US007041487B2

(12) United States Patent

(10) Patent N0.:

(45) Date of Patent:

Kakkis et a1.
(54) RECOMBINANT (X-L-IDURONIDASE,
METHODS FOR PRODUCING AND
PURIFYING THE SAME AND METHODS
FOR TREATING DISEASES CAUSED BY
DEFICIENCIES THEREOF

(76)

Inventors: Emil D. Kakkis, 2572 Laguna Vista

Dr., Novato, CA (US) 94949; Becky

Tanamachi, 3343 Walnut Ave., Signal
Hill, CA (US) 90809
(*)

Notice:

Jul. 25, 2002

et

al.

Recombinant

Human

Iduronate-2

Sulphatase: Correction Of Mucopolysaccharidosis-Type Ii

Transplantation

Prior Publication Data

For

Lysosomal

Storage

Diseases,

Lanceti345:l398 (1995).

Jan. 16, 2003

Ioannou, et al. Overexpression Of Human ot-Galactosidase

A Results In Its Intracellular Aggregation, Crystallization In
Lysosomes, And Selective Secretion, J. Cell Bi0l.i119

Continuation of application No. 09/439,923, ?led on

Nov. 12, 1999, noW Pat. No. 6,426,208.

(5):1137-1150 (1992).
Kakkis, et al. Strong Transcriptional Activation Of
Translocated C-Myc Genes Occurs Without A Strong
Nearby Enhancer Or Promoter, Nucleic Acids Res.i16

Int. Cl.
C12N 9/00
C12N 9/26
C07H 21/04
C12P 21/06

(2006.01)
(2006.01)
(2006.01)
(2006.01)

US. Cl. ............................ ..

(1):77-96 (1988).
Kakkis, et al. Overexpression Of The Human Lysosomal
Enzyme A-L-Iduronidase In Chinese Hamster Ovary Cells,

Pr0t. Exp. Purifi5:225-232 (1994).

435/201; 435/4; 435/6;

435/69.1; 435/183; 435/252.3; 435/320.1;

536/232; 424/90.1; 530/350
Field of Classi?cation Search ............. .. 435/69.1,

435/183, 25243, 320.1, 201; 424/901; 530/350;

536/2342

See application ?le for complete search history.

(56)

Barton, et al. The Hurler Corrective Factor, J. Biol.

Chem.i246(4):7773-9 (1971).

Related US. Application Data

(58)

794 (1992).

Fibroblasts And Characterization Of The Puri?ed Enzyme,

Biochem. Ji289:241-6 (1993).
Clements, et al. Human Alpha-L-Iduronidase 1. Puri?ca
tion, Monoclonal Antibody Production, Native And Subunit
Molecular Mass, Eur. J. 0fBi0chem.i152(1):43-9 (1994).
Friedman, et al. Progress ToWard Human Gene Therapy,
Sciencei244:l275-l28l (1989).
Hoogerbrugge, et al. Allogeneic Bone MarroW

US 2003/0013179 A1

(52)

pmeWsWire.com/cgi-.
Anson, et al. Correction Of Human Mucopolysaccharidosis
TypeiVI
Fibroblasts
With
Recombinant
N
Acetylgalactosamine-4-Sulphatase, Biochem Ji284:789

U.S.C. 154(b) by 326 days.

(65)

(51)

BioMarin and Genzyme Report positive One Year Sum

mary Data For MPS-l Patients Genzyme General (Sep. 14,
1999), Retrieved from the Internet: <URL:http://WWW.

Bielicki,

(21) Appl. N0.: 10/206,443

(63)

*May 9, 2006

Subject to any disclaimer, the term of this

patent is extended or adjusted under 35
This patent is subject to a terminal dis
claimer.

(22) Filed:

US 7,041,487 B2

References Cited

Kakkis, et al. Long-Tenn and High-Dose Trials Of Enzyme

Replacement Therapy In The Canine Model Of
Mucopolysaccharidosisl, Biochem. Mol. Med.i58(2):156
1 67 (1 996).
Kakkis, et al. Enzyme-Replacement Therapy In
Mocopolysaccharidosis I., New England J. Med., 344:182
8 (2001).
Ledley, et al. Clinical Application Of Somatic Gene
Therapy In Inborn Errors Of Metabolism, J. Inherit. Metab.

Dis.i13:597-616 (1990).
U.S. PATENT DOCUMENTS

10/1969 Stoughton
6/1975 Higuchi

5,270,051 A

12/1993 Harris

(74) Attorney, Agent, or FirmiMarshall, Gerstein & Borun

6,149,909 A
6,238,662 B1

11/2000
5/2001

LLP

Scott ..................... .. 424/9461

Scott ..................... .. 424/9461

FOREIGN PATENT DOCUMENTS

GB
GB
W0
W0
W0
W0

(Continued)

3,472,931 A
3,891,757 A

1001949
1 029 548 A
WO 93/10244
WO 97/10353
WO 99/51724
WO 99/58691

8/1965
8/2002
5/1993
3/1997
10/1999
11/1999

OTHER PUBLICATIONS

Primary ExamineriManjunath N. Rao

(57)

ABSTRACT

The present invention provides a recombinant ot-L-idu

ronidase and biologically active fragments and mutants
thereof, methods to produce and purify this enzyme as Well
as methods to treat certain genetic disorders including-ot-L

iduronidase de?ciency and mucopolysaccharidosis I (MPS

1).

Franchimont, et al., Induction of In?ammation by Immu

nological Reactions, Agents and Actions, 6(1-3):2-4, 1976.

33 Claims, 11 Drawing Sheets

US 7,041,487 B2
Page 2
OTHER PUBLICATIONS

LoWry, et al. An Update On The Frequency Of Muco

polysaccharide Syndromes In British Columbia, Hum.

Gen.i85:389-390 (1990).
MyeroWitz, et al. Maturation Of ot-L-Iduronidase In

Cultured Human Fibroblasts, J. Biol. Chem.i256(6):

3044-8 (1981).
MoskoWitz, et al. Cloning And Expression Of cDNA
Encoding The Human Lysosomal Enzyme, ot-L
Iduronidase, FASEB Ji6zA77 (1992).

Scott, et al. Multiple Polymorphisms Within the ot-L

Iduronidase Gene (Idua): Implications For A Role In
Modi?cation Of Mps-I Disease Phenotype, Hum. Mol.

Genez.i2(9):1471-3 (1993).
Shull, et al. Enzyme Replacement In A Canine Model Of
Hurler Syndrome, Proc. Natl. Acad. Sc., USAi9lzl2937

12941 (1994).
Stoltzfus, et al. Mucopolysaccharidosis I: Cloning And
Characterization

Of cDNA Encoding

Canine

ot-L

Iduronidase, Am. J Hum. GeneZ.i47(3):A167 (1990).

