Note: Descriptions are shown in the official language in which they were submitted.
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METHOD FOR THE PRODUCTION OF rDSPA a1
Field of the Invention
The present invention is directed to a method for the isolation and
purification of recombinant
Desmondus rotundas salivary ptasminogen activator a1 (rDSPA a,1), to rDSPA a1
purified by
this process, to pharmaceutical compositions containing rDSPA a1 so purified,
and to methods
for treatment using rDSPA a1 so purified.
BACKGROUND OF THE INVENTION
Thromboses are produced by the formation of a blood clot in blood vessels.
One distinguishes between venous thromboses including pulmonary embolisms and
arterial
thromboses including acute myocardial infarction. Pulmonary embolism and
cardiac infarction
are fife-threatening events requiring immediate medical intervention.
A popular form of therapy for such arterial and venous thromboses is the use
of
ptasminogen activators to perform enzymatic thrombolysis (Collen et al., Ann.
Rev. Med.,
{1988), 39: 405-423). These substances, called thrombolytics, convert
plasminogen, the
inactive proenzyme of the fibrtnolysis system in the blood, into the active
proteolytic enzyme,
plasmin. Plasmin, in tam, dissolves the fibrous substance fibrin, which is a
substantial
component of a blood clot; this leads to reopening of the blocked vessels and
restoration of
blood flaw. However, because plasmin is a relatively nonspecific protease, it
can also destroy,
through proteolysis, components in the blood indispensable for intact
hemostasis (e.g.
. fibrinogen) and thus increase the risks of hemorrhaging.
The frst enzymatic thrombolytics, streptokinase and urokinase, are compounds
which, once injected into the circulation, systemically convert plasminogen
into plasmtn and
induce systemic proteoiysis. Thus, thromboiysis therapies which use these
compounds are
accompanied by the complications related to hemorrhage. Newer thrombolytic
therapies,
based on the use of plasminogen activators of the tissue type, commonly
referred to as t-PA,
have been developed but they are also beset by a number of drawbacks,
including serious
bleeding complications, a relatively frequent incidence of reocctusion, an
inability to be
uniformly effective, and susceptibility to inactivation by plasminogen
activator inhibitors such as
Type 1 plasminogen activator inhibitor (PAI-1 ) (Loskutoff, Seminars in
Thrombosis and
Hemostasis, Vol. 14, No. 1 {1988)).
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More recently, plasminogen activator proteins have been purifed from vampire
bat (Desmondus rotundus) saliva and salivary glands (European Published Patent
Application
0 383 417 (Baldus et al.); European Published Patent Application 0 352 119
(Duong et aL)).
These plasminogen activators {referred to as DSPA) are serine proteases which
catalyze the
conversion of plasminogen to plasmin but they exhibit greater selectivity
towards fibrin-bound
plasminogen and, hence, may be associated with decreased severity and
frequency of
bleeding when used for thrombolytic therapy. Furthermore, DSPA is not readily
inactivated by
plasma inhibitors such as PAI-1, and therefore, may be associated with a lower
frequency of
reocctusion.
Two high motecutar weight forms of DSPA (designated a.1 and a.2) can be found
in bat
saliva, both of which consist of several domains, including a protease domain,
and both of
which are capable of tightly binding to ptasminogen in the presence of fibrin.
The various
fom3s of DSPA have been produced in mammalian cell culture by recombinant
biotechnology
{Kratzschmer et al., Gene (1991), 105: 229-237; European Published Patent
Application 0 352
119 (Duong et at.)) and smaif scale purification of recombinantly produced
DSPA (rDSPA) has
been described tWitt et al., Blood (1992), 79: 1213-1217). However, the
isolation and
purification of rDSPA on a commercial scale and in a state of purity suitable
for pharmaceutical
formulations has not been disctosed.
The instant application is concerned with isolation and purification of
recombinant DSPA a,1 {rDSPA cc1) on a commercial scale. The invention as
described results
in rDSPA a1 sufficiently pure and stable to be sold commercially and to be
clinically usable.
SUMMARY OP THE INVENTION
The present invention provides a method for the isolation and purification of
rDSPA cc1
on a commercial scale, and results in product which is suitable for clinical
use.
Accordingly, an aspect of the invention is directed to a method for purifying
rDSPA a1 from a biological medium, said method comprising the following steps:
(a) applying the medium to a cation exchange resin under loading conditions
which
result in selective binding of rDSPA a1 to the cation exchange resin;
(b) optionally, washing the cation exchange resin to remove non-rDSPA cci
proteins
and non-protein contaminants;
- {c) selectively eluting the bound rDSPA al from the cation exchange resin;
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(d) applying the rDSPA al-containing eluent from step (c) to a hydrophobic
interaction
resin under loading conditions which result in selective binding of rDSPA al
to the hydrophobic
- interaction resin;
(e) optionatly, washing the hydrophobic interaction resin to remove non-rDSPA
al
protein and non-protein contaminants;
' (f) selectively eluting the bound rDSPA al from the hydrophobic interaction
resin;
(g) applying the rDSPA al-containing eluent from step (f) to an affinity
chromatography resin under loading conditions which result in the selective
binding of rDSPA
al to the affnity chromatography resin;
(h) optionally, washing the affiinity chromatography resin to remove
non-DSPA al protein and non-protein contaminants;
(i) selectively eluting the bound rDSPA al from the affnity chromatography
resin to
produce substantially pure rDSPA al in an aqueous solution.
