Note: Descriptions are shown in the official language in which they were submitted.
1 3331 63
-- 1 --
1 ThiB ln~ention relate~ to the recov-ry of ti~su- plasminogen
activator (t-PA) from liguid media and more ~pecifically, to an
improved method for recovering intact single-chain t-PA substan-
tially free of degraded t-PA and other non-homologous proteins.
5 BACXGROUND AND PRIOR ART
Plasminogen activator~ have received attention for their
role in the fibrinolytic system. These enzyme~ catalyze the
conversion of the proenzyme plasminogen into the proteolytic
enzyme plasmin; plasmin can, in turn, degrade fibrin, a ma~or
10 component of blood clots. Thus, plasminogen activators are
potentially useful for the therapeutic treatment of blood clot~.
The known plasminogen activator~ include streptokinAFe,
which is of bacterial origin, urokinase (u-PA), which has been
i601ated from urine and culture fluids, and tissue plasminogen
activator (t-PA), which is now becoming available from cultured
human cell~ (Rifkin et al., J. Exp. Med. 139:1317-1328 (1974);
Wilson et al, CAncer Res. 40:933-938 (1980)). Streptokinase and
U-PA are available commerci~lly, but appear not to posses~ the
therapeutic efficacy of t-PA.
la
;
1 333 1 63
- 2 -
1 Intact t-PA ~8 a gly~o~I~Lein having a molecular weight of
about 66,000 daltons, and exists as either a one chain
polypeptide (Binder et al., J. Biol. Chem. 254:1998-2003 (1979))
or it may be cleaved by plasmin (Wallen et al., Prog. in
Fibrinolysis 5:16-23 (1981)), into a two-chain form, wherein the
two polypeptides are linked by a disulfide bond (Rijken et al.,
Biochem. Biophys. Acta 580:140-153 (1979)). Non-glycosylated,
enzymatically active t-PA has been produced in eukaryotic cells
grown in the presence of drugs that prevent glycosylation
lo (Little et al., Biochemistry 23:6991-699S (1985)); and in
bacteria (Pennica et al., Nature (London) 301:214-221 (1983)).
Degraded forms of t-PA, having molecular weights of approximately
50,000 and 32,000, have been found coexisting with intact, one-
chain and two-chain t-PA (Granelli - Piperino & Reich, J. Exp.
Med. 148:223-234 (1978)). Prior art methods for isolating t-PA
have not been particularly effective at separating the degraded
forms of t-PA from the intact t-PA.
In pharmaceutical formulations of t-PA, the availability of
substantial quantities of pure intact single-chain enzyme is
important and desired. The strong fibrin binding exhibited by
t-PA (Thorsen et al., Throm. Diath. Haemorrh. 28:65-74 (1972)) is
believed to be important for its therapeutic efficacy. The lower
molecular weight degraded forms, which have aberrant fibrin binding
properties (Banyai et al., FEBS ~ett. 163:37-41 (1983)), do not
appear to display the specificity and clot localization properties
':
~ 333 ~ 63
1 of intact one-chain and two-chain t-PA. Further, it is believed
that single-chain t-PA ic more desirable in pharmaceutical formu-
lation than the two-chain form due to the much slower rate at
which the single-chain form is inactivated by specific inhibitors
of t-PA found in plasma (Lecander et al., Brit. J. Haematol.
57:407-412 (1984)).
Various protocols have been described for the purification
of t-PA using chromatographic, electrophoretic, and selective
extraction and precipitation methods. Most of these methods,
lo including a widely used purification (Rijken and Collen, J. Biol
Chem. 256:7035-7041 (1981)), are not appropriate for the large-
scale production of t-PA as they are inefficient in product
recovery, only partially effective in removing impurities, or use
adsorbants which may introduce toxic, mitogenic, tumorogenic or
immunogenic ligands into the t-PA preparation (Reagan et al.,
Throm. Research 40:1-9 (1985)). Large scale purification methods
employing immunoaffinity chromatography (Wallen et al Eur. J.