Nelson, et al. Incidence Of The Mucopolysaccharidoses In

Northern Ireland, Hum. Gen.i101:355-8 (1997).
Neu?eld, et al. The Mucopolysaccharidoses, The

Stoltzfus, et al. Cloning And Characterization Of cDNA

Encoding Canine ot-L-Iduronidase, J. Biol. Chem.i267

Metabolic Basis ofInherited Disease, Scriver, Beaudet, Sly,

Taylor,

and Valle, Eds. McGraW Hill, NeW Yorki1565-1587

MucopolysaccharidosisiType-I human Skin Fibroblasts,

Biochem Ji274z263-8 (1991).

(1989).

(10):6570-5 (1992).
et

al.

ot-L-Iduronidase

In

Normal

And

Rome, et al. ot-L-Iduronidase For Human Kidney Melh.

Tolstosbev, et al. Gene Expression Using Retroviral Vec

Enzymoli83z578-582 (1982).

tors, Current Opinions BioZech.il:55-6l (1990).

Tucker, et al. Mouse IgA Heavy Chain Gene Sequence:
Implications For Evolution Of Immunoglobullin Hinge
Exons,Proc. Natl. Acad. Sci. USAi78(12):7684-8 (1981).

Schuchman, et al. Human A-L-Iduronidase: Puri?cation

And Properties Of The High Uptake (Higher Molecular

Weight) And The LoW Uptake (Processed) Forms J. Biol.

Chem,i259(5):3132-3140 (1984).
Scott, et al. Chromosomal Localization Of The Human
ot-L-Iduronidase Gene (Idua) To 4p16.3 Am. J. Hum.

Unger, et al. Recombinant ot-L-Iduronidase: Characteriza

tion Of The Puri?ed Enzyme And Correction Of

GeneLA7z802-7 (1990).

Mucopolysaccharidosis Type I Fibroblasts, Biochem. Ji

304:43-9 (1994).

Scott, Human ot-L-Iduronidase: cDNA Isolation And

Expression, Proc. Natl. Acad. Sci. USAi88z9695-9

Zhao, et al. Carbohydrate Structures Of Recombinant Hu

man ot-L-Iduronidase Secreted By Chinese Hamster Ovary

(1991).

Cells, J. Biol. Chem.i273(36):22758-22765 (1997).

U S. Patent

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US 7,041,487 B2
1

RECOMBINANT ot-L-IDURONIDASE,

lack of suitable donors. An alternative therapy available to

METHODS FOR PRODUCING AND

PURIFYING THE SAME AND METHODS
FOR TREATING DISEASES CAUSED BY
DEFICIENCIES THEREOF

through in treating and managing this disease.

EnZyme replacement therapy has long been considered a
potential therapy for MPS I folloWing the discovery that

all affected patients Would provide an important break

ot-L-iduronidase can correct the enzymatic defect in Hurler

cells in culture. In this corrective process, the enZyme

This application is a continuation application of US.

patent application Ser. No. 09/439,923, ?led Nov. 12, 1999,

containing a mannose-6-phosphate residue is taken up into

now US. Pat. No. 6,426,208, issued Jul. 30, 2002, Which is

cells through receptor-mediated endocytosis and transported

incorporated herein by reference.

to the lysosomes Where it clears the stored substrates,

heparan sulfate and derrnatan sulfate. Application of this
therapy to humans has previously not been possible due to
inadequate sources of ot-L-iduronidase in tissues. The
enZyme replacement concept Was ?rst effectively applied to
Gaucher patients in a modi?ed placental glucocerebrosidase.

FIELD OF THE INVENTION

The present invention is in the ?eld of molecular biology,

enZymology, biochemistry and clinical medicine. In particu

lar, the present invention provides a recombinant ot-L
iduronidase, methods to produce and purify this enZyme as
Well as methods to treat certain genetic disorders including

The delivery and effective uptake of glucocerebrosidase in

Gaucher patients demonstrated that an enZyme could be
taken up in vivo in suf?cient quantities to provide effective

ot-L-iduronidase de?ciency and mucopolysaccharidosis I

(MPS I).

therapy.
20

BACKGROUND OF THE INVENTION

Carbohydrates play a number of important roles in the

functioning of living organisms. In addition to their meta
bolic 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 pro
vides important properties to the bodys structure.

25

30

A genetic de?ciency of the carbohydrate-cleaving, lyso

35

(Scriver, C. R., Beaudet, A. L., Sly, W. S., and Valle, D.,

FIG. 2 represents the results from an SDS-PAGE run of

bovine serum albumin in a concentration of 50 pg. Lanes 5

40

dation, clouding of the cornea, coarsened facial features,

cardiac disease, respiratory disease, liver and spleen enlarge

ment, hernias, and joint stiffness. Patients suffering, from
Hurler syndrome usually die before age 10. In an interme
diate form knoWn as Hurler-Scheie syndrome, mental func

FIG. 1 represents the nucleotide and deduced amino acid

sequences of cDNA encoding ot-L-iduronidase (SEQ ID

Nos: 1 and 2). Nucleotides 1 through 6200 are provided.
Amino acids are provided starting With the ?rst methionine
in the open reading frame.
eluate obtained according to the procedure set forth in
Example 1. Lane 1 is blank. Lane 2 contained high molecu
lar Weight standards. Lane 3 is a blank. Lane 4 contained

doses in The Metabolic Basis of Inherited Disease

Eds.), pp. 156541587, McGraW-Hill, NeW York). In a severe

form, MPS I is commonly knoWn as Hurler syndrome and
is associated With multiple problems such as mental retar

therapeutically su?icient supplies of the enZyme. The mam

malian enZyme Was cloned in 1990 (StoltZfus et al., J. Biol.
Chem. 2671657046575 (1992), and the human enZyme Was
cloned in 1991 (MoskoWitZ et al., FASEB J 6:A77 (1992)).
DESCRIPTION OF THE FIGURES

somal enZyme ot-L-iduronidase causes a lysosomal storage

disorder knoWn as mucopolysaccharidosis I (MPS I) (Neu

feld, E. F., and MuenZer, J. (1989). The mucopolysacchari

For ot-L-iduronidase enZyme therapy in MPS I, a recom

binant source of enZyme has been needed in order to obtain

45

through 10 represent eluate containing recombinantly pro

duced human ot-L-iduronidase in amounts of 1 pg, 2 pg, 5
pg, 5 pg, 5 pg and 5 pg, respectively.
FIG. 3 reveals the urinary GAG levels in 16 MPS I
patients in relation to normal excretion values. There is a
Wide range of urine GAG values in untreated MPS I patients.
A greater than 50% reduction in excretion of undegraded

tion is generally not severely affected, but physical problems

GAGs folloWing therapy With recombinant ot-L-iduronidase

may lead to death by the teens or tWenties. Scheie syndrome

is the mildest form of MPS I. It is compatible With a normal

therapy.

life span, but joint stiffness, corneal clouding and heart valve
disease cause signi?cant problems.
The frequency of MPS I is estimated to be 1:100,000

is a valid means to measure an individuals response to

50

according to a British Columbia survey of all neWboms

(LoWry et al., Human Genetics 85:3894390 (1990)) and

1:70,000 according to an Irish study (Nelson, Human Genet

55

ics 10113554358 (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 neWbom screening for MPS I