Another aspect of the invention is rDSPA al protein which has been isolated
and purified by the method of the instant application.
Another aspect of the invention is directed toward a pharmaceutical
composition containing rDSPA al which has been isolated and purified by the
method of the
instant application, and a pharmaceutically acceptable excipient.
Another aspect of the invention is a method of using rDSPA ocl isolated and
purified by the method of the instant application to treat a human with
arterial or venous
thromboses.
DETAILED DESCRIPTION OF THE INVENTION
- As used in the specification and appended claims, unless specified to the
contrary, the following terms have the meaning indicated:
"Biological medium" refers to a precise recipe of salts and nutrients used to
propagate cells in culture.
"Conditioned medium" refers to a biological medium in which cells have been
grown. The medium has, therefore, been conditioned by the growth of the cells
and contains
products excreted into the medium during cell growth. These can be both waste
products
produced during growth or proteins which have been made and secreted into the
medium by
the cells during growth.
"Cation exchange resin" refers to a natural or artificial substance, usually a
solid, which is able to exchange bound ions for ions from the surrounding
liquid medium. A
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cation exchange resin has negative functionaf fixed ions and exchanges
positive counter-ions.
The anchor groups (exchange-active components) in commercially available canon
exchangers are usually -C6H50-, -S03 , -COO-, -P03 , or -As03 . Weaker cation
exchange
resins are those in which the binding strength of the cation is not high, such
as those with
carboxyl or carboxyalkyt functionalities. Furthermore, weaker cation exchange
resins are
usually not fully dissociated at acidic pH. A particular weak canon exchange
resin used in the
invention is comprised of a matrix of silica particles covalently bound to
pofyethyfeneimine
silane, wherein the amino groups of the polyethyleneimine have been
derivatized with carboxyl
groups. Such a resin is commercially available from J.T. Baker, under the
trade name
Widepore CBX~ chromatography resin.
"Hydrophobic interaction resin" refers to a natural or artificial substance,
usually
a solid, which contains uncharged groups, such as methyl, ethyl, or other
alkyl groups. These
groups form hydrophobic bonds with groups on protein moieties which are passed
through the
resin and result in separation of proteins based on the strength of
interaction between the
protein and resin groups. A particular hydrophobic interaction resin is
composed of semi-rigid
spherical beads synthesized by a copolymerization of ethylene glycol and
methacrylate type
polymers derivatized with butyl groups. Such a resin is commercially avaitabte
from Toso-
Haas, under the trade name Toyo-Pearl~ 650M C4.
"Affinity chromatography resin" refers to a natural or artificial substance,
usually
a solid, which is used for the puriftcation of proteins. The resin separates
proteins based on
the affinity which occurs befween groups on the protein and groups on the
resin. in the instant
invention, the resin used as an affinity chromatography resin is usually used
as a size
exclusion resin to separate proteins based on their size. A particular
affinity chromatography
resin is a cross-linked co-polymer of altyl dextran and N,N'-methylene
bisacrytamide in the
form of beads which are capable of fractionating globular proteins between
20,000 and
8,000,000 kDa. Such a resin is commercially available from Pharmacia, under
the trade name
of Sephacryl~ S-400.
"Alkyl" refers to a straight or branched chain monovatent radical consisting
solely of carbon and hydrogen, containing no unsaturation and having from one
to eighteen
carbon atoms, preferably from one to six carbon atoms, e.g. methyl, ethyl, n-
propyi, isopropyl
(1-methylethyl), n-butyl, t butyl (1,1-dimethylethyl), sec-butyl (1-
methylpropyl), n-pentyt, n-
hexyl, and the like.
"Substantially pure" as applied to the purity of the rDSPA ccl product
following
the purification scheme detailed in this application means that greater than
80% of the total
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protein in the final purification product is rDSPA al, preferably greater than
90% of the total
protein isolated is rDSPA al and most preferably 98% of the total protein
isolated is rDSPA
al. Protein content and purity are based on reverse phase HPLC and SDS-page
gel analysis.
"Non-rDSPA al protein and non-protein contaminants" refers to all material
other than rDPSA al found within the biological media from which rDSPA ocl is
being purified.
"Pharmaceutically acceptable excipient" refers to an acceptable carrier, and
any pharmaceutically acceptable auxiliary substance as required to be
compatible with
physiological conditions, which are non-toxic and do not adversely effect the
biological activity
of the pharmaceutical composition suspended or included within it. Suitable
excipients would
be compounds such as mannitol, succinate, glycine, or serum albumin.