Biochem 132:681-686 (1983); Nielsen et al, EMB0 J. _:115-119
(183)) are limited by the cost of the antibody resin, the
difficulty in sterilizing or sanitizing this resin and by the
potential for the antibody or fragments of the antibody leaching
into the recovered t-PA. In addition, the published methods do
not provide procedures to concentrate t-PA to give useful
therapeutic formulations. Furthermore, the presence of degraded
(:
1 333 1 63
- 4 -
1 forms of t-PA in preparations of the purified enzyme remains
problematic to those skilled in the art (Rruithof et al.,
Biochem. J. 226:631-636 (1985)). Degraded t-PA is commonly found
in fermentation broth. Degraded t-PA not only dilutes the intact
t-PA, but in addition, as mentioned above, i6 not specific and is
less able to localize clots as the intact t-PA. Therefore,
contamination of final t-PA product with degraded t-PA provides
serious drawbacks to the product as a therapeutic agent.
However, chromatographic methods for the specific recovery of
intact t-PA free from degraded forms have not been known, so that
the method disclosed by Rijken and Collen, gupra, fails to
separate intact t-PA from its degraded forms, and the two forms
have consistently co-purified together.
Most tissue culture cells require serum supplementation of
media for optimal growth and survival. The known methods for
recovery of t-PA from conditioned tissue culture media are
generally effective only when serum-free media is used. In those
examples wherein 6erum containing production medium is used
(Reagen et al, 6upra; Cederholm-Williams & Porter, Brit J.
Dermatology 110:423-429 (1984), Kluft et al., Adv. Biotechnol.
Processes 2:97-110 (1983)) only partially pure t-PA or t-PA
containing degradation products is recovered. This degradation
is attributed to serum components and may be only partially
blocked by the addition of proteinase inhibitors (Reagen et al,
supra).
1 333 1 63
-- 5
1 SUMMARY OF THE INVENTION
The pre6ent lnvention provldes a rapid, efficient method for
the recovery of intact, ~ingle-chain ti6sue pla6minogen activator6
(t-PA) from liguid media, e.g., 6erum-free and serum-6upplemented
media u6ed to culture cell6 which secrete intact t-PA or from
extract6 of cell6 which intracellularly deposit t-PA or non-
glycosylated t-PA polypeptide. The novel method of the present
invention effect6 the recovery of t-PA 6ubstantially free of
degraded t-PA by contacting a liquid medium with at least one
substrate capable of effecting a 6eparation of intact t-PA from
degraded t-PA.
The present invention also provides method6 for further
adsorbing t-PA onto additional adsorbant 6ubstrate6, e.g.
adsorbant 6ubstrates comprising at least one aminocarboxylic
acid, followed by eluting and recovering the t-PA. Such
additional ad60rption and elution can precede or follow the novel
method6, whlle retaining the benefit6 of the present invention.
The present invention al60 provides a method for minimizing
the amount of degraded t-PA and two-chain t-PA recovered from
serum- or serum fraction-supplemented media by pre-treating the
serum with an additional 6ubstrate 6uch as, e.g., lysine-Sepha-
rosetm (Pharmacia Fine Chemical6, Piscataway, N.J.)
chromatography.
,
1 333 1 63
- 5a -
1 The present invention further provides a method
for recovering intact tissue plasminogen activator (t-PA)
comprising the steps of: a) providing a liquid medium
selected from serum-free medium, serum-supplemented medium,
serum-fraction supplemented medium and albumin-supplemented
medium; b) pretreating said serum-supplemented or serum-
fraction supplemented medium with a first adsorbant
substrate capable of removing substantially all plasminogen
present in the serum-supplemented or serum-fractions
supplemented medium; c) adding to said liquid medium a
plasminogen inhibitor; d) contacting said liquid medium with
a metal chelate adsorbant substrate selected from divalent
cation chelates; e) subjecting said metal chelate adsorbant
substrate to a first solution which selectively dissociates
therefrom degraded t-PA but not said intact t-PA; f)
subjecting said metal chelate adsorbant of step e to at
least one second solution which selectively dissociates
therefrom the intact t-PA; g) contacting said liquid medium
with a substrate comprising an immoblized aminocarboxylic
acid; h) subjecting said immobilized aminocarboxylic acid
substrate to at least one third solution that dissociates
from said substrate degraded t-PA but not said intact t-PA;
and i) subjecting said immobilized aminocarboxylic acid
substrate of step h to at least one fourth solution that
dissociates from said substrate said intact t-PA.