60

FIG. 6 demonstrates in three patients that a substantial

shrinkage of liver and spleen together With signi?cant clini

cal improvement in joint and soft tissue storage Was asso
ciated With a greater than 65% reduction in undegraded
GAG after only 8 Weeks of treatment With recombinant
enzyme.
FIG. 7 demonstrates that there is substantial normaliza

tion of livers (FIG. 7A) and spleens (FIG. 7B) in patients

Would likely ?nd some previously undetected patients.

treated With recombinant enZyme after only 12 Weeks of

Except for bone marroW transplantation, there are no

signi?cant therapies available for MPS I. Bone marroW

transplants can be effective in treating some of the symptoms

FIG. 4 demonstrates leukocyte iduronidase activity before

and after enZyme therapy in MPS I patients.
FIG. 5 demonstrates the buccal iduronidase activity
before and after enZyme therapy.

therapy.
65

FIG. 8 demonstrates a precipitous drop in urinary GAG

of the disorder but have high morbidity and mortality in

excretion over 22 Weeks of therapy With recombinant

MPS I and often are not available to patients because of a

enZyme in 6 patients.

US 7,041,487 B2
3

BRIEF SUMMARY OF THE INVENTION

In a fourth aspect, the present invention provides novel

ot-L-iduronidase produced in accordance With the methods
of the present invention and thereby present in amounts

In one aspect, the present invention features a method to

produce ot-L-iduronidase in amounts Which enable using the

enzyme therapeutically. In a broad embodiment, the method
comprises the step of transfecting a cDNA encoding for all

Which enable using the enZyme therapeutically. The speci?c

activity of the ot-L-iduronidase according to the present
invention is in excess of 200,000 units per milligram protein.
Preferably, it is in excess of about 240,000 units per milli
gram protein. The molecular Weight of the ot-L-iduronidase
of the present invention is about 82,000 daltons, about
70,000 daltons being amino acid and about 12,000 daltons

or a part of an ot-L-iduronidase into a cell suitable for the

expression thereof. In some embodiments, a cDNA encoding

for a complete ot-L-iduronidase is used, preferably a human
ot-L-iduronidase. HoWever, in other embodiments, a cDNA
encoding for a biologically active fragment or mutant

being carbohydrates.

thereof may be used. Speci?cally, one or more amino acid

In a ?fth aspect, the present invention features a novel

method to purify ot-L-iduronidase. According to a ?rst

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.

embodiment, a cell mass may be groWn in about 10% serum

In one particularly preferred embodiment, the cDNA is

protein-free production medium Without any signi?cant

adaptation to produce a high speci?c activity starting mate

containing medium folloWed by a sWitch to a modi?ed

transfected into a Chinese hamster ovary cell to create cell

line 2.131. In yet other preferred embodiments, the produc

tion procedure features one or more of the folloWing char

20

rial for puri?cation. Preferably, a concentration/dia?ltration

scheme is employed that alloWs for the removal of exog

acteristics Which have demonstrated particularly high pro

duction levels: (a) the pH of the cell groWth culture may be

enous materials that may be required for recombinant pro

duction of the same such as, for example, Pluronics 13-68, a

loWered to about 6.5 to 7.0, preferably to about 6.7*6.8

commonly used surfactant for protecting cells from sparging

damage. Such exogenous materials should normally be
separated from the crude bulk to prevent fouling of the

during the production process, (b) about 2/3 to 3/4 of the

medium may be changed approximately every 12 hours, (c)
oxygen saturation may be optimiZed at about 80% using
intermittent pure oxygen sparging, (d) microcarriers With

25

columns. In another preferred embodiment, a ?rst column

load is acidi?ed to minimize the competitive inhibition
effect of uronic acids found in protein-free medium formu

about 10% serum 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 PF4CHO product may be optimiZed to

30

include supplemental amounts of one or more ingredients

selected from the group consisting of glutamate, aspartate,

glycine, ribonucleosides and deoxyribonucleosides, (f) a
perfusion Wand such as a Bellco perfusion Wand may be
used in a frequent batch-feed process rather than a standard

column steps are used to eliminate less desirable ot-L

35

intended perfusion process, and (g) a mild sodium butyrate

induction process may be used to induce increased ot-L

iduronidase expression.
In a second aspect, the present invention provides a

40

transfected cell line Which features the ability to produce

ot-L-iduronidase in amounts Which enable using the enZyme
invention features a recombinant Chinese hamster ovary cell

the cell line may contain at least about 10 copies of a an

expression construct. In even more preferred embodiments,
the cell line expresses recombinant ot-L-iduronidase in

iduronidase that is nicked or degraded. In yet other preferred

embodiments, a three step column chromatography may be
used to purify the enZyme. Such a three step column
chromatography may include using a blue sepharose FF, a

Cu++ chelating sepharose chromatography and a phenyl

sepharose HP chromatography. In another preferred embodi
ment, an acid pH treatment step is used to inactivate

potential viruses Without harming the enZyme.

therapeutically. In preferred embodiments, the present

line such as the 2.131 cell line that stably and reliably
produces amounts of ot-L-iduronidase Which enable using
the enZyme therapeutically. In some preferred embodiments,

lations. In other preferred embodiments, a heparin, phenyl

and siZing column puri?cation scheme is used to produce
pure enZyme using automatable steps and validatable media.
In other preferred embodiments, the heparin and phenyl

45

In a sixth aspect, the present invention features novel

methods of treating diseases caused all or in part by a
de?ciency in ot-L-iduronidase. In one embodiment, this
method features administering a recombinant ot-L-idu
ronidase or a biologically active fragment or mutant thereof
alone or in combination With a pharmaceutically suitable

carrier. In other embodiments, this method features trans

50

ferring a nucleic acid encoding all or a part of an ot-L

amounts of at least about 20410 micrograms per 107 cells per

iduronidase into one or more host cells in vivo. Preferred

day.

embodiments include optimiZing the dosage to the needs of

the organism to be treated, preferably mammals or humans,
to effectively ameliorate the disease symptoms. In preferred
embodiments, the disease is mucopolysaccharidosis I (MPS
I), Hurler syndrome, Hurler-Scheie syndrome or Scheie

In a third aspect, the present invention provides novel

vectors suitable to produce ot-L-iduronidase in amounts

Which enable using the enZyme therapeutically. In preferred

55

embodiments, the present invention features an expression

vector comprising a cytomegalovirus promoter/enhancer

syndrome.
In a seventh aspect, the present invention features novel

element, a 5' intron consisting of a murine Ca intron, a

pharmaceutical compositions comprising ot-L-iduronidase

cDNA encoding all or a fragment or mutant of an ot-L

iduronidase, and a 3' bovine groWth hormone polyadenyla

tion site. Also, preferably the cDNA encoding all or a

60

useful for treating a disease caused all or in part by a

de?ciency in ot-L-iduronidase. Such compositions may be

fragment or mutant of an ot-L-iduronidase is about 2.2 kb in

suitable for administration in a number of Ways such as

length. This expression vector may be transfected at, for

parenteral, topical, intranasal, inhalation or oral administra

example, a 50 to 1 ratio With any appropriate common

selection vector such as, for example, pSV2NEO, to

enhance multiple copy insertions. Alternatively, gene ampli

?cation may be used to induce multiple copy insertions.