'Therapeutically effective amount" refers to that amount of rDSPA a 1 which,
when administered to a human in need thereof, is suffcient to effect
treatment, as defined
below, for disease-states characterized by thrombosis. The amount of a
compound which
constitutes a "therapeutically effective amount" wilt vary depending on the
compound, the
disease-state and its severity, but can be determined routinely by one of
ordinary skill in the art
having regard to his own knowledge and to this disclosure.
'Treating" or "treatment" as used herein cover the treatment of a disease-
state
in a human, which disease-state is characterized by thrombosis, and include:
(i) preventing the disease-state from occurring in a human, in particular when
such human is predisposed to the disease-state but has not yet been diagnosed
as having it;
(ii) inhibiting the disease-state, i.e., arresting its development; or
(iii) relieving the disease-state, i.e., causing regression of the disease-
state.
"Optional" or "optionally" means that the subsequently described event of
circumstances may or may not occur, and that the description includes
instances where said
event or circumstance occurs and instances in which it does not.
Description of the Preferred Embodiments
The present invention is directed to a method for the isolation and
purification of
rDSPA «.l on a commercial scale and in a form suitable for use in
pharmaceutical
formulations. The rDSPA al is produced by fermenting a mammalian cell line
capable of
secreting the rDSPA al product into the culture media. The conditioned media,
i.e. that media
obtained from the bioreactor containing the mammalian cells, is harvested, and
the rDSPA al
is separated from other proteins and contaminants by a series of
chromatographic steps,
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beginning with the use of a cation exchange resin, followed by selective
elution of the rDSPA
al from the resin. The rDSPA al fraction obtained by selective elution from
the cationic
exchange resin is then applied to a hydrophobic interaction resin, where a
selective elution
from the matrix provides a second level of purification. The eluted rDSPA a 1
fraction is then
applied to an affinity chromatography resin, where selective elution provides
a further level of
purification. The purified rDSPA al is next concentrated by conventional
techniques, such as '
ultrafiltration, and then lyophilized.
Specifically, the invention is practiced as described below.
A. Isolation and Purification
1. Culture Media and Cell Lines
The culture medium comprises a base medium suitable for mammalian cell
growth, such as DMEM or Ham's F12. A particular medium is William's E medium
(Williams,
G.M. and Gunn, J.M, Exp. Cell Res., (1974) 89:739). For the inoculation growth
phase, the
base medium will usually be supplemented with a serum source, typically bovine
serum (BS)
or newborn calf serum (CS), present at a concentration in the range from about
0.1 % to 10%
by weight, usually being present at about 1 % to 5% by weight. Other growth
factors or
buffers, such as HEPES, may also be added. During the perfusion growth phase,
the serum
concentration is usually maintained at the same concentration, typically being
in the range
from about 3% to 8%, usually being about 5%.
Cell lines suitable for use in the present invention include mammalian cell
tines
capable of non-adherent growth in suspension culture and/or adherent growth on
microcarrier
beads. Particular cell lines which meet these requirements include Chinese
hampster ovary
(CHO) cell lines, BHK cells, or the HEK293 cell tine (Kratzschmer et al.,
Gene, (1991 ) 116:
281-284; Petri, T., J. BioTechnology, (1995) 39: 75-83).
A particularly preferred CHO cell line is DKB11, which is described in Urlaub,
G. and Chasin, L.A., Proc. NatL Acad Sci. USA, (1980) 77: 4216-4220. These
cells have
been co-transfected with the expression vectors pSVPAI1 and pUDHFR1, which
contain the
coding sequences for DSPA ocl and mouse dihydrofolate reductase, respectively
(Petri, T.,
ibid). The transformed CHO cell Line used in the present invention is
designated CD16 and '
has been deposited with the American Type Culture Collection, Rockviile,
Maryland (ATCC)
and have been given ATCC # CRL 12023.
2. Expansion of Culture and Production Phase
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After inoculation with either.conffuent spinner cultures or portions of other
bioreactor cultures, the cell culture will be expanded to production density,
typically in the
- range from about 1-40x106 cellslml.
~ After the desired cell density on microcarrier beads is achieved, perfusion
of
fresh media supplemented with serum is initiated. Alternatively, cells may be
grown in
' suspension culture. Typically, the concentration of the serum in the fresh
media wilt be in the
range from about 2% to 10% by weight, more typically in the range from about
3% to 8% by
weight, and more normally being about 5% by weight. Initially, the perfusion
rate will be in the
range from about 0.25 to 0.75 culture voiumes/day, typicatly being about 0.5
culture
volumesJday. As the cell growth increases, the perfusion rate is increased to
a final rate in the
range from about 1.5 to 2.5 culture voiumes/day, typically over a period of
about 2 to 10 days.
During the expansion, sterite, pre-equilibrated microcarrier beads are added
to the reactor to
maintain the microcarrier bead to cell density ratio in the range from about
0.5 to 1.0 grams of
microcarrier beads to 109 cells. Conveniently, the microcarrier beads are
added to the reactor
using an aspirator through the sample line.