The present invention further provides a method of
pretreating serum or fractionated serum for a medium in
- 5b - 1 333 1 63
1 which cultured cells can be grown for the production of a
protein, comprising contacting the serum or fractionated
serum with an insoluble matrix containing pendant lysine
groups, whereby substances selected from the group
comprising proteolytic substances and other substances
having affinity for lysine are removed from the serum or
fractionated serum.
The present invention further provides a method of
producing a protein in cultured cells grown in a medium
containing serum or fractionated serum, comprising
pretreating the serum or fractionated serum by contacting
the serum or fractionated serum with an insoluble matrix
containing pendant lysine groups, whereby substances
selected from the group comprising proteolytic substances
and other substances having affinity for lysine are removed
from the serum or fractionated serum.
Untreated serum used in growth media for culture
cells contains plasminogen and plasmin which are known to
1 3331 63
- 6 -
1 proteolytically cleave t-PA (Wallen et al, 6upra). Lysine-
Sepharose chromatography has been 6hown to be effective in the
removal of these proteins from 6erum (Wu et al, Exp. Cell
Research 96:37-46 (1975) Quigley et al J. Biol. Chem. Vol. 249,
pg. 4306-4311 tl974)). Such depleted 6erum is capable of
supporting the growth of tissue culture cells (Wu et al, 6upra;
Kaufman et al, Nolec. Cellular Biology 5:1750-1759 (1985)). The
present invention provides improved methods for the removal of
plasminogen and plasmin from 6erum, and further provides a novel
use of "scrubbed 6erum" in combination with aprotonin (an
inhibitor of t-PA proteases) as an essential reagent if intact
single-chain t-PA is to be recovered from serum 6upplemented
media.
Also provided are compounds and compositions obtained by
practicing the present invention, 6aid compounds and compositions
comprising intact t-PA, 6ubstantially free from degraded t-PA and
other unrelated proteins, as well as methods for using such
compounds and compositions.
One substrate useful in the present invention, Zn++ chelate,
has previously been employed for recovering t-PA (Rijken et al.,
supra.) However, the prior art protocols differ significantly from
those disclosed here. The modified zinc column protocol disclosed
here provides the advantages of better separation of intact from
degraded t-PA, and increasing the efficiency of purification by
1 3~3 1 63
1 separating the bulk of the contaminating proteins, as well a8 the
degraded t-PA, from the desired single-chain t-PA.
The literature teaches the use of high ionic strength solu-
tions for chromatography, greater than 0.5 M salt concentrations
when using metal-chelate resins to minimize non-specific adsorption
effects. (Riiken et al, supra; Porath et al, Nature 258:598-599
(1975)). The present invention includes the unexpected observation
that the use of a low ionic strength washing condition (under 100
mM salt, and preferably NaCl) allows for the elution of degraded
lo t-PA and the majority of other proteins bound to the column while
retaining intact t-PA. This results in the ultimate recovery of
t-PA free of degraded t-PA and unrelated proteins which is not
possible if traditional methods (Rijken et al, supra, Rijken &
Collen, supra) are used.
An additional substrate useful in certain embodiments of the
present invention, immobilized lysine, has also been used to
recover plasminogen activator activity from human plasma and
homogenized human veneous tissue (Radcliffe and Heinze, Arch.
Biochem. Biophys. 189:185-194 (1978)), cadaveric perfusates
(Allen and Pepper, Thrombos. Haemostas. 45:43-50 (1981), and from
medium conditioned by incubation with a guinea pig tumor cell line
(Oerstein et al., Cancer Res. 43:1783-1789)). This substrate,
however, has been reported ineffective for the purification of t-PA
found in human uterine tissue (Rijken et al., supra).