65

tion. Within the scope of this aspect are embodiments

featuring nucleic acid sequences encoding all or a part of an
ot-L-iduronidase Which may be administered in vivo into
cells affected with an ot-L-iduronidase de?ciency.

US 7,041,487 B2
5

DETAILED DESCRIPTION OF THE

INVENTION

ot-L-iduronidase expression Without a substantial effect on

the carbohydrate processing and cellular uptake of the

enZyme. Such an induction process may provide about a

tWo-fold increase in production Without signi?cantly alter

In one aspect, the present invention features a method to

ing post-translational processing.

produce ot-L-iduronidase in amounts Which enable using the

enzyme therapeutically. In general, the method features
transforming a suitable cell line With the cDNA encoding for

Particularly preferred embodiments of the method for

producing ot-L-iduronidase according to the present inven

all of ot-L-iduronidase or a biologically active fragment or

mutant thereof. Those of skill in the art may prepare expres

tion feature one, more than one or all of the optimizations

described herein. The production method of the present

invention may therefore provide a production culture pro
cess having the folloWing features:
1. A microcarrier based culture using Cytodex 2 beads or
an equivalent thereof is preferably used in large scale culture
?asks With overhead Wand stirring using a Bellco perfusion

sion constructs other than those expressly described herein

for optimal production of ot-L-iduronidase in suitable cell

lines transfected thereWith. Moreover, skilled artisans may

easily design fragments of cDNA encoding biologically

active fragments and mutants of the naturally occurring

Wand or an equivalent thereof. Attachment to these beads

may be achieved by culture in a 10% fetal bovine serum in

ot-L-iduronidase Which possess the same or similar biologi

cal activity to the naturally occurring ?ll-length enZyme.

DME/F12 1:1 medium modi?ed With ingredients including

ribonucleosides, deoxyribonucleosides, pyruvate, non-es

To create a recombinant source for ot-L-iduronidase, a

large series of expression vectors may be constructed and

tested for expression of a ot-L-iduronidase cDNA. Based on
transient transfection experiments as Well as stable transfec
tions, an expression construct may be identi?ed that pro

20

sential amino acids, and HEPES and at a pH of about

6746.9. After about 3 days in this medium, a Washout

procedure is begun in Which protein-free medium replaces

vides particularly high level expression. In one embodiment

approximately 2/3 of the medium approximately every 12

of the present invention, a Chinese hamster cell line 2.131

hours for a total of about 344 Washes. Subsequently and

developed by transfection of the ot-L-iduronidase expression

construct and selection for a high expression clone provides
particularly high level expression. Such a Chinese hamster
cell line according to this embodiment of the present inven

25

2. The culture conditions are preferably maintained at a

dissolved oxygen of 80% of air saturation at a pH of about
6.7 and at a temperature of about 370 C. This may be

tion may secrete about 5,000 to 7,000 fold more ot-L

iduronidase than normal. The ot-L-iduronidase produced

thereby may be properly processed, taken up into cells With
high af?nity and is corrective for ot-L-iduronidase de?cient
cells such as those from patients suffering from Hurlers

achieved using a control toWer, service unit and appropriate

30

35

production process speci?cally designed to produce the

enZyme in high quantities. According to preferred embodi

The dissolved oxygen may be supplied by intermittent pure

equivalent thereof. A pH of about 6.7 is optimal for the

scalable surface on Which to groW adherent cells. In espe

40

microporous.
According to other preferred embodiments of the method
for producing ot-L-iduronidase according to the present
invention, a culture system is optimiZed. In a ?rst embodi
ment, the culture pH is loWered to about 6.5 to 7.0, prefer
ably to about 6.7468 during the production process. One
advantage of such a pH is to enhance accumulation of

45

50

this procedure is to enhance the secretion rate of recombi

nant ot-L-iduronidase and capture more active enZyme. In a

55

microcarriers With about 10% serum initially are used to

produce a cell mass folloWed by a rapid Washout shift to a

protein-free medium for production. In a ?fth embodiment,

from JRH Biosciences called Excell PF CHO. This medium

supports levels of secretion equivalent to that of serum using
a cell line such as the 2.131 cell line. It may be preferably
modi?ed to include an acidic pH of about 6.7 (+/0.1), and
it may be buffered with HEPES at 7.5 mM. The medium may
contain 0.05 to 0.1% of Pluronics F-68 (BASE), a non-ionic
surfactant or an equivalent thereof Which features the advan

tage of protecting cells from shear forces associated With

sparging. The medium may further contain a proprietary
supplement that proves to be important in increasing the
productivity of the medium over other protein-free mediums
that are presently available. Those skilled in the art Will
readily understand that the choice of culture medium may be

a groWth medium such as a JRH Biosciences PF4CHO

product may be optimiZed to include supplemental amounts

beloW a pH of about 6.5. The culture is therefore maintained

optimally betWeen a pH of about 6.6 to 6.8.
3. The production culture medium may be a modi?ed

form of the commercially available proprietary medium

second embodiment, about 2/3 to 3A of the medium is

third embodiment, oxygen saturation is optimiZed at about

80% using intermittent pure oxygen sparging rather than
continuous sparging. In a fourth embodiment, cytodex 2

accumulation of the ot-L-iduronidase enZyme. The enZyme

is particularly unstable at pHs above about 7.0. BeloW a pH

of about 6.7, the secretion rate may decrease, particularly

lysosomal enZymes that are more stable at acidic pH. In a

changed approximately every 12 hours. One advantage of

manufacturers. An air saturation of about 80% results in

improved ot-L-iduronidase secretion over 40% and 60% air
saturation. HoWever 90% air saturation does not provide
signi?cantly enhanced secretion over 80% air saturation.
oxygen sparging using a 5 micron stainless steel sparger or

ments of such a process, microcarriers are used as a loW cost

cially preferred embodiments, such microcarriers are

probes such as those produced by Wheaton. HoWever,

skilled artisans Will readily appreciate that this can easily be

achieved by equivalent control systems produced by other

Syndrome.
The method for producing ot-L-iduronidase in amounts
that enable using the enZyme therapeutically features a

throughout the entire remaining culture period, the cells are

cultivated in protein-free medium.