After cell density has reached the production level, the serum addition to the
ire~h nr.I+r rre rr.eriir am urili 4,e rcrlr marl hmir~liv tn o nnnnonfr~finn
in fhc r~nnc frnr~, ohm r+ f1 1
a cw m.uum c ~ ~ ~cmn ~ ~ vvr.r ~r~ . vuuww, iy rm'....~ ... ...
.,......,....w ..w.~.. . ... ~. .... . w .y. .. m .. s=..wu«.. .
to 2.0 weight percent, typically 1 %.
The culture in production phase requires attention to multiple fermentation
parameters: temperature, pH, and the level of dissolved oxygen are monitored
daily.
Additional culture media, serum, and alkali need to be provided as the supply
tanks are
depleted. Samples of the conditioned media, defined as media which contains
product, are
analyzed routinely, at least every two days to assure that production
continues free from
contamination.
The procedure for collection of the conditioned media from bioreactors
minimizes the harvesting of cells by using screens with approximately 100-150
micron pore
diameters. Suspension cultures use a vortex flow filter to retain cells in the
bioreactor. The
harvest is collected in sterile containers and stored for up to 38 days before
further processing.
rDSPA al found in the bioreactor harvest has been secreted from the CHO cells
in a
~ 30 processed form which has full biological activity and is ready for
purification.
3. Cation Exchange Chromatography
After pH adjustment to the range from 4 to 6, preferably to about 5, the
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rDSPA al in the conditioned media is applied to a cation exchange matrix
(usually packed in
the form of a column) under conditions selected to provide essentially
complete binding of
- rDSPA oc 1 to the matrix. While other proteins will also be bound, the
initial binding stage
provides a fcrst level of separation as a number of the undesired or
contaminating proteins and
other compounds, such as phenol red, in the conditioned media will be unable
to bind to the
matrix and thus will flow through the matrix. The rDSPA al is further purified
by selective
elution from the matrix, where the elution may be accomplished by either a
stepwise elution or
linear gradient elution by increasing the ionic strength of the buffer. tn
either case, the rDSPA
al is collected for further purification as described below.
Suitable cation exchange matrices include a wide variety of resins derivatized
with cationic functionalities which are able to bind rDSPA al. Preferred are
synthetic resins,
such as those comprised of silica gel particles, cross-linked agarose, or
cross-linked
polymethacrylate polymers, derivatized with cationic functionalities such as
carboxyl,
carboxymethyl, sutfonyl, phosphoryl, and the like. Particularly useful are
relatively weak
resins, such as those having carboxyl or carboxyatkyl functionatities, such as
carboxymethyt or
carboxyethyl. A particularly preferred resin is comprised of a matrix of
silica particles
covalentty bound to polyethyleneimine silane, with amino groups of the
polyethyleneimine
sitane derivatized with carboxyl groups. Such a resin is Baker Widepore CBX~
(45mm bead
size), which is commercially available from J.T. Baker.
The binding and elution conditons will vary depending on the binding strength
of the cationic resin. For weak cationic resins, such as Baker Widepore CBX,
binding may be
effected at low ionic strength under slightly acidic conditions, typically pH
4-7, preferably about
pH 5. After washing the matrix, the rDSPA al may be selectively eluted by
exposing the
matrix to a mobile phase having an elevated ionic strength, employing either
linear or step-
wise elution. For the Baker Widepore CBX resin, the rDSPA ccl will elute at a
pH of about 7.5,
with a salt concentration between about 100mM and 500mM NaCI. The column may
then be
stripped and regenerated for subsequent use.
Preferrably, with the Baker Widepore CBX matrix, the resin will initially be
equilibrated with a buffer of 100mM sodium acetate, pH 5. Buffer is applied to
the column at a
flow rate of 0.2 to 2.0 column volumes per minute until the pH of the effluent
stabilizes at 5.
The conditioned media containing rDSPA al is filtered with a 1.2 um filter and
then titrated to a
pH between 4 and 7, most preferrably 5, using glacial acetic acid. The media
is then applied
to the column, typically using a gear pump, at a flow rate no greater than 2.0
column volumes
per minute. A filter is provided to remove particulates which might plug the
column matrix.
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The column matrix is then re-equilibrated with the acetate equilibration
buffer until the pH -
stabilizes at 5, typically requiring from about 2 to 7 column volumes. A wash
buffer containing
- - 50mM sodium phosphate, pH 7.5 is next applied to the column at about 0.1
to 0.2 column
volumes per minute until the pH of the eluent stabilizes at 7.5. rDSPA al is
then eluted from
the column using an elution buffer containing 50mM sodium phosphate, 500mM
sodium
chloride, pH 7.5. The elution buffer is run until no more protein is found
eluting from the
column. A stripping buffer containing 2.0 M sodium acetate, pH 8 is then
applied to the
column in order to regenerate the matrix. The storage buffer contains 10%
acetic acid and
45% ethanol.
