Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Recombinant alpha-L-iduronidase, methods for producing and purifying the same and methods for treating diseases caused by deficiencies thereof

a technology of recombinant alpha-l-iduronidase and purification method, which is applied in the field of recombinant alpha-l-iduronidase, can solve the problems of high morbidity and mortality, low production level, and low clinical efficacy, and achieves preservation or enhancement of the biological activity of the enzyme and high production level

Inactive Publication Date: 2006-12-21
BIOMARIN PHARMA INC
View PDF5 Cites 16 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides a method to produce α-L-iduronidase in amounts that can be used therapeutically. The method involves transfecting a cDNA encoding for α-L-iduronidase into a cell and growing the cells in a specific medium. The resulting enzyme is then purified and its specific activity is enhanced. The invention also provides a recombinant Chinese hamster ovary cell line that stably produces α-L-iduronidase. The invention also includes novel expression vectors and a novel method to purify α-L-iduronidase. The technical effects of the invention include the ability to produce a therapeutically effective amount of α-L-iduronidase and the use of specific techniques to optimize its production and purification.

Problems solved by technology

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.
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.
Except for bone marrow transplantation, there are no significant therapies available for MPS I. Bone marrow transplants can be effective in treating some of the symptoms of the disorder but have high morbidity and mortality in MPS I and often are not available to patients because of a lack of suitable donors.
Application of this therapy to humans has previously not been possible due to inadequate sources of α-L-iduronidase in tissues.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Recombinant alpha-L-iduronidase, methods for producing and purifying the same and methods for treating diseases caused by deficiencies thereof
  • Recombinant alpha-L-iduronidase, methods for producing and purifying the same and methods for treating diseases caused by deficiencies thereof
  • Recombinant alpha-L-iduronidase, methods for producing and purifying the same and methods for treating diseases caused by deficiencies thereof

Examples

Experimental program
Comparison scheme
Effect test

example 1

Producing Recombinant Iduronidase

[0072] Standard techniques such as those described by Sambrook et al. (1987) “Molecular Cloning: A Laboratory Manual”, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. may be used to clone cDNA encoding human α-L-iduronidase. The human α-L-iduronidase cDNA previously cloned was subcloned into PRCCMV (InVitrogen) as a HindIII-XbaI fragment from a bluescript KS subclone. An intron cassette derived from the murine immunoglobulin Cot intron between exons 2 and 3 was constructed using PCR amplification of bases 788-1372 (Tucker et al., Proc. Natl. Acad. Sci. USA 78: 7684-7688 (1991) of clone pRIR14.5 (Kakkis et al., Nucleic Acids Res. 16:7796 (1988)). The cassette included 136 bp of the 3′ end of exon 2 and 242 bp of the 5′ end of exon 3, which would remain in the properly spliced cDNA. No ATG sequences are present in the coding, region of the intron cassette. The intron cassette was cloned into the HindIII site 5′ of the α-L-iduronidase ...

example 2

[0077] Seed train: A vial of working cell bank (WCB) CHO cells 2.131 is partially thawed in a 37° C. water bath. The thawed cells are added to seven mL of fresh cell culture medium (DMEM / F12) containing thymidine (7.6 mg / L), hypoxanthine (13.6 mg / L), G418 (375 mg / mL) and fetal bovine serum (5%). The cells are collected by gentle centrifugation (400 r.p.m. for 5 minutes). The pelleted cells are resuspended in fresh DMEM / F12 medium substituted as described above and the cells are placed in a T-75 cm flask in 25 mL of the medium.

[0078] After 2 to 3 days of incubation at 37° C. and 5% carbon dioxide, the cells are placed in a T-225 flask with 50 mL of fresh medium. The next split of cells is into a 250 mL spinner (100 mL of cells and medium) and the agitator (spinner) is rotated at 50 r.p.m. The inoculum volume and cell number is increased by a series of incubations (cell density increases) and subculturing (cell volume increases). The subculturing is performed such that the final cell...

example 3

[0083] Recombinant iduronidase over-expressed in a Chinese Hamster Ovary (CHO) cell line, has been purified to near homogeneity following a 3 step column chromatography process. The first column involves an affinity chromatography step using Blue Sepharose 6 FF. The Blue eluate is then further purified by another affinity chromatography step using Cu++ Chelating Sepharose FF. The final polish of the highly purified enzyme is achieved by hydrophobic interaction chromatography using Phenyl Sepharose High Performance (HP). The over-all yield ranges from 45 to 55 percent and the purity of the final product is >99%. The process is robust, reproducible, and scalable for large-scale manufacturing. The purified enzyme has been characterized with respect to its enzymatic activity using a fluorescence-based substrate, and its functional uptake by fibroblast cells. The enzyme has also been characterized for substrate specificity, carbohydrate profiles, and isoelectric focusing (IEF) profiles. ...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The present invention provides a recombinant α-L-iduronidase and biologically active fragments and mutants thereof, methods to produce and purify this enzyme as well as methods to treat certain genetic disorders including—α-L-iduronidase deficiency and mucopolysaccharidosis I (MPS 1).

Description

[0001] This application is a continuation application of U.S. patent application Ser. No. 09 / 439,923, filed Nov. 12, 1999, now U.S. Pat. No. 6,426,208, issued Jul. 30, 2002, which is incorporated herein by reference.FIELD OF THE INVENTION [0002] The present invention is in the field of molecular biology, enzymology, biochemistry and clinical medicine. In particular, the present invention provides a recombinant α-L-iduronidase, methods to produce and purify this enzyme as well as methods to treat certain genetic disorders including α-L-iduronidase deficiency and mucopolysaccharidosis I (MPS I). BACKGROUND OF THE INVENTION [0003] 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, c...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(United States)
IPC IPC(8): A61K38/47C07H21/04C12P21/06C12N9/36C12N15/09A61K9/08A61K38/00A61K38/43A61K47/02A61K47/10A61K48/00A61P3/00A61P19/00A61P43/00C12N9/24C12N9/88
CPCA61K38/47A61K48/00C12N9/88C12N9/2402C12Y302/01075C12Y402/02004C12Y302/01076C12Y302/01014A61P1/16A61P19/00A61P3/00A61P37/02A61P37/08A61P43/00
Inventor KAKKIS, EMILTANAMACHI, BECKY
Owner BIOMARIN PHARMA INC
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com