60

optimiZed continually according to particular commercial

of one or more ingredients selected from the group consist

embodiments available at particular points in time. Such

ing of glutamate, aspartate, glycine, ribonucleosides and

changes encompass no more than routine experimentation

and are intended to be Within the scope of the present
invention.

deoxyribonucleosides. In a sixth embodiment, a perfusion

Wand such as a Bellco perfusion Wand may be used in a

frequent batch-feed process rather than a standard intended

perfusion process. In a seventh embodiment, a mild sodium
butyrate induction process may be used to induce increased

65

4. The production medium may be analyZed using an

amino acid analyZer comparing spent medium With starting

medium. Such analyses have demonstrated that the 2.131

US 7,041,487 B2
7

cell line depletes a standard PF CHO medium of glycine,

glutamate and aspartate to a level of around 10% of the

may produce approximately 25 mg per liter of culture per

day, or more at peak culturing density.

starting concentration. Supplementation of these amino

In a second aspect, the present invention provides a

acids to higher levels may result in enhanced culture density

and productivity that may lead to a 243 fold higher produc
tion than at baseline. Skilled artisans Will appreciate that
other cell lines Within the scope of the present invention may

transfected cell line Which possesses the unique ability to

produce ot-L-iduronidase in amounts Which enable using the

enZyme therapeutically. In preferred embodiments, the

be equally useful for producing ot-L-iduronidase according

present invention features a recombinant Chinese hamster

ovary cell line such as the 2.131 cell line that stably and

to the present method. Hence, more or less supplemental

reliably produces amounts of ot-L-iduronidase. In preferred

nutrients may be required to optimiZe the medium. Such

embodiments, the cell line may contain at least about 10

copies of an expression construct comprising a CMV pro

optimizations are intended to be Within the scope of the

present invention and may be practiced Without undue

moter, a Ca intron, a human ot-L-iduronidase cDNA, and a

experimentation.

bovine groWth hormone polyadenylation sequence. In even

more preferred embodiments, the cell line expresses ot-L
iduronidase at amounts of at least about 2(k40 micrograms

5. The medium may be supplemented With ribonucleo

sides and deoxyribonucleosides to support the dihydrofolate

per 107 cells per day in a properly processed, high uptake

form appropriate for enZyme replacement therapy. Accord

reductase de?cient cell line 2.131. Skilled artisans Will

appreciate that other cell lines Within the scope of the present

ing, to preferred embodiments of this aspect of the inven

tion, the transfected cell line adapted to produce ot-L

invention may be equally useful for producing ot-L-idu

ronidase according to the present method. Hence, more or

less ribonucleosides and deoxyribonucleosides may be

required to optimiZe the medium. Such optimiZations are
intended Within the scope of the present invention and may

20

be practiced Without undue experimentation.

6. After reaching con?uence at about 344 days of culture,
approximately 2/3 of the medium may be changed out
approximately every 12 hours. The change out of medium
may be accomplished using, for instance, a Bellco perfusion

25

Wand Which is a stirring device With a holloW center and

screen ?lter at its tip. By pumping out medium through the

holloW interior of the Wand through the 40 micron screen.
The microcarriers With the 2.131 cell mass are separated

from supernatant containing the enZyme.

7. The rapid and frequent turnover of the medium has
been shoWn by productivity studies to result in improved

30

35

40

more enZyme. The method of this embodiment has proven to

batch culture or daily or every other day batch/feed strate

45

cells may be maintained in excellent condition With high

According to one embodiment Wherein the Chinese hamster

ovary cell line 2.131 is provided, there are approximately 10
copies of the expression vector for ot-L-iduronidase. Such a

cell line has demonstrated the ability to produce large

quantities of human ot-L-iduronidase (minimum 20 micro
grams per 10 million cells per day). Particularly preferred
embodiments such as the 2.131 cell line possess the ability

to produce properly processed enZyme that contains

N-linked oligosaccharides containing high mannose chains

degrees of viability and a high level of productivity.

8. Production of ot-L-iduronidase may be enhanced by the
use of sodium butyrate induction of gene expression. Sys

modi?ed With phosphate at the 6 position in suf?cient

50

quantity to produce an enZyme With high af?nity (K-uptake

of less than 3 nM).
3. The enZyme produced from the cell lines of the present
invention such as a Chinese hamster ovary cell line 2.131 is

e?fects on carbohydrate processing. LoWer levels of butyrate

rapidly assimilated into cells, eliminates glycosaminoglycan

have not been shoWn to induce as Well, and substantially

55

storage and has a half-life of about 5 days in cells from

patients su?fering from ot-L-iduronidase de?ciency.

4. The cell line of preferred embodiments such as a 2.131

cell line adapts to large scale culture and stably produces

gest that tWo-fold or greater induction results in less pro

cessing of the carbohydrates and less phosphate addition to

the enZyme as Well as increasing toxicity. One particularly
preferred method uses 2 mM butyrate addition every 48
hours to the culture system. This embodiment results in
about a tWo-fold induction of enZyme production using this
method Without signi?cant e?fect on the uptake af?nity of the
enZyme, (K-uptake of less than 30 U/ml or 2.0 mM). Using
embodiments of the present method featuring all of the
above modi?cations and induction, a 15 liter culture system

common selection vector such as pSV2NEO. The selection

vector pSV2NEO in turn confers G418 resistance on suc

enhances the acquisition of multiple copy number inserts.

be superior to perfusion culture and far superior to strict

higher levels may result in higher induction but declining

af?nity of the produced enZyme for cells from patients
su?fering from ot-L-iduronidase de?ciency. The results sug

murine Ca intron betWeen exons 2 and 3, a human cDNA of

ments, a ratio of about 50 to 1 is used since this ratio

enZyme. Studies of the secretion rate of the enZyme during

a 12 hour culture cycle demonstrate that the cells are actively

tematic studies of a 2.131 cell line demonstrated that about

2 mM butyrate can be applied and result in about a tWo-fold
or greater induction of enZyme production With minimal

groWth rate and until they readily attach to substrates.

2. The cell line of preferred embodiments is transfected
With an expression vector containing the cytomegalovirus
promoter/enhancer element, a 5' intron consisting of the

cessfully transfected cells. In particularly preferred embodi

frequent changes result in less total accumulation of

gies. Using the every approximately 12 hour change, the

features:
1. The cell line of preferred embodiments is derived from
a parent cell line Wherein the cells are passaged in culture
until they have acquired a smaller siZe and more rapid

about 2.2 kb in length, and a 3' bovine groWth hormone

polyadenylation site. This expression vector may be trans
fected at, for example, a 50 to 1 ratio With any appropriate

overall collection of enZyme from the cell culture. Less

secreting enZyme for the majority of the culture period.

More frequent changes are unlikely to yield substantially

iduronidase in amounts Which enable using the enZyme

therapeutically possesses one or more of the folloWing

60

human ot-L-iduronidase under these conditions. The cells of

preferred embodiments are able to groW and secrete ot-L
iduronidase at the acid pH of about 6.6 to 6.8 at Which
enhanced accumulation of ot-L-iduronidase can occur.

5. Particularly preferred embodiments of the cell line

according to the invention, such as a 2.131 cell line are able
65

to secrete human ot-L-iduronidase at levels exceeding 2,000

units per ml (8 micrograms per ml) tWice per day using a

specially formulated protein-free medium.

