text.