U.S. patent application number 11/135264 was filed with the patent office on 2005-12-29 for precursor n-acetylgalactosamine-4 sulfatase, methods of treatment using said enzyme and methods for producing and purifying said enzyme.
This patent application is currently assigned to BIOMARIN PHARMACEUTICAL INC.. Invention is credited to Chan, Wai-Pan, Chen, Lin, Fitzpatrick, Paul A., Henstrand, John M., Qin, Minmin, Starr, Christopher M., Swiedler, Stuart, Wendt, Dan J., Zecherle, Gary N..
Application Number | 20050287133 11/135264 |
Document ID | / |
Family ID | 32685968 |
Filed Date | 2005-12-29 |
United States Patent
Application |
20050287133 |
Kind Code |
A1 |
Qin, Minmin ; et
al. |
December 29, 2005 |
Precursor N-acetylgalactosamine-4 sulfatase, methods of treatment
using said enzyme and methods for producing and purifying said
enzyme
Abstract
The present invention provides a highly purified recombinant,
human precursor N-acetylgalactosamine-4-sulfatase and biologically
active mutants, fragments and analogs thereof as well as
pharmaceutical formulations comprising highly purified recombinant
human precursor N-acetylgalactosamine-4-sulfatase. The invention
also provides methods for treating diseases caused all or in part
by deficiencies in human N-acetylgalactosamine-4-sulfatase
including MPS VI and methods for producing and purifying the
recombinant precursor enzyme to a highly purified form.
Inventors: |
Qin, Minmin; (Pleasanton,
CA) ; Zecherle, Gary N.; (Navato, CA) ; Chan,
Wai-Pan; (Castro Valley, CA) ; Fitzpatrick, Paul
A.; (Albany, CA) ; Swiedler, Stuart; (Oakland,
CA) ; Henstrand, John M.; (Davis, CA) ; Wendt,
Dan J.; (Walnut Creek, CA) ; Chen, Lin; (San
Francisco, CA) ; Starr, Christopher M.; (Sonoma,
CA) |
Correspondence
Address: |
MARSHALL, GERSTEIN & BORUN LLP
233 S. WACKER DRIVE, SUITE 6300
SEARS TOWER
CHICAGO
IL
60606
US
|
Assignee: |
BIOMARIN PHARMACEUTICAL
INC.
Novato
CA
|
Family ID: |
32685968 |
Appl. No.: |
11/135264 |
Filed: |
May 23, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11135264 |
May 23, 2005 |
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10704365 |
Nov 7, 2003 |
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10704365 |
Nov 7, 2003 |
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10290908 |
Nov 7, 2002 |
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6866844 |
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10704365 |
Nov 7, 2003 |
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10658699 |
Sep 9, 2003 |
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10658699 |
Sep 9, 2003 |
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09562427 |
May 1, 2000 |
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Current U.S.
Class: |
424/94.6 |
Current CPC
Class: |
C12N 9/16 20130101; C12Y
301/06012 20130101; B01D 15/327 20130101; A61K 38/00 20130101; B01D
15/3828 20130101; B01D 15/3804 20130101; A61K 48/00 20130101 |
Class at
Publication: |
424/094.6 |
International
Class: |
A61K 038/46 |
Claims
1-23. (canceled)
24. A method for producing a recombinant precursor
N-acetylgalactosamine-4- -sulfatase enzyme comprising the steps of:
(a) growing a cell transfected with a DNA encoding all or a
biologically active fragment or mutant of a human
N-acetylgalactosamine-4-sulfatase enzyme, (b) introducing the
transfected cells into a bioreactor, (c) supplying a growth medium
to the bioreactor, (d) harvesting said medium containing said
enzyme; and (e) substantially removing the transfected cells from
the said harvest medium.
25. The method of claim 24 wherein said cell is a mammalian
cell.
26. The method of claim 25 wherein said mammalian cell is a Chinese
Hamster Ovary cell.
27. The method of claim 24 wherein the transfected cells are grown
on a growth medium comprising a JRH Excell 302 medium supplemented
with one or more agents selected from the group consisting of
L-glutamine, glucose, hypoxanthine/thymidine, serine, asparagine
and folic acid.
28. The method of claim 24 wherein said growth medium does not
contain G418:
29. The method of claim 24 wherein the transfected cells are grown
in a bioreactor for up to about 45 days.
30. The method of claim 24 wherein the transfected cells are grown
in a bioreactor for up to about 90 days.
31. The method of claim 24 wherein the transfected cells are
substantially separated from the media containing the enzyme
through successive membranes.
32. The method of claim 31 wherein the successive membranes are
4.0-0.75 .mu.m nomimal, 0.45 .mu.m and 0.2 .mu.m.
33-61. (canceled)
62. A method to purify a N-acetylgalactosamine-4-sulfatase or
biologically active fragment, analog or mutant thereof comprising:
(a) obtaining a fluid containing said
N-acetylgalactosamine-4-sulfatase or biologically active fragment,
analog or mutant thereof; (b) reducing the proteolytic activity of
any protease in said fluid able to cleave said
N-acetylgalactosamine-4-sulfatase or biologically active fragment,
analog or mutant thereof, wherein said reducing does not harm said
N-acetylgalactosamine-4-sulfatase or biologically active fragment,
analog or mutant thereof; (c) contacting the fluid with a Cibracon
blue dye interaction chromatography resin; (d) contacting the fluid
with a copper chelation chromatography resin; (e) contacting the
fluid with a phenyl hydrophobic interaction chromatography resin;
(f) recovering said N-acetylgalactosamine-4-sulfatase or
biologically active fragment, analog or mutant thereof; wherein
steps (c), (d) and (e) can be performed in any temporal
sequence.
63. A pharmaceutical composition comprising precursor
N-acetylgalactosamine-4-sulfatase and a polyoxyethylenesorbitan at
a concentration ranging from about 0.002% to about 0.008%
(weight/volume).
64. The pharmaceutical composition of claim 63 wherein said
polyoxyethylene sorbitan is polyoxyethylene sorbitan 80 at a
concentration of 0.005% (weight/volume).
65. A method for treating a disease caused all or in part by a
deficiency in N-acetylgalactosamine-4-sulfatase activity comprising
the step of administering to a human subject in need of such
treatment an effective amount of a composition comprising
recombinant human N-acetylgalactosamine-4-sulfatase (rhASB).
66. The method of claim 65 wherein the amount of rhASB is effective
to provide one or more beneficial effects selected from the group
consisting of joint mobility, joint pain, joint stiffness, exercise
tolerance, exercise endurance, pulmonary function, visual acuity,
and activities of daily living.
67. A method for treating mucopolysaccharidosis VI (MPS VI)
comprising the step of administering to a human subject in need of
such treatment an effective amount of a composition comprising
recombinant human N-acetylgalactosamine-4-sulfatase (rhASB).
68. The method of claim 67 wherein the amount of rhASB is effective
to provide one or more beneficial effects selected from the group
consisting of joint mobility, joint pain, joint stiffness, exercise
tolerance, exercise endurance, pulmonary function, visual acuity,
and activities of daily living.
69.-74. (canceled)
Description
[0001] This is a continuation-in-part of U.S. Ser. No. 10/290,908
filed Nov. 7, 2002 and a continuation-in-part of U.S. Ser. No.
10/658,699 [Attorney Docket No. 30610/30013A] filed Sep. 9, 2003,
which in turn is a divisional of U.S. Ser. No. 09/562,427 filed May
1, 2000, each of which is incorporated by reference herein in its
entirety.
FIELD OF THE INVENTION
[0002] The present invention is in the field of clinical medicine,
biochemistry and molecular biology. The present invention features
therapeutics and methods for treating mucopolysaccharidosis VI as
well as production and purification procedures for producing such
therapeutics.
BACKGROUND OF THE INVENTION
[0003] MPS VI (Maroteaux-Lamy syndrome) is a lysosomal storage
disease in which the affected patients lack the enzyme
N-acetylgalactosamine-4-sulfa- tase (ASB). The enzyme metabolizes
the sulfate moiety of glycosaminoglycan (GAG) dermatan sulfate
(Neufeld, et al., "The mucopolysaccharidoses" The Metabolic Basis
of Inherited Disease, eds. Scriver et al., New York:McGraw-Hill,
1989, p. 1565-1587). In the absence of the enzyme, the stepwise
degradation of dermatan sulfate is blocked and the substrate
accumulates intracellularly in the lysosome in a wide range of
tissues. The accumulation causes a progressive disorder with
multiple organ and tissue involvement in which the infant appears
normal at birth, but usually dies before puberty. The diagnosis of
MPS VI is usually made at 6-24 months of age when children show
progressive deceleration of growth, enlarged liver and spleen,
skeletal deformities, coarse facial features, upper airway
obstruction, and joint. deformities. Progressive clouding of the
cornea, communicating hydrocephalus, or heart disease may develop
in MPS VI children. Death usually results from respiratory
infection or cardiac disease. Distinct from MPS I, MPS VI is not
typically associated with progressive impairment of mental status,
although physical limitations may impact learning and development.
Although most MPS VI patients have the severe form of the disease
that is usually fatal by the teenage years, affected patients with
a less severe form of the disease have been described which may
survive for decades.
[0004] Several publications provide estimates of MPS VI incidence.
A 1990 British Columbia survey of all births between 1952 and 1986
published by Lowry et al (Lowry, et al., Human Genet 85:389-390
(1990)) estimates an incidence of just 1:1,300,000. An Australian
survey (Meikle et al., JAMA 281(3):249-54) of births between
1980-1996 found 18 patients for an incidence of 1:248,000. A survey
in Northern Ireland (Nelson, et al., Hum. Genet. 101:355-358
(1997)) estimated an incidence of 1:840,000. Finally, a survey from
The Netherlands from 1970-1996 calculated a birth prevalence of
0.24 per 100,000 (Poorthuis, et al., Hum. Genet. 105:151-156
(1999)). Based on these surveys, it is estimated that there are
between 50 and 300 patients in the U.S. who are diagnosed with all
forms of this syndrome.
[0005] There is no satisfactory treatment for MPS VI although a few
patients have benefited from bone marrow transplantation (BMT)
(Krivit, et al., N. Engl. J. Med. 311(25):1606-11 (1984); Krivit,
et al., Int. Pediat. 7:47-52 (1992)). BMT is not universally
available for lack of a suitable donor and is associated with
substantial morbidity and mortality. The European Group for Bone
Marrow Transplantation reported transplant-related mortality of 10%
(HLA identical) to 20-25% (HLA mismatched) for 63 transplantation
cases of lysosomal disorders (Hoogerbrugge, et al., Lancet 345:
1398-1402 (1995)). Other than BMT, most patients receive
symptomatic treatment for specific problems as their only form of
care. It is an object of the present invention to provide enzyme
replacement therapy with recombinant human
N-acetylgalactosamine-4-sulfatase (rhASB). No attempts to treat
humans with rhASB have been made. Likewise, no acceptable clinical
dosages or medical formulations have been provided. Several enzyme
replacement trials in the feline MPS VI model have been
conducted.
SUMMARY OF THE INVENTION
[0006] The present invention encompasses the production,
purification, and the use of a composition comprising a highly
purified N-acetylgalactosamine-4-sulfatase in the precursor form.
The DNA and encoded amino acid sequence of the precursor form is
set forth in SEQ ID NOS: 1 and 2, respectively. The signal sequence
is predicted to be amino acids 1-38 of SEQ ID NO: 2, and
recombinant production has resulted in product commencing at either
amino acid residue 39 or 40 of SEQ ID NO: 2.
[0007] In a first aspect, the present invention features novel
methods of treating diseases caused all or in part by a deficiency
in N-acetylgalactosamine-4-sulfatase (ASB). A method comprises
administering an effective amount of a pharmaceutical composition
to a subject in need of such treatment. In the preferred
embodiment, the pharmaceutical composition comprises highly
purified N-acetylgalactosamine-4-sulfatase in the precursor form,
or a biologically active fragment, mutant or analog thereof alone
or in combination with a pharmaceutically suitable carrier. The
subject suffers from a disease caused all or in part by a
deficiency of N-acetylgalactosamine-4-sulfatase. In other
embodiments, this method features transferring a nucleic acid
encoding all or a part of an N-acetylgalactosamine-4-sulfatase
(ASB) or a biologically active mutant or analog thereof into one or
more host cells in vivo. Preferred 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 VI
(MPS V1) or Maroteaux-Lamy syndrome.
[0008] This first aspect of the invention specifically provides
methods of treating humans suffering from diseases caused all or in
part by a deficiency in ASB activity by administering a
therapeutically effective amount of human ASB, preferably
recombinant human ASB. Thus, the invention contemplates use of
human ASB in preparation of a medicament for the treatment of a
deficiency in ASB activity, as well as a pharmaceutical composition
containing human ASB for use in treating a deficiency in ASB
activity. The deficiency in ASB activity can be observed, e.g., as
activity levels of 50% or less, 25% or less, or 10% or less
compared to normal levels of ASB activity and can manifest as a
mucopolysaccharidosis, for example mucopolysaccharidosis VI (MPS
VI) or Maroteaux-Lamy syndrome. The therapeutically effective
amount is an amount sufficient to provide a beneficial effect in
the human patient and preferably provides improvements in any one
of the following: joint mobility, pain, or stiffness, either
subjectively or objectively; exercise tolerance or endurance, for
example, as measured by walking or climbing ability; pulmonary
function, for example, as measured by FVC, FEV.sub.1 or FET; visual
acuity; or activities of daily living, for example, as measured by
ability to stand up from sitting, pull clothes on or off, or pick
up small objects. The human ASB is preferably administered as a
highly purified recombinant preparation as described herein.
Preferred preparations contain rhASB with a purity greater than
95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, or
99.9%, as measured by reverse-phase HPLC. Preferred preparations
also contain the precursor form of ASB at high purity, so that
processed forms of ASB are not detected on Coomassie-stained
SDS-PAGE. Most preferably the purity of the precursor form of ASB
is greater than 95%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%,
99.7%, 99.8%, or 99.9%, as measured by size exclusion
chromatography (SEC)-HPLC. The human ASB is also preferably
formulated with a surfactant or non-ionic detergent as described
herein, optionally excluding a formulation with polyoxyethylene
sorbitan 20 or 80 at 0.001%.
[0009] Administration of rhASB at doses below 1 mg/kg per week,
e.g. at 0.2 mg/kg per week, has been found to have a beneficial
effect. The invention contemplates doses of at least 0.1 mg/kg, 0.2
mg/kg, 0.5 mg/kg, 0.75 mg/kg, 1 mg/kg, or 1.5 mg/kg per week, and
may range up to 2 mg/kg, 4 mg/kg, 5 mg/kg or higher per week. The
preferred dose is 1 mg/kg/week. Such doses are preferably delivered
once weekly but optionally may be divided in equal amounts over
more frequent time periods such as biweekly or daily. A variety of
parenteral or nonparenteral routes of administration, including
oral, transdermal, transmucosal, intrapulmonary (including
aerosolized), intramuscular, subcutaneous, or intravenous that
deliver equivalent dosages are contemplated. Administration by
bolus injection or infusion directly into the joints or CSF is also
specifically contemplated, such as intrathecal, intracerebral,
intraventricular, via lumbar puncture, or via the cistema magna. A
variety of means are known in the art for achieving such
intrathecal administration, including pumps, reservoirs, shunts or
implants. Preferably the doses are delivered via intravenous
infusions lasting 1, 2 or 4 hours, most preferably 4 hours, but may
also be delivered by an intravenous bolus.
[0010] Other means of increasing ASB activity in the human subjects
are also contemplated, including gene therapy that causes the
patient to transiently or permanently increase expression of
exogenous or endogenous ASB. Transfer of an exogenous hASB gene or
a promoter that increases expression of the endogenous hASB gene is
possible through a variety of means known in the art, including
viral vectors, homologous recombination, or direct DNA
injection.
[0011] In a second aspect, the present invention features novel
pharmaceutical compositions comprising an
N-acetylgalactosamine-4-sulfata- se (ASB) or a biologically active
fragment, mutant or analog thereof useful for treating a disease
caused all or in part by a deficiency in
N-acetylgalactosamine-4-sulfatase (ASB). In the preferred
embodiment, the N-acetylgalactosamine-4-sulfatase is precursor
N-acetylgalactosamine-4-su- lfatase. Such compositions may be
suitable for administration in a number of ways such as parenteral,
topical, intranasal, inhalation or oral administration. Within the
scope of this aspect are embodiments featuring nucleic acid
sequences encoding all or a part of an
N-acetylgalactosamine-4-sulfatase (ASB) which may be administered
in vivo into cells affected with an
N-acetylgalactosamine-4-sulfatase (ASB) deficiency.
[0012] In a third aspect, the present invention features a method
to produce an N-acetylgalactosamine-4-sulfatase (ASB) or a
biologically active fragment, mutant or analog thereof in amounts
which enable using the enzyme therapeutically. In a broad
embodiment, the method comprises the step of transfecting a cDNA
encoding for all or a part of a N-acetylgalactosamine-4-sulfatase
(ASB) or a biologically active mutant or analog thereof into a cell
suitable for the expression thereof. In the preferred embodiment,
the cells are grown in a constant or continuous culture or in
perfusion. In another embodiment, the cells are grown in a medium
that lacks G418; In some embodiments, a cDNA encoding for a
complete N-acetylgalactosamine-4-sulfatase (ASB) is used,
preferably a human N-acetylgalactosamine-4-sulfatase (ASB).
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 (CHO) cell, such as the CHO-K1 cell line. In
yet other preferred embodiments, the production procedure comprises
the following steps: (a) growing cells transfected with a DNA
encoding all or a biologically active fragment or mutant of a human
N-acetylgalactosamine-4-sulfatase in a suitable growth medium to an
appropriate density, (b) introducing the transfected cells into a
bioreactor, (c) supplying a suitable growth medium to the
bioreactor, and (d) separating the transfected cells from the media
containing the enzyme.
[0013] In a fourth aspect, the present invention provides a
transfected cell line which features the ability to produce
N-acetylgalactosamine-4-s- ulfatase (ASB) in amounts which enable
using the enzyme therapeutically. In a preferred embodiment, the
N-acetylgalactosamine-4-sulfatase is precursor
N-acetylgalactosamine-4-sulfatase. In preferred embodiments, the
present invention features a recombinant CHO cell line such as the
CHO K1 cell line that stably and reliably produces amounts of an
N-acetylgalactosamine-4-sulfatase (ASB) or a biologically active
fragment, mutant or analog thereof which enable using the enzyme
therapeutically. Especially preferred is the transgenic CHO-K1 cell
line designated CSL4S-342. In some preferred embodiments, the
transgenic cell line contains one or more copies of an expression
construct. Preferably, the transgenic cell line contains about 10
or more copies of the expression construct. In even more preferred
embodiments, the cell line expresses the recombinant
N-acetylgalactosamine-4-sulfatase (ASB) or a biologically active
fragment, mutant or analog thereof in amounts of at least about
20-40 micrograms per 10.sup.7 cells per day.
[0014] In a fifth aspect, the present invention provides novel
vectors suitable to produce N-acetylgalactosamine-4-sulfatase (ASB)
or a biologically active fragment, mutant or analog thereof in
amounts which enable using the enzyme therapeutically.
[0015] In a sixth aspect, the present invention provides novel
N-acetylgalactosamine-4-sulfatase (ASB) or a biologically active
fragment, mutant or analog thereof produced in accordance with the
methods of the present invention and thereby present in amounts
which enable using the enzyme therapeutically. The specific
activity of the N-acetylgalactosamine-4-sulfatase (ASB) according
to the present invention is preferably in the range of 20-90 units,
and more preferably greater than about 50 units per mg protein. In
the preferred embodiment, the N-acetylgalactosamine-4-sulfatase is
highly purified precursor N-acetylgalactosamine-4-sulfatase.
[0016] In a seventh aspect, the present invention features a novel
method to purify N-acetylgalactosamine-4-sulfatase (ASB) or a
biologically active fragment, mutant or analog thereof. According
to a first embodiment, a transfected cell mass is grown and removed
leaving recombinant enzyme. Exogenous materials should normally be
separated from the crude bulk to prevent fouling of the columns.
Preferably, the growth medium containing the recombinant enzyme is
passed through an ultrafiltration step. In another preferred
embodiment, the method to purify the precursor
N-acetylgalactosamine-4-sulfatase comprises: (a) obtaining a fluid
containing precursor N-acetylgalactosamine-4-sulfatase; (b)
reducing the proteolytic activity of a protease in said fluid able
to cleave the precursor N-acetylgalactosamine-4-sulfatase, wherein
said reducing does not harm said precursor
N-acetylgalactosamine-4-sulfatase; (c) contacting the fluid with a
Cibracon blue dye interaction chromatography resin; (d) contacting
the fluid with a copper chelation chromatography resin; (e)
contacting the fluid with a phenyl hydrophobic interaction
chromatography resin; (f) recovering said precursor
N-acetylgalactosamine-4-sulfatase. Preferably, steps (c), (d) and
(e) can be performed sequentially. Those skilled in the art readily
appreciate that one or more of the chromatography steps may be
omitted or substituted, or that the order of the chromatography
steps may be changed within the scope of the present invention. In
other preferred embodiments, the eluent from the final
chromatography column is ultrafiltered/diafiltered, and an
appropriate step is performed to remove any remaining viruses.
Finally, appropriate sterilizing steps may be performed as
desired.
DESCRIPTION OF THE FIGURES
[0017] FIG. 1 provides a flow diagram of the method for producing a
human N-acetylgalactosamine-4-sulfatase (ASB) according to the
present invention.
[0018] FIG. 2 provides a flow diagram of the method for purifying a
human N-acetylgalactosamine-4-sulfatase (ASB) according to the
batch process method (Table 14).
[0019] FIG. 3 provides the flow diagram of the method for purifying
a human N-acetylgalactosamine-4-sulfatase (ASB) according to the
perfusion process method (Tables 14 and 15).
[0020] FIGS. 4A-4C depicts results obtained for the chromatograms
of the Blue Sepharose Column (FIG. 4A), Copper Chelating Sepharose
Column (FIG. 4B) and Phenyl Sepharose Column (FIG. 4C).
[0021] FIG. 5 depicts a 4-20% polyacrylamide gradient gel showing
the result of a silver-stained SDS-PAGE of the perfusion process
purification method (Tables 14 and 15).
[0022] FIGS. 6A-6F depicts the results on 4-20% polyacrylamide
gradient SDS gels of the following samples: lane 1, NEB broad range
prestained molecule weight standards (MW in kDa); lane 2, 5 .mu.g
ASB from lot AS60001 (old batch process); lane 3, 5 .mu.g ASB from
lot AP60109 UF4 (perfusion process); lane 4, 5 .mu.g ASB from lot
AP60109 UF10 (perfusion process); lane 5, 5 .mu.g ASB from lot
AP60109 UF15 (perfusion process); lane 6, 5 .mu.g ASB from lot
AP60109 AUF18 (perfusion process); lane 7, 5 .mu.g ASB from lot
AP60109 AUF22 (perfusion process); lane 8, 5 .mu.g ASB from lot
AP60109 AUF25 (perfusion process); and, lane 9, 5 .mu.g ASB from
lot AP60109-AUF27 (perfusion process). The gels are stained either
with Coomassie R-250 or silver-stained.