US 7,041,487 B2
10
In a third aspect, the present invention provides novel
vectors suitable to produce ot-L-iduronidase in amounts

for such correction (half-maximal correction at 0.7 pM) may

be very important for the success of enzyme replacement

Which enable using the enzyme therapeutically. The produc

tion of adequate quantities of recombinant ot-L-iduronidase

therapy.
The human cDNA of ot-L-iduronidase predicts a protein
of 653 amino acids and an expected molecular Weight of

is a critical prerequisite for studies on the structure of the

enzyme as Well as for enzyme replacement therapy. The cell

70,000 daltons after signal peptide cleavage. Amino acid

sequencing reveals alanine 26 at the N-terminus giving an

lines according to the present invention permit the produc

tion of signi?cant quantities of recombinant ot-L-idu
ronidase that is appropriately processed for uptake. Over
expression in Chinese hamster ovary (CHO) cells has been
described for three other lysosomal enzymes, ot-galactosi
dase (loannou et al., J. Cell. Biol. 119:113741150 (1992)),

expected protein of 629 amino acids. Human recombinant

ot-L-iduronidase has a Histidine at position 8 of the mature

protein. The predicted protein sequence comprises six

potential N-linked oligosaccharide modi?cation sites. All of
these may be modi?ed in the recombinant protein. The third
and sixth sites have been demonstrated to contain one or

iduronate 2-sulfatase (Bielicki et al., Biochem. J. 289:

more mannose 6-phosphate residues responsible for high

2414246 (1993)), and N-acetylgalactosamine 4-sulfatase

(Amson et al., Biochem. J. 284:7894794 (1992)), using, a
variety of promoters and, in one case, ampli?cation. The

af?nity uptake into cells. The folloWing peptide corresponds

to Amino Acids 26445 of Human Recombinant ot-L-idu
ronidase With an N-ter'minus alanine and the folloWing
sequence:

present invention features a dihydrofolate reductase-de?

cient CHO cell line, but according to preferred embodiments

of the invention ampli?cation is unnecessary. Additionally,
the present invention provides a high level of expression of
the human ot-L-iduronidase using the CMV immediate early
gene promoter/ enhancer.
The present invention features in preferred embodiments
an expression vector comprising a cytomegalovirus pro
moter/ enhancer element, a 5' intron consisting of the murine
Ca intron derived from the murine long chain immunoglo

ala-glu-ala-pro-his-leu-val-his-val-asp-ala-ala-arg-ala
leu-trp-pro-leu-arg-arg

25

84, and 89 kDa, is comparable to 87 kDa, found for urinary

bulin CO. gene betWeen exons 2 and 3, a human cDNA of

about 2.2 kb in length, and a 3' bovine groWth hormone

polyadenylation site. This expression vector may be trans
fected at, for example, a 50 to 1 ratio With any appropriate
common selection vector such as, for example, pSV2NEO.

30

corrective factor (Barton et al., J. Biol. Chem. 246:

777347779 (1971)), and to 76 kDa and 82 kDa, found for
enzyme secreted by cultured human ?broblasts (MyeroWitz
et al., J. Biol. Chem. 256: 304443048 (1991); Taylor et al.,
Biochem. J 2742634268 (1991)). The differences Within

35

measurements. The pattern of intracellular processing of the

recombinant enzyme-a sloW decrease in molecular size and
the eventual appearance of an additional band smaller by 9

The selection vector such as pSV2NEO in turn confers G418

resistance on successfully transfected cells. In particularly

preferred embodiments, a ratio of about 50 to 1 expression
vector to selection vector is used since this ratio enhances

and betWeen the studies are attributed to imprecision of the

the acquisition of multiple copy number inserts. According

kDa is the same as for the human ?broblast enzyme. This

to one embodiment Wherein the Chinese hamster ovary cell

faster band arises by proteolytic cleavage of 80 N-terminal

line 2.131 is provided, there are approximately 10 copies of

the expression vector for ot-L-iduronidase. Such an expres

sion construct has demonstrated the ability to produce large

quantities of human ot-L-iduronidase (minimum 20 micro
grams per 10 million cells per day) in a suitable cell line such
as, for example, a Chinese hamster ovary cell line 2.131.
In a fourth aspect, the present invention provides novel

40

method to purify ot-L-iduronidase. In preferred embodi

45

produce a rapid and e?icient puri?cation With validatible

chromatography resins and easy load, Wash and elute opera
tion. The method of purifying ot-L-iduronidase of the
present invention involves a series of column chromatogra

phy steps Which alloW the high yield puri?cation of enzyme

from protein-free production medium.
50

effective in therapy in vivo.

The speci?c activity of the ot-L-iduronidase according to

According to a ?rst embodiment, the cell mass is groWn

in about 10% serum containing medium folloWed by a
sWitch to a modi?ed protein-free production medium With

out any signi?cant adaptation to produce a high speci?c

activity starting material for puri?cation. In a second

the present invention is in excess of about 200,000 units per

milligram protein. Preferably, it is in excess of about 240,

000 units per milligram protein. The molecular Weight of the
full length ot-L-iduronidase of the present invention is about
82,000 daltons comprising about 70,000 daltons of amino
acids and 12,000 daltons of carbohydrates. The recombinant

55

enzyme of the present invention is endocytosed even more

60

embodiment,

concentration/dia?ltration

scheme

is

employed that alloWs for the removal of such exogenous

materials as Pluronics F-68 from the crude bulk to prevent
fouling of columns. In a third embodiment, a ?rst column

ef?ciently than has been previously reported for a partially

puri?ed preparation of urinary enzyme. The recombinant

load is acidi?ed to minimize the competitive inhibition

effect of such compounds as uronic acids found in protein
free medium formulations. In a fourth embodiment, a hep

arin, phenyl and sizing column puri?cation scheme is used

enzyme according to the present invention is effective in

reducing the accumulation of radioactive S-labeled GAG in

ot-L-iduronidase-de?cient ?broblasts, indicating that it is

transported to lysosomes, the site of GAG storage. The
remarkably loW concentration of ot-L-iduronidase needed

amino acids.
In a ?fth aspect, the present invention features a novel

ments, the present invention features a method to purify

recombinant ot-L-iduronidase that has been optimized to

ot-L-iduronidase produced in accordance With the methods

of the present invention and thereby present in amounts that
enable using the enzyme therapeutically. The methods of the
present invention produce a substantially pure ot-L-idu

ronidase that is properly processed and in high uptake form

appropriate for enzyme replacement therapy and that is

The overexpression of the ot-L-iduronidase of the present

invention does not result in generalized secretion of other
lysosomal enzymes that are dependent on mannose-6-P
targeting. The secreted recombinant ot-L-iduronidase is
similar to normal secreted enzyme in many respects. lts
molecular size, found in various determinations to be 77, 82,

to produce pure enzyme using automatable steps. In a ?fth

embodiment, the heparin and phenyl column steps are used

65

to eliminate less desirable ot-L-iduronidase that is nicked or

degraded. In a sixth embodiment, an acid pH treatment step

is used to inactivate potential viruses Without harming the

US 7,041,487 B2
11

12

enzyme. In a seventh embodiment, a 3 step column chro

matography process is followed. The ?rst column involves

In a sixth aspect, the present invention features novel

methods of treating diseases caused all or in part by a

an af?nity chromatography step using Blue Sepharose 6 PP.

