Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
2000-11-09
2003-05-27
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S004000, C435S183000, C530S350000, C530S412000, C530S416000
Reexamination Certificate
active
06569661
ABSTRACT:
FIELD OF THE INVENTION
The present invention is in the field of molecular biology, enzymology, biochemistry and clinical medicine. In particular, the present invention provides a human recombinant &agr;-L-iduronidase, methods of large-scale production and purification of commercial grade human recombinant &agr;-L-iduronidase enzyme, and methods to treat certain genetic disorders including &agr;-L-iduronidase deficiency and mucopolysaccharidosis I (MPS I).
BACKGROUND OF THE INVENTION
Carbohydrates play a number of important roles in the functioning of living organisms. In addition to their metabolic roles, carbohydrates are structural components of the human body covalently attached to numerous other entities such as proteins and lipids (called glycoconjugates). For example, human connective tissues and cell membranes comprise proteins, carbohydrates and a proteoglycan matrix. The carbohydrate portion of this proteoglycan matrix provides important properties to the body's structure.
A genetic deficiency of the carbohydrate-cleaving, lysosomal enzyme &agr;-L-iduronidase causes a lysosomal storage disorder known as mucopolysaccharidosis I (MPS I) (Neufeld and Muenzer, pp. 1565-1587, in
The Metabolic Basis of Inherited Disease,
Eds., C. R. Scriver, A. L. Beaudet, W. S. Sly, and D. Valle, McGraw-Hill, New York (1989)) In a severe form, MPS I is commonly known as Hurler syndrome and is associated with multiple problems such as mental retardation, clouding of the cornea, coarsened facial features, cardiac disease, respiratory disease, liver and spleen enlargement, hernias, and joint stiffness. Patients suffering from Hurler syndrome usually die before age 10. In an intermediate form known as Hurler-Scheie syndrome, mental function is generally not severely affected, but physical problems may lead to death by the teens or twenties. Scheie syndrome is the mildest form of MPS I. It is compatible with a normal life span, but joint stiffness, corneal clouding and heart valve disease cause significant problems.
The frequency of MPS I is estimated to be 1:100,000 according to a British Columbia survey of all newborns (Lowry, et al.,
Human Genetics
85:389-390 (1990)) and 1:70,000 according to an Irish study (Nelson,
Human Genetics
101:355-358 (1990)). There appears to be no ethnic predilection for this disease. It is likely that worldwide the disease is underdiagnosed either because the patient dies of a complication before the diagnosis is made or because the milder forms of the syndrome may be mistaken for arthritis or missed entirely. Effective newborn screening for MPS I would likely find some previously undetected patients.
Except for a few patients which qualify for bone marrow transplantation, there are no significant therapies available for all MPS I patients. Hobbs, et al. (
Lancet
2: 709-712 (1981)) first reported that bone marrow transplantation successfully treated a Hurler patient. Since that time, clinical studies at several transplant centers have shown improvement in physical disease and slowing or stabilizing of developmental decline if performed early. (Whitley, et al.,
Am. J. Med. Genet.
46: 209-218 (1993); Vellodi, et al.,
Arch. Dis. Child.
76: 92-99 (1997); Peters, et al.,
Blood
91: 2601-2608 (1998); Guffon, et al.,
J. Pediatrics
133: 119-125 (1998)) However, the significant morbidity and mortality, and the need for matched donor marrow, limits the utility of bone marrow transplants. An alternative therapy available to all affected patients would provide an important breakthrough in treating and managing this disease.
Enzyme replacement therapy has been considered a potential therapy for MPS I following the discovery that &agr;-L-iduronidase can correct the enzymatic defect in Hurler cells in culture, but the development of human therapy has been technically unfeasible until now. In the corrective process, the enzyme containing a mannose-6-phosphate residue is taken up into cells through receptor-mediated endocytosis and transported to the lysosomes where it clears the stored substrates, heparan sulfate and dermatan sulfate. Application of this therapy to humans has previously not been possible due to inadequate sources of &agr;-L-iduronidase in tissues.