[0023] FIG. 7A depicts the results on silver-stained 4-20%
polyacrylamide gradient SDS gels. FIG. 7B depicts the results on
Coomassie stained 4-20% polyacrylamide gradient SDS gels. Lane 1,
lot AP60202 UF4; lane 2, lot AP60202 UF10; lane 3, lot AP60202
UF18; lane 4, lot AP60202 (BMK); lane 5, lot 102PD0139x B3; lane 6,
lot 102PD0139x B5; lane 7, perfusion reference standard
rhASB-202-002; lane 8, lot 102PD0139 P1; lane 9, lot 102PD0139 P2;
and, lane 10, Mark 12 standard (MW in kDa).
[0024] FIGS. 8A-8C depicts profiles obtained for the Blue Sepharose
Column (FIG. 8A), Copper Chelating Sepharose Column (FIG. 8B) and
Phenyl Sepharose Column (FIG. 8C). In FIG. 8B, cathepsin activity
is indicated by the red line.
[0025] FIG. 9A depicts the results of a silver-stained 4-20%
polyacrylamide gradient SDS. gels. FIG. 9B depicts the results of
the proteins transferred from the gel of FIG. 9A transferred onto
nitrocellulose and probed by anti-rhASB antibodies. FIG. 9C depicts
the results of the proteins transferred from the gel of FIG. 9A
transferred onto nitrocellulose and probed by anti-cathepsin
antibodies. BioLabPreStain indicates molecular weight standards (MW
in kDa).
[0026] FIG. 10 shows serum anti-ASB antibody levels over 96 weeks
of treatment with rhASB in a Phase 1/2 clinical study in
humans.
[0027] FIG. 11 shows the reduction in total urinary GAG levels over
96 weeks of treatment with rhASB in a Phase 1/2 clinical study in
humans.
[0028] FIG. 12 shows a comparison of urinary GAG levels at week 96
in humans treated with rhASB to age-appropriate normal levels.
[0029] FIG. 13 shows the improvement in results of the 6-minute
walk test over 96 weeks of treatment with rhASB in a Phase 1/2
clinical study in humans.
[0030] FIG. 14 shows serum anti-ASB antibody levels over 48 weeks
of treatment in a Phase 2 clinical study in humans.
[0031] FIG. 15 shows the reduction in total urinary GAG levels over
48 weeks of treatment in a Phase 2 clinical study in humans.
[0032] FIG. 16 shows a comparison of levels at week 48 in humans
treated with rhASB to age-appropriate normal levels.
[0033] FIG. 17 shows the improvement in the 12-minute walk test
results over 48 weeks of treatment in a Phase 2 clinical study in
humans.
[0034] FIG. 18 shows the improvement in the 3-minute stair climb
results over 48 weeks of treatment in a Phase 2 clinical study in
humans.
[0035] FIG. 19 shows results of the Expanded Timed Get Up and Go
Test over 48 weeks of treatment in a Phase 2 clinical study in
humans.
[0036] FIGS. 20 and 21, respectively, show results of joint pain
and joint stiffness questionnaires over 48 weeks of treatment in a
Phase 2 clinical study in humans.
DETAILED DESCRIPTION OF THE INVENTION
[0037] The present invention encompasses the production,
purification, and the use of a composition comprising a highly
purified N-acetylgalactosamine-4-sulfatase in the precursor form.
The purity of N-acetylgalactosamine-4-sulfatase in the precursor
form is at least equal to or greater than 95, 96, 97 or 98% by
total protein as determined by the reverse-phase HPLC method.
Preferably, the purity is at least equal to or greater than 99%.
More preferably, the purity is at least equal to or greater than
99.1, 99.2, 99.3 or 99.4%. Even more preferably, the purity is at
least equal to, or greater than 99.5, 99.6, 99.7 or 99.8%. Even
much more preferably, the purity is at least equal to or greater
than 99.9%. The purity of precursor
N-acetylgalactosamine-4-sulfatase is measured using the
reverse-phase HPLC method (see Example 9). The purity of precursor
N-acetylgalactosamine-4-sulfatase is that whereby the composition
essential free of any contaminating cell proteins or degraded or
mature or processed N-acetylgalactosamine-4-sulfatase that is
detectable by the reverse-phase HPLC method. All percent purity is
based on total protein as determined by the reverse-phase HPLC
method. The consistently repeatable high purity obtainable using
the purification process disclosed herein makes it possible to
treat these patients that require long-term, chronic treatment with
high purity rhASB preparations administered at every treatment,
e.g., weekly. Thus, the invention contemplates administering
precursor rhASB of such high purity over a long period of time,
e.g. 12 weeks, 24 weeks, 48 weeks, 96 weeks or more.
[0038] In a first aspect, the present invention features novel
methods of treating diseases caused all or in part by a deficiency
in N-acetylgalactosamine-4-sulfatase (ASB). In one embodiment, this
method features administering a recombinant
N-acetylgalactosamine-4-sulfatase (ASB) or a biologically active
fragment, mutant or analog thereof alone or in combination with a
pharmaceutically suitable carrier. In other embodiments, this
method features transferring a nucleic acid encoding, all or a part
of an N-acetylgalactosamine-4-sulfatase (ASB) or a biologically
active mutant thereof into one or more host cells in vivo.
Preferred 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 VI (MPS V1),
Maroteaux-Lamy syndrome.
[0039] The purity of precursor N-acetylgalactosarine-4-sulfatase is
that whereby the composition is essentially free of any
contaminating cell proteins which can cause an immunological or
allergic reaction by the subject who is administered precursor
N-acetylgalactosamine-4-sulfatase. A composition is essentially
free of such contaminating host cell proteins if the composition,
when administered to a subject, does not cause any immunological or
allergic reaction. The high purity of the precursor
N-acetylgalactosamine-4-sulfatase is important for avoiding an
immunological or allergic reaction by the subject to the impurities
present in the pharmaceutical composition. This is especially-true
of proteins of the cells from which the precursor
N-acetylgalactosamine-4-su- lfatase is purified. When recombinant
precursor N-acetylgalactosamine-4-su- lfatase is expressed and
purified from Chinese Hamster Ovary cells, the Chinese Hamster
Ovary proteins can cause immunological or allergic reactions (e.g.
hives) in the subject. The only means to avoid this type of
reaction is to ensure that the precursor
N-acetylgalactosamine-4-sulfa- tase is sufficiently pure so that
the contaminating Chinese Hamster Ovary proteins are not of
sufficient amount to cause such reaction(s). The purity of the
pharmaceutical composition is especially important as subjects
include patients suffering from MPS VI and are thus already
immunologically compromised.
[0040] Also preferably the purity of the precursor
N-acetylgalactosamine-4- -sulfatase (ASB) is such that the
composition has only trace amounts of processed or degraded forms.
Proteases present in the host cells cleave the
N-acetylgalactosamine-4-sulfatase into lower molecular weight
forms. While some of these forms may also be enzymatically active,
the precursor form is preferable for cellular uptake and lysosomal
targeting and thus a higher amount of non-processed precursor ASB
in the final preparation is desirable. Moreover, degraded forms of
ASB in the drug product may also engender higher incidence or
amounts of antibodies to ASB itself, which is highly undesirable in
cases such as this where long-term therapy of patients is required.
The perfusion purification process described herein results in a
highly pure preparation that is essentially free of contaminating
host cell proteins or processed/aggregated forms of ASB as assayed
by the combination of SDS-PAGE, RPHPLC and SEC-HPLC. When
administered to humans, the product produced by this process
appears to have a longer half-life.
[0041] The indication for recombinant human
N-acetylgalactosamine-4-sulfat- ase (rhASB) is for the treatment of
MPS VI, also known as Maroteaux-Lamy Syndrome. According to
preferred embodiments, an initial dose of 1 mg/kg (.about.50 U/kg)
is provided to patients suffering from a deficiency in
N-acetylgalactosamine-4-sulfatase. Preferably, the
N-acetylgalactosamine-4-sulfatase is administered weekly by
injection. According to other preferred embodiments, patients who
do not demonstrate a reduction in urinary glycosaminoglycan
excretions of at least fifty percent are changed to a dosage of 2
mg/kg (.about.100 U/kg) within about three months of initial
dosage. Preferably, the N-acetylgalactosamine-4-s- ulfatase (rhASB)
or a biologically active fragment, mutant or analog thereof is
administered intravenously over approximately a four-hour period
once weekly preferably for as long as significant clinical symptoms
of disease persist. Also, preferably, the
N-acetylgalactosamine-4-sulfatase (rhASB) is administered by an
intravenous catheter placed in the cephalic or other appropriate
vein with an infusion of saline begun at about 30 cc/hr. Further,
preferably the N-acetylgalactosamine-4-sulfatase (rhASB) is diluted
into about 250 cc of normal saline.
[0042] In a second aspect, the present invention features novel
pharmaceutical compositions comprising human
N-acetylgalactosamine-4-sulf- atase (rhASB) or a biologically
active fragment, mutant or analog thereof useful for treating a
deficiency in N-acetylgalactosamine-4-sulfatase. The recombinant
enzyme may be administered in a number of ways in addition to the
preferred embodiments described above, 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 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 application such as ointments,
jellies, creams and suspensions, 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 intended for
oral administration.
[0043] To produce pharmaceutical compositions in this form of
dosage units for oral application containing a therapeutic enzyme,
the enzyme may be mixed with a solid, pulverulent carrier, for
example lactose, saccharose, sorbitol, mannitol, a starch such as
potato starch, corn starch, amylopectin, laminaria powder or citrus
pulp powder, a cellulose derivative or gelatine and also may
include lubricants such as magnesium or calcium stearate or a
Carbowax or other polyethylene glycol waxes and compressed to form
tablets 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, or
alternatively with a film 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. 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 Carbowax or a suitable
oil as e.g., sesame oil, olive oil, 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 potato starch, corn starch or
amylopectin, cellulose derivatives or gelatine, and may also
include magnesium stearate or stearic acid as lubricants.
[0044] Therapeutic enzymes of the present invention may also be
administered parenterally such as by subcutaneous, intramuscular or
intravenous injection either by single injection or pump infusion
or by sustained release subcutaneous implant, and therapeutic
enzymes may be administered by inhalation. In subcutaneous,
intramuscular and intravenous injection the therapeutic enzyme (the
active 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, preferably of
the vegetable oil variety such as peanut oil, cottonseed oil and
the like. Other parenteral vehicles such as organic compositions
using solketal, glycerol, formal, and aqueous parenteral
formulations may also be used.
[0045] For parenteral application by injection, compositions may
comprise an aqueous solution of a water soluble pharmaceutically
acceptable salt of the active acids according to the invention,
desirably in a concentration of 0.5-10%, and optionally also a
stabilizing agent and/or buffer substances in aqueous solution.
Dosage units of the solution may advantageously be enclosed in
ampoules.
[0046] 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.
[0047] For topical application, the pharmaceutical compositions are
suitably in the form of an ointment, cell, suspension, cream or the
like. The amount of active substance may vary, for example between
0.05-20% by weight of the active substance. Such pharmaceutical
compositions for topical application may be prepared in known
manner by mixing, the active substance with known carrier materials
such as isopropanol, glycerol, paraffin, stearyl alcohol,
polyethylene glycol, etc. The pharmaceutically acceptable carrier
may also include a known chemical absorption promoter. Examples of
absorption promoters are, e.g., dimethylacetamide (U.S. Pat. No.
3,472,931), trichloro ethanol or trifluoroethanol (U.S. 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 described in the British patent specification
No. 1,464,975, which discloses a carrier material consisting of a
solvent comprising 40-70% (v/v) isopropanol and 0-60% (v/v)
glycerol, the balance, if any, being an inert constituent of a
diluent not exceeding 40% of the total volume of solvent.
[0048] The dosage at which the therapeutic enzyme containing
pharmaceutical compositions are administered may vary 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 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.
[0049] The pharmaceutical compositions containing the therapeutic
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 a therapeutic enzyme (or
therapeutic enzymes), the subject formulations may contain one or
more substrates or cofactors for the reaction catalyzed by the
therapeutic enzyme in the compositions. Therapeutic enzyme
containing, compositions may also contain more than one therapeutic
enzyme. Likewise, the therapeutic enzyme may be in conjugate form
being bound to another moiety, for instance PEG. Additionally, the
therapeutic enzyme may contain one or more targeting moieties or
transit peptides to assist delivery to a tissue, organ or organelle
of interest.
[0050] The recombinant enzyme employed in the subject methods and
compositions may also be administered by means of transforming
patient cells with nucleic acids encoding the
N-acetylgalactosamine-4-sulfatase or a biologically active
fragment, mutant or analog thereof. The nucleic acid sequence so
encoding may be incorporated into a vector for transformation into
cells of the patient to be treated. Preferred embodiments of such
vectors are described herein. The vector may be designed so as to
integrate into the chromosomes of the subject, e.g., retroviral
vectors, or to replicate autonomously in the host cells. Vectors
containing encoding N-acetylgalactosamine-4-sulfatase nucleotide
sequences may be designed so as to provide for continuous or
regulated expression of the enzyme. Additionally, the genetic
vector encoding the enzyme may be designed so as to stably
integrate into the cell genome or to only be present transiently.
The general methodology of conventional genetic therapy may be
applied to polynucleotide sequences encoding-
N-acetylgalactosamine-4-sulfatase. Reviews of conventional genetic
therapy techniques can be found in Friedman, Science 244:1275-1281
(1989); Ledley, J. Inherit. Aletab. Dis. 13:587-616 (1990); and,
Tososhev, et al., Curr. Opinions Biotech. 1:55-61 (1990).
[0051] A particularly preferred method of administering the
recombinant enzyme is intravenously. A particularly preferred
composition comprises recombinant
N-acetylgalactosamine-4-sulfatase, normal saline, phosphate buffer
to maintain the pH at about 5-7, and human albumin. The composition
may additionally include polyoxyethylenesorbitan, such as
polysorbate 20 or 80 (Tween-20 or Tween-80) to improve the
stability and prolong shelf life. Alternatively, the composition
may include any surfactant or non-ionic detergent known in the art,
including but not limited to polyoxyethylene sorbitan 40 or 60;
polyoxyethylene fatty acid esters; polyoxyethylene sorbitan
monoisostearates; poloxamers, such as poloxamer 188 or poloxamer
407; octoxynol-9 or octoxynol 40.
[0052] Preferably the surfactant or non-ionic detergent is present
at a concentration of at least 0.0001%, or at least 0.0005%, or at
least 0.001%, 0.002%, 0.003%, 0.004% or 0.005% (w/v). Preferably
the concentration is the lowest necessary to achieve the desired
stability, but may be up to 0.005%, 0.006%, 0.007%, 0.008%, 0.009%,
0.01%, or 0.02% (w/v). Most preferably the composition comprises a
polysorbate at a concentration of 0.005%.+-.0.003% (e.g. 0.002% to
0.008%). These composition ingredients may preferably be provided
in the following amounts:
1 N-acetylgalactosamine-4-sulfatase 1-5 mg/ml or 50-250 units/ml
Sodium chloride solution 150 mM in an IV bag, 50-250 cc total
volume Sodium phosphate buffer 10-100 mM, pH 5.8, preferably 10 mM
Human albumin, optional 1 mg/mL Tween -20 or Tween -80
0.001%-0.005% (w/v)
[0053] In a preferred embodiment, the ASB is formulated as 1 mg/mL
in 150 mM NaCl, 10 mM NaPO.sub.4, pH 5.8, 0.005% polysorbate
80.
[0054] In a third aspect, the present invention features a method
to produce N-acetylgalactosamine-4-sulfatase (ASB) or a
biologically active fragment, mutant or analog thereof in amounts
which enable using the enzyme therapeutically. In a broad
embodiment, the method comprises the step of transfecting a cDNA
encoding for all or a part of a N-acetylgalactosamine-4-sulfatase
(ASB) or a biologically active mutant or analog thereof into a cell
suitable for the expression thereof. In some embodiments, a cDNA
encoding for a complete N-acetylgalactosamine-4-- sulfatase (ASB)
is used, preferably a human N-acetylgalactosamine-4-sulfat- ase
(ASB). 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.
[0055] 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, such as the
CHO-K1 cell line. In yet other preferred embodiments, the
production procedure comprises the following steps: (a) growing
cells transfected with a DNA encoding all or a biologically active
fragment or mutant of a human N-acetylgalactosamine-4-sulfatase a
suitable growth medium to an appropriate density, (b) introducing
the transfected cells into a bioreactor, (c) supplying a suitable
growth medium to the bioreactor, (d) harvesting said medium
containing the recombinant enzyme, and (e) substantially removing
the transfected cells from the harvest medium.
[0056] A suitable medium for growing the transfected cells is a JRH
Excell 302 medium supplemented with L-glutamine, glucose and
hypoxanthine/thymidine, optionally with or without G418. In a
preferred medium, the JRH Excell 302 medium is further supplemented
with folic acid, serine, and asparagine, and there is no G418
present in the medium. Using this preferred medium to culture cells
provides a higher purity precursor rhASB compared to using the
medium supplemented with G418 but not with folic acid, serine, and
asparagine (see lane 2 of FIG. 6). It is preferred to grow the
cells in such a medium to achieve a cell density of about
1.times.10.sup.7 cells/ml resulting in 10-40 mg/ml of active
enzyme. Moreover, it is preferable to grow the transfected cells in
a bioreactor for about 5 to 15 days. More preferably, it is about 9
days. Preferably, the transfected cells are grown in a bioreactor
using a perfusion-based process with collections continuing up to
35 days. More preferably, the transfected cells are grown in a
bioreactor using a perfusion-based process with collections
continuing up to 45 days. Even more preferably, the transfected
cells are grown in a bioreactor using a perfusion-based process
with collections continuing up to 60 days. Even much more
preferably, the transfected cells are grown in a bioreactor using a
perfusion-based process with collections continuing up to 90
days.
[0057] According to preferred embodiments, the transfected cells
may be substantially removed from the bioreactor supernatant by
filtering them through successive membranes such as a 10 .mu.m
membrane followed by a 1 .mu.m membrane followed by a 0.2 .mu.m.
Any remaining harvest medium may be discarded prior to
filtration.
[0058] Recombinant human N-acetylgalactosamine-4-sulfatase may be
produced in Chinese hamster ovary cells (Peters, et al. J. Biol.
Chem. 265:3374-3381). Its uptake is mediated by a high affinity
mannose-6-phosphate receptor expressed on most, if not all, cells
(Neufeld et al., "The mucopolysaccharidoses" The Metabolic Basis of
Inherited Disease, eds. Scriver, et al. New York:McGraw-Hill (1989)
p. 1565-1587). Once bound to the mannose-6-phosphate receptor, the
enzyme is endocytosed through coated pits and transported to the
lysosomes. At the pH of lysosomes, the enzyme is active and begins
removing sulfate residues from accumulated dermatan sulfate. In MPS
VI fibroblasts, the clearance of storage is rapid and easily
demonstrated within 92 hours of enzyme exposure (Anson, et al., J.
Clin. Invest. 99:651-662 (1997)). The recombinant enzyme may be
produced at a 110-L (approximately 90 L working volume)
fermentation scale according to a process according to the flow
diagram outlined in FIG. 1.
[0059] The recombinant enzyme can be produced using the following
method as set forth in Table 1A-C.
2TABLE 1A Cell Culture Process by the Fed Batch Process Step
Process In-Process Testing 1. Thawing of the Inoculate the thawed
cells into one T-75 Cell count Working Cell Bank flask with 25 mL
of JRH Exell 302 medium Cell viability (WCB) supplemented with 4 mM
L-glutamine, 4.5 g/L glucose and 10 mg/L hypoxanthine/thymidine;
further supplemented with folic acid, serine and asparagine (no
G418) Culture for 3 days to achieve 1 .times. 10.sup.10 cell
density .dwnarw. 3. 250 mL Spinner Add cells to 175 mL of
supplemented Cell count Flask medium (no G418) Cell viability
Culture for 3 days .dwnarw. 4. 1 L Spinner Flask Add cells to 800
mL of supplemented Cell count medium (no G418) Cell viability
Culture for 1-2 days .dwnarw. 5. 8 L Spinner Flask Add cells to 4 L
of supplemented medium Cell count (no G418) Cell viability Culture
for 1-2 days .dwnarw. 6. 2 .times. 8 L Spinner Split working volume
into 2 8 L Spinner Cell count Flask Flasks Cell viability Add cells
to 5.5 L of supplemented medium (no G418) to each 8 L Spinner Flask
Culture for 1-2 days .dwnarw. 7. Inoculation of 110 L Add cells to
7 mL of supplemented Cell count Bioreactor medium Cell viability
Culture 9 days .dwnarw. 8. Production Approximately 9 days of
growth in Cell Count bioreactor Cell viability Activity .dwnarw. 9.
Harvest Harvest is pumped into 100 L bag, Supernatant refrigerated
overnight .dwnarw. 10. Cell Removal Cells are removed from the
harvest QC Release Point medium by filtration through a 10 .mu.m
Activity membrane cartridge followed by 1 .mu.m and Bioburden 0.2
.mu.m cartridges. Since the cells have been Endotoxin allowed to
settle overnight the final 5 to Mycoplasma 10% of the harvest
medium is discarded In vitro advent. Agents prior to
filtration.
[0060] In one embodiment, the transfected cells are grown in a cell
culture process that is a perfusion-based process with collections
continuing for up to 35 or more days with a collection rate of
approximately 400 L per day from one 110 L bioreactor. Preferably,
the collection rate is approximately 800 L per day from one 110 L
bioreactor. A process flow diagram comparing the perfusion cell
culture process with the batch cell culture process is shown in
Table 1B. Comparisons to the fed batch process as well as details
of the specific changes implemented for the perfusion-based cell
culture process are summarized in Table 1C.
3TABLE 1B Cell Culture Process Comparison Between Fed Batch and
Perfusion Processes Batch Process Perfusion Process 1 Working Cell
Bank (WCB) Vial 1 WCB Vial .dwnarw. .dwnarw. T75 cm.sup.2 flask T75
cm.sup.2 flask .dwnarw. .dwnarw. 250 mL Spinner Flask 250 mL
Spinner Flask .dwnarw. .dwnarw. 2 .times. 250 mL Spinner Flask 2
.times. 250 mL Spinner Flask .dwnarw. .dwnarw. 2 .times. 3 L
Spinner Flask 2 .times. 3 L Spinner Flask .dwnarw. .dwnarw. 2
.times. 8 L Spinner Flask 2 .times. 8 L Spinner Flask .dwnarw.
.dwnarw. 2 .times. 110 L bioreactors 1 .times. 110 L bioreactors
(80-95 L working volume) (75-85 L working volume) Batch Mode 11-12
days Batch Mode 1-3 days 1 .times. 110 L bioreactors (75-85 L
working volume) Culture duration up to 35 days .dwnarw. .dwnarw.