The Blue eluate is then further puri?ed by another affinity

de?ciency in ot-L-iduronidase. Recombinant ot-L-idu

ronidase provides enzyme replacement therapy in a canine

chromatography step using Cu++ Chelating Sepharose FF.

Finally hydrophobic interaction chromatography using Phe
nyl Sepharose High Performance (HP) is used.

model of MPS 1. This canine model is de?cient in ot-L

iduronidase due to a genetic mutation and is similar to

Particularly preferred embodiments of the method for

purifying ot-L-iduronidase according to the present inven

Was administered intravenously to 11 dogs. In those dogs

treated With Weekly doses of 25,000 to 125,000 units per kg

tion feature more than one or all of the optimizations

for 3, 6 or 13 months, the enzyme Was taken up in a variety

human MPS 1. Puri?ed, properly processed ot-L-iduronidase

according to the folloWing particular embodiments. The

of tissues and decreased the lysosomal storage in many

puri?cation method of the present invention may therefore

provide a puri?ed ot-L-iduronidase having the characteris

tissues. The long term treatment of the disease Was associ

ated With clinical improvement in demeanor, joint stiffness,

coat and groWth. Higher doses of therapy (125,000 units per
kg per Week) result in better ef?cacy and including normal

tics described herein.

1. Concentration/dia?ltration: Crude supernatant is pro
cessed With a holloW ?ber concentrator (A/G Technologies,
30K cuto?) to reduce ?uid volume by about 75% and is then

ization of urinary GAG excretion in addition to more rapid

clinical improvement in demeanor, joint stiffness and coat.

Enzyme therapy at even small doses of 25,000 units (0.1
mg/kg/Wk) resulted in signi?cant enzyme distribution to

dia?ltrated With a heparin load buffer (10 mM NaPO4, pH

5.3, NaCl 200 mM). The dia?ltration is an important step
that eliminates undesirable compounds such as Pluronics
F-68 from the supernatant, a surfactant needed in many cell
cultures of the present invention that can foul columns. The
dia?ltration may also partly remove competitor inhibitors

20

evident in terms of activity, mobility, groWth and overall

health. The therapy at this dose did not improve other tissues
that are important sites for disease in this entity such as

that may prevent binding to the heparin column. These

inhibitors may be found in PFiCHO medium and are

25

as 2 Weeks. Studies at this increased dose are ongoing in tWo

about 5.0 before loading on Heparin Sepharose CL-6B.

30

dogs for six months to date. These MPS I dogs are shoWing

signi?cant clinical improvement and substantial decreases in

urinary GAG excretion into the normal range. Other than an
immune reaction controlled by altered administration tech

Without the dia?ltration and pH adjustment, heparin col

umns cannot be run using PFiCHO medium Without

cartilage and brain. Higher doses of 125,000 units (0.5

mg/kg) given 5 times over tWo Weeks demonstrate that
improved tissue penetration can be achieved, and a thera
peutic effect at the tissue level Was accomplished in as little

believed to be uronic acids derived from a soybean hydroly

sate present in this particular medium.
2. Heparin column: The load may be adjusted to a pH of
Other types of heparin columns such as a heparin FF
(Pharmacia) have different linkages and do not bind ot-L
iduronidase as ef?ciently. A loWer pH neutralizes uronic
acids to some extent Which lessens their competitive effect.

some tissues and decreases in GAG storage. If continued for

over 1 year, signi?cant clinical effects of the therapy Were

35

niques, the enzyme therapy has not shoWn signi?cant clini

cal or biochemical toxicity. Enzyme therapy at this higher

having substantial enzyme ?oWthrough. The column may be

Weekly dose is effective at improving, some clinical features

Washed With a pH of about 5.3 buffer and then eluted in 0.6

M NaCl. The narroW range of binding and elution salt
concentration leads to an e?icient puri?cation step and
enzyme that is often greater than 90% pure after one step.

pharmaceutical compositions comprising human ot-L-idu

of MPS I and decreasing storage Without signi?cant toxicity.

In a seventh aspect, the present invention features novel
40

The recombinant enzyme may be administered in a number

3. Phenyl column: A Phenyl-Sepharose BP (Pharmacia)

may be used in the next step. The heparin eluate may be
adjusted to about 1.5 M NaCl and loaded on the column. The
choice of resin is important as is the salt concentration in

ensuring that the enzyme binds completely (no How

through) and yet elutes easily and completely With about
0.15 M NaCl. The eluate obtained is nearly pure ot-L
iduronidase.
4. A pH inactivation may be performed to provide a robust

step for the removal of potential viruses. The phenyl pool is

adjusted to a pH of about 3.3 using Citrate pH 3.0 and held

of Ways such as parenteral, topical, intranasal, inhalation or

oral administration. Another aspect of the invention is to

provide for the administration of the enzyme by formulating

45

it With a pharmaceutically-acceptable carrier Which may be

solid, semi-solid or liquid or an ingestable capsule.

Examples of pharmaceutical compositions include tablets,

drops such as nasal drops, compositions for topical appli
cation such as ointments, jellies, creams and suspensions,
50

aerosols for inhalation, nasal spray, liposomes. Usually the

recombinant enzyme comprises betWeen 0.05 and 99% or

betWeen 0.5 and 99% by Weight of the composition, for

example, betWeen 0.5 and 20% for compositions intended
for injection and betWeen 0.1 and 50% for compositions

at room temperature for about 4 hours. The enzyme may

then be neutralized. Embodiments featuring this step have

been shoWn to eliminate viruses at a minimum of about 5 log
units. The step does not substantially inactivate or affect the

ronidase useful for treating a de?ciency in ot-L-iduronidase.

55

intended for oral administration.

enzyme activity.

To produce pharmaceutical compositions in this form of

dosage units for oral application containing a therapeutic

5. The enzyme may then be concentrated and injected

onto a Sephacryl S-200 column and the peak of enzyme
collected.
Enzyme puri?ed in this manner has been shoWn to contain

carrier, for example lactose, saccharose, sorbitol, mannitol,

enzyme, the enzyme may be mixed With a solid, pulverulent

60

mannose-6-phosphate residues of suf?cient quantity at posi

a starch such as potato starch, corn starch, amylopectin,

laminaria poWder or citrus pulp poWder, a cellulose deriva
tive or gelatine and also may include lubricants such as

tions 3 and 6 of the N-linked sugars to give the enzyme

magnesium or calcium stearate or a CarboWax or other

uptake af?nity of less than 30 units per ml (less than 2 nM)

enzyme. The enzyme is substantially corrective for gly

polyethylene glycol Waxes and compressed to form tablets

cosamino glycan storage disorders and has a half-life inside

cells of approximately 5 days.