4. Hydrophobic Interaction Chromatography
The rDSPA al fraction collected from the canon exchange matrix is next
applied to a hydrophobic interaction matrix (usually in the form of a column)
under conditions
which allow binding of the rDSPA ocl to the matrix, typically high ionic
strength and low pH.
The rDSPA al is then selectively eluted by increasing the organic solvent
concentration of a
mobile phase applied to the column, using a linear or a step-wise gradient.
The rDSPA a.1
fraction is collected for further purification. This step reduces DNA
contamination by 100 to
1000 fold and inactivates potential viral contaminants.
Suitable hydrophobic interaction matrices include a wide variety of uncharged
resins having covalently attached hydrophobic groups, such as propyl, butyl,
octyl, phenyl, and
the like. The resins may be cross-linked organic polymers, such as styrene-
divinyfbenzene,
silica, agarose, polymethacryfate, or any one of a wide variety of other
suitable particulate
supports. A particularly preferced resin is comprised of semi-rigid spherical
beads
synthesized by a copolymerization of ethylene glycol and methacrylate type
polymers
~ derivatized with butyl groups. Such a resin is Toyo-Pearl~ 650M (40-90p,m
beads) which is
commercially available from Toso-Haas.
Binding to the hydrophobic interaction column is effected under conditions of
high ionic strengfh, usually at an acidic pH from 3-5, more usually about 4.
Substantially all
the protein contained in the rDSPA al fraction which had been eluted from the
cation
exchange resin is bound to the hydrophobic interaction column. The various
proteins may be
selectively eluted based on the differing strengths of hydrophobic interaction
with the
hydrophobic groups on the matrix, i.e., in order of increasing hydrophobicity
of the protein.
Elution may be performed with a step-wise or linear gradient, usually with an
alcohol efuant,
such as ethanol or isopropanol. A particularly preferred alcohol is ethyl
alcohol.
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Preferrabiy, with the Toyo-Pearl C4 matrix, equilibration may be performed
with
an equilibration buffer having 50mM sodium acetate, 500mM sodium chloride, pH
4. After the
rDSPA al fraction from the ion exchange column is titrated to pH 4, it is
applied to the C4
matrix, and the matrix is then re-equilibrated with the equilibration buffer
described above. The
column is then washed sequentially with two buffers, as follows. The first
wash (Wash 1 ) uses
at least two column volumes of a buffer containing 20mM HCI. Washing is
continued until the '
effluent contains no further protein, as evidenced by a steady UV absorbance.
The matrix is
then washed (Wash 2) with not less than 10 column volumes of a buffer
containing 19%
ethanol, 20mM HCI, and washing is continued with a buffer containing 20.5%
ethanol, 20 mM
HCI unfit no further protein is eluted. Elution of the rDSPA al product is
effected using a buffer
containing 29% ethanol, 20mM HCI, pH 2.5. After eluting the product, the
column is stripped
with not less than two column volumes of a buffer containing 100mM NaOH. The
matrix is
stored in Wash 1 buffer.
5. Affinity chromatography
The rDSPA ccl fraction collected from the hydrophobic interaction resin is
next
applied to an affenity matrix (usually in the form of a column) under
conditions which allow
binding of the rDSPA al to the affinity matrix. While the rDSPA ccl remains
bound to the
column, contaminants are eluted by washing the column with 2 to 3 column
volumes of 20 mM
HCI. This step facilitates a buffer exchange to aid pharmaceutical formulation
and solubility
and also helps in the inactivation/removat of potential viral contaminants.
Preferably, 2.5
column volumes of the buffer is used. The rDSPA al is then selectively eluted
using a buffer
containing 200mM glyctne, free base at a pH between 4 and 5. The rDSPA al
fraction is
collected for concentration.
In This invention, the resins used as affinity resins are ones which are
normally
used as size exclusion resins. Suitable affinity matrices include resins
comprised of a cross-
linked polymer of atlyl dextran and N,N'-methylene btsacrylamide in the form
of beads with a
diameter between 25 and 75 ~,m. A particularly preferred resin is Sephacryt
400~, which is
commercially available from Pharmacia and is capable of fractionating globular
proteins
between 20,000 and 8,000,000 kDa. "
Binding of rDSPA al to the affinity resin is achieved under conditions of low
ionic strength and low pH. Substantially all the rDSPA al collected from the
hydrophobic
interaction resin is bound to the column. The rDSPA al may be selectively
eluted by raising
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the pH andior ionic strength. Elution may be performed with a step-wise or
linear gradient,
usually with an eiuent containing 200mM giycine, free base.
- Preferrably, with the Sephacryl S-400 matrix, equilibration may be performed
with a buffer having 19% ethanol, 20mM HCI, until the pH of the effluent
remains unchanged.
The matrix is loaded with the rDSPA al fraction from the hydrophobic
interaction cotumn, and
- then washed with a buffer containing 20mM HCt until the UV signal from the
effluent is steady.