1 33s 1 63
1 Previously, the identities of the isolated activators were
not rigorously determined, nor were the purities of the enzymes
established. Further, the previously reported methods for
elution of t-PA from the immobilized lysine substrates did not
provide a system to concentrate t-PA. It is important to obtain
t-PA in concentrations useful for therapeutic formulation and
subsequent admini~tration. The present invention provides a
method for recovering t-PA from lysine-Sepharose in very pure
form, using either basic or acidic eluting conditions. Acidic
lo elution provides a product with higher solubility which is more
suitable for pharmaceutical formulation. This formulation
provides methods for concentrating the t-PA which include alone,
or in combination, dialysis, diafiltration, cationic exchange
chromatography on S-Sepharose, and freeze-drying.
One possible detergent used in purification, Zwittergent 3-
12tm (Calbiochem, La Jolla, California), can be removed from t-PA
by dialysis or diafiltration. Alternatively, Pluronictm F-68
(BASF) can be used. Either of these detergents have the
desireable property that they can be freeze-dried to a powder
along with the t-PA.
It is thus an ob;ect of the present invention to provide a
rapid, simple method for the recovery of tissue plasminogen
activators which increases the recovery of intact t-PA, substan-
tially free of degraded t-PA and other undesirable proteins and
polypeptides, from a variety of liquid media such as those used
(, . ~
1 333 1 63
1 in the culture of eukaryotic or bacterial cells, or from extracts
of 6uch cells, which express the intact t-PA polypeptide.
It is a further object of the present invention to provide a
method which maximizes the amount of single-chain enzyme relative
to the amount of two-chain recovered.
It is a further object of the present invention to provide a
method for the recovery of intact t-PA substantially free of
other proteins including other plasminogen activators, such as u-
PA, and non-homologous proteins.
lo It is a yet another ob;ect of the present invention to
provide a method for the recovery of intact t-PA which provides a
product suitable for the subsequent formulation as an effective
pharmaceutical composition for therapeutic use.
It is yet another object of the present invention to provide
a method for formulating t-PA in a concentration suitable for
therapeutic use.
It is yet another ob;ect of the present invention to provide
a method for formulating t-PA in a concentration ~uitable for
therapeutic use.
It is yet another object of the present invention to provide
a method for formulating t-PA useful for large scale commercial
production of the desired form of t-PA.
1 333 1 63
l BRIEF DESCRIPTION OF THE DRAWINGS
Figure l shows chromatography studies of tissue plasminogen
activator. Conditioned serum-free medium or medium supplemented
with serum which had been pretreated by adsorption with lysine-
Sepharose was clarified and applied to a column of Zn-chelate
Sepharosetm. This column was developed as described in the text
of Example l. Figure (A) shows the elution pattern of total
protein (A 280 nm) and t-PA activity (histograph). A 5-50 micro-
liter (ul) aliquiot of each fraction was incubated at 37 C with
200 ul of 0.01 M Tris-HCl (pH 8.5), 0.1% Tween 80 and 0.2 mM S-
2288tm (Kabi~. The change in adsorbancy at 405 nm was monitored
to measure the amidolytic activity of t-PA. The t-PA contained
in the "Zn B" fractions was applied to a lysine-Sephaosetm
column, and eluted either at pH 8 (Figure B) or at pH 4.0 (Figure
C) as described in the text of Example l. In each of the figure
panels the arrows at the top indicated the application of a
different wash or elution buffers to the columns.
Figure 2 shows an SDS - polyacrylamide gel electrophoresis of
tissue plasminogen activator. The figure shows a coomassie blue
stained gel (Laemmli, Nature (London) 227:680-685 (1970) ) of three
independent preparations of t-PA recovered using the procedures
described in Example l from conditioned medium supplemented with
pretreated serum. The left most lane contains a mixture of reduced
and alkylated standard proteins, from top to bottom:
phosphorylase b (94,000 mw), albumin (67,000 mw), ovalbumin
1 333 1 63
-- 11 --
1 (43,000 mw), carbonic anhydrase (30,000 mw~. The remaining lanes
each contain 5mg of tissue plasminogen activator. Lanes marked
with a (+) contain t-PA which had been chemically reduced with
DTT before electrophoresis.