65

or cores for dragees. If dragees are required, the cores may

be coated for example With concentrated sugar solutions

Which may contain gum arabic, talc and/or titanium dioxide,

US 7,041,487 B2
13

14

or alternatively With a ?lm forming agent dissolved in easily

volatile organic solvents or mixtures of organic solvents.
Dyestuffs can be added to these coatings, for example, to
distinguish betWeen different contents of active substance.

may be administered per day be mentioned from about 0.1

mg to about 2000 mg or from about 1 mg to about 2000 mg.

The pharmaceutical compositions containing the thera

peutic enZyme may suitably be formulated so that they
provide doses Within these ranges either as single dosage
units or as multiple dosage units. In addition to containing

For the composition of soft gelatine capsules consisting of

gelatine and, for example, glycerol as a plasticiZer, or similar
closed capsules, the active substance may be admixed With

a therapeutic enZyme (or therapeutic enzymes), the subject

a CarboWax or a suitable oil as e.g., sesame oil, olive oil,

formulations may contain one or more substrates or cofac

or arachis oil. Hard gelatine capsules may contain granulates

of the active substance With solid, pulverulent carriers such
as lactose, saccharose, sorbitol, mannitol, starches such as

tors for the reaction catalyZed by the therapeutic enZyme in

tions may also contain more than one therapeutic enZyme.

potato starch, corn starch or amylopectin, cellulose deriva

tives or gelatine, and may also include magnesium stearate

The recombinant enZyme employed in the subject meth

ods and compositions may also be administered by means of

or stearic acid as lubricants.

transforming patient cells With nucleic acids encoding the

Therapeutic enZymes of the subject invention may also be

administered parenterally such as by subcutaneous, intra

recombinant ot-L-iduronidase. The nucleic acid sequence so

the compositions. Therapeutic enZyme containing composi

muscular or intravenous injection or by sustained release

encoding may be incorporated into a vector for transforma

tion into cells of the subject to be treated. Preferred embodi

subcutaneous implant. In subcutaneous, intramuscular and

intravenous injection, the therapeutic enZyme (the active

be designed so as to integrate into the chromosomes of the

ingredient) may be dissolved or dispersed in a liquid carrier

vehicle. For parenteral administration, the active material
may be suitably admixed With an acceptable vehicle, pref
erably of the vegetable oil variety such as peanut oil,

ments of such vectors are described herein. The vector may

20

ronidase nucleotide sequences may be designed so as to

provide for continuous or regulated expression of the

enZyme. Additionally, the genetic vector encoding the

cottonseed oil and the like. Other parenteral vehicles such as

organic compositions using solketal, glycerol, formal, and

25

aqueous parenteral formulations may also be used.

cell genome or to only be present transiently. The general

comprise an aqueous solution of a Water soluble pharma

ceutically acceptable salt of the active acids according to the

invention, desirably in a concentration of 0.5410%, and
optionally also a stabiliZing agent and/or bulfer substances

niques can be found in Friedman, Science 244:127541281

(1989); Ledley, J. Inherit. Metab. Dis. 13:5874616 (1990);

and Tososhev et al., Curr Opinions Biolech. 1:55461 (1990).

in aqueous solution. Dosage units of the solution may

advantageously be enclosed in ampules.

A particularly preferred method of administering the

35

at about 5.8 and human albumin. These ingredients may be

40

For topical application, the pharmaceutical compositions

ot-L-iduronidase

0.05402 mg/mL or l2,500450,000 units per

mL
Sodium chloride solution 150 mM in an IV bag, 504250 cc total volume

cream or the like. The amount of active substance may vary,

substance. Such pharmaceutical compositions for topical

recombinant enZyme is intravenously. A particularly pre

ferred composition comprises recombinant ot-L-idu
ronidase, normal saline, phosphate buffer to maintain the pH

provided in the folloWing amounts:

are suitably in the form of an ointment, gel, suspension,

for example, betWeen 0.05420% by Weight of the active

enZyme may be designed so as to stably integrate into the

methodology of conventional genetic therapy may be

applied to polynucleotide sequences encoding ot-L-idu
ronidase. RevieWs of conventional genetic therapy tech

For parenteral application by injection, compositions may

When therapeutic enZymes are administered in the form

of a subcutaneous implant, the compound is suspended or
dissolved in a sloWly dispersed material knoWn to those
skilled in the art, or administered in a device Which sloWly
releases the active material through the use of a constant
driving force such as an osmotic pump. In such cases,
administration over an extended period of time is possible.

subject, e.g., retroviral vectors, or to replicate autonomously

in the host cells. Vectors containing encoding ot-L-idu

Sodium phosphate buffer 10450 mM, pH 5.8

1 mgmL

45 Human albumin

application may be prepared in knoWn manner by mixing the

The invention having been described, the folloWing

active substance With knoWn carrier materials such as iso

propanol, glycerol, paraf?n, stearyl alcohol, polyethylene

glycol, etc. The pharmaceutically acceptable carrier may

50

examples arc offered to illustrate the subject invention by

Way of illustration, not by Way of limitation.

also include a knoWn chemical absorption promoter.

Examples of absorption promoters are, e.g., dimethylaceta

mide (US. Pat. No. 3,472,931), trichloro ethanol or tri?uo
roethanol (US. Pat. No. 3,891,757), certain alcohols and
mixtures thereof (British Patent No. 1,001,949). A carrier
material for topical application to unbroken skin is also

EXAMPLE 1

Producing Recombinant Iduronidase

55

Standard techniques such as those described by Sambrook

described in the British patent speci?cation No. 1,464,975,

etldal. (1987) Molecular Cloning: A Laboratory Manual,

Which discloses a carrier material consisting of a solvent

2 ed., Cold Spring Harbor Laboratory, Cold Spring Harbor,

comprising 4(k70% (v/v) isopropanol and 0460% (v/v)

glycerol, the balance, if any, being an inert constituent of a
diluent not exceeding 40% of the total volume of solvent.

60

cloned Was subcloned into PRCCMV (Invitrogen) as a

The dosage at Which the therapeutic enZyme containing

HindIII-XbaI fragment from a bluescript KS subclone. An

intron cassette derived from the murine immunoglobulin Cot

pharmaceutical compositions are administered may vary

intron betWeen exons 2 and 3 Was constructed using PCR

Within a Wide range and Will depend on various factors such

as, for example, the severity of the disease, the age of the
patient, etc., and may have to be individually adjusted. As a
possible range for the amount of therapeutic enZyme Which

N.Y. may be used to clone cDNA encoding human ot-L

iduronidase. The human ot-L-iduronidase cDNA previously

65

ampli?cation of bases 78841372 (Tucker et al., Proc. Natl.

Acad. Sci. USA 78: 768447688 (1991) of clone pRIR14.5
(Kakkis et al., Nucleic Acids Res. 16:7796 (1988)). The