Harvest Collection stored in 200 L Harvest Collection stored in 200
L Polyethylene Bags Polyethylene Bags
[0061]
4TABLE 1C Summary Description of the Differences between the Fed
Batch and Perfusion Processes Description Description Production
Step (Batch) (Perfusion) CELL CULTURE One batch is the result of
one Change: One batch is the result production run with two 110 L
of one production run with one bioreactors. 110 L bioreactor
Thawing of WCB One vial for each production batch. Change: vial
Tests: Expected cell viability >95% Cell Viability: >90%
Expected .gtoreq.1 .times. 10.sup.6 cells recovered Viability of
>90% on thaw has proven to yield successful runs and product
that meets specifications .dwnarw. T75 cm.sup.2 flasks
Approximately 5 .times. 10.sup.6 cells plated into No Change 1
flask. Length of step: .about.3 days Tests: Expected cell viability
>90% Expected .gtoreq.2 .times. 10.sup.7 cells recovered
.dwnarw. 250 mL Spinner Cells from T75 is split onto a 250 mL No
Change Flask spinner flask. Length of step: .about.2 days Tests:
Expected cell viability >90% Expected .gtoreq.2 .times. 10.sup.8
cells recovered .dwnarw. 2 .times. 250 mL Cells from 1 .times. 250
mL spinner flask are No Change Spinner Flask split into two 250 mL
spinner flask. Length of step: .about.3 days Tests: Expected cell
viability >90% Expected .gtoreq.4 .times. 10.sup.8 cells
recovered .dwnarw. 2 .times. 3 L Spinner Cells from 2 .times. 250
mL spinner flasks No Change Flask are split into two 3 L spinner
flask Length of step: .about.4 days Tests: Expected cell viability
>90% Expected .gtoreq.4.8 .times. 10.sup.9 cells recovered
.dwnarw. 2 .times. 8 L Spinner Cells from 2 .times. 3 L spinner
flasks are No Change Flask split into two 8 L spinner flask. Length
of step: .about.2 days Tests: Expected cell viability >90%
Expected .gtoreq.1.6 .times. 10.sup.10 cells recovered .dwnarw.
Inoculation of Inoculum of .gtoreq.0.8 .times. 10.sup.10 cells are
added Change: culture flask to each of two bioreactors containing
32 L Inoculum of .gtoreq.1.6 .times. 10.sup.10 cells culture medium
each. added to one bioreactor Culture monitored with PC-interfaced
containing 64 L culture medium. control system. (Reflects the use
of one bioreactor versus two.) .dwnarw. Production Growth: 3 days
of growth until culture Changes: reaches density of >3.2 .times.
10.sup.10 cells. Growth/Transition/Harvesting: Vertical Split:
Culture media added to When cell density reaches >8 .times.
10.sup.10 cells/mL, final volume of 95 L. perfusion is Harvesting:
Supernatant was harvested started. The perfusion rate is at day 11
or when cell viability fell gradually increased to 5 vessel below
70%. Supernatant is filtered and volumes per day based on stored.
glucose levels. When the cell density is >1.28 .times. 10.sup.12
the pH set point is adjusted to 7.35. Once increases in perfusion
rates cease, glucose level is maintained by reducing cell density
with a cell bleed. Harvested supernatant is collected and filtered.
Termination Run is terminated at harvest. Change: Run is terminated
after 35 or more days is reached, or activity falls below 2 mg/L
for three consecutive days or after adequate harvested supernatant
is collected.
[0062] The inoculum preparation for scale-up process is the same
for the fed batch and perfusion processes. In one embodiment, the
rhASB cell culture is initiated by thawing a single vial from the
Working Cell Bank and transferring its contents (approximately 1
mL) to approximately 25 mL of EX-CELL 302 Medium (Modified
w/L-Glutamine, No Phenol Red) in a T75 cm.sup.2 cell culture flask.
In each expansion step, the cell culture is incubated until a
viable cell count of approximately 0.8.times.10.sup.6 cells/mL is
achieved. Each cell expansion step is monitored for cell growth
(cell density) and viability (via trypan blue exclusion). All
additions of EX-CELL 302 Medium (Modified w/L-Glutamine, No Phenol
Red) medium and cell transfers are preformed aseptically in a
laminar flow hood. The cell culture is expanded sequentially from
the T75 cm.sup.2 flask to a 250 mL spinner flask, to two 250 mL
spinner flasks, to two 3 L spinner flasks, and finally to two 8 L
spinner flasks. The entire scale-up process lasts approximately 14
days. When the two 8 L spinner flasks are at a density of at least
1.0.times.10.sup.6 cells/mL, the flasks are used to seed one 110 L
bioreactor.
[0063] It is preferred that bioreactor operations for rhASB
expression or production or manufacture utilize a perfusion-based
cell culture process. Preferably, the bioreactor, using the
perfusion process, can control cell densities up to as high as 37
million cells per mL; compared to 4-5 million cells per mL using
the fed batch process.
[0064] The perfusion-based process runs longer (35 days) than the
fed batch process (11-12 days) and produces a greater volume of
harvested cell culture fluid (approximately 400 L/day at a
perfusion rate of 5 vessel volumes per day) compared to the fed
batch process (190 L/run). Preferably, harvesting is performed up
to 35 days for a total collection of approximately 8400 L of
supernatant.
[0065] End of Production Cells (EPC) are evaluated for genetic
stability, identity, sterility and adventitious agent contamination
per ICH guideline. Preferably, EPC results, obtained from a 35-day
long bioreactor run, AC60108, produced under cGMP conditions, show
no growth or negative results or no detection of the presence of
bacteria and fungi, mycoplasma, adventitious viral contaminants,
murine viruses, or like contaminants or particles.
[0066] In a fourth aspect, the present invention provides a
transgenic cell line which features the ability to produce
N-acetylgalactosamine-4-s- ulfatase (ASB) or a biologically active
fragment, mutant or analog thereof 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 CHO K1 cell line that stably and reliably produces
amounts of N-acetylgalactosamine-4-sulfatase (ASB) which enable
using the enzyme therapeutically. Especially preferred is the
CHO-K1 cell line designated CSL4S-342. In some preferred
embodiments, the cell line contains one or more of an expression
construct. More preferably, the cell line contains contains about
10 or more copies of the expression construct. In even more
preferred embodiments, the cell line expresses recombinant
N-acetylgalactosamine-4-sulfatase (ASB) in amounts of at least
about 20-80 or 40-80 micrograms per 10.sup.7 cells per day.
[0067] Recombinant human N-acetylgalactosamine-4-sulfatase (rhASB)
may be produced from a stable transfected CHO-K1 (Chinese hamster
ovary) cell line designated CSL4S-342. The cell line is described
in the literature (Crawley, J. Clin. Invest. 99:651-662 (1997)).
Master Cell Bank (MCB) and Working Cell Bank (WBC) were prepared at
Tektagen Inc. (Malvem, Pa.). The cell banks have been characterized
per ICH recommended guidelines for a recombinant mammalian cell
line.
[0068] In a fifth aspect, the present invention provides novel
vectors suitable to produce N-acetylgalactosamine-4-sulfatase (ASB)
or a biologically active fragment, mutant or analog thereof in
amounts which enable using the enzyme therapeutically.
[0069] In a sixth aspect, the present invention provides novel
N-acetylgalactosamine-4-sulfatase (ASB) or a biologically active
fragment, mutant or analog thereof produced in accordance with the
methods of the present invention and thereby present in amounts
which enable using the enzyme therapeutically. The preferred
specific activity of the N-acetylgalactosamine-4-sulfatase (ASB)
according to the present invention is about 20-90 Unit, and more
preferably greater than 50 units per milligram protein. Preferably,
the enzyme has a deglycosylated weight of about 55 to 56 kDa, most
preferably about 55.7 kDa. Preferably, the enzyme has a
glycosylated weight of about 63 to 68 kDa, most preferably about 64
to 66 kDa. The present invention also includes biologically active
fragments including truncated molecules, analogs and mutants of the
naturally-occurring human N-acetylgalactosamine-4-sulfatase.
[0070] The human cDNA for N-acetylgalactosamine-4-sulfatase
predicts a protein of 533 amino acids with a signal peptide of 41
amino acids (Peters, et al. J. Biol. Chem. 265:3374-3381). The
predicted molecular weight is about 55.9 kDa after signal peptide
cleavage. The recombinant enzyme has an apparent molecular weight
of 64 kDa on SDS-PAGE due to carbohydrate modifications. The
predicted protein sequence contains six potential N-linked
oligosaccharide modification sites of which four may be used based
on a 2,000 kDa average mass and 8,000 kDa difference between
predicted and apparent mass. A mature form of the intracellular
protein has three peptides attached by cystine bonds. The largest
peptide has a molecular weight of 47 kDa; the other two has a
molecular weight of 6 and 7 kDa respectively.
[0071] A description of a drug product produced and purified
according to the methods of the present invention is provided in
Table 2.
5TABLE 2 Drug Product Preliminary Specifications Test Procedure
Specification Activity Fluorescence assay 20,000-120,000 mUnits
Adventitious Viruses* In Vitro Assay Pass Appearance Visual Clear,
colorless to pale yellow solution Bacterial Endotoxin LAL .ltoreq.2
EU/mL Chloride Atomic Absorption Report Value ASB fibroblast Uptake
TBD .ltoreq.40 nmol Assay Mycoplasma* Points to Consider Pass 1993
Particulates USP .ltoreq.600/vial at 25 .mu.m &
.ltoreq.6000/vial at 10 .mu.m PH USP 5.5-6.8 Phosphate Atomic
Absorption Report Value Protein Concentration UV 280 0.8-1.2 mg/ml
Purity SDS PAGE 1 major band between 65-70 kDa RP-HPLC >95%
Residual Blue Dye TBD Report Value Residual Copper TBD Report Value
Sodium Atomic Absorption Report Value Specific Activity Calculation
40,000-80,000 mUnits/mg Sterility 21 CFR 610 Pass *Tested on
harvested supernatant from bioreactor (after cell removal by
filtration).
[0072] In a seventh aspect, the present invention features a novel
method to purify N-acetylgalactosamine-4-sulfatase (ASB) or a
biologically active fragment, mutant or analog thereof. According
to a first embodiment, a transfected cell mass is grown and removed
leaving recombinant enzyme. Exogenous materials should normally be
separated from the crude bulk to prevent fouling of the columns.
Preferably, the growth medium containing the recombinant enzyme is
passed through an ultrafiltration and diafiltration step. In one
method, the filtered solution is passed through a DEAE Sepharose
chromatography column, then a Blue Sepharose chromatography column,
then a CuFF Chelating Sepharose chromatography column, and then a
Phenyl Sepharose chromatography column. Such a four step column
chromatography including using a DEAE Sepharose, a Blue Sepharose,
a Cur Chelating Sepharose and a Phenyl Sepharose chromatography
column sequentially results in especially highly purified
recombinant enzyme. Those of skill in the art appreciate that one
or more chromatography steps may be omitted or substituted or the
order of the steps altered within the scope of the present
invention. In other preferred embodiments, the eluent from the
final chromatography column is ultrafiltered/diafiltered, and an
appropriate step is performed to remove any remaining viruses.
Finally, appropriate sterilizing steps may be performed as desired.
The recombinant enzyme may be purified according to a process
outlined in FIG. 2. The quality of the recombinant enzyme is key to
patients. The rhSB produced by this method is substantially pure
(>95%).
[0073] In a preferred embodiment, the ultrafiltration/diafiltration
step is performed with a sodium phosphate solution of about 10 mM
and with a sodium chloride solution of about 100 mM at a pH of
about 7.3. In another embodiment, the DEAE Sepharose chromatography
step is performed at a pH of about 7.3 wherein the elute solution
is adjusted with an appropriate buffer, preferably a sodium
chloride and sodium phosphate buffer. In additional preferred
embodiments, the Blue Sepharose chromatography step is performed at
a pH of about 5.5 wherein the elute solution is adjusted with an
appropriate buffer, preferably a sodium chloride and sodium acetate
buffer. Also, in preferred embodiments, the Cu++ Chelating
Sepharose chromatography step is performed with an elution buffer
including sodium chloride and sodium acetate. In especially
preferred embodiments, a second ultrafiltration/diafiltration step
is performed on the eluate from the chromatography runs wherein the
recombinant enzyme is concentrated to a concentration of about 1
mg/ml in a formulation buffer such as a sodium chloride and sodium
phosphate buffer to a pH of about 5.5 to 6.0, most preferably to a
pH of 5.8. Phosphate buffer is a preferred buffer used in the
process because phosphate buffer prevents critical degradation and
improves the stability of the enzyme.
[0074] A more detailed description of particularly preferred
purification methods within the scope of the present invention is
set forth in Table 3.
6TABLE 3 Purification Process Overview Step Process 1. UF/DF
Filtered harvest fluid (HF) is concentrated ten fold and then
diafiltered with 5 volumes of 10 mM Sodium Phosphate, 100 mM NaCl,
pH 7.3 using a tangential flow filtration (TFF) system. .dwnarw. 2.
DEAE Pre-wash 1 buffer: 0.1 N NaOH Sepharose FF Pre-wash 2 buffer:
100 mM NaPO4 pH 7.3 (flow through) Equilibration buffer: 100 mM
NaCl, 10 mM NaPO4, pH 7.3 Load: Product from Step 1 Wash buffer:
100 mM NaCl, 10 mM NaPO4, pH 7.3 Strip buffer: 1 M NaCl, 10 mM
NaPO4, pH 7.3 Sanitization buffer: 0.5 N NaOH Storage buffer: 0.1 N
NaOH .dwnarw. 3. Blue Sepharose Pre-wash 1: 0.1 N NaOH FF Pre-wash
2: H.sub.2O Pre-wash 3: 1 M NaAc, pH 5.5 Equilibration buffer: 150
mM NaCl, 20 mM NaAc, pH 5.5 Load: DEAE flow through Wash buffer:
150 mM NaCl, 20 mM NaAc, pH 5.5 Elution buffer: 500 mM NaCl, 20 mM
NaAc, pH 5.5 Regeneration buffer: 1 M NaCl, 20 mM NaAc, pH 5.5
Sanitization buffer: 0.1 N NaOH, 0.5-2 hours Storage buffer: 500 mM
NaCl, 20 mM NaAc, pH 5.5, 20% ETOH .dwnarw. 4. Cu++ Sanitization
buffer: 0.1 N NaOH Chelating Wash buffer: H.sub.2O Sepharose FF
Charge Buffer: 0.1 M Copper Sulfate Equilibration buffer: 20 mM
NaAc, 0.5 M NaCl, 10% Glycerol, pH 6.0 Load: Blue Sepharose Eluate
Wash Buffer 1: 20 mM NaAc, 0.5 M NaCl, 10% Glycerol, pH 6.0 Wash
Buffer 2: 20 mM NaAc, 1 M NaCl, 10% Glycerol, pH 4.0 Wash Buffer 3:
20 mM NaAc, 1 M NaCl, 10% Glycerol, pH 3.8 Elution Buffer: 20 mM
NaAc, 1 M NaCl, 10% Glycerol, pH 3.6 Strip Buffer: 50 mM EDTA, 1 M
NaCl Sanitization Buffer: 0.5 N NaOH, 0.5-2 hours Storage Buffer:
0.1 N NaOH .dwnarw. 5. Phenyl Pre-wash 1 Buffer: 0.1 N NaOH
Sepharose HP Pre-wash 2 Buffer: H.sub.2O Equilibration buffer: 3 M
NaCl, 20 mM NaAc, pH 4.5 Load: Cu.sup.++ Chelating Sepharose Eluate
Wash Buffer 1: 3.0 M NaCl, 20 mM NaAc, pH 4.5 Wash Buffer 2: 1.5 M
NaCl, 20 mM NaAc, pH 4.5 Elution buffer 1: 1.0 M NaCl, 20 mM, NaAc,
pH 4.5 Strip Buffer: 0 M NaCl, 20 mM NaAc, pH 4.5 Sanitization
Buffer: 0.5 N NaOH Storage Buffer: 0.1 N NaOH .dwnarw. 6. UF/DF The
purified rhASB is concentrated and diafiltered to a final
concentration of 1.5 mg/ml in formulation buffer (150 mM NaCl, 10
mM NaPO4, pH 5.8) using a TFF system. .dwnarw. 7. Formulation (if
Dilute with additional formulation buffer to 1.0 mg/ml necessary)
.dwnarw. 8. Viral 0.02 .mu.m filtration into sterile container
Reduction/ Sterile filtration .dwnarw. 9. Vialing Product filled
into 5 cc Type 1 glass vials, manually stoppered, crimped and
labeled.
[0075] The formulated bulk drug substance can be sterilized through
a 0.04 micron or preferably a 2 micron filter in a class 100
laminar flow hood into Type 1 glass vials. The vials may be filled
to a final volume of about 5 mL using a semi-automatic liquid
filling machine. The vials may then be manually stoppered, sealed
and labeled.
[0076] A more preferred method to purify a precursor
N-acetylgalactosamine-4sulfatase comprises: (a) obtaining a fluid
containing precursor N-acetylgalactosamine-4-sulfatase; (b)
reducing the proteolytic activity of a protease in said fluid able
to cleave the precursor N-acetylgalactosamine-4-sulfatase, wherein
said reducing does not harm said precursor
N-acetylgalactosamine-4-sulfatase; (c) contacting the fluid with a
Cibracon blue dye interaction chromatography resin; (d) contacting
the fluid with a copper chelation chromatography resin; (e)
contacting the fluid with a phenyl hydrophobic interaction
chromatography resin; and (f) recovering said precursor
N-acetylgalactosamine-4-sulfatas- e. Preferable, steps (c), (d) and
(e) are performed sequentially. This method requires no more than
three chromatography steps or columns. In order to obtain highly
purified precursor N-acetylgalactosamine-4-sulfata- se, no further
chromatography steps or columns are required. This method does not
comprise the fluid contacting a DEAE Sepharose resin. The recovered
precursor N-acetylgalactosamine-4-sulfatase has a purity of at
least equal to or greater than 99%. The overall recovery yield can
be at least about 40-60%.
[0077] Preferably, obtaining the fluid containing the precursor
N-acetylgalactosamine-4-sulfatase comprises growing a culture of
cells transformed with a gene encoding
N-acetylgalactosamine-4-sulfatase; preferably, the gene encodes
human N-acetylgalactosamine-4-sulfatase. Preferably, the cells are
mammalian cells. More preferably, the mammalian cells are Chinese
Hamster Ovary cells. The obtaining step can further comprise
harvesting the fluid from said culture of cells. The obtaining step
can further comprise concentrating said fluid to about
20.times..
[0078] A feature of this method is an early separation of protease
activity and the precursor N-acetylgalactosamine-4-sulfatase. This
separation can comprise either (1) the reduction, inhibition, or
inactivation of the protease activity, or (2) the physical
separation of the protease(s) from the precursor
N-acetylgalactosamine-4-sulfatase. Preferably, this separation
occurs as early as possible during the purification process. The
purpose is to keep to a minimum the number of molecules of
precursor N-acetylgalactosamine-4-sulfatase being cleaved into the
mature or processed form and/or other degraded form(s). The
precursor form of N-acetylgalactosamine-4-sulfatase is the
preferred form, as opposed to the mature or processed form, because
it is more readily taken up into the target tissue and for
subsequent targeting to the lysosome. The earlier or sooner the
protease activity is separated from the precursor
N-acetylgalactosamine-4-sulfatase: the fewer the number of
molecules of the precursor form would be cleaved into the mature or
processed form.
[0079] The activity of the protease is reduced or inhibited by
adjusting the fluid to a pH value between about 4.8 to 8.0.
Preferably, the pH value is between about 4.8 to 5.5. More
preferably, the pH value is between about 4.8 and 5.2. The specific
protease activity that is desired to be reduced is protease
activity that specifically cleaves precursor form of
N-acetylgalactosamine-4-sulfatase into the mature or processed
forms. The protease activity is found in one or more cysteine
protease. A cysteine protease that specifically cleaves precursor
N-acetylgalactosamine-4-sulfatase is cathepsin L. This cathepsin L
has a molecular weight of about 36 kDa in its inactive form that is
converted to its active forms of 21-29 kDa in size upon exposure to
pH of less than 5.0 (see FIG. 9C). The pH can be adjusted into any
value whereby the protease(s) is not converted from its inactive
form to its active form and the desired precursor
N-acetylgalactosamine-4-sulfatase, or biological activity thereof,
is not harmed or not irreversibly harmed.
[0080] Preferably, step (c) comprises passing the fluid through a
Cibracon blue dye interaction chromatography column. More
preferably, the Cibracon blue dye interaction chromatography column
is a Blue Sepharose 6 Fast Flow column. Preferably, step (d)
comprises passing the fluid through a copper chelation
chromatography column. More preferably, the copper chelation
chromatography column is a Chelating Sepharose Fast Flow column.
Preferably, step (e) comprises passing the fluid through a phenyl
hydrophobic interaction chromatography column. More preferably, the
phenyl hydrophobic interaction chromatography column is a Phenyl
Sepharose 6 Fast Flow High Sub column. Preferably, the temporal
sequence of steps (c), (d) and (e) is step (c), step (d) and step
(e).
[0081] The recovering step can comprise ultrafiltration and/or
diafiltration of the fluid. The recovering can comprise filtering
the fluid to remove DNA and/or filtering the fluid to remove virus.
The filtering, for removing virus, can comprise passing said fluid
through a 0.02 .mu.m filter.
[0082] This method can also be used to purify a
N-acetylgalactosamine-4-su- lfatase or biologically active
fragment, analog or mutant thereof.
[0083] The purity of rhASB is measured or determined using
reverse-phase high performance liquid chromatography ("RP-HPLC"),
which separates proteins based on differences in hydrophobicity.
This assay uses a C4 column (Phenomenex Jupiter) as the stationary
phase and a gradient of water:acetonitrile as the mobile phase. The
protein samples are initially injected onto the column in water;
under these conditions, all proteins will bind to the column. A
gradually increasing concentration of acetonitrile is then infused
through the column. This acetonitrile gradient increases the
hydrophobicity of the mobile phase, to the point where individual
proteins become soluble in the mobile phase and elute from the
column. These elution times are accurately reproducible for each
individual protein in a mixture. Proteins are detected as peaks on
a chromatogram by ultraviolet absorbance at 210 nm. The areas of
each peak are calculated, and the sample purity can be calculated
as the ratio of the rhASB peak area to the total area of all peaks
in the chromatogram. RP-HPLC is a proven high-resolution,
reproducible method of determining the purity of rhASB.
[0084] Studies prior to this application indicate that the purity
of ASB was determined by performing an impurity protein ELISA. This
method of using impurity protein ELISA (the details of which are
not disclosed) probably used antibodies raised against a mixture of
potential host-cell impurity proteins. The ELISA would likely be
performed using a standard curve of the same mixture of potential
impurity proteins used to generate the antibodies. Test samples
would likely be quantitated for impurity levels, relative to this
standard mixture. These assays are valuable tools in protein
purification, but are less accurate than RP-HPLC for determining
the product purity for the following reasons:
[0085] (1) In the RP-HPLC assay, the proportions of rhASB and
impurities are both determined by the same measurement (UV
absorbance). In the ELISA, the impurity concentration is determined
by antibody binding whereas the target protein content is
determined by another assay method (usually UV absorbance or
Bradford). The "percent purity" value should be calculated as the
ratio of two quantities with the same units, experimentally
determined by the same method.