Elution of bound rDSPA al is effected using a buffer containing 200mM glycine.
After eluting
the product, the column is stripped with not less than two column volumes of a
buffer
containing 100mM NaOH. Following washing of the matrix with at least two
column volumes
of the wash buffer (20mM HCl}, the matrix can be stored in this buffer.
6. Concentration
The purified protein of the present invention may then be concentrated,
typically by filtration, or the like, and may eventually be lyophilized or
otherwise incorporated
into conventional pharmaceutical preparations. Useful concentration and
lyophilization
methods are well described in the scientific literature.
B. Formulation
1. Pharmaceutical compositions
This invention provides a reagent with significant therapeutic value. rDSPA
cc1,
purified by the methods described, which should be useful in the treatment of
arterial and
venous thromboses.
Recombinant DSPA al, purified as described herein, can be administered to a
patient. This reagent can be combined for therapeutic use with other
ingredients, e.g., in
conventional pharmaceutically acceptable carriers or diluents, along with
physiologically
innocuous stabilizers and excipients. These combinations can be sterile
filtered and placed
into dosage forms as by lyophilization in dosage vials or storage in
stabilized aqueous
preparations.
The quantities of reagent necessary for effective therapy will depend upon
many different factors, including means of administration, physiological state
of the patient,
and other medicants administered. Thus, treatment dosages should be titrated
to optimize
safety and efficacy. Typically, dosages used in vifro may provide useful
guidance in the
amounts useful for in situ administration of these reagents. Animal testing of
effective doses
~ ;- i ~ ;~ fj~ ;. ,. i
~ t l r' ~ ~..~ ', ; ~.. .
CA 02245554 1998-08-04
-12-
for treatment will provide further predictive indication of human dosage.
Various consideration
are described, e.g., in Gilman et al. (eds) (1990) Goodman and Gilman's: The
Pharmacological Bases of Therapeutics, 8th Ed., Pergamon Press; and
Remington's
Pharmaceutical Sciences, 18th ed. (1990), Mack Publishing Co., Easton, Penn.;
- Methods for administration are discussed therein and
below, e.g., for oral, intravenous, intraperitoneal, or intramuscular
administration, transdermal
diffusion, and others. Pharmaceutically acceptable carriers will include
water, saline, buffers.
and other compounds described, e.g., in the Merck Index, Merck & Co., Rahway,
New Jersey.
Based on the dosages required of other plasminogen activators, the need for a
total dosage
of between 80 and 110 mg would be expected (Physicians' Desk Reference, 49th
ed. (1995),
Medical Economics Data Production Company, pp 1083-1085).
Therapeutic formulations may be administered in any conventional dosage
formulation. While it is possible for the active ingredient to be administered
alone, it is -
preferable to present it as a pharmaceutical formulaticn. Formulations
comprise at least cne
active ingredient, as defined above, together with one or more acceptable
carriers thereof.
Each carrier must be both pharmaceutically and physiologically acceptable in
the sense of
being compatible with the other ingredients and not injurious to the patient.
Formulation
inc;ude those suitable for oral or parenteral (including subcutaneous,
intramuscular,
intravenous and intradermal) administration. The formulations may conveniently
be presented
in unit dosage form and may be prepared by any methods well known in the art
of pharmacy.
See, e.g. Gilman et. al., ibid, and Remington ibid..
In summary, the preferred embodiments of the invention as described in the
Summary of the Invention are as follows:
a method for the isolation and purification of rDSPA al from a biological
medium, said
method comprising the following steps:
(a) applying the medium at a pH of 5 to a cotton exchange resin comprised of a
matrix
of silica particles covalently bound to polyethyleneimine silane, wherein
the.amino groups have
been derivatized with carboxyl groups, which results in selective binding of
rDSPA al to the
cotton exchange resin;
(b) washing the cotton exchange resin at pH 5 with 100mM NaOAc and 50 mM
NaPO.~
at pH 7.5, to remove non-rDSPA al protein and non-protein contaminants;
(c) eluting the bound rDSPA al from the catien exchange resin with a buffer
containing
50 mM sodium phosphate and 500 mM sodium chloride at pH 7.5;
AMENDED SHEET
IPEA/EP
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{d) applying the rDSPA al-containing eluent from step (c) at pH 4 to a
hydrophobic -
interaction resin comprised of semi-rigid spherical beads synthesized by a
copolymerization of
- - ethylene glycol and methacrylate type polymers derivatized with butyl
groups, which results in
selective binding of rDSPA al to the hydrophobic interaction resin;
(e) washing the hydrophobic interaction resin with 20 mM HCl and then with
20 mM HCI containing 19% EtOH to remove non-rDSPA al protein and non-protein
contaminants;
(f) eluting the bound rDSPA al from the hydrophobic interaction resin with a
buffer
containing 29% ethanol and 20 mM hydrochloric acid at pH 2.5;
(g) applying the rDSPA al-containing eiuent from step (f) at pH 2.5 to an
affinity
chromatography resin comprised of a cross-linked polymer of allyl dextran and
N,N'-methylene
bisacrylamide which fractionates globular proteins between 20,000 and
8,000,000 kDa, which
results in the selective binding of rDSPA ccl to the affinity chromatography
resin;
(h) washing the affinity chromatography resin wi#h 20 mM HCI to remove non-
rDSPA
a 1 protein and non-protein contaminants;
(i) eluting the bound rDSPA al from the affinity chromatography resin with a
bufFer
containing 200 rnM giycine a# a pH beiween ~+ and 5, to produce substantially
pure rDSPA al
in an aqueous solution.