Figure 3 6hows a zymograph of t-PA recovered by a method of
the present invention from Zn-chelate Sepharose. Each lane
contains one unit of t-PA. The samples were mixed with Laemmli
sample buffer (no DTT), but not heat denatured, and
electrophoresed at 4C through a 0.75 mm thick 8.7% SDS
lo polyacrylamide gel using the Hoeffer "Mighty Smalltm"
electrophoresis unit. Electrophoresis was carried out at a
constant 150 V. After electrophoresis, the gel was soaked for 15
minutes each in two changes of 100 ml of phosphate buffered
~ ~ saline (PBS) + 2.5% (v/v) Triton X-100, followed by two washed
with PBS. The gel is placed onto a standard plasminogen-enriched
fibrin plate and incubated at 37. Zones of clearing are
detected within 2 hours. Lane "A" was obtained from samples
eluted with 20 mM Tris-HCl (pH 7.5), 25 mM NaCl, 0.1 M imidazole,
0.01% Tween 80 (termed Zn A), and indicates more rapidly
migrating (i.e., degraded) t-PA near 50,000 and 32,000 daltons.
Lane "B" was recovered by elution with 20 mM Tris-HCl (pH 7.5),
1.0 M NaCl, 50 mM NaEDTA, 0.01% Tween 80 (termed Zn B).
Figure 4 shows the separation of intact and degraded t-PA.
The figure shows a commassie blue stained gel of a non-reduced
c~r~
1 333 1 63
- 12 -
1 sample of partially purified t-PA which contained intact (65,000
mw) and degraded (50,000 and 32,000 mw~ t-PA ("Load") and
samples in which a substantial 6eparation of these forms into the
"A pool" ("Zn A~) and "B pool" ("Zn B") had been effected through
chromatography on Zn-chelate Sepharose using the protocols
described herein. Other experimental details were as described
in Figure 2.
Figure 5 6hows the inhibition by aprotinin of the conversion
of one-chain to two-chain t-PA in various tissue culture media.
lo Increasing amounts of aprotinin were added to tissue culture
media used for the production of t-PA. The t-PA synthesized
during 48 hours of incubation who analyzed by "Western Blot"
analysis as described in the text. _ shows t-PA produced in
serum-free medium; B, medium supplemented with 0.5% serum, and C,
medium supplemented with 0.5% serum which had been preadsorbed
with lysine Sepharose.
DETAILED DESCRIPTION
A rapid, efficient procedure has been developed for the
recovery of intact, single-chain tissue plasminogen activator
(t-PA) from a liquid medium. The method of the present invention
comprises contacting liguid medium which contains t-PA with at
least one substrate capable of effecting a separation of intact
t-PA from degraded t-PA, and with additional substrates capable
(
~ 37J ~ 1 63
1 of effecting a separation of the intact t-PA from other unrelated
proteins.
The pre6ent invention also provides methods for treating
serum, which is to supplement the nutrient medium used for the
production of t-PA by tissue culture cells, by contacting this
serum with lysine-Sepharose. This pre-treatment was found to be
essential to minimize the proteolytic degradation of t-PA and
further effects the removal of serum proteins which otherwise co-
purify with t-PA.
The present invention also provides compounds and
compositions obtained by practicing the present invention, as
well as compounds and compositions comprising intact t-PA, and
other unrelated proteins and methods for their use.
The liquid media used in one aspect of the invention have
generally been conditioned by incubation with cells which
actively produce intact t-PA, herein exemplified by, but not
limited to, a Bowes melanoma cell-line which has been genetically
engineered to express higher levels of t-PA than does the
parental cell line. Any eukaryotic or procaryotic cell culture
or cell line which secretes t-PA or non-glycosylated t-PA, such
as tunicamycin treated Bowes melanoma cells (Little et al.,
supra), or lysates of cells, such as E. coli (Pennica et al,
supra), which deposit the t-PA or the non-glycosylated t-PA
polypeptide intracellularly, would be appropriate conditioning
agents for liquid media useful in the present invention.
1 333 1 63
- 14 -
1 Such liquid media will generally contain a mixture of intact
t-PA and degraded t-PA. Degraded t-PA includes those forms of t-
PA which have been proteolytically cleaved to produce lower
molecular weight forms, such a6 the 50,000 and 32,000 species.