[0086] (2) For the ELISA to work well, the sample detected by the
antibody should have the same protein composition as the standard.
This is very unlikely to be the case in an impurity protein ELISA.
The assay standard for this type of assay would be a mixture of
many individual proteins, against which the antibodies were
generated. However, only a small subset of impurity proteins should
be present in purified rhASB product. Therefore, the antibody
reagent will now be detecting a different mixture of proteins, and
the response versus the standard will probably be quite nonlinear.
When this occurs, the assay yields a different net value for each
sample dilution, so one does not know which dilution (if any) is
giving the correct value.
[0087] (3) In addition, not all potential impurity proteins are
immunogenic or immunogenic to the same degree in animals such as
rabbits, used to generate the antibodies. Therefore, the impurity
level determined by ELISA may only reflect a subset of the
impurities present in the purified products. It is entirely
possible to have one or more major impurities in the product that
are totally undetectable. In contrast, RP-HPLC detects all proteins
because UV absorbance is a universal property of protein
molecules.
[0088] (4) Finally, there are two types of impurities in a purified
drug product: product-unrelated impurities (host cell proteins as
discussed above) and product-related impurities (degradation
products including processed forms and aggregates). The latter
cannot be detected by the impurity ELISA but can be readily
detected by RP-HPLC.
[0089] Therefore, actual numbers obtained from the impurity protein
ELISA are open to question, and RP-HPLC numbers are based on a
firmer foundation.
[0090] In addition, SDS-PAGE analysis permits detection of both
host cell impurities and processed or degraded forms of the desired
drug protein product. When used in conjunction with Western blot,
product-unrelated impurities from host cell contaminants can be
differentiated from product-related impurities. Finally, SEC-HPLC
permits a level of quantification of product-related impurities
because it can detect impurities of different molecular weights,
including lower molecular weight processed or degraded forms of the
protein as well as monomers, dimers and other multimers.
[0091] An embodiment of this method of purification is depicted in
Table 4.
7TABLE 4 Method of Purification Step Process Harvest Filtration
Filtration through Clarification filters, 0.45 .mu.m filters and
finally 0.2 .mu.m filter. Filtered polled harvests are stored in
polypropylene bags UF Concentration Equilibration and flush: 100 mM
sodium phosphate, pH 7.3 Load: filtered harvest fluid
Concentration: Concentration to 20X Filtration: Filter the diluted
product through a 0.2 .mu.m filter into storage container pH
adjustment and pH adjustment: Add 10% glacial acetic acid to pooled
20X concentrates to a final filtration pH of equal to or less than
about 7.3; preferably, the pH is about 4.0 to 7.3; more preferably,
the pH is about 4.5 to 5.5; even more preferably, the pH is about
5.0 Load: pooled 20X concentrates Rinse: Water-for Injection (WFI)
Filtration through Clarification filters and 0.2 .mu.m filter.
Flush: 20 mM sodium acetate, 120 mM sodium chloride, pH 5.0 The
recovery yield can be at least about 83% Cibracon blue dye
Pre-Wash: 0.1 N sodium hydroxide interaction Wash:
Water-for-Injection (WFI) chromatography Equilibration: 10 mM
sodium phosphate, pH is less than about 6.5; preferably, pH is
column about 5.0 to 6.5; more preferably, pH is about 6.45 (Blue
Sepharose 6 Load: pH adjusted and filtered pooled 20X concentrates
FF) Wash: 10 mM sodium phosphate, pH 6.45 (Blue, Blue Elution: 10
mM sodium phosphate, 125 mM sodium chloride, pH 6.45 Sepharose)
Regeneration: 10 mM sodium phosphate, 1.0 M sodium chloride, pH
6.45 Sanitization: 0.1 N sodium hydroxide Wash 1:
Water-for-Injection (WFI) Wash 2: 10 mM sodium phosphate, 1.0 M
sodium chloride, pH 6.45 Storage: 20% Ethanol The recovery yield
can be at least about 84% Copper chelation Pre-Wash: 0.1 N sodium
hydroxide chromatography Wash: Water-for-Injection (WFI) column
Charge Buffer: 0.1 M cupric sulfate (Chelating Equilibration: 20 mM
sodium acetate, 0.5 M sodium chloride, 10% glycerol, pH is
Sepharose FF) less than about 6.0; preferably, pH is about 3.6 to
5.5; more preferably, pH is (Copper, CC, about 5.5
Copper-Chelating) Load: Adjust glycerol content of pooled Blue
Eluates to 10%, by adding 100 mM sodium acetate, 2.0 M sodium
chloride, 50% glycerol, pH 5.2 Wash 1: 20 mM sodium acetate, 0.5 M
sodium chloride, 10% glycerol, pH 5.5 Wash 2: 20 mM sodium acetate,
0.5 M sodium chloride, 10% glycerol, pH 3.9 Elution: 20 mM sodium
acetate, 0.5 M sodium chloride, 10% glycerol, pH 3.6 Eluate hold
for 30-120 minutes prior to adjustment to pH 4.5 with 0.5 M NaOH
Regeneration: 50 mM EDTA, 1.0 M sodium chloride, pH 8.0
Sanitization: 0.5 M sodium hydroxide Storage: 0.1 M sodium
hydroxide The recovery yield can be at least about 86% Phenyl
Pre-Wash: 0.1 N sodium hydroxide hydrophobic Wash:
Water-for-Injection (WFI) interaction Equilibration: 20 mM sodium
acetate, 2.0 M sodium chloride, pH is about 4.5 to chromatography
7.1; preferably, pH is about 4.5 column Load: Adjust sodium
chloride content of Copper Eluate to 2 M, by adding 20 mM (Phenyl
Sepharose sodium acetate, 5 M sodium chloride, pH 4.5 6 FF High
Sub) Wash 1: 10 mM sodium phosphate, 2.0 M sodium chloride, pH 7.1
(Phenyl, Phenyl Wash 2: 20 mM sodium acetate, 2.0 M sodium
chloride, pH 4.5 High Sub) Elution: 20 mM sodium acetate, 250 mM
sodium chloride, pH 4.5 Regeneration: 20 mM sodium acetate, pH 4.5
Sanitization: 0.5 N sodium hydroxide Storage: 0.1 N sodium
hydroxide The recovery yield can be at least about 88% UF/DF, DNA
Equilibration: 10 mM sodium phosphate, 150 mM sodium chloride, pH
5.8 Filtration, Viral Concentration: Concentration to NMT 1.5 mg/mL
Filtration, Diafiltration: 10 mM sodium phosphate, 150 mM sodium
chloride, pH 5.8 Formulation DNA Filtration: Product filtered
through a DNA filter Viral Filtration/Dilution: Product filtered
through a 0.02 .mu.m filter and diluted to 1.0 mg/ml Formulation:
Polysorbate 80 at a concentration of 50 .mu.g/mL is added
Filtration: Filter the diluted product through a 0.2 .mu.m filter
into storage container
[0092] The components of the drug product thus obtained are set
forth in Table 5. The components of the drug product composition
within the scope of the present invention are set forth in Table
6.
8TABLE 5 Drug Product Component Component Description Active
Recombinant human N-acetylgalactosamine-4-sulfatase Ingredient
Excipients Sodium Phosphate, Monobasic, 1 H.sub.20 Sodium
Phosphate, Dibasic, 7 H.sub.20 Sodium Chloride Container Kimble
Glass, Type I 5 ml clear glass vial, Borosilitcate West
pharmaceuticals, S-127 4432150 Grey stopper
[0093]
9TABLE 6 Drug Product Composition Component Amount RhASB 1 mg/mL
Sodium Phosphate, Monobasic, 1 H.sub.20 9 mM Sodium Phosphate,
Dibasic, 7 H.sub.20 1 mM Sodium Chloride 150 mM
[0094] The invention having been described, the following-examples
are offered to illustrate the subject invention by way of
illustration, not by way of limitation.
EXAMPLE 1
Clinical Evaluation with Recombinant Human
N-acetylgalactosamine-4-sulfata- se
[0095] Summary
[0096] The indication for recombinant human
N-acetylgalactosamine-4-sulfat- ase (rhASB) is the treatment of MPS
VI, also known as Maroteaux-Lamy Syndrome. We propose a clinical
development program for rhASB consisting of an initial open-label
clinical trial that will provide an assessment of weekly infusions
of the enzyme for safety, pharmacokinetics, and initial response of
both surrogate and defined clinical endpoints. The trial will be
conducted for a minimum of three months to collect sufficient
safety information for 5 evaluable patients. At this time, should
the initial dose of 1 mg/kg not produce a reasonable reduction in
excess urinary glycosaminoglycans or produce a significant direct
clinical benefit, the dose will be doubled and maintained for an
additional three months to establish safety and to evaluate further
efficacy.
[0097] Objectives
[0098] Our primary objective is to demonstrate safety of a weekly
infusion of rhASB in patients with MPS VI for a minimum of a
three-month period. Measurements of safety will include adverse
events, immune response and allergic reactions (complement
activation, antibody formation to recombinant enzyme), complete
clinical chemistry panel (kidney and liver function), urinalysis,
and CBC with differential.
[0099] One secondary objective is to evaluate efficacy by
monitoring changes in several parameters known to be affected in
MPS VI. These include a six-minute walk test (as a measure of
exercise tolerance), full pulmonary function (PFT) evaluation,
reduction in levels of urinary glycosaminoglycans and hepatomegaly
(as measures of kidney and liver GAG storage), growth velocity,
joint range of motion, Children's Health Assessment Questionnaire
(CHAQ), visual acuity, cardiac function, sleeping studies, and two
different global assessments; one performed by the investigator,
one performed by the patient/caregiver. A second secondary
objective is to determine pharmacokinetic parameters of infused
drug in the circulation, and general distribution and half-life of
intracellular enzyme using leukocytes and buccal tissue as sources
of tissue. It is anticipated that these measures will help relate
dose to clinical response based on the levels of enzyme delivered
to the lysosomes of cells.
[0100] Methods
[0101] We will conduct a single center, open-labeled study to
demonstrate safety and to evaluate clinical parameters of treatment
with rhASB in patients with MPS VI. Patients will be admitted for a
two week baseline evaluation that will include a medical history
and physical exam, psychological testing, endurance testing
(treadmill), a standard set of clinical laboratory tests (CBC,
Panel 20, CH50, UA), a MRI or CAT scan of the body (liver and
spleen volumetric determination, bone and bone marrow evaluation,
and lymph node and tonsillar size), a cardiology evaluation
(echocardiogram, EKG, CXR), an airway evaluation (pulmonary
function tests), a sleep study to evaluate for obstructive events
during sleep, a joint restriction analysis (range of motion will be
measured at the elbows and interphalangeal joints), a LP with CNS
pressure, and biochemical studies (buccal
N-acetylgalactosamine-4-sulfatase activity on two occasions,
leukocyte N-acetylgalactosamine-4-sulfatase activity on two
occasions, urinary GAG on three occasions, serum generation for
ELISA of anti-rhASB antibodies and 24 hour urine for creatinine
clearance). In addition to the above evaluations, each patient will
be photographed and videotaped performing some physical movements
such as attempting to raise their hands over their heads and
walking. Patients will be titrated with antihistamines such that
pretreatment with these agents could be effectively employed prior
to infusion of enzyme. The proposed human dose of 1 mg/kg (50 U/kg)
will be administered weekly by i.v. infusion over 4 hours. The
patient will remain in the hospital for the first two weeks,
followed by short stays for the next four weeks. Treatment for the
final six weeks will be conducted at a facility close to the
patient's home. Patients will return to the hospital for a complete
evaluation at three months. Should dose escalation to 2 mg/kg be
required, the patients will follow the same schedule outlined above
for the first twelve weeks. Under either scenario, a complete
evaluation will also occur at 6 months from the time of entering
the trial. Safety will be monitored throughout the trial. Patients
completing the trial will be continued on therapy following an
extended protocol for as long as safety and efficacy conditions
warrant it until BLA approval.
[0102] Patient Number and Enrollment Rate
[0103] A single patient will be enrolled at the onset of the trial,
with two additional patients one month later, and two more patients
two weeks later barring any unforeseen complications related to
treatment. Additional patients will be admitted should any of the
enrolled patients become critically ill, or if a child is in need
of an acute clinical procedure for life threatening or harmful
conditions.
[0104] Diagnosis and Inclusion/Exclusion Criteria
[0105] The patient may be male or female, aged five years or older
with a documented diagnosis of MPS VI confirmed by measurable
clinical signs and symptoms of MPS VI, and supported by a
diminished fibroblast or leukocyte ASB enzyme activity level.
Female patients of childbearing potential must have a negative
pregnancy test (urine .beta.-hCG) just prior to each dosing and
must be advised to use a medically accepted method of contraception
throughout the study. A patient will be excluded from this study if
the patient has previously undergone bone marrow transplantation;
is pregnant or lactating; has received an investigational drug
within 30 days prior to study enrollment; or has a medical
condition, serious intercurrent illness, or other extenuating
circumstance that may significantly decrease study compliance.
[0106] Dose, Route and Regimen
[0107] Patients will receive rhASB at a dose of 1 mg/kg (.about.50
U/kg) for the first 3 months of the study. In the event that excess
urine GAGs are not decreased by a reasonable amount and no clinical
benefit is observed, the dose will be doubled. Dose escalation will
occur only after all 5 patients have undergone 3 months of therapy.
This rhASB dosage form will be administered intravenously over
approximately a four-hour period once weekly for a minimum of 12
consecutive weeks. A peripheral intravenous catheter will be placed
in the cephalic or other appropriate vein and an infusion of saline
begun at 30 cc/hr. The patient will be premedicated with up to 1.25
mg/kg of diphenylhydramine i.v. based on titration experiments
completed prior to the trial. rhASB will be diluted into 100 cc of
normal saline supplemented with 1 mg/ml human albumin. The diluted
enzyme will be infused at 1 mg/kg (about 50 units per kg) over a 4
hour period with cardiorespiratory and pulse oximeter monitoring.
The patients will be monitored clinically as well as for any
adverse reaction to the infusion. If any unusual symptoms are
observed, including but not limited to malaise, shortness of
breath, hypoxemia, hypotension, tachycardia, nausea, chills, fever,
and abdominal pain, the infusion will be stopped immediately. Based
on clinical symptoms and signs, an additional dose of
diphenylhydramine, oxygen by mask, a bolus of i.v. fluids or other
appropriate clinical interventions such as steroid treatment may be
administered. If an acute reaction does occur, an assessment for
the consumption of complement in the serum will be tested. A second
i.v. site will be used for the sampling required for
pharmacokinetic analysis.
[0108] Evaluable Patients
[0109] The data from any given patient will be considered evaluable
as long as no more than two non-sequential infusions are missed
during the 12 weeks of therapy. The initial, midpoint and final
evaluations must be completed.
[0110] Safety
[0111] The enzyme therapy will be determined to be safe if no
significant acute reactions occur that cannot be prevented by
altering the rate of administration of the enzyme, or acute
antihistamine or steroid use. The longer-term administration of the
enzyme will be determined to be safe if no significant
abnormalities are observed in the clinical examinations, clinical
labs, or other appropriate studies. The presence of antibodies or
complement activation will not by themselves be considered unsafe,
but such antibodies will require monitoring by ELISA, and by
clinical assessments of possible immune complex disease.
[0112] Efficacy
[0113] One purpose of this study is to evaluate potential endpoints
for the design of a pivotal trial. Improvements in the surrogate
and clinical endpoints are expected as a result of delivery of
enzyme and removal of glycosaminoglycan storage from the body. Dose
escalation will be performed if mean excess urinary
glycosaminoglycan levels are not reduced by a reasonable amount
over three months and no significant clinical benefit is observed
at 3 months. Improvements are expected to be comparable to those
observed in the recently completed MPS I clinical trial and should
include improved airway index or resolution of sleep apnea,
improved joint mobility, and increased endurance.
EXAMPLE 2
[0114] A comprehensive review of the available information for the
MPS VI cat and relevant pharmacology and toxicology studies is
presented below: Enzyme replacement therapy has been established as
a promising treatment for a variety of inherited metabolic
disorders such as Gaucher Disease, Fabry Disease and
Mucopolysaccharidosis I. In some of these disorders a natural
animal model offers the ability to predict the clinical efficacy of
human treatment during pre-clinical studies. This was found to be
true in MPS I (canine model). With this in mind, studies have been
performed with the MPS VI cat prior to the commencement of human
studies for this disease. Sufficient safety and efficacy data exist
to proceed with a clinical trial in human MPS VI patients.
[0115] Studies of rhASB MPS VI cats indicate that no cat has died
as a result of drug administration. As predicted, experiments in
MPS VI cats also indicate that rhASB uptake is dependent on the
presence of mannose 6-phosphate modified carbohydrate sidechains.
RhASB in MPS VI cats has also been shown to clear storage from a
variety of major organs and moderately alters bone density.
Long-term dose-ranging efficacy studies suggest that a dose of 1
mg/kg/week is the lowest concentration to see significant clinical
benefits. Studies has also been performed to compare enzyme
distribution, clearance of tissue glycosaminoglycan storage, and
decrease of urinary glycosaminoglycan levels after bolus and slow
(2 hour) infusion. Studies in progress continue to evaluate the
safety of weekly infusions of the projected clinical dose of 1
mg/kg of rhASB in cats suffering from MPS VI.
[0116] A spontaneous form of MPS VI in several families of Siamese
cats was identified in the 1970's (Jezyk, Science 198:834-36
(1977)), and detailed reports of the pathological changes in these
animals have been published (Haskins, et al., Am. J. Pathol.
101:657-674 (1980); Haskins, et al., J. Am. Vet. Med. Assoc.
182:983-985 (1983); Konde, et al., Vet. Radiol. 28:223-228 (1987)).
Although the clinical presentation of these cats is somewhat
variable, they all exhibit general changes that have been reported
in the literature (Jezyk, et al., Science 198:834-36 (1977); Konde,
et al, Vet Radiol. 28:223-228 (1987); Crawley, "Enzyme replacement
therapy in a feline model of mucopolysaccharidosis type VI" PhD
thesis, University of Adelaide, Adelaide, S. Australia, (1998)).
Table 6 has been constructed from these sources to provide the
"average" changes one would expect to observe in an untreated MPS
VI cat:
10TABLE 7 MPS VI Cat Model Changes Relative to Human Clinical
Observation Timing of Onset disease (independent of time) Facial
dysmorphia: 2 months Similar to human disease Small head, Broad
maxilla, Small ears Diffuse corneal clouding 2 months Similar to
human disease Bone abnormalities: First signs at 2 Similar to human
disease - Epiphyseal dysplasia, months - progressive alterations in
enchondral Subluxations, calcification Pectus excavatum Reduced
body weight 3 months Similar to human disease Reduced cervical
spine Normal cat value is Similar to human disease flexibility
180.degree. at all ages. In MPS VI: 3 months: 130-170.degree. 5
months: 45-130.degree. 6 months: 30-100.degree. 11 months:
20-80.degree. Osteoporosis/Degenerative 1 year or more Similar to
human disease Joint Disease Hind limb gait defects See table below
Carpal tunnel syndrome Hind limb paresis or paralysis
C.sub.1-C.sub.2 subluxation, (thoracolumbar cord Cervical cord
compression compressions) secondary to thickened dura more typical
Grossly normal liver and Liver and spleen enlarged spleen in humans
Thickened cardiac valves Similar to human disease No CNS lesions -
mild lateral May be comparable to ventricle enlargement
hydrocephalus in human disease
[0117] Other biochemical/morphological determinations indicate that
by 35 days, organs of untreated cats have maximal storage of
glycosaminoglycans in tissues (Crawley, et al., J. Clin. Invest.
99:651-662 (1997)). Urinary glycosaminoglycan levels are elevated
at birth in both normal and MPS VI cats but after approximately 40
days, normal cats have decreased levels. MPS VI cats urinary
glycosaminoglycans remain elevated or continue to increase until
reaching steady state after approximately 5 months.
[0118] Variability in clinical presentation is seen in affected
littermates. In addition to some variability in the timing of onset
of particular abnormalities, the time course of progression for
some of the clinical and pathological changes is also variable. In
general, the bone lesions are typically progressive (Konde et al.,
Vet. Radiol. 28:223-228 (1987)), while the corneal clouding is not.
In addition, some paralyzed cats have been noted to improve to
severe paresis with time. Studies detailing disease progression in
individual cats are limited to clinical (or radiographic)
observations. Some of these have distinct pathological
correlations, such as neurological deficit and cord compression
secondary to proliferation of bony tissue in the thoracolumbar
region (Haskins, et al., J. Am. Vet. Med. Assoc. 182:983-985
(1983)).
[0119] A six-month efficacy study enzyme replacement therapy using
recombinant feline ASB in newborn MPS VI cats was conducted. This
was prompted by the observation that several treated MPS VI cats
developed antibodies to the human enzyme (refer to section 6.5).
These antibodies may alter uptake and stability of the enzyme
(Brooks, et al., Biochim. Biophys. Acta 1361:203-216 (1997)).
Feline enzyme was infused at 1 mg/kg weekly. The major conclusions
of the study were that urinary GAG, body weight/growth, bone
morphometry and clearance of stored material from several tissues
was improved relative to the same dose of human recombinant enzyme
used in the previous study, that antibodies were not detected
beyond the range observed in normal cats, and that the feline
enzyme dose at 1 mg/kg was comparable in reversing disease as the
human enzyme dose at 5 mg/kg in a head-to-head comparison
(Bielicki, et al., J. Biol. Chem., 274:36335-43 (1999)). These
studies indicate that an incremental improvement in endpoints and
immunogenicity is possible when the cat-derived enzyme is given to
cats. This provides additional support to dosing human patients
with the human enzyme at 1 mg/kg/week. The results of this study
are set forth in Table 7.
11TABLE 8 Efficacy of Weekly Bolus Injections of CHO-derived
Recombinant Feline ASB in Newborn MPS VI Cats Results Dose 1 mg/kg
Duration 6 months (n = 2) 3 months (n = 3) Urinary GAGS Decreased
to 2x normal Decreased to 2x normal Antibody titers Within range
observed in To be completed normal cats Clinical Appearance
Persistent corneal clouding Persistent corneal Some resolution of
facial clouding dysmorphia Some resolution of Improved body shape
facial dysmorphia, Improved body shape Weight Heavier than normal
Slightly lighter than normal Spine Flexibility
160.degree.-180.degree. Not examined (normal = 180.degree.)