*****
The following specific preparations and examples are provided as a guide to
assist in the practice of the invention, and are not intended as a limitation
on the scope of the
invention.
EXAMPLE 1
Cell Culture Production of rDSPA al
A vial from the DSPA Working Celt Bank was thawed and inoculated into a roller
bottle
with 5% calf serum in growth media {WEC 5.0 media, Williams E modified). Over
a two week
period the cells were expanded into four roller bottles, the cells were
trypsinized from the roller
bottles and inoculated into four 1 L spinner flasks at 4 x 1 O8 cells each in
1 L of growth media.
' ' After four days, the cultures from the four spinners were used #o
inoculate a 10L bioreactor.
An additional 1 L of growth media was added to the bioreactor culture on the
day of inoculation.
The next day, the bioreactor was filled to 10L with growth media. Culturing
both with and
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without addition of microcarrier beads followed this protocol. The medium pH
was controlled
at 7.0-7.4 and oxygenation was maintained at all times by a sparging system.
The
temperature was maintained at 34-39°C.
The cell density on the day of inoculation in the bioreactor was 8.1 x 105
ceps per mL
and after two days the cell density had doubled, at which time the perfusion
rate was set at 0.5
culture volumes per day {cvlday). The perfusion rate was increased in relation
to cell density
such that by nine days post inoculation of the bioreactor the density had
reached
6.2 x 106 celIsImL and perfusion increased to 2 cv/day.
The bioreactor was then switched to production media (1 % calf serum in WEC
5.0
media) and two days later collection of production harvest began and continued
for as long as
10 weeks. During the production phase, the average cell density was
x 106 cells per mL with approximately 75% viability. The average rDSPA al
production
was 40 milligrams rDSPA «.l per liter and average specific production rate was
6 picograms
rDSPA al per cell per day.
15 Expansion of fermentation volumes was achieved by transferring half the
contents of
one reactor to a new vessel. Both bioreactors were placed in production media,
perfused at
2 cv/day, and collection of production harvest began immediately.
EXAMPLE 2
Purification of rDSPA al
1. Canon Exchange Chromatography (Column A}. The bioreactor harvest is stored
at
ambient temperature (15-25°C). !t is then filtered through a 0.45
micron filter prior to loading
onto a cation exchange column. Purification of the harvest begins with a
column
chromatography step using the CBX resin made by J.T. Baker (Column A). This
step
facilitates a significant purification of the protein.
The Column A buffers consist of Buffer A1: Equilibration buffer {50mM NaoAc,
pH 5.0),
Buffer A2: Wash buffer (50mM NaP04, pH 7.5), Buffer A3: Elution buffer (50mM
NaP04, 500
mM NaCI, pH 7.5), Buffer A4: Strip buffer {2 M NaAc, pH 8.0), and Buffer A5:
Storage buffer
(45% EtOH, 10% HoAo).
The following column operating parameters are appropriate for a column packed
with
6 kg of resin and has a column volume of 15 liters. Not more than 4.5 grams of
rDSPA al can
be loaded on the column per run. The column is loaded at a flow rate of not
more than 2 liters
per minute and not more than 20 psi column pressure. The column and monitor
specifications
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are checked prior to each run. The load material and chromatographic buffers
are degassed -
by sparging with helium prior to use.
The bioreactor harvest is filtered with a 1.2 mm filter then titrated to pH
5.00 (t 0.10)
using glacial acetic acid. Prior to loading, the column is equilibrated with
not less than 30 liters
of Buffer A1 until the effluent is pH 5.00 (t 0.20). Once the column is
equilibrated, the harvest
is loaded on the column. The column is re-equilibrated with not less than 80
liters of Buffer A1.
The column is properly re-equilibrated when the effluent is pH 5.00 (t 0.20)
and when a stable
UV baseline is reached. The column is washed with not less than 145 liters of
Buffer A2. The
column is property washed when the effluent is pH 7.50 (t 0.20) and when a
stable UV
baseline is reached.
The product is eluted from the column with not less than 30 liters of Buffer
A3 and
collected in a separate vessel. Elution is considered complete when a stable
UV baseline is
reached. After eluting the product, the column is stripped until a stable UV
baseline is
achieved, using not less than 30 liters of Buffer A4. The column is then
cleaned for reuse (not
to exceed 50 cycles of use). The Column A product (or eluate) is stored at 2-
8°C . The
average recovery was 93% and the average (protein) purity of the eiuate was 81
%.