Also included are those forms of t-PA which have been modified to
alter their fibrin b1n~in~ or fibrin activation characteristic6,
resulting in decreased thrombolytic activity or decreased
6pecificity.
Ligands employed in the present invention are capable of
effecting a separation of intact t-PA from degraded t-PA.
Examples of such ligands include an adsorbant substrate
comprising the general formula:
CH -COOH
~ upport- (CH2)n ~ N
substrate ~``~CH2-COOH
where n is greater than or equal to zero. These molecules
chelate metal ions such as Zn++, Cu++, Ni++ or Co++. Other
chelating agents capable of complexing divalent cations may be
useful in the present invention as well.
Additional benefits can be obtained in the practice of this
invention by employing a plurality of ligands, ~uch as lysine and
propylsulfonate to further separate intact t-PA from undesirable
contaminants.
For ease of use, the ligands effecting 6eparations are
generally immobilized on support substrates. These support
1 333 1 63
1 substrates can compri~e any ~upport materials known to the art
which do not interfere with the ~eparations as disclosed herein.
Such support 6ubstrates can be linked, e.g., convalently bound,
to the 6eparation ligands by any conventional means to provide
increased ease in handling and washing such substrate to improve
the efficiency of the method of the present invention. Support
substrates known to the art include dextrans, agarose, cellulose,
polyacrylamide, silica, etc. When an adsorbant substrate is
linked to a support 6ubstrate, the term resin is used.
lo Certain preferred embodiments of the present invention
produce higher yields of intact one-chain t-PA, substantially
free from intact, two-chain t-PA and degraded t-PA. In the
preferred embodiment, the liquid medium is serum-free nutrient
medium incubated with Bowes melanoma cells. This medium usually
contains low levels of degraded t-PA and unrelated proteins in
mixture with intact t-PA. However, tissue culture cells
freguently reguire for optimal growth or viability media with
serum, fractionated serum, or defined proteins, such as albumin,
transferrin, insulin, cell attachment, growth factors, etc. It
is reported in the literature (Reagan et al, supra; Cederholms-
Williams and Porter, supra; Kluft et al, supra) and observed by
us that the presence of ~erum in the medium used for the
production of t-PA results in increased levels of degraded and
two-chain t-PA or decreases the purity of the t-PA recovered.
1 333 1 63
In the preferred embodiment ~f the present invention where
serum or fractionated ~erum was used to formulate the liquid
medium, it was generally pretreated by adsorption to lysine-
Sepharose. This pre-adsorbed serum ~upported survival and growth
of the cell cultures equivalent to untreated serum (Wu et al,
supra; Kaufman et al, supra.) This pre-treatment removed ~;ub-
stantially all the plasminogen or plasmin from the serum (Deutsch
and Mertz, Science 170:1095-1096 (1970). Plasminogen, when
converted to plasmin by plasminogen activators, i8 known to
lo catalyze the degradation of t-PA (Banyai et al, supra;
Wallen et al., Prog. Chem. Fibrinolysis Thrombolysis 5:16-23
(1983)). This removal of plasminogen was essential for the
recovery of high yields of single-chain t-PA. It is reported
that the inclusion of protease inhibitors in t-PA production
medium is only partially effective in preventing the degradation
of t-PA (Reagan et al, supra). We have furthermore observed that
the pre-treatment of serum removes other materials having
affinity for lysine, and which may otherwise co-purify with the
t-PA in certain embodiments of the present invention. The use of
pre-adsorbed serum is therefore essential for the recovery of
intact t-PA free of degraded t-PA and other unrelated protein
from serum-supplemented medium.