Neurological Normal Normal Radiology Improved quality Not examined
Density and dimensions of bone (similar to 1 mg/kg rh4S in ref. 10)
Gross Bone/Cartilage Variable; decreased cartilage Not examined
Thickness thickness more uniform subchondral bone (similar to 1
mg/kg rh4S.sup.a) Spinal Cord No compressions present Not examined
Cellular Level Liver (Kupffer) Complete lysosomal storage Complete
lysosomal clearing storage clearing Skin Almost complete reduction
is Mild reduction storage Cornea/Cartilage No clearance of
lysosomal No clearance (ear, articular) storage compared with of
lysosomal untreated MPS VI controls storage Heart Valves
Significant reduction in To be completed lysosomal storage Aorta
Almost complete reduction in Mild reduction in lysosomal storage
lysosomal storage
[0120] Table 9 provides a summary of all studies performed using
recombinant human ASB in the MPS VI cat model.
12TABLE 9 RhASB Study Results Duration Route of No. Cat Dose (Mo.)
Administration Urinary GAGS Histopathology 1 0/8 mg/kg/14 d 7-22
Bolus i.v. Decreased 50% Normalization of vacuolization in liver
1.5 mg/kg/7 d 22-27 Bolus i.v. compared to untreated Significant
reduction in kidney and skin cat 1 0.5 mg/kg/14 d 12-23 Bolus i.v.
Decreased to near No correction in cornea and chondrocytes 1.4
mg/kg/7 d 23-27 Bolus i.v. normal No kidney immune complex
deposition 1 0.8 mb/kg/14 d 2-15 Bolus i.v. 1 0.2 mg/kg/8 d 6 Bolus
i.v. Marginal reduction N/A compared to untreated 4 1 mg/kg/7 d 5/6
Bolus i.v. Decreased and Complete lysosomal storage clearing in
liver cells maintained at 3x No evidence or renal impairment or
glomerular immune complex normal compared to deposition untreated
at 10x Significant reduction of lysosomal storage in heart valves
normal Gradient storage content from media to adventia in aorta 1
11 Bolus i.v. Mild reduction of lysosomal storage of skin (hip
joint, dura, kidney) No evidence of renal impairment or glomerular
deposition No significant changes in lysosomal storage of
cornea/cartilage 2 5 mg/kg/7 d 5/6 Bolus i.v. Decreased and
Complete lysosomal storage in clearing in liver and skin (hip
joint, maintained at 2x dura, kidney) normal compared to No
evidence of renal impairment or glomerular deposition untreated at
10x Near complete reduction in lysosomal storage in heart valves
normal Thin band of vacuolated cells in outer tuncia media 1 11
Bolus i.v. No evidence of renal impairment or glomerular deposition
Near complete reduction of lysosomal storage in heart valves Thin
band of vacuolated cells in outer tuncia media 2 0.5 mg/kg 6 Bolus
i.v. Decreased to 3x Complete lysosomal clearing in liver 2 .times.
weekly normal Mild to moderate reduction in skin Variable reduction
of lysosomal storage of heart valves Mild reduction of lysosomal
storage in aorta 2 1 mg/kg/7 d 1 Long infusion Reduced after first
or Reduction of lysosomal storage in reticuloendothelial cells and
very (2 hr) second infusion to mild in heat valve and aorta after 5
infusion 2 1 Short infusion below untreated MPS (10 min) VI cats 5
1 mg/kg/7 d 6 Long infusion (2 hr)
EXAMPLE 3
Distribution and Feasibility
[0121] An initial study was performed to document enzyme uptake and
distribution, and to serve as a pilot study of potential endpoints
for future efficacy studies (Crawley, et al., J. Clin. Invest.
97.1864-1873 (1996)). Recombinant human ASB was administered by
bolus injection to affected cats once per week or once every two
weeks at 0.5 up to 1.5 mg/kg. Evaluation of one untreated MPS VI
cat (Cat D), and one normal cat provided the values from which
comparisons were drawn. The data from the one untreated cat was
further supported by historical assessment of 38 additional
untreated cats. The acute uptake and distribution studies were
conducted in normal cats using an immune assay technique that
allowed the detection of human ASB in the presence of normal cat
enzyme.
[0122] The major conclusions of these studies demonstrated wide
uptake of enzyme with the expected predominance of liver and spleen
uptake as observed in other enzyme replacement studies in MPS
animal models. The uptake efficiency was dependent on the presence
of mannose 6-phosphate modified carbohydrate side-chains on the
enzyme. The half-life of the enzyme was determined to be 2-4 days.
Therapeutically, the enzyme did clear storage from a variety of
major organs and did moderately alter bone density. The cornea,
bone morphology and cartilage defects were not effectively treated
in older MPS VI cats. The study results are summarized in Table
10.
13TABLE 10 Summary: Distribution/Feasibility MPS VI Cat Study
Findings Cat A B C Parameter Treated MPS VI Treated MPS VI Treated
MPS VI Dose 0.8 mg/kg 1.5 mg/kg 0.5 mg/kg 1.4 mg/kg 0.8 mg/kg per
14 d per 14 d per 7 d per 14 d per 7 d Age at dose (mo.) 7*-22
22-27 12*-33 23-27 2*-15 Infusion Parameters 2-10 ml (PBS) via
cephalic v. for 5-20 minutes Plasma t.sub.1/2 (i.v. bolus) 13.7
.+-. 3.2 min @ 1 mg/kg 45 min @ 7.5 mg/kg All values relative to
endogenous feline ASB enzyme four hours after infusion of 1 mg/kg
rhASB in normal cats Liver: 495x Spleen: 6x Lung: 22.3x Heart: 4.3x
Aorta: 4x Skin: 31x Cartilage: 0x Cornea: 0x Tissue t.sub.1/2 2-4
days @ 1 mg/kg in most organs (detectable enzyme in most tissues of
cat B, but only in liver of A after 7 days) Neurological Ambulation
N/A Marginal progression fluctuated, but to paretic gate by end of
improved on higher study dose Corneal Opacity Did not change with
therapy (slit lamp exam 3x late in rx) Skeletal (x-rays) Lesions
progressed (no radiographic improvement 4 views every 3 mo.
Increased bone volume/trabecular # in cat C (received earlier rx)
Vertebral compression in cat C Anaphylaxis No anaphylaxis, minimal
distress on infusion; Antibody response 1 .times. 10.sup.6 64,000
64,000 (Ig titers) (plasma could inhibit Untreated MPS VI =
4,000-32,000 enzyme activity in vitro) Urinary GAGS Decreased 50%
Decreased to near normal (at .about.400 days) compared to untreated
cat Urinary dermatan Midway for all 3 cats (relative to untreated
control D and normal) sulfate (.about.400 days) Body Weight 2.5-3.0
kg vs. normal 4-7 kg Liver/Spleen Grossly normal Heart Valves
Grossly normal Cartilage Abnormal thickness and formation
Microscopy Normalization of vacuolization in liver, (vacuolization)
Significant reduction in kidney and skin, No correction in cornea
and chondrocytes Kidney immune Absent complex deposition
EXAMPLE 4
Efficacy in MPS VI Cats Treated from Birth
[0123] A long term dose-ranging efficacy study was performed in MPS
VI cats starting at birth (Crawley, et al., J. Clin. Invest.
99:651-662 (1997)), and is summarized in Table 10. MPS VI cats were
treated weekly with bolus i.v. injections of 0.2, 1 and 5 mg/kg of
rhASB beginning at birth. A total of 9 cats were treated for 5, 6
or 11 months. In addition, 12 MPS VI and 9 normal cats were
included as untreated controls. The major conclusions are that 0.2
mg/kg dose did not alter disease progression in the one cat
studied, and the only documented clinical benefit was a reduction
in the storage in liver Kupffer cells. Urinary GAG levels decreased
to near normal during the trial in the higher dose groups. In
addition to improvements in the major organs, the higher doses of
therapy from birth were able to prevent or ameliorate the bony
deformity of the spine and the abnormal form of many bones. There
was a dose-dependent effect on improvement in L-5 vertebral bone
mineral volume, bone trabecular thickness, and bone surface density
between the 1 and 5 mg/kg doses, although both were equivalent in
improving bone formation rate at 5 to 6 months of ERT (Byers, et
al., Bone 21:425-431 (1997)). The mitral valve and aorta was
dependent on dose and was less complete at 1 mg/kg but nearly
complete at 5 mg/kg. No improvement of storage in cartilage and
cornea was observed at any dose. The study suggests that the 1
mg/kg/week dose is the lowest concentration to see significant
clinical benefit. The study results are summarized in Table 11.
14TABLE 11 Efficacy of Weekly Bolus Injections of CHO-derived
Recombinant Human ASB in Newborn MPS VI Cats (Study PC-BM102-002)
Results Dose 1 mg/kg 5 mg/kg Duration 5/6 mo 11 mo 5/6 mo 11 mo N 4
1 2 1 Biochemical Urinary GAGs Decreased and maintained at 3x
Decreased and maintained at 2x normal compared to untreated at 10x
normal compared to untreated at 10x normal normal Clinical
Appearance Variable changes; Variable changes; Persistent corneal
clouding by slit Persistent corneal clouding by slit lamp lamp
Weight Intermediate (no rx vs. normal) Intermediate (no rx vs.
normal) Spine Flexibility 130-160.degree. 90.degree. 180.degree.
160.degree. (normal = 180) (untreated MPS VI = 90.degree.)
Neurological 1 of 4 mild No deficits No deficits No deficits
hindlimb paralysis Radiology Improved bone quality, density and
Improved bone quality, density dimensions and dimensions Possibly
superior to 1 mg/kg Gross Bone/Cartilage Variability, but
Degenerative Variability, but Degenerative Thickness improved joint
disease improved joint disease present present Spinal Cord 1 of 4
with No No cord compressions several mild compressions compressions
Cellular Level Liver (Kupffer) Complete Maintained Complete
Maintained lysosomal storage lysosomal storage clearing clearing
Skin (hip Joint, No evidence of Mild reduction Complete Maintained
Dura, Kidney) renal impairment or in lysosomal lysosomal storage No
evidence glomerular immune storage clearing of renal complex
deposition No evidence of No evidence of impairment or renal
impairment or renal impairment or glomerular glomerular glomerular
deposition deposition deposition Cornea/Cartilage NA No significant
NA No (ear, articular) changes in significant lysosomal storage
changes in lysosomal storage Heart Valves Significant Significant
Near complete reduction in (Variable) (variable) reduction
lysosomal storage reduction in in lysosomal lysosomal storage
storage near complete Aorta Gradient of storage content from Thin
band of vacuolated cells in media to adventitia outer tunica
media
EXAMPLE 5
Efficacy of Twice Weekly Infusions of Recombinant Human ASB in
Newborn MPS VI Cats
[0124] A six-month study was performed in newborn cats to evaluate
a 0.5 mg/kg infusion given twice weekly. In addition, the enzyme
used in this study was derived exclusively from the CSL-4S-342 cell
line. The major conclusions of the study include that compared with
the previously reported 1 mg/kg weekly dose, this study produced
similar improvements in physical, biochemical, neurological and
radiographic parameters. The most notable differences were slightly
worsened cervical spine flexibility, and less clearance of
lysosomal storage in the denser connective tissues such as the
heart valves and aorta. The results are summarized in Table 12.
15TABLE 12 Efficacy of Twice Weekly Bolus Injections of CHO-derived
Recombinant Human ASB in Newborn MPS VI Cats Parameter Results Dose
0.5 mg/kg Duration 2x weekly: 6 months (n = 2; cats 225f, 226m)
Urinary GAGs Decreased to 3x normal Antibody titres Within range
observed in normal cats Clinical Appearance Persistent corneal
clouding Some resolution of facial dysmorphia Improved body shape
Weight Intermediate (between no treatment and normal) Spine
Flexibility 90.degree.-150.degree. (normal = 180.degree.)
Neurological No hind limb paralysis Radiology Improved quality,
density and dimensions of bone (similar to 1 mg/kg rh4S in ref. 110
Gross Bone/Cartilage Variable; decreased cartilage thickness and
more Thickness uniform subchondral bone (similar to 1 mg/kg
rh4S.sup.a) Spinal Cord No compressions present Cellular Level
Liver (Kupffer) Complete lysosomal clearing Skin Mild to moderate
reduction in storage Cornea/Cartilage No clearance of lysosomal
storage compared (ear, articular) with untreated MIPS VI controls
Heart Valves Variable reduction in lysosomal storage (complete in
225f; no change from untreated in 226m) Aorta Mild reduction in
lysosomal storage
EXAMPLE 6
Evaluation of Enzyme Uptake and Distribution as a Function of the
Rate of Enzyme Infusion in MPS VI Cats
[0125] The primary goal of this study was to compare enzyme
distribution, clearance of tissue GAG storage, and decrease of
urinary GAG levels after bolus infusion and after slow (2 hour)
infusion of an identical 1 mg/kg dose. The slow administration
proposal is based on experience from preclinical and clinical
studies of .alpha.-L-iduronidase for the treatment of MPS I. In
addition, the study provided the first data that enzyme produced at
BioMarin from cell line CSL4S-342 is biologically active and safe.
Major conclusions of the study include that all four cats (two per
group) treated in this study showed no acute adverse reaction to
either the slow or fast infusion, and no detrimental effects of
repeated enzyme infusions. However, bolus infusion results in high
liver uptake which is not preferred. Slow infusion provides better
distribution into tissues and therefore is a preferred method for
clinical trial.
[0126] The tissue distribution of rhASB obtained in the study
suggested that 2-hour infusions might increase enzyme levels in
other organs apart from the liver, including increased activity in
the brain. Reduction in urinary GAG was observed immediately after
the first or second infusion to levels below the range observed in
untreated MPS VI cats. Correction of lysosomal storage was observed
in reticuloendothelial cells and very mild in some fibroblasts
(heart valve) and smooth muscle cells (aorta) after 5 infusions. No
other significant clinical response to infusions was observed in
either group, however this was not unexpected due to the short
duration of the study, and due to therapy starting after
significant disease changes had already developed. The extended
2-hour infusion was safe and well tolerated relative to the shorter
protocols used in previous studies. The 2-hour infusion may provide
improvement in enzyme distribution based on the one cat that was
evaluable for enzyme tissue distribution.
EXAMPLE 7
6 Month Safety Evaluation of Recombinant Human
N-acetylgalactosamine-4-sul- fatase in MPS VI Affected Cats
[0127] Two 6 month studies in MPS VI cats have been initiated using
the enzyme produced by the manufacturing process according to the
present invention. The purpose of these studies is to evaluate the
safety and efficacy of weekly infusions of the projected human
clinical dose of rhASB in cats suffering from MPS VI. Study 6
involves kittens dosed initially at 3 to 5-months of age. Study 7
involves kittens treated from birth with weekly infusions of the
projected human clinical dose of rhASB. The studies are intended to
access potential toxicology. Cats will be observed for changes in
behavior during infusion of the recombinant enzyme to assess
possible immune responses. Serum will be monitored for complement
depletion and for the formation of antibody directed against the
recombinant enzyme. General organ function will be monitored by
complete clinical chemistry panels (kidney and liver function),
urinalysis, and complete blood counts (CBC) with differential.
Urinary glycosaminoglycan levels will be monitored on a weekly
basis at a set time points relative to enzyme infusion. Evidence of
clinical improvements in disease will be documented. These data
will provide additional assessment of the potential efficacy of the
treatment and will validate the activity and uptake of the enzyme
in vivo. The studies have and will be conducted in a manner
consistent with the principles and practices of GLP regulations as
much as possible.
[0128] Preliminary results of the first study indicate that
administration of rhASB has not had any detrimental effects on any
of the animals, with bodyweights and clinical chemistries generally
maintained within reference ranges. However, both of the cats with
significantly elevated antibody titers developed abnormal clinical
signs during infusions, however both animals behaved normally once
enzyme infusions ceased and did not appear to suffer any longer
tern ill effects. Extended infusion times (4 hours) and increased
premedication antihistamines have allowed continued therapy in the
cats without any abnormal clinical signs. Mild reduction in urinary
GAG levels suggest some efficacy of therapy in reducing stored
glycosaminoglycans in tissues or circulation, however fluctuations
in these levels were observed over time making interpretation
difficult. None of the 5 cats have shown obvious clinical
improvements in response to ERT, but this will require at least 6
month treatment based on previous studies.sup.23. Antibody titers
have developed in four out of the five cats, with noticeable
increases in titers observed after 2 months of ERT. Two of these
cats have developed significantly elevated titers after 2 or 3
months.
EXAMPLE 8
Safety Profile for MPS VI Cats Treated with rhASB
[0129] A study has commenced enrolling affected cats that were
treated within 24 hours of birth. Forty-one MPS VI cats have been
treated using rhASB. Administration of enzyme to normal cats has
been restricted to one to two cats to confirm acute safety of new
batches prior to exposure of the valuable affected animals to
therapy. In summary, no MPS VI cat has died as a result of drug
administration, although four cats have died as a result of viral
infection or an underlying congenital abnormality. Enzyme for the
studies was produced according to the production methods of the
present invention. The preliminary data are set forth in Table
13.
16TABLE 13 MPS VI Cat Efficacy Study Summary from Hopwood
Laboratory Rx Length Study # # of Cats Dose/wk (mg/kg) (mos.)
Mortality 1 2 Variable 13-21 None 1 2 1 0.2 5 None 2 1 0.2 1 Died:
congenital heart defect -- 2 0.5 3-5 1 died parvovirus 2 4 1 3-11 1
died parvovirus 2 -- 1 1 6 None (s.c.) -- 4 1 6 None 2 2 5 3-11 1
died parvovirus 2 4 2 0.5 (twice) 5 None -- 3 0.5 (twice) 5 None 5
4 1 1 None 6 5 1 Started None 7 5 1 Started None
EXAMPLE 8A
Tissue Distribution with Slow Infusion
[0130] A separate study was undertaken to compare the rate of
uptake and tissue distribution of rhASB in young MPS VI cats
(<10 weeks old at initiation) using a long- (two hours) or a
short-infusion (10-15 minutes) protocol (ASB-PC-004). Seven normal
cats (12-24 months old) were sedated and had femoral artery
cannulae implanted to allow blood sampling. rhASB at a dose of 1.0
mg/kg or 7.3 mg/kg was infused into the cephalic vein in a volume
of 7 mL over 40 seconds. Serial heparinized arterial blood samples
were obtained at approximately 2, 4, 6, 8, 10, 20, 30, 40, 60 and
120 minutes post-dose. Plasma was collected by centrifugation and
frozen until analysis. After four hours, animals were euthanized
and tissues were collected, weighed and frozen until analysis.
[0131] In a separate study, three normal cats (8-66 months old)
received a 1.0 mg/kg IV bolus dose of rhASB in a 7 mL dose volume.
One cat per time point was euthanized at 2, 4 or 7 days post-dose.
Selected tissues were collected, weighed and frozen until analysis.
One additional normal cat, not exposed to rhASB, was euthanized to
provide feline ASB levels in tissues for analytical comparisons.
rhASB or feline ASB were quantitated in plasma and tissues was
performed using an ELISA assay technique. rhASB in samples was
adsorbed to an immobilized anti-rhASB monoclonal antibody and
feline ASB was absorbed to a polyclonal anti-rhASB antibody that
cross-reacts with feline ASB. The absorbed ASB was then quantitated
using afluorescent substrate.
[0132] Plasma concentration analyses showed that the t1/2 of rhASB
at the 1.0 mg/kg dose was 13.7.+-.3.2 minutes and was .congruent.45
minutes for the 7.3 mg/kg dose. Tissue t1/2 following the 1.0 mg/kg
dose ranged from 2.4-4.2 days in the liver, spleen, lung, kidney
and heart. Low, but detectable levels were seen in these tissues
(except heart) up to seven days following dosing. Four hours after
a dose of 1.0 mg/kg ASB, the majority of the dose delivered to the
liver, was detected in all tissues measured except cartilage and
cornea, with the majority in the liver. With the exception of the
cerebrum and cerebellum, levels were 4- (aorta) to 495- (liver)
fold higher than those of feline ASB in the untreated control cat.
The tissue half-lives determined in this study (2.4-4.2 days at 1.0
mg/kg) support the weekly clinical dosing frequency.
[0133] The primary objective of this study was to evaluate the
effects of infusion rate on enzyme distribution, clearance of
tissue GAG storage, and decrease of urinary GAG levels. Groups
(n=2/group) of 10-week-old MPS VI cats were administered short
(10-15 minutes) or long (two hours) IV infusions of rhASB, 1.0
mg/kg once weekly for five weeks. Animals were euthanized two days
after the last infusion and selected tissues (liver, spleen, heart,
lung, kidney, skin, aorta, cerebrum, cerebellum, cartilage, cornea
and lymph nodes) were collected for determination of ASB activity
and for histopathological evaluation. Tissues were stored frozen
until analysis. The study employed the same immune assay to
determine the tissue ASB levels as described above (ASB-PC-001).
One cat in the two-hour infusion group had reduced tissue enzyme
levels compared to the other treated cats. High levels of enzyme
were detected around the catheterization site following the last
dose while low levels were seen in the contralateral limb. These
findings suggest that the catheter had dislodged prematurely and
had contributed to the low tissue levels of ASB seen in the tissues
from this animal. Comparison of enzyme activities in the remaining
three cats showed increased enzyme activity in the liver, lung,
kidney, cerebrum and cerebellum and reduced activity in the
mesenteric lymph nodes of the two-hour infusion cat relative to the
10-minute infusion animals. No enzyme was found in the skin,
cartilage and corneas. Although the data from this study is
limited, the findings from this study suggest that two-hour
infusions may result in increased enzyme levels in the tissues
(apart from the liver) relative to 10-minute infusions, indicating
that the extended infusions provide better tissue distribution.
EXAMPLE 9
Method of Manufacture and Purification (Perfusion Process)
[0134] A process flow diagram comparing the purification processes
for the fed batch and perfusion-based cell culture processes is
provided in Table 14. Comparisons to the fed batch purification
process as well as details of the specific changes implemented for
the purification of the perfusion process material are summarized
in Table 15. Table 16 depicts the purification method used for the
perfusion-based cell cultures.
17TABLE 14 Purification Process Comparison between Batch and
Perfusion Processes Batch Process Perfusion Process Harvest
Filtration Harvest Filtration .dwnarw. .dwnarw. 10X Concentration
and Diafiltration 20X Concentration .dwnarw. .dwnarw. DEAE
Sepharose Chromatography pH adjustment and filtration .dwnarw.
.dwnarw. Blue SepharoseFF Chromatography Blue SepharoseFF
Chromatography .dwnarw. .dwnarw. Copper Chelating Sepharose FF
Chromatography Copper Chelating Sepharose FF Chromatography
.dwnarw. .dwnarw. Phenyl Sepharose HP Chromatography Phenyl
Sepharose FF Hi Sub Chromatography .dwnarw. .dwnarw.
Ultrafiltration/Diafiltration Ultrafiltration/Diafiltration DNA
removal Viral reduction through a 0.04 .mu.m filter Viral reduction
through a 0.02 .mu.m filter Formulation Formulation w/Polysorbate
80 0.2 .mu.m filter 0.2 .mu.m filter Formulated Bulk Drug Substance
is stored until QA Formulated Bulk Drug Substance is stored until
QA release. Formulated Bulk Drug Substance lots that meet release.