2. Hydrophobic Interaction Chromatography (Column B). Following chromatography
on the CBX resin, the rDSPA al product is chromatographed using a C4
hydrophobic
interaction resin (Toso-Haas) (Column B). This step facilitates significant
removal of non-
protein contaminants and also helps in the inactivationlremoval of possible
viral contaminants.
The Column B buffers consist of Buffer B1: Equilibration buffer (50mM NaoAc,
500mM NaCI,
pH 4.0), Buffer B2: Wash and Storage buffer (ZOmM HCI), Buffer B3: Wash buffer
(20mM
HCI, 19% EtOH), Buffer B4: Wash buffer (20mM HCI, 20.5% EtOH), Buffer B5:
Elution buffer
(20mM HCI, 29.5% EtOH), and Buffer B6: Strip buffer (0.1 N NaOH). The
following column
operating parameters are appropriate for a column volume of 15 liters. Not
more than 9 grams
of -
rDSPA al can be loaded on the column per run. The column is loaded at a flow
rate of not
more than 2 liters per minute and not more than 15 psi column pressure. The
column and
monitor specifications are checked prior to each run.
Prior to loading, the column is equilibrated until the effluent is pH 4.00 (t
0.20). The
Column A eluate is titrated to pH 4.00 (t 0.10) using glacial acetic acid,
degassed, and loaded.
This column is re-equilibrated with not less than 30 liters of Buffer B1 until
the effluent is pH
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4.00 (t 0.20) and a stable UV baseline is reached. The column is washed with
three buffers.
The first wash is with not less than 45 liters of Buffer B2. The column is
properly washed
when the effluent is pH 1.90 (t 0.20) and when a stable UV baseline is
reached. The column
is washed a second time with not less than 145 liters of Buffer B3 until a
stable UV baseline is
reached. The column is washed a third time with not less than 30 liters of
Buffer B4 and is
complete when a stable UV baseline is reached.
The rDSPA a,1 is eluted from the column with not less than 30 liters of Buffer
B5 and
collected in a separate vessel. Elution is continued untit a stable UV
baseline is reached. The
column is then cleaned for reuse (not to exceed 50 cycles of use). The Column
B product (or
eiuate) is stored at 2-8°C for not more than 15 days before further
processing. The average
recovery was 91 % and the average (protein) purity of the eluate was 97%.
3. Affinity chromatography (Column C). The Column B product is next
chromatographed using Sephacryl S-400 (Pharmacia) (Column C). This step
facilitates a
buffer exchange to aid formulation and also helps in the inactivation/removal
of viral
contaminants. The Column C buffers consist of Buffer C1: Equilibration buffer
(20mM HCf,
19% EtOH), Buffer C2: Wash buffer (20mM HCI), Buffer C3: Elution and Storage
buffer
(200mM giycine, sterile filtered), and Buffer C4: Strip buffer (0.1 N NaOH).
The following
column operating parameters are appropriate for a column volume of 10 liters.
Not more than
20 grams of rDSPA al can be loaded on the column per run. The column is loaded
at a flow
rate of not more than 0.5 titers per minute and not more than 15 psi column
pressure. The
eluates from one or more Column B runs are pooled, then diluted with one part
Buffer C2 and
two parts Buffer 5 eluate. The final ethanol concentration will be
approximately 19%.
Prior to loading, the column is equilibrated with not less than 15 liters of
Buffer C1 until
~ the effluent is pH 1.80 (t 0.20). After Loading the column is washed with
not less than 30 liters
of Buffer C2. The column is properly washed when the UV signal is stable. The
product is
eluted from the column with not less than 20 liters of Buffer C3 and collected
in a separate
vessel. Elution is-continued until a stabte UV baseline is reached.
After eluting the product, the column is stripped with not less than 12 liters
of Buffer C4
and stored until reuse not to exceed 20 cycles. The Column C product (or
eluate) is diluted to
a concentration of < 1 mg per milliliter with Buffer C3 and stored at 2-
8°C for further
.. processing. The average recovery of rDSPA al was 97% and the average
(protein) purity of
the eluate was 98%.
~ CA 02245554 1998-08-04
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The concentration and formulation steps followed the completion of the
purification
steps above and was performed on the S-400 affinity column eluate, which had
been stored
for not more than 70 days. Column C eluates were pooled and concentrated by a
spiral-
wound ultrafiitration membrane that removes low molecular weight substances
(30,000 dalton
cut-off). The product was collected into a pyrogen free container at a
concentration greater
than
8 mglmL. Mannitol was added to a 4% final concentration.
Final formulation buffer (200mM glycine, 4% mannitol (wlv) was added to bring
the
concentration of the bulk formulated product to 7.5mgImL. The bulk formulated
product is
then sterile filtered, dispensed into vials and lyophilized.
20
ANEEhI~E~ SHEE'E'
IPEA/EP