For &agr;-L-iduronidase enzyme therapy in MPS I, a recombinant source of enzyme has been needed in order to obtain therapeutically sufficient supplies of the enzyme. The cDNA for the canine enzyme was cloned in 1991 (Stoltzfus, et al.,
J. Biol. Chem.
267:6570-6575 (1992) and for the human enzyme in the same year. (Scott, et al.,
Proc. Natl. Acad. Sci. U.S.A.
88:9695-9699 (1991), Moskowitz, et al.,
FASEB J
6:A77 (1992)). Following the cloning of cDNA for &agr;-L-iduronidase, the production of adequate quantities of recombinant &agr;-L-iduronidase allowed the study of enzyme replacement therapy in canine MPS I. (Kakkis, et al.,
Protein Expr. Purif.
5: 225-232 (1994)) Enzyme replacement studies in the canine MPS I model demonstrated that intravenously-administered recombinant &agr;-L-iduronidase distributed widely and reduced lysosomal storage from many tissues. (Shull, et al.,
Proc. Natl. Acad. Sci. U.S.A.
91: 12937-12941 (1994); Kakkis, et al.,
Biochem. Mol. Med.
58: 156-167 (1996)).
BRIEF SUMMARY OF THE INVENTION
In one aspect, the present invention features a method to mass produce human recombinant &agr;-L-iduronidase in large scale amounts with appropriate purity to enable large scale production for long term patient use of the enzyme therapy. In a broad embodiment, the method comprises the step of transfecting a cDNA encoding for all or part of an &agr;-L-iduronidase into a cell suitable for the expression thereof. In some embodiments, a cDNA encoding for a complete &agr;-L-iduronidase is used, preferably a human &agr;-L-iduronidase. However, in other embodiments, a cDNA encoding for a biologically active fragment or mutant thereof may be used. Specifically, one or more amino acid substitutions may be made while preserving or enhancing the biological activity of the enzyme. In other preferred embodiments, an expression vector is used to transfer the cDNA into a suitable cell or cell line for expression thereof. In one particularly preferred embodiment, the cDNA is transfected into a Chinese hamster ovary cell to create cell line 2.131. In yet other preferred embodiments, the production procedure features one or more of the following characteristics which have demonstrated particularly high production levels: (a) the pH of the cell growth culture may be lowered to about 6.5 to 7.0, preferably to about 6.8-7.0 during the production process, (b) as many as 2 to 3.5 culture volumes of the medium may be changed during each 24-hour period by continuous perfusion, (c) oxygen saturation may be optimized to about 40% but may be as high as 80%, (d) macroporous cellulose microcarriers with about 5% serum in the medium initially, may be used to produce cell mass followed by a rapid washout shift to protein-free medium for production, (e) a protein-free or low protein-medium such as a JRH Biosciences PF-CHO product may be optimized to include supplemental amounts of one or more ingredients selected from the group consisting of: glutamate, aspartate, glycine, ribonucleosides, and deoxyribonucleosides; (f) a stirred tank suspension culture may be perfused in a continuous process to produce iduronidase.
In a second aspect, the present invention provides a transfected cell line which features the ability to produce &agr;-L-iduronidase in amounts which enable using the enzyme therapeutically. In preferred embodiments, the present invention features a recombinant Chinese hamster ovary cell line such as the 2.131 cell line that stably and reliably produces amounts of &agr;-L-iduronidase which enable using the enzyme therapeutically. In some preferred embodiments, the cell line may contain more than 1 copy of an expression construct. In even more preferred embodiments, the cell line expresses recombinant &agr;-L-iduronidase in amounts of at least 20 micrograms per 10
7
cells per day.
In a third aspect, t
Chan Wai-Pan
Chen Lin
Fitzpatrick Paul A.
Hendstrand John M.
Kakkis Emil D.
Achutamurthy Ponnathapu
BioMarin Pharmaceutical Inc.
Chiang Robin C.
Halluin Albert P.
Howrey Simon Arnold & White , LLP
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