Formulated Bulk Drug Substance lots that meet release specifcations
may be pooled at this step release specifcations may be pooled at
this step .dwnarw. .dwnarw. Released Formulated Bulk Drug Substance
shipped to Released Formulated Bulk Drug Substance shipped to
Hollister-Stier Laboratories for filling, vialing and
Hollister-Stier Laboratories for filling, vialing and labeling
labeling.
[0135]
18TABLE 15 Summary Description of Purification Changes Production
Description Description Step (Batch) (Perfusion) PURIFICATION
PROCESS Concentration/ Tangential Flow Filtration: 2.8 m.sup.2
Changes: Diafiltration Albumin retentive MWCO filters Tangential
Flow Filtration: 5.6 m.sup.2 Albumin Operation: retentive MWCO
filters Concentration to 10X Operation: Diafiltered against 5
volumes 10 mM Concentration to 20X sodium phosphate, 100 mM sodium
No diafiltration, system rinsed with 100 mM chloride, pH 7.3 sodium
phosphate, pH 7.3 Rationale: Reduction of volume for storage at
4.degree. C. .dwnarw. DEAE Sepharose Column: Step deleted FF
Flow-Through 2 .times. 30 cm diameter .times. 33 cm height
Chromatography Pre-Wash: 0.1 N sodium hydroxide Wash:
Water-for-Injection (WFI) Neutralize: 100 mM sodium phosphate, pH
7.3 Equilibration: 10 mM sodium phosphate, 100 mM sodium chloride,
pH 7.3 Load: concentrated/diafiltered harvest Wash: 10 mM sodium
phosphate, 100 mM sodium chloride, pH 7.3 Strip: 10 mM sodium
phosphate, 1.5 M sodium chloride, pH 7.3 Regeneration: 10% glacial
acetic acid Wash: Water-for-Injection (WFI) Sanitization: 0.5 N
sodium hydroxide Storage: 0.1 N sodium hydroxide .dwnarw. pH
adjustment pH adjustment: addition of 10% glacial and filtration
acetic acid to pooled 20X concentrates to a final pH 5.0 Load:
pooled 20X concentrates Filtration through clarification filters
and 0.2 .mu.m filter Flush: 20 mM sodium acetate, 120 mM sodium
chloride, pH 5.0 Rationale: Increases rhASB binding capacity of the
subsequent Blue Sepharose resin column. .dwnarw. Blue Sepharose
Column: Change: FF 20 cm diameter .times. 11 cm height Column:
Chromatography Pre-Wash: 0.1 N sodium hydroxide 45 cm diameter
.times. 16 cm height Wash: Water-for-Injection (WFI)
Neutralization: omitted Neutralization: 1 M sodium acetate, pH
Equilibration: 10 mM sodium phosphate, 5.5 pH 6.45 Equilibration:
20 mM sodium acetate, Load: pooled 20X concentrates, pH 150 mM
sodium chloride, pH 5.5 adjusted to 5.0 and filtered. Load: DEAE
flow through adjusted to Wash: 10 mM sodium phosphate, pH 6.45 20
mM sodium acetate, 150 mM sodium Elution: 10 mM sodium phosphate,
125 mM chloride, pH 5.5 sodium chloride, pH 6.45 Wash: 20 mM sodium
acetate, 150 mM Regeneration: 10 mM sodium phosphate, sodium
chloride, pH 5.5 1.0 M sodium chloride, pH 6.45 Elution: 20 mM
sodium acetate, 500 mM Wash 2: 10 mM sodium phosphate, 1.0 M sodium
chloride, pH 5.5 sodium chloride, pH 6.45 Regeneration: 20 mM
sodium acetate, Rationale: 1.0 M sodium chloride, pH 5.5 Conditions
allow improved removal of Sanitization: 0.1 N sodium hydroxide
potential CHO impurities allowing for Wash 1: Water-for-Injection
the deletion of DEAE Sepharose Wash 2: 1 M sodium acetate, pH 5.5
Chromatography. Storage: 20% Ethanol .dwnarw. Copper Chelating
Column: Changes: Sepharose FF 14 cm diameter .times. 9 cm height
Column: Chromatography Pre-Wash: 0.1 N sodium hydroxide 40 cm
diameter .times. 16 cm height Wash: Water-for-Injection (WFI)
Equilibration: 20 mM sodium acetate, 0.5 M Charge Buffer: 0.1 M
cupric sulfate sodium chloride, 10% glycerol, pH 5.5 Equilibration:
20 mM sodium acetate, Load: Adjust glycerol content of pooled 0.5 M
sodium chloride, 10% glycerol, Blue Eluate to 10%, by adding 100 mM
pH 6.0 sodium acetate, 2.0 M sodium chloride, Load: Adjust glycerol
content of Blue 50% glycerol, pH 5.2 Eluate to 10%, by adding 20 mM
sodium Wash 1: 20 mM sodium acetate, 0.5 M acetate, 500 mM sodium
chloride, 50% sodium chloride, 10% glycerol, pH 5.5 glycerol, pH
6.0 Wash 2: 20 mM sodium acetate, 0.5 M Wash 1: 20 mM sodium
acetate, 0.5 M sodium chloride, 10% glycerol, pH 3.9 sodium
chloride, 10% glycerol, pH 6.0 Wash 3: omitted Wash 2: 20 mM sodium
acetate, 0.5 M Rationale: sodium chloride, 10% glycerol, pH 4.0
Consistent and reproducible product Wash 3: 20 mM sodium acetate,
0.5 M purity obtained with modified step sodium chloride, 10%
glycerol, pH 3.8 Elution: 20 mM sodium acetate, 0.5 M sodium
chloride, 10% glycerol, pH 3.6 Regeneration: 50 mM EDTA, 1.0 M
sodium chloride, pH 8.0 Sanitization: 0.5 M sodium hydroxide
Storage: 0.1 M sodium hydroxide .dwnarw. Viral Inactivation Pooled
Eluate fractions held for 30-120 No Change minutes prior to
adjustment to pH 4.5 with 0.5 M NaOH .dwnarw. Phenyl Sepharose
Column: Change: Chromatography 10 cm diameter .times. 16 cm height
Column: Resin: Phenyl Sepharose High Performance 40 cm diameter
.times. 16 cm height Pre-Wash: 0.1 N sodium hydroxide Resin: Phenyl
Sepharose High Sub Fast Wash: Water-for-Injection (WFI) Flow
Equilibration: 20 mM sodium acetate, Equilibration: 20 mM sodium
acetate, 2.0 M 3.0 M sodium chloride, pH 4.5 sodium chloride, pH
4.5 Load: Adjust sodium chloride content of Load: Adjust sodium
chloride content of Copper Eluate to 3 M, by adding 20 mM Copper
Eluate to 2 M, by adding 20 mM sodium acetate, 5 M sodium chloride,
pH sodium acetate, 5 M sodium chloride, pH 4.5 4.5 Wash 1: 20 mM
sodium acetate, 3.0 M Wash 1: 10 mM sodium phosphate, 2.0 M sodium
chloride, pH 4.5 sodium chloride, pH 7.1 Wash 2: 20 mM sodium
acetate, 1.6 M Wash 2: 20 mM sodium acetate, 2.0 M sodium chloride,
pH 4.5 sodium chloride, pH 4.5 Elution: 20 mM sodium acetate, 1 M
Elution: 20 mM sodium acetate, 250 mM sodium chloride, pH 4.5
sodium chloride, pH 4.5 Regeneration: 20 mM sodium acetate,
Rationale: pH 4.5 Alternate resin has more favorable flow
Sanitization: 0.5 N sodium hydroxide characteristics and capacity
for rhASB. Storage: 0.1 N sodium hydroxide Wash 1 allows more
robust clearance of potential protein impurities. .dwnarw. UF/DF
Final Tangential Flow Filtration: <0.4 m.sup.2 Change: MWCO 10
kDa Filters. Tangential Flow Filtration: 2.8 m.sup.2 MWCO
Equilibration: 10 mM sodium 10 kDa Filters. phosphate, 150 mM
sodium chloride, pH 5.8 Concentration: Concentration to NMT 1.5
mg/mL Diafiltration: 10 mM sodium phosphate, 150 mM sodium
chloride, pH 5.8 .dwnarw. DNA Removal Not Done New Step: DNA
Filtration: Product filtered through an ion exchange-based DNA
filter. Rationale: Additional DNA clearance to compensate for
deletion of DEAE Flow-Through Chromatography step. .dwnarw.
Formulation Diluted to 1.0 mg/ml with 10 mM sodium phosphate, 150
mM sodium chloride, pH 5.8 .dwnarw. Viral filtration Viral
Filtration: Product filtered through Change: a 0.04 .mu.m filter
Viral Filtration: Product filtered through a 0.02 .mu.m filter
Rationale: Use of smaller pore size to enhance viral clearance.
.dwnarw. Formulation Step occurs earlier in process Change: (Prior
to viral filtration) Diluted to 1.0 mg/ml with 10 mM sodium
phosphate, 150 mM sodium chloride, pH 5.8 Formulation: Polysorbate
80 is added to a concentration of 50 .mu.g/mL. .dwnarw. Filtration
Filtration: Filter the diluted product No Change through a 0.2
.mu.m filter into storage container
[0136]
19TABLE 16 rhASB Purification Method (Perfusion Process) Step
Process Harvest Filtration Filtration through Clarification
filters, 0.45 .mu.m filters and finally 0.2 .mu.m filter. Filtered
polled harvests are stored in polypropylene bags UF Concentration
Equilibration and flush: 100 mM sodium phosphate, pH 7.3 Load:
filtered harvest fluid Concentration: Concentration to 20X
Filtration: Filter the diluted product through a 0.2 .mu.m filter
into storage container pH adjustment and pH adjustment: Add 10%
glacial acetic acid to pooled 20X concentrates to a final
filtration pH of 5.0 Load: pooled 20X concentrates Rinse: Water-for
Injection (WFI) Filtration through Clarification filters and 0.2
.mu.m filter. Flush: 20 mM sodium acetate, 120 mM sodium chloride,
pH 5.0 Blue Sepharose 6 Pre-Wash: 0.1N sodium hydroxide FF Wash:
Water-for-Injection (WFI) (Blue, Blue Equilibration: 10 mM sodium
phosphate, pH 6.45 Sepharose) Load: pH adjusted and filtered pooled
20X concentrates Wash: 10 mM sodium phosphate, pH 6.45 Elution: 10
mM sodium phosphate, 125 mM sodium chloride, pH 6.45 Regeneration:
10 mM sodium phosphate, 1.0 M sodium chloride, pH 6.45
Sanitization: 0.1 N sodium hydroxide Wash 1: Water-for-Injection
(WFI) Wash 2: 10 mM sodium phosphate, 1.0 M sodium chloride, pH
6.45 Storage: 20% Ethanol Chelating Pre-Wash: 0.1N sodium hydroxide
Sepharose FF Wash: Water-for-Injection (WFI) (Copper, CC, Charge
Buffer: 0.1M cupric sulfate Copper-Chelating) Equilibration: 20 mM
sodium acetate, 0.5 M sodium chloride, 10% glycerol, pH 5.5 Load:
Adjust glycerol content of pooled Blue Eluates to 10%, by adding
100 mM sodium acetate, 2.0M sodium chloride, 50% glycerol, pH 5.2
Wash 1: 20 mM sodium acetate, 0.5 M sodium chloride, 10% glycerol,
pH 5.5 Wash 2: 20 mM sodium acetate, 0.5 M sodium chloride, 10%
glycerol, pH 3.9 Elution: 20 mM sodium acetate, 0.5 M sodium
chloride, 10% glycerol, pH 3.6 Eluate hold for 30-120 minutes prior
to adjustment to pH 4.5 with 0.5 M NaOH Regeneration: 50 mM EDTA,
1.0M sodium chloride, pH 8.0 Sanitization: 0.5M sodium hydroxide
Storage: 0.1M sodium hydroxide Phenyl Sepharose Pre-Wash: 0.1 N
sodium hydroxide 6 FF High Sub Wash: Water-for-Injection (WFI)
(Phenyl, Phenyl Equilibration: 20 mM sodium acetate, 2.0 M sodium
chloride, pH 4.5 High Sub) Load: Adjust sodium chloride content of
Copper Eluate to 2 M, by adding 20 mM sodium acetate, 5 M sodium
chloride, pH 4.5 Wash 1: 10 mM sodium phosphate, 2.0 M sodium
chloride, pH 7.1 Wash 2: 20 mM sodium acetate, 2.0M sodium
chloride, pH 4.5 Elution: 20 mM sodium acetate, 250 mM sodium
chloride, pH 4.5 Regeneration: 20 mM sodium acetate, pH 4.5
Sanitization: 0.5 N sodium hydroxide Storage: 0.1 N sodium
hydroxide UF/DF, DNA Equilibration: 10 mM sodium phosphate, 150 mM
sodium chloride, pH 5.8 Filtration, Viral Concentration:
Concentration to NMT 1.5 mg/mL Filtration, Diafiltration: 10 mM
sodium phosphate, 150 mM sodium chloride, pH 5.8 Formulation DNA
Filtration: Product filtered through a DNA filter Viral
Filtration/Dilution: Product filtered through a 0.02 .mu.m filter
and diluted to 1.0 mg/ml Formulation: Polysorbate 80 at a
concentration of 50 .mu.g/mL is added Filtration: Filter the
diluted product through a 0.2 .mu.m filter into storage
container
[0137] All purification columns are regenerated prior to use,
sanitized after use and stored in the appropriate buffers as
indicated in Tables 15 and 16.
[0138] Purification Raw Materials
[0139] All materials are supplied by qualified vendors.
20TABLE 17 Raw Materials for Purification Ingredient Grade Glacial
Acetic Acid USP Cupric Sulfate Pentahydrate USP Edetate Disodium
USP Dehydrated Alcohol, USF (Ethanol, 200 Proof) USP Glycerine USP
Sodium Acetate, Trihydrate USP Sodium Chloride USP Sodium Hydroxide
50% w/w Solution Reagent Grade Sodium Phosphate, Dibasic,
Heptahydrate USP/EP Sodium Phosphate, Monobasic, Monohydrate USP
Hydrochloric Acid, 6 N Volumetric Solution Reagent Grade
Polysorbate 80, MF/EP (CRILLE 4 HP) NF/EP Water-for-Injection,
Packaged in Bulk USP
[0140] The glycerine to be utilized is to be derived from a
synthetic process. All raw materials used are to be in compliance
with the latest version of the CPMP/CVMP Note for Guidance
entitled, "Minimising the Risk of Transmitting Animal Spongiform
Encephalopathy Agents Via Human and Veterinary Medicinal Products,"
in which tallow derivatives such as glycerol and fatty acids
manufactured by rigorous processes involving high temperatures and
pressure conditions or chemical reactions known to be terminally
hostile to the bovine spongiform encephalopathic (BSE) agent are
thought unlikely to be infectious. Thus, the risk of BSE
transmission from the glycerine is considered to be low.
[0141] The viral safety of rhASB is confirmed by a combination of
selection and qualification of vendors, raw material testing, cell
bank characterization studies, viral removal studies and
inactivation capacity of the rhASB purification process, and
routine lot release testing. Relevant US, EU, and ICH regulations
and guidelines have been referenced to ensure the viral safety of
rhASB.
[0142] Column Chromatography, DNA Removal and Viral Filtration
[0143] RhASB is now purified using a series of chromatography and
filtration steps. The harvest fluid is concentrated to 20.times. by
ultrafiltration, pH adjusted, filtered and loaded onto a Blue
Sepharose Fast Flow chromatography column (45 cm.times.16 cm). The
Blue Sepharose Fast Flow eluate is filtered prior to loading on to
a Copper Chelating Sepharose column. The Copper eluate is filtered
prior to loading on to a Phenyl Sepharose Fast Flow High Sub
column. All three column chromatography purification steps are run
in a bind and elute mode. The Phenyl Sepharose column eluate is
passed through an anion exchange filter and viral reduction filter
prior to concentration and buffer exchange by ultrafiltration.
[0144] Viral Removal/Inactivation Studies
[0145] Two studies were conducted to assess the viral reduction
capacity of the modified rhASB purification process. Studies were
performed at BioReliance (Rockville, Md.) using two model virus
systems, Xenotropic murine leukemia virus (XMuLV) and Murine Minute
Virus (MMV). XMuLV is an enveloped single-stranded RNA retrovirus
with low resistance to physico-chemical inactivation. MMV is a
small, non-enveloped single-stranded DNA virus with high resistance
to physico-chemical agents.
[0146] These studies evaluated two chromatographic steps (Copper
Chelating Sepharose FF and Phenyl Sepharose FF High Sub) and the
viral filter (0.02 .mu.m) used in the rhASB purification process.
Spike and recovery studies were performed using scaled down
versions of the process steps. The critical parameters maintained
were retention times and matrix-solution interactions. This was
achieved by replicating the buffers, linear flow rates and column
heights but adjusting for column diameter. Materials used in the
study (product and buffers) were collected from actual full scale
production.
[0147] Chromatography columns (Copper Chelating Sepharose and
Phenyl Sepharose) were packed and pre-run with either typical rhASB
loads (blank) or loads spiked with viral buffer prior to shipping
to BioReliance. Chromatograms and product yields in the presence of
viral buffers were comparable to blank runs. Identical column loads
were spiked with either XMuLV or MMV immediately prior to
chromatography. The amount of viral reduction for each of the
evaluated steps was determined by comparing the viral burden in the
column loads and eluates. A summary of the results for this study
are shown in Table 18.
21TABLE 18 Reduction Factors for XmuLV and MMV MMV Log Process Step
XMuLV Log Reduction Reduction Blue Sepharose not tested not tested
Copper Chelating .gtoreq.3.51 .+-. 0.52 .gtoreq.2.71 .+-. 0.52
(+low pH hold) Phenyl Sepharose .gtoreq.3.54 .+-. 0.36 .gtoreq.1.72
.+-. 0.64 DNA Filtration not tested not tested Viral Filtration
.gtoreq.5.51 .+-. 0.43 .gtoreq.4.76 .+-. 0.00 Total log Reduction
.gtoreq.12.56 .gtoreq.9.19
[0148] In-Process Testing
[0149] In-process testing is performed throughout the process and
is illustrated in FIG. 3. In-process testing of the harvest cell
culture fluid and the purification intermediates is described in
Tables 19 and 20.
22TABLE 19 In-Process Testing of the Harvested Cell Culture Fluid
Test Action Levels Bacterial Endotoxin by LAL .gtoreq.2 EU/mL
(USP/EP) Bioburden .gtoreq.1 cfu/10 mL (USP/EP) Total Protein by
Bradford Results used for the calculation of the and Activity Blue
Sepharose column load Mycoplasma.sup.1 Negative (Release
Specification) In vitro Assay For The Negative Presence of Viral
(Release Specification) Contaminants.sup.1 .sup.1Sampling is
performed at multiple time points during the cell culture harvest
stage of manufacturing. The last sample removed prior to the
termination of the cell culture process is tested and a negative
result is required for lot release.
[0150]
23TABLE 20 In-Process Testing of Purification Intermediates Test
Action Levels Bacterial Endotoxin by .gtoreq.3 EU/mL in column
eluates.sup.1 LAL (USP/EP) Bioburden .gtoreq.20 cfu/mL
pre-filtration.sup.1 (USP/EP) .gtoreq.1 cfu/100 mL in Formulated
Bulk Drug Substance (FBDS).sup.2 Activity Result used for the
calculation of the Copper Chelating and Phenyl Sepharose column
loads Total Protein by Result used for the calculation of the
protein UV-Vis concentration for the UF/DF Spectrophotometry
.sup.1Results exceeding action levels are investigated per standard
operating procedures. .sup.2If results exceed action level, the
FBDS will be 0.2 .mu.m filtered into appropriate sterile storage
containers and then quarantined pending release for shipment to
filling sites.
[0151] Results of Perfusion Method of Purifying Precursor rhASB
[0152] Table 21 provides data on the degree of purity of rhASB
obtained using the purification methods described in Tables 15 and
16. The "Purity by RP-HPTC" column indicates the degree of purity
obtained for the combined amount of both the precursor and mature
forms of rhASB.
24TABLE 21 rhASB Lot Release Results Purity by RP- Presence of the
Manufacturing Lot HPLC processed forms* process AP60028 99.6 -
Batch process AP60029 98.8 + Batch process AP60030 98.7 + Batch
process AP60031 99.1 - Batch process AP60032 99.8 - Batch process
AP60033 99.4 - Batch process AP60035 98.7 - Batch process AP60036
99.4 - Batch process AP60038 99.6 - Batch process AP60039 99.3 -
Batch process AP60040 99.1 - Batch process AP60101 99.3 - Batch
process AP60102 98.9 - Batch process AP60103 99.0 - Batch process
AP60104 99.0 + Batch process AP60105 99.0 - Batch process AP60106
99.2 - Batch process ALP60107 99.4 - Batch process AP60108 99.7 -
Perfusion process AP60109 99.8 - Perfusion process AP60201 99.0 N/A
Perfusion process AP60202 100 - Perfusion process *"+" indicates
the presence of the processed forms (estimated at 1-15%) in the
formulated bulk drug substance; "-" indicates the absence of the
processed forms.
[0153] FIGS. 4A-4C provide the representative chromatograms of the
three chromatographic steps of the perfusion method of purification
(Table 16). Table 22 provides the average recoveries of precursor
rhASB for each of these three chromatographic steps. FIG. 5
provides the data for the purity analysis of in-process samples at
each chromatographic step and of the purified final product
(precursor rhASB).
25TABLE 22 Summary of Purification Recoveries. Step Average
Recovery (%) pH Adjustment to 5.0 83 Blue Sepharose Column 84
Copper Chelating Sepharose Column 86 Copper to Phenyl Transition 86
Phenyl Sepharose Column 88 Overall 50
[0154] When purified products obtained using the batch process and
the perfusion process are compared, the perfusion process clearly
produces a purer precursor rhASB. Even attempts to further optimize
the batch processes by selecting different wash fractions or
varying the conditions did not result in the consistently pure
product obtainable by the perfusion process. The perfusion process
is able to produce consistent precursor rhASB purity of 99% or
more, while the batch process does not (Table 21). FIG. 6 provides
a comparison between the products obtained using the old batch
method wherein the cell culture is cultured using a medium that is
supplemented with G418 and not supplemented with folic acid, serine
and asparagine (lane 2), and the purified products purified using
the perfusion process (lanes 3-9)). The samples of rhASB were
denatured in SDS with a reducing agent and subjected to
electrophoresis through 4-20% PAGE in SDS running buffer. The batch
process purified products clearly contain far more impurities
(especially near the 48 kDa size) than the perfusion process
purified products. The batch process rhASB appear to detectable
amounts of processed forms (estimated at 1-15%) (lane 2). The
perfusion process purified lots are highly comparable to each other
and are significantly less complex than the batch process purified
rhASB standard, indicating a higher degree of purity for the
perfusion process material.
[0155] Table 23 provides percent purity of precursor rhASB purified
using the perfusion process purification method.
26TABLE 23 Purity of Final Product. Purity by RP-HPLC Lot (% main
peak) AC60109 UF #4 99.8 AC60109 UF #10 99.6 AC60109 UF #1 99.7
AG60109 UF #18 99.9 AC60109A UF #22 99.7 AG60109A UF #25 99.8
AC60109A UF #27 100 AG60109 99.8 BMK Manufacturing AC60202 UF #4 ND
AC60202 UF #10 ND AC60202 UF #18 ND AC60202 100 BMX Manufacturing
Average 99.8 ND = not determined.
[0156] Table 24 provides percent purity of lots of precursor rhASB
purified using the perfusion process purification method, where
purity has been evaluated by RP-HPLC and SEC-HPLC (which allows
quantification of impurities of different molecular weights such as
processed forms or multimers).
27TABLE 24 Purity by RP-HPLC and SEC-HPLC Purity by RP-HPLC Purity
by SEC-HPLC Lot (% Main Peak) (% Main Peak) AP60109 99.8 99.8
AP60202 100 >99 AP60204 99.7 99.8 AP60206 100 99.8 AP60301 100
99.6 AP60302 100 99.6 AP60303 100 99.5 AP60304 100 ND* Average 99.9
99.6 *Not Determined
[0157] Protease Removal in the Perfusion Process
[0158] At the 180 L batch scale two relatively large 24 L DEAE
Sepharose runs are required per lot. The perfusion process
generates 10-fold larger harvest volumes that impose severe
impractical DEAE Sepharose column sizes and buffer volumes for
practical large scale purification. Therefore, elimination of the
particularly untenable DEAE Sepharose step was evaluated in an
effort to streamline the new perfusion purification train. As a
consequence, the three column train (as described in Table 16)
overcomes this large scale production obstacle.
[0159] The DEAE Sepharose chromatography step in the batch process
(described in Table 15) was employed primarily to remove total
protein (including proteases) and the pH indicator dye, Phenol Red.
The latter issue is addressed in the new process by removal of
Phenol Red from the 302 media formulation. The removal of total
protein by DEAE sepharose step resulted in higher Blue sepharose
capacity at pH 5.5. In the new process, Blue Sepharose conditions
needed to be identified that balanced high rhASB capacity while
limiting protease activity. Both capacity and proteolysis are
enhanced at low pH. In addition, clearance of proteases should be
demonstrable to improve the robustness of the subsequent Copper
Chromatography step, where clearance of the protease, cathepsin,
has been found problematic in the Batch process. Conditions were
found which yielded the desired results, whereby, loading was
performed at low pH, while wash and elution conditions were at
higher pH, conditions that efficiently clear the protease,
cathepsin, with an acceptable load of 0.8 mg rhASB/ml resin.
Removal of the protease activity (e.g. cathepsin) is demonstrated
in the chromatograms of FIG. 8.
[0160] Comparison of rhASB purified from perfusion run, 102PD0055,
using either the new process (without DEAE FT chromatography) or
old Batch purification process (PS Eluate (OP) indicate that the
old process material purified (using the batch method) from the
same reactor run, lane PS Eluate (OP), has significant more
proteolysis, as demonstrated by the additional bands on
silver-stained gel and detectible by anti-ASB antibodies (see FIG.
9). The new process is much purer relative to the batch standard.
The cathepsin protease is cleared by Blue chromatography.
Anti-cathepsin cross-reactive material is seen in the Blue Strip
fraction using the new process.
[0161] The pharmacokinetic results described below in Example 11
show that the purer rhASB preparation results in a longer half-life
than the previous batch standard.
EXAMPLE 10
Surfactant Effects on Particle Formation
[0162] An experimental protocol was carried out to determine the
effect of Polysorbate-80 on particle generation in vials of rhASB
made with the new perfusion cell culture process. Visual and
particle count assays were employed to determine the extent of
particle generation. In addition, activity testing, polyacrylamide
gel electrophoresis (PAGE), immunoblotting and size exclusion
chromatography (SEC) were performed to confirm product quality in
the presence of Polysorbate-80 and/or shaking relative to untreated
controls. Vials and stoppers used for the rhASB batch process (Old,
Wheaton 5 ml, 2905-B24B/ West S-127 (4401/45)) were also compared
to the proposed vials and stoppers to be used for the
perfusion-derived material (New, Wheaton 5 ml, 2905-B8BA/ West
S-127 (4432/50)).
[0163] Vials were filled after rhASB had been passed through a 0.2
.mu.m filter using a 10 ml disposable pipette. Two vials were
filled for each rhASB-containing test. Both were used for the
visual assay with one dedicated for particle counting and the other
saved for the remainder of assays. Only one buffer control
vial/test was filled. Few particles were introduced due to set-up,
sample handling, filtration or filling. From visual and particle
count analysis (results shown in Tables 25A-C and 26A-C below), on
days 0, 1 and 14 days, no significant particulate formation was
detected in unshaken vials of formulated drug relative to
buffer-only controls.
[0164] After 18 hr of shaking at 4 degrees C., a large increase in
particles was observed in both vial configurations with rhASB in
formulation buffer alone (10 mM sodium phosphate, 150 mM sodium
chloride, pH 5.8), without polysorbate. Fine flakes were clearly
visible.
[0165] The particle counting was done using the HIAC/ROYCO Model
9703 Particle Counter. The liquid particle counter is capable of
sizing and counting particles ranging from 2.0 .mu.m to 150 .mu.m.
Three aliquots of not less than 5 mL each are put into the light
obscuration sensor. The average particle counts from runs 2 and 3
are reported as particle counts per mL. Based on HIAC-Royko
quantification of particles 10 .mu.m in size, 2069 and 1190
particles/mL were counted in old and new vials configurations,
respectively. In the presence of 0.001% Polysorbate-80, a markedly
lower count was found with 21 and 49 particles/ml. At 0.005%
Polysorbate-80, essentially no difference between shaken and
unshaken vials could be detected.
[0166] In old vials/seals without Polysorbate-80, there was an
unexpected drop in particle counts/ml from 2069 to 268 from days 1
to 14 days after shaking. The visual assay indicated larger flakes
in the same 14 day vials in which lower counts were found; it is
possible that the larger particles interfered with the HIAC-Royko
assay, or that the old vial configuration contributes to increased
particle formation with eventual coalescence and aggregation into
larger, albeit fewer, particles.
[0167] Additional testing was done to confirm the integrity of the
protein after shaking. Specific activity was unchanged. Size
Exclusion Chromatography, a method that can detect changes in
protein aggregation, which might be influenced by mechanical
agitation, showed no increase in larger molecular weight species.
In order to detect breakdown products, SDS-PAGE gels of rhASB were
either silver stained or transferred onto nitrocellulose and
blotted with Anti-rhASB antibody. No additional bands were detected
in any of the samples.
28TABLE 25A Visual Assay. Day 0 102PD0089-01 Buffer Vial
[Polysorbate-80] % No Shake No Shake Old 0 (-) (-) (-) Old 0.005
(-) (-) (-) Old 0.001 (-) (-) (-) New 0 (-) (-) (-) New 0.005 (-)
(-) (-) New 0.001 (-) (-) (-)
[0168]
29TABLE 25B Visual Assay. Day 1 102PD0089-08 Buffer No No Vial
[Polysorbate-80] % Shake Shake Shake Shake Old 0 (++) (++) (-) (-)
(-) (-) Old 0.005 (-) (-) (-) (-) (-) (-) Old 0.001 (+/-) (-) (-)
(-) (-) (-) New 0 (++) (++) (+/-) (-) (-) (-) New 0.005 (-) (-) (-)
(-) (-) (-) New 0.001 (+/-) (-) (+/-) (-) (-) (-)
[0169]
30TABLE 25C Visual Assay. Day 14 102PD0089-01 Buffer No No Vial
[Polysorbate-80] % Shake Shake Shake Shake Old 0 (+++) (+++) (-)
(-) (-) (-) Old 0.005 (-) (-) (-) (-) (-) (-) Old 0.001 (+/-) (+)
(-) (-) (-) (-) New 0 (++) (++) (+/-) (-) (-) (-) New 0.005 (-) (-)
(-) (-) (-) (-) New 0.001 (+/-) (-) (+/-) (-) (-) (-)
[0170]
31 Visual Assay: No visible precipitation - Borderline, uncertain
about observation -/+ Small amount of fine precipitate +
Significant precipitation, cloudy ++ Very cloudy, large flakes of
precipitate +++
[0171]
32TABLE 26A Particulate Analysis, HIAC-Royko (Particles/mL). Day 0
102PD0089-01 Buffer No Shake No Shake Vial [Polysorbate-80] % 10
.mu.m 25 .mu.m 10 .mu.m 25 .mu.m Old 0 4.5 0.5 2.5 0.0 Old 0.005
2.0 0.5 1.5 0.0 Old 0.001 4.5 1.5 2.5 1.0 New 0 6.5 2.5 6.0 4.0 New
0.005 1.5 0.5 3.0 1.0 New 0.001 19.5 5.0 2.0 0.0
[0172]
33TABLE 26B Particulate Analysis, HIAC-Royko (Particles/mL). Day 1
102PD0089-01 Buffer Shake No Shake Shake No Shake Vial
[Polysorbate-80] % 10 .mu.m 25 .mu.m 10 .mu.m 25 .mu.m 10 .mu.m 25
.mu.m 10 .mu.m 25 .mu.m Old 0 2069.0 74.5 3.0 0.5 0.5 0.5 2.5 2.0
Old 0.005 3.5 0.0 2.0 0.5 3.5 2.5 1.0 0.5 Old 0.001 21.5 2.5 2.0
1.0 2.5 1.0 1.5 1.5 New 0 1190.0 34.5 5.0 0.5 0.0 0.5 2.5 3.5 New
0.005 19.0 0.0 4.5 0.0 2.5 0.0 17.0 0.0 New 0.001 49.0 0.5 9.0 2.5
9.5 0.5 6.0 1.0
[0173]
34TABLE 26C Particulate Analysis, HIAC-Royko (Particles/mL). Day 14
102PD0089-01 Buffer Shake No Shake Shake No Shake Vial
[Polysorbate-80] % 10 .mu.m 25 .mu.m 10 .mu.m 25 .mu.m 10 .mu.m 25
.mu.m 10 .mu.m 25 .mu.m Old 0 268.0 18.0 0.5 0.0 4.5 2.0 0.0 0.5
Old 0.005 2.0 2.5 1.0 1.0 2.5 0.5 6.0 3.0 Old 0.001 72.0 6.5 7.0
3.0 7.0 1.0 1.0 2.0 New 0 1335.0 29.0 2.0 0.0 1.5 0.0 1.5 0.5 New
0.005 7.0 0.5 16.0 0.0 13.5 0.0 13.0 2.0 New 0.001 38.0 1.5 5.5 0.5
16.0 0.5 8.0 0.0
[0174] The results indicated that the manual fill procedure
generates very few particles regardless of formulation. After
constant and vigorous shaking at 180 rpm, particles were clearly
visible in vials containing rhASB formulated without
Polysorbate-80.
[0175] The presence of Polysorbate-80 was highly effective in
inhibiting mechanically-induced particle formation. Protection was
dramatic, with particle counts kept down to essentially background
levels in shaken vials at 0.001% and 0.005% Polysorbate-80. The
0.005% Polysorbate-80 concentration shows slight improvement in
reducing particle counts over the 0.001% Polysorbate-80
concentration.
[0176] Shaking or formulation was not observed to cause any product
instability as evaluated by activity, protein concentration, purity
or percent aggregation.
EXAMPLE 11
Clinical Study in Humans
[0177] A Phase 1/2, randomized, double-blinded clinical trial of
recombinant human N-acetylgalactosamine-4-sulfatase (rhASB) in
patients with mucopolysaccharidosis VI (MPS VI), Maroteaux-Lamy
Syndrome, was conducted using drug product produced according to
the batch process described herein. The rhASB was formulated at a
concentration of 1 mg/mL, pH 5.8, 9 mM monobasic sodium phosphate.1
H.sub.20, 1 mM dibasic sodium phosphate.7 H.sub.20, 150 mM sodium
chloride. When administered to patients, the rhASB was prepared by
diluting the appropriate amount of drug solution in an intravenous
(IV) bag with normal saline at room temperature. All patients were
premedicated with an antihistamine prior to infusion to reduce the
potential for infusion-associated reactions.
[0178] The objectives of this study were to evaluate the safety,
efficacy and pharmacokinetic profile of two doses of rhASB as. ERT
in patients with MPS VI. Patients were randomized in a double-blind
fashion to one of two dose groups, 0.2 and 1.0 mg/kg. Drug was
given once per week as a four-hour IV infusion. Measures of safety
included complete chemistry panel, urinalysis, complete blood count
(CBC) with differential, and tracking of AEs. Immune response and
infusion-associated reactions, complement activation and antibody
formation are assessed for all patients. Efficacy parameters
included exercise tolerance/endurance (6-minute walk test),
respiratory capacity (pulmonary function tests), joint range of
motion (JROM), functional status (Childhood Health Assessment
Questionaire [CHAQ]), levels of urinary GAGs, and changes in
hepatomegaly, visual acuity, cardiac function, and sleep apnea.
[0179] Pharmacokinetic evaluations were conducted during infusions
at Weeks 1, 2, 12, 24 and periodically thereafter to measure
antigen (enzyme) levels during and after infusion. Pharmacokinetic
evaluations were also conducted at Weeks 83, 84 and 96 of the
open-label extension of the study, when patients were switched
commencing at Week 84 to the new perfusion-process purified rhASB
in a formulation with 0.005% polysorbate80.
[0180] A total of seven patients was enrolled, and included
patients with a range of characteristics consistent with rapidly
advancing or moderately advancing disease. One patient dropped out
of the study at Week 3 for personal reasons and was replaced. A
pre-specified interim analysis was conducted following the
completion of 24 weeks of treatment for the remaining six patients.
Data from the Week 24 interim analysis showed that rhASB was well
tolerated and the 1.0 mg/kg dose appeared to produce greater
clinical benefit and to have a safety profile comparable to that of
the 0.2 mg/kg dose.
[0181] Patients in the low-dose group were offered continued
treatment at the 1.0 mg/kg dose level. Two patients initiated
therapy at the higher dose level at intervals after Week 48 (Weeks
59 and 69). Patients treated through Week 96 have received the
majority of planned rhASB infusions. No patient has missed more
than two infusions total during this period.
[0182] Safety assessments performed during the initial 24, then 48
weeks of treatment showed that weekly treatment with rhASB at 0.2
or 1.0 mg/kg was well tolerated without significant differences
between the two dose groups. Longer term treatment (through Week
96) at 1.0 mg/kg weekly also was well tolerated.
[0183] Anti-rhASB Antibody Development and Complement Levels
[0184] All six patients on study drug at Week 30 had developed
antibody to rhASB. Levels for four of the six patients remained
relatively low and declined from peak levels by Week 60. Of the two
patients that developed higher levels of antibody than the other
four patients, one patient's antibody levels declined from the peak
level at Week 24, and the other patient's levels fell rapidly from
the peak level at Week 60. No consistent changes in CH50, C3, or C4
complement levels have been observed during 96 weeks of treatment.
Several patients have had modest intermittent declines in CH50
levels; however, no evidence of complement consumption or
correlation between these low levels and infusion-associated
symptoms were noted. FIG. 10 shows antibody levels over 96 weeks of
treatment.
[0185] Urinary Glycosaminoglycans
[0186] The level of urinary GAGs is a biochemical marker of the
degree of lysosomal storage in MPS-affected individuals. By Week
24, urinary GAGs were reduced by an average of 70% in the high-dose
group versus 55% in the low-dose group. The percentage of DS, the
primary storage product of MPS VI, was similarly decreased by 44%
and 18%, respectively. Examination of the time course of the change
in urinary excretion of GAGs as a function of weeks on treatment
showed a more pronounced drop in total urinary GAGs by Week 6 in
the high-dose group. These data confirm that the higher dose
produced a larger change in both urinary GAGs and DS. Urinary GAGs
have continued to decline through Week 96 for the patients in both
treatment groups who remained in the study. FIG. 11 shows the
reduction in total urinary GAG levels over the 96 weeks and FIG. 12
shows a comparison of levels at week 96 to age-appropriate normal
levels. Note that the two patients in the 0.2 mg/kg dose group who
remained in the study, Patients 41 and 45, rolled over to the 1.0
mg/kg dose on Weeks 69 and 59, respectively. At Week 96, these two
patients had reductions in GAGs from screening of 85% and 63%.
These were similar to reductions seen in the patients on 1.0 mg/kg
through the entire 96 weeks. Patients 42, 43, and 44 had reductions
of 64%, 77%, and 86% from screening, respectively. In both groups,
the patients who had higher baseline GAG levels had higher percent
reductions than patients with GAG levels that were closer to the
normal range (<5 times upper limit of normal, i.e., mean plus 2
SD).
[0187] Endurance
[0188] The 6-minute walk test served as the primary clinical
measure of endurance. The two patients unable to walk >100 m at
baseline, who were randomized to receive 1.0 mg/kg of study drug
(#43 and #44), had large improvements in the total distance walked
by Week 24. All patients except #44, who developed-C1-C2 cord
compression, showed improvement in their distance walked by Weeks
48 and 96. FIG. 13 shows the improvement in results of the 6-minute
walk test over 96 weeks of treatment.
[0189] Additional Measures of Efficacy
[0190] Modest improvements in a number of other efficacy parameters
were seen during the 96 weeks of study drug treatment. Shoulder
ROM, particularly for flexion, improved in five of the six study
patients at the Week 24 assessment. Three of the four patients
(#41, 43 and 45) with assessments at Week 96 continued to show
increases in shoulder flexion. Slight improvements were also seen
in the measures of respiratory function, forced vital capacity
(FVC) and forced expiratory volume for one second (FEV 1), in three
of four patients, at Week 96. Visual acuity, measurable in four out
of six patients enrolled, improved by one or more lines in three of
these four patients, although one patient had changes in his
prescription lenses during this period. Hepatomegaly measured by CT
scan, although not a prominent feature of this disease, also
declined modestly, particularly in those patients with larger
livers at baseline.
[0191] Little change was seen in the remaining efficacy parameters,
including cardiac function by ECG, grip and pinch strength, CHAQ
questionnaire, height and weight, bone mineral density, and sleep
studies. No significant deterioration was observed in any of these
parameters over the 96 weeks.
[0192] Pharmacokinetics
[0193] Pharmacokinetic evaluations were conducted during Weeks 1,
2, 12 and 24 of the double-blind study and during Weeks 83, 84 and
96 of the open-label extension. Beginning with Week 84, patients
began to receive rhASB manufactured by the perfusion process
described in Example 9.
[0194] Blood samples were collected during the infusion at 0, 15,
30, 60, 90 and 180 minutes, at the end of infusion at 240 minutes,
and post-infusion at 5, 10, 15, 30, 45, 60, 90, 120 and 240
minutes. Pharmacokinetic parameters for rhASB were calculated using
non-compartmental methods. Areas under the plasma
concentration-time curve to infinity and first moment were
calculated using the linear trapezoidal method to the last time
point with a concentration above the qualified limit of
quantitation.
[0195] For patients administered 0.2 mg/kg/week, the mean values
for AUC.sub.0-1 were relatively consistent from Week 1 through Week
24 (10,009.+-.5,107 at Week 1, 11,232.+-.3,914 at Week 2,
13,812.+-.9,230 at Week 12, and 13,812.+-.9,230 at Week 24). For
the 1.0 mg/kg/week cohort, the mean values for AUC were
94,476.+-.13,785 at Week 1, 180,909.+-.46,377 at Week 2,
157,890.+-.45,386 at Week 12 and 251,907.+-.201,747 at Week 24. For
this 1.0 mg/kg/week group the increase in values for AUC.sub.0-4
and AUC.sub..infin. and large standard deviation was due to a
single patient whose AUCs were about 2-fold higher than the other
two patients in the group.
[0196] Comparison of the mean values for AUC.sub.0-1 between the
0.2 and 1.0 mg/kg/week cohorts showed an increase far in excess of
the 5-fold increase in dose, indicating that the pharmacokinetics
of rhASB are not linear over this dose range.
[0197] Pharmacokinetic parameters at Weeks 83, 84 and 96 are shown
in Table 27 below.
35TABLE 27 Parameter Week 83 Week 84 Week 96 Cmax (ng/mL) 1,143
.+-. 284 1,367 .+-. 262 1,341 .+-. 523 Tmax (min) 180 120 121
AUC.sub.0-t (min .multidot. ng/mL) 172,423 .+-. 49,495 213,713 .+-.
45,794 200,116 .+-. 76,506 AUC.sub..infin. (min .multidot. ng/mL)
173,570 .+-. 49,969 215,383 .+-. 47,018 201,157 .+-. 77,248 CL
(mL/min/kg) 6.23 .+-. 2.10 4.81 .+-. 0.99 5.54 .+-. 2.09 Vz (mL/kg)
67.6 .+-. 22.0 122 .+-. 60.2 123 .+-. 17.4 Vss (mL/kg) 266 .+-.
52.3 236 .+-. 21.1 233 .+-. 27.6 t1/2 (min), half-life 8.49 .+-.
4.68 19.0 .+-. 13.2 17.3 .+-. 8.26 MRT (min) 44.4 .+-. 7.61 50.9
.+-. 12.9 46.1 .+-. 16.1 Arithmetic mean and standard deviation are
reported except for Tmax which is the median. Individual patient
values were reported if n < 3.
[0198] Parameters for Week 83 were comparable to those obtained
from Weeks 2 through 24 (excluding one patient with high AUCs),
indicating consistency in rhASB pharmacokinetics after weekly
exposure for approximately 18 months. From Week 83 to Week 84, mean
AUC values trended upward by approximately 25% while total body
clearance trended downward by approximately 25%. From Week 83 to
Week 84, the half-life increased from about 8.5 minutes to 17-19
minutes. Parameters from Week 96 were comparable to those from Week
84. Although this pharmacokinetic data was from a small number of
patients and there was substantial overlap of individual values,
these results indicated that the high purity rh ASB preparation
obtained from the modified process described in Example 9 had a
longer half-life than the original rhASB preparation.
EXAMPLE 12
Further Clinical Study in Humans
[0199] A Phase 2 open-label clinical trial of recombinant human
N-acetylgalactosamine-4-sulfatase (rhASB) in patients with
mucopolysaccharidosis VI (MPS VI), Maroteaux-Lamy Syndrome, was
conducted using drug product produced according to the new
perfusion process described in Example 9. The rhASB was formulated
at a concentration of 1 mg/mL, pH 5.8, 9mM sodium phosphate,
monobasic, .1 H.sub.20, 1 mM sodium phosphate, dibasic, .7 H20, 150
mM sodium chloride, 0.005% polysorbate 80.
[0200] The objectives of this study were to evaluate the safety,
efficacy and pharmacokinetics of 1.0 mg/kg rhASB given as a weekly
IV infusion. The inclusion criteria were modified for this study to
include a relatively uniform set of patients with impaired
endurance. Patients had to be able to walk at least 1 m but no more
than 250 m in the first six minutes of a 12-minute walk test at
baseline. The other inclusion and exclusion criteria were similar
to those of the Phase 1/2 Study in that patients had to be at least
five years old and have a documented biochemical or genetic
diagnosis. In addition to the walk test, a number of endpoints not
previously studied in MPS patients were included in this study.
[0201] Measures of safety included clinical laboratory evaluations,
urinalysis, CBC with differential, ECG and tracking of AEs.
Infusion-associated reactions as well as complement consumption and
antibody formation are being assessed for all patients. Efficacy
parameters included measures of endurance and mobility, including a
12-minute walk test, the Expanded Timed Get Up and Go test, the
3-minute stair climb test and physical activity. Subjective
effects, such as joint pain and joint stiffness, were assessed with
a questionnaire, and other measures in the Denver Developmental
scale such as time to tie shoes, touch top of head, pull over
sweater, and pick up coins and put them in a cup were also
assessed. Additional efficacy measures included measures of visual
acuity, bone density studies, assessment of grip and pinch
strength, shoulder ROM, functional status, pulmonary function,
urinary GAG excretion, visual exams, cardiac function, and oxygen
saturation during sleep. Pharmacokinetic evaluations were conducted
during infusions at Weeks 1, 2, 12 and 24 to measure antigen
(enzyme) levels during and after infusion. Ten patients were
enrolled--five in the U.S. and five in Australia. All patients
completed Week 24 of treatment. There were no deaths or
discontinuations. All patients received all 24 study drug
infusions; therefore, all patients were included in the safety and
efficacy analyses.
[0202] As in the Phase 1/2 study, there was wide heterogeneity of
disease severity among the MPS VI patients enrolled in the Phase 2
Study. Several patients also had neurological features of the
disease, including communicating hydrocephalus, cervical spine
instability, and spinal disc disease.
[0203] Clinical Laboratory Assessments
[0204] No clinically significant laboratory abnormalities were
observed in these patients over the 24-week period.
[0205] Anti-rhASB Antibodies and Complement Studies
[0206] Eight of the 10 patients developed antibody to rhASB by Week
24. Of the three patients that developed higher levels of antibody
than the other patients, two patients' antibody levels continued to
rise after Week 24 but appeared to have stabilized by Week 48.
These two patients were either the "null" genotype or had a
mutation in the presumed major antigenic site for ASB. The
reduction in urinary GAGs was less for these two patients compared
to the others. FIG. 14 shows antibody levels over 48 weeks of
treatment.
[0207] At Week 24, one patient had decreased C4 and CH50 values,
both pre- and post-Week 12 infusion, which were considered to be
clinically significant by the investigator. C4 and CH50 were
slightly below the lower limit of normal prior to infusion and
showed a greater decrease following the infusion. Although these
results suggested the possibility of complement consumption, no
clinical signs or symptoms consistent with such consumption were
observed. Clinically significant abnormalities in complement levels
were not observed in any of the other patients in the study.
[0208] Urinary Glycosaminoglycans
[0209] The mean reduction in urinary GAG levels was rapid, reaching
a nadir at Week 6, and remaining relatively constant from Week 6
through Week 24. By Week 24, urinary GAGs were reduced by an
average of 71%, and by. Week 48 urinary GAGs were reduced by an
average of 76%. All ten patients showed a decrease in urinary GAG
levels that approached the normal range after 24 weeks of
treatment. These data are consistent with the results seen for the
seven patients in the Phase 1/2 study. FIG. 15 shows the reduction
in total urinary GAG levels over the 48 weeks and FIG. 16 shows a
comparison of levels at week 48 to age-appropriate normal
levels.
[0210] Measures of Endurance
[0211] Distance walked was assessed in a 12-minute walk test for
all patients at baseline and every six weeks thereafter. Distance
was recorded as the mean of two separate measures at each
evaluation and was determined at both 6 minutes and 12 minutes.
[0212] At baseline, the mean distances walked at 6 and 12 minutes
were 152.4 (.+-.75.0) and 264.0 (.+-.161.7) meters, respectively.
At Week 24 the mean distance walked at 6 minutes had improved by
57.3 (.+-.59.0) meters (62% per patient), while the mean distance
walked at 12 minutes had improved by an average of 155 meters (98%
per patient). All patients improved their distance walked at 12
minutes, and all but one patient improved in the first 6 minutes of
the walk. At Week 48, the average improvement in the 12-minute walk
test was 212 meters (138% per patient). FIG. 17 shows the
improvement in the 12-minute walk test results over the 48
weeks.
[0213] The 3-minute stair climb was the second measure of endurance
tested. At baseline, patients could climb a mean of 50.3 (.+-.29.5)
stairs. By 24 weeks, the mean number of stairs climbed had
increased to 98.1 (.+-.62.5) stairs, an improvement of 48 stairs or
110% per patient (.+-.116%). By 48 weeks, the average number of
stairs climbed had increased by 61 stairs to a total of 111 stairs,
an improvement of 147% per patient. FIG. 18 shows the improvement
in the 3-minute stair climb results over the 48 weeks. Patients
varied greatly in terms of the number of stairs climbed, and
performance at baseline did not necessarily correlate with the
degree of improvement at Week 24.
[0214] Additional Efficacy Measures
[0215] A number of additional efficacy parameters were evaluated
during the first 24 weeks of this study. The Expanded Timed Get Up
and Go Test was used as a measure of general functional ability.
Overall, there was no significant reduction in total time needed
for this test between baseline and Week 24, but there was a modest
improvement by Week 48. FIG. 19 shows results of the Expanded Timed
Get Up and Go Test over 48 weeks of treatment.
[0216] Forced Expiratory Time (FET), FVC and FEV1 were performed to
assess pulmonary function. Table 28 below displays pulmonary
function values at baseline, Week 24 and Week 48 and also shows
improvement in height and organomegaly at Week 48. No significant
changes in these parameters were seen over the 24-week period. At
Week 48, 5/10 patients improved in FVC and FEVY, of whom 3 of the 5
showed improvement despite less than 2% change in growth. At Week
48 there was also an average 43% improvement in Forced Expiratory
Time (>1 second).
36TABLE 28 Pulmonary Function versus Growth & Organomegaly
Relative Relative Baseline Baseline Wk Wk Change in % .DELTA. %
.DELTA. % .DELTA. % .DELTA. Height FVC 24 48 Height [cm] Liver
Liver Spleen Spleen Patient Age (cm) (liters) FVC FVC.sup.1 (%) Wk
48.sup.2 Wk 48 Wk 48.sup.3 Wk 48 200 18 96.2 0.47 0.44 0.48 -0.05
(<1) -6.3 -15.2* -5.88 -13.90 201 9 93.2 0.52 0.54 0.60 6.65
(7.1) -2.3 -14.4* -8.82 -20.17 202 17 120 0.75 ND 0.92 0.55 (<1)
-6.7 -5.3 -27.81 -26.69 203 15 107 0.28 0.27 0.38 1.9 (1.8) -8.6
-12.5 -16.45 -20.22 204 7 87 0.52 0.60 0.50 3.8 (4.4) -1.3 -4.5
-6.74 -9.71 300 22 102.4 0.37 0.39 0.37 2.5 (2.4) -15.5 -20* -16.58
-21.09 301 9 121.1 1.40 1.46 1.55 4.9 (4.0) +26.3 +3 19.67 -2.56
302 6 99.7 0.81 0.85 0.83 4.8 (4.8) +5.7 -13 -1.92 -19.99 303 8
84.6 0.16 0.27 0.31 1.1 (1.3) -5.8 -13+ -45.33 -49.38 304 16 124.7
0.83 0.74 0.83 1.3 (1.0) -2.8 -15* 6.93 -6.03 .sup.1Bolded values
indicate FVC values that represent clinically meaningful increases
.sup.2mean liver size at baseline 681.6 .+-. 118 cc .sup.3mean
spleen size at baseline 146.8 .+-. 40 cc *Liver volumes above 95%
limit age-adjusted to body weight at baseline, and within normal
limits at week 48 +Liver volumes above 95% limit age-adjusted to
body weight at baseline and week 48
[0217]
37TABLE 29 Shoulder Range of Motion Change @ Week 24 (degrees of
Change @ Shoulder Motion Baseline function) Week 48 All patients (n
= 10) Active Flexion 102 6 3 Passive Flexion 116 4 (1) Active
Extension 52 5 5 Passive Extension 63 9 (6) Active Lateral Rotation
59 7 6 Passive Lateral Rotation 69 9 (6) Patients with baseline
flexion <90.degree. (n = 3) Active Flexion 85 9 5 Active
Extension 50 6 4 Active Lateral Rotation 52 7 7
[0218] Shoulder ROM (flexion, extension, rotation) was assessed
both actively and passively. Overall for the group, a modest
percent increase was seen in both active and passive ROM; however,
some patients showed a decrease in some measures at 24 weeks. At 48
weeks, there was great variability in results due to several
outliers; however, there continued to be improvement seen in
shoulder ROM particularly in patients that had less than 90 degrees
of flexion at baseline. Table 29 above displays the degrees of
shoulder range of motion at baseline, Week 24 and Week 48.
[0219] Pain and stiffness were assessed using a questionnaire.
While two patients had no change in pain score, seven patients
showed improvement in pain by Week 24 (one patient had no baseline
evaluation). All of the nine patients evaluated showed improvement
in stiffness at Week 24. Pain and stiffness evaluated using the
questionnaire continued to improve by Week 48. Results of the joint
pain questionnaire and joint stiffness questionnaire over 48 weeks
of treatment are shown in FIGS. 20 and 21, respectively.
[0220] Significant improvements in grip strength were seen across
the entire group of patients. Seven patients had improvements in
grip strength for one or both hands. Little change was seen in the
pinch test over the 24-week period. There was little significant
change seen in the other quality of life assessments, except that
at Week 48, there was a modest improvement in Dexterity and
Sensation (as measured by the coin pick-up test).
[0221] Activity levels were measured using the belt-attached
ActiTrac.RTM. device. No significant changes in activity level were
observed.
[0222] No significant changes were observed in cardiac function
assessments (ECGs) or in standing height and supine length. Sleep
studies performed in a subset of patients showed no clinically
significant changes.
[0223] Although the invention has been described with reference to
the presently preferred embodiments, it should be understood that
various modifications can be made without departing from the spirit
of the invention. Accordingly, the invention is limited only by the
following claims.
Sequence CWU 1
1
2 1 1656 DNA Homo sapiens CDS (45)..(1643) 1 tagctacagt cggaaaccat
cagcaagcag gtcattgttc caac atg ggt ccg cgc 56 Met Gly Pro Arg 1 ggc
gcg gcg agc ttg ccc cga ggc ccc gga cct cgg cgg ctg ctc ctc 104 Gly
Ala Ala Ser Leu Pro Arg Gly Pro Gly Pro Arg Arg Leu Leu Leu 5 10 15
20 ccc gtc gtc ctc ccg ctg ctg ctg ctg ctg ttg ttg gcg ccg ccg ggc
152 Pro Val Val Leu Pro Leu Leu Leu Leu Leu Leu Leu Ala Pro Pro Gly
25 30 35 tcg ggc gcc ggg gcc agc cgg ccg ccc cac ctg gtc ttc ttg
ctg gca 200 Ser Gly Ala Gly Ala Ser Arg Pro Pro His Leu Val Phe Leu
Leu Ala 40 45 50 gac gac cta ggc tgg aac gac gtc ggc ttc cac ggc
tcc cgc atc cgc 248 Asp Asp Leu Gly Trp Asn Asp Val Gly Phe His Gly
Ser Arg Ile Arg 55 60 65 acg ccg cac ctg gac gcg ctg gcg gcc ggc
ggg gtg ctc ctg gac aac 296 Thr Pro His Leu Asp Ala Leu Ala Ala Gly
Gly Val Leu Leu Asp Asn 70 75 80 tac tac acg cag ccg ctg tgc acg
ccg tcg cgg agc cag ctg ctc act 344 Tyr Tyr Thr Gln Pro Leu Cys Thr
Pro Ser Arg Ser Gln Leu Leu Thr 85 90 95 100 ggc cgc tac cag atc
cgt aca ggt tta cag cac caa ata atc tgg ccc 392 Gly Arg Tyr Gln Ile
Arg Thr Gly Leu Gln His Gln Ile Ile Trp Pro 105 110 115 tgt cag ccc
agc tgt gtt cct ctg gat gaa aaa ctc ctg ccc cag ctc 440 Cys Gln Pro
Ser Cys Val Pro Leu Asp Glu Lys Leu Leu Pro Gln Leu 120 125 130 cta
aaa gaa gca ggt tat act acc cat atg gtc gga aaa tgg cac ctg 488 Leu
Lys Glu Ala Gly Tyr Thr Thr His Met Val Gly Lys Trp His Leu 135 140
145 gga atg tac cgg aaa gaa tgc ctt cca acc cgc cga gga ttt gat acc
536 Gly Met Tyr Arg Lys Glu Cys Leu Pro Thr Arg Arg Gly Phe Asp Thr
150 155 160 tac ttt gga tat ctc ctg ggt agt gaa gat tat tat tcc cat
gaa cgc 584 Tyr Phe Gly Tyr Leu Leu Gly Ser Glu Asp Tyr Tyr Ser His
Glu Arg 165 170 175 180 tgt aca tta att gac gct ctg aat gtc aca cga
tgt gct ctt gat ttt 632 Cys Thr Leu Ile Asp Ala Leu Asn Val Thr Arg
Cys Ala Leu Asp Phe 185 190 195 cga gat ggc gaa gaa gtt gca aca gga
tat aaa aat atg tat tca aca 680 Arg Asp Gly Glu Glu Val Ala Thr Gly
Tyr Lys Asn Met Tyr Ser Thr 200 205 210 aac ata ttc acc aaa agg gct
ata gcc ctc ata act aac cat cca cca 728 Asn Ile Phe Thr Lys Arg Ala
Ile Ala Leu Ile Thr Asn His Pro Pro 215 220 225 gag aag cct ctg ttt
ctc tac ctt gct ctc cag tct gtg cat gag ccc 776 Glu Lys Pro Leu Phe
Leu Tyr Leu Ala Leu Gln Ser Val His Glu Pro 230 235 240 ctt cag gtc
cct gag gaa tac ttg aag cca tat gac ttt atc caa gac 824 Leu Gln Val
Pro Glu Glu Tyr Leu Lys Pro Tyr Asp Phe Ile Gln Asp 245 250 255 260
aag aac agg cat cac tat gca gga atg gtg tcc ctt atg gat gaa gca 872
Lys Asn Arg His His Tyr Ala Gly Met Val Ser Leu Met Asp Glu Ala 265
270 275 gta gga aat gtc act gca gct tta aaa agc agt ggg ctc tgg aac
aac 920 Val Gly Asn Val Thr Ala Ala Leu Lys Ser Ser Gly Leu Trp Asn
Asn 280 285 290 acg gtg ttc atc ttt tct aca gat aac gga ggg cag act
ttg gca ggg 968 Thr Val Phe Ile Phe Ser Thr Asp Asn Gly Gly Gln Thr
Leu Ala Gly 295 300 305 ggt aat aac tgg ccc ctt cga gga aga aaa tgg
agc ctg tgg gaa gga 1016 Gly Asn Asn Trp Pro Leu Arg Gly Arg Lys
Trp Ser Leu Trp Glu Gly 310 315 320 ggc gtc cga ggg gtg ggc ttt gtg
gca agc ccc ttg ctg aag cag aag 1064 Gly Val Arg Gly Val Gly Phe
Val Ala Ser Pro Leu Leu Lys Gln Lys 325 330 335 340 ggc gtg aag aac
cgg gag ctc atc cac atc tct gac tgg ctg cca aca 1112 Gly Val Lys
Asn Arg Glu Leu Ile His Ile Ser Asp Trp Leu Pro Thr 345 350 355 ctc
gtg aag ctg gcc agg gga cac acc aat ggc aca aag cct ctg gat 1160
Leu Val Lys Leu Ala Arg Gly His Thr Asn Gly Thr Lys Pro Leu Asp 360
365 370 ggc ttc gac gtg tgg aaa acc atc agt gaa gga agc cca tcc ccc
aga 1208 Gly Phe Asp Val Trp Lys Thr Ile Ser Glu Gly Ser Pro Ser
Pro Arg 375 380 385 att gag ctg ctg cat aat att gac cca aac ttc gtg
gac tct tca ccg 1256 Ile Glu Leu Leu His Asn Ile Asp Pro Asn Phe
Val Asp Ser Ser Pro 390 395 400 tgt ccc agg aac agc atg gct cca gca
aag gat gac tct tct ctt cca 1304 Cys Pro Arg Asn Ser Met Ala Pro
Ala Lys Asp Asp Ser Ser Leu Pro 405 410 415 420 gaa tat tca gcc ttt
aac aca tct gtc cat gct gca att aga cat gga 1352 Glu Tyr Ser Ala
Phe Asn Thr Ser Val His Ala Ala Ile Arg His Gly 425 430 435 aat tgg
aaa ctc ctc acg ggc tac cca ggc tgt ggt tac tgg ttc cct 1400 Asn
Trp Lys Leu Leu Thr Gly Tyr Pro Gly Cys Gly Tyr Trp Phe Pro 440 445
450 cca ccg tct caa tac aat gtt tct gag ata ccc tca tca gac cca cca
1448 Pro Pro Ser Gln Tyr Asn Val Ser Glu Ile Pro Ser Ser Asp Pro
Pro 455 460 465 acc aag acc ctc tgg ctc ttt gat att gat cgg gac cct
gaa gaa aga 1496 Thr Lys Thr Leu Trp Leu Phe Asp Ile Asp Arg Asp
Pro Glu Glu Arg 470 475 480 cat gac ctg tcc aga gaa tat cct cac atc
gtc aca aag ctc ctg tcc 1544 His Asp Leu Ser Arg Glu Tyr Pro His
Ile Val Thr Lys Leu Leu Ser 485 490 495 500 cgc cta cag ttc tac cat
aaa cac tca gtc ccc gtg tac ttc cct gca 1592 Arg Leu Gln Phe Tyr
His Lys His Ser Val Pro Val Tyr Phe Pro Ala 505 510 515 cag gac ccc
cgc tgt gat ccc aag gcc act ggg gtg tgg ggc cct tgg 1640 Gln Asp
Pro Arg Cys Asp Pro Lys Ala Thr Gly Val Trp Gly Pro Trp 520 525 530
atg taggatttca ggg 1656 Met 2 533 PRT Homo sapiens 2 Met Gly Pro
Arg Gly Ala Ala Ser Leu Pro Arg Gly Pro Gly Pro Arg 1 5 10 15 Arg
Leu Leu Leu Pro Val Val Leu Pro Leu Leu Leu Leu Leu Leu Leu 20 25
30 Ala Pro Pro Gly Ser Gly Ala Gly Ala Ser Arg Pro Pro His Leu Val
35 40 45 Phe Leu Leu Ala Asp Asp Leu Gly Trp Asn Asp Val Gly Phe
His Gly 50 55 60 Ser Arg Ile Arg Thr Pro His Leu Asp Ala Leu Ala
Ala Gly Gly Val 65 70 75 80 Leu Leu Asp Asn Tyr Tyr Thr Gln Pro Leu
Cys Thr Pro Ser Arg Ser 85 90 95 Gln Leu Leu Thr Gly Arg Tyr Gln
Ile Arg Thr Gly Leu Gln His Gln 100 105 110 Ile Ile Trp Pro Cys Gln
Pro Ser Cys Val Pro Leu Asp Glu Lys Leu 115 120 125 Leu Pro Gln Leu
Leu Lys Glu Ala Gly Tyr Thr Thr His Met Val Gly 130 135 140 Lys Trp
His Leu Gly Met Tyr Arg Lys Glu Cys Leu Pro Thr Arg Arg 145 150 155
160 Gly Phe Asp Thr Tyr Phe Gly Tyr Leu Leu Gly Ser Glu Asp Tyr Tyr
165 170 175 Ser His Glu Arg Cys Thr Leu Ile Asp Ala Leu Asn Val Thr
Arg Cys 180 185 190 Ala Leu Asp Phe Arg Asp Gly Glu Glu Val Ala Thr
Gly Tyr Lys Asn 195 200 205 Met Tyr Ser Thr Asn Ile Phe Thr Lys Arg
Ala Ile Ala Leu Ile Thr 210 215 220 Asn His Pro Pro Glu Lys Pro Leu
Phe Leu Tyr Leu Ala Leu Gln Ser 225 230 235 240 Val His Glu Pro Leu
Gln Val Pro Glu Glu Tyr Leu Lys Pro Tyr Asp 245 250 255 Phe Ile Gln
Asp Lys Asn Arg His His Tyr Ala Gly Met Val Ser Leu 260 265 270 Met
Asp Glu Ala Val Gly Asn Val Thr Ala Ala Leu Lys Ser Ser Gly 275 280
285 Leu Trp Asn Asn Thr Val Phe Ile Phe Ser Thr Asp Asn Gly Gly Gln
290 295 300 Thr Leu Ala Gly Gly Asn Asn Trp Pro Leu Arg Gly Arg Lys
Trp Ser 305 310 315 320 Leu Trp Glu Gly Gly Val Arg Gly Val Gly Phe
Val Ala Ser Pro Leu 325 330 335 Leu Lys Gln Lys Gly Val Lys Asn Arg
Glu Leu Ile His Ile Ser Asp 340 345 350 Trp Leu Pro Thr Leu Val Lys
Leu Ala Arg Gly His Thr Asn Gly Thr 355 360 365 Lys Pro Leu Asp Gly
Phe Asp Val Trp Lys Thr Ile Ser Glu Gly Ser 370 375 380 Pro Ser Pro
Arg Ile Glu Leu Leu His Asn Ile Asp Pro Asn Phe Val 385 390 395 400
Asp Ser Ser Pro Cys Pro Arg Asn Ser Met Ala Pro Ala Lys Asp Asp 405
410 415 Ser Ser Leu Pro Glu Tyr Ser Ala Phe Asn Thr Ser Val His Ala
Ala 420 425 430 Ile Arg His Gly Asn Trp Lys Leu Leu Thr Gly Tyr Pro
Gly Cys Gly 435 440 445 Tyr Trp Phe Pro Pro Pro Ser Gln Tyr Asn Val
Ser Glu Ile Pro Ser 450 455 460 Ser Asp Pro Pro Thr Lys Thr Leu Trp
Leu Phe Asp Ile Asp Arg Asp 465 470 475 480 Pro Glu Glu Arg His Asp
Leu Ser Arg Glu Tyr Pro His Ile Val Thr 485 490 495 Lys Leu Leu Ser
Arg Leu Gln Phe Tyr His Lys His Ser Val Pro Val 500 505 510 Tyr Phe
Pro Ala Gln Asp Pro Arg Cys Asp Pro Lys Ala Thr Gly Val 515 520 525
Trp Gly Pro Trp Met 530
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