As an example, the pre-treatment of serum was accomplished
by first diluting the serum with three volumes of cold sterile
water. The diluted serum was passed at 4C through a column of
1 333 1 63
- 17 -
1 lysine-Sepharose resin at a flow rate of about one column volume
per hour. The effluent, herein referred to as "scrubbed serum",
was collected, assayed for plasminogen (Wu et al, supra), filter
sterilized and stored frozen until used in the formulation of the
liquid medium. Approximately one milliliter of resin was used to
treat each milliliter equivalent of undiluted serum. The level
of plasminogen in sera varies 6ignificantly. It therefore is
sometimes necessary to use amounts of resin greater than that
specified above. With all ~erum tested, it was found that less
lo resin was required for the complete removal of plasminogen if the
serum is diluted as described here, than if undiluted 6erum is
used as described in the literature (Wu et al, supra). It may be
necessary with the diluted serum to adjust the osmotic ~trength
by adding NaCl before using it in to supplement tissue culture
media. The resin is then regenerated by washing it with a
solution comprising 5 M urea, 1 N NaCl, 50 mM Na EDTA (pH 7.5),
followed by sterile water. The resin column was ~anitized by
washing with 20% ethanol and then storing the column with ethanol
for at least 18 hours. The resin was thoroughly washed with
sterile water before re-use.
To further exemplify a presently preferred embodiment of one
aspect of the present invention, 80wes melanoma cells, adsorbed
to tissue culture flasks (Rijken and Collen, 6upra) or
microcarriers tKluft et al., ~upra) were used to condition liquid
1 333 1 63
- 18 -
1 media which contained O to 0.5% scrubbed 6erum. Aprotinin at a
concentration of 5 to lO0 RIU/ml, And typically 10 RIU/ml was
included ~n the t-PA production medium. The cells were removed by
centrifugation or filtration. Filters used for clarification
should be of low-protein binding materials. It is useful to pre-
treat the filters by passing a solution of 0.1% Pluronic F-68tm
(BASF) or Tweentm 80 (Atlas Chemical Company, Inc.) therethrough.
These conditioned media were chilled to approximately 4C,
adjusted to between approximately pH 7 and 8 with 1 M HCl or
NaOH, supplemented with 0.01% (w/v) Tween 80 or Pluronic F-68
and passed through a first column comprising Zn++ Chelate
Sepharosetm or Zn++ Chelate Fast Flowtm resin. These resins were
prepared as recommended by the manufacturer.
Routinely, the t-PA from 200 liter of 0.5% serum-
supplemented conditioned medium could be completely adsorbed onto
1 liter of resin. Medium may be passed over the resin at the
maximal flow rate recommended by the manufacturer, with
substantially all the detectable t-PA activity retained on the
resin.
The column employed in this embodiment of the present
invention desirably has a high binding capacity and flow
properties such that the t-PA could be rapidly concentrated from
the culture medium. Desirably, the medium should be passed
through the column without significant depletion of essential
nutrients, modifications of pH or ionic ~trength nor addition of
1 333 1 63
-- 19 --
1 compounds toxic to ti6sue culture cells, so that the medium may
be recycled into the cuiture, thus reducing the production costs
related to media use. Optimal binding and recovery of t-PA was
achieved when chromatography was performed at 4C using buffers
of approximately pH 7-8, e.g., 20 mM Tris-HCl (pH 7.5 measured at
20C), and supplemented with 10 KIU aprotinin/ml and with 0.01%
(w/v) Tween 80 or Pluronic F-68.
The t-PA-charged resin was washed with buffer containing
approximately 1.0 M NaCl to remove non-specifically adsorbed
material, and then with a buffer containing approximately 25 mM
NaCl to decrease the ionic strength of the aqueous phase of the
resin. The decreased ionic strength of the aqueous phase of the
intermediate washes, generally less than the equivalent of 100 mM
NaCl, and desirably below 25 mM NaCl, is an important feature of
the embodiments of the present invention employing metal chelate
adsorbant ~ubstrates such as Zn++ chelate. It has been
discovered that, employing medium at the ionic strengths taught
by the prior art (1 M NaCl) (Rijken et al, supra;
Rijken & Collen, supra), intact t-PA is not separated from
degraded t-PA or from the bulk of protein adsorbed to the column.
At the ionic ~trength of 25 mM NaCl, degraded forms of t-PA and
the bulk o~ the non-related proteins adsorbed to the resin can be
eluted as described below without significant elution of the
desired intact form of t-PA.
1 333 1 63
- 20 -
1 Plasminogen activator6 which have been adsorbed during the
practice of the present invention can be eluted from the
6ubstrate. When employing an adsorbant 6ubstrate, an agent which
is capable of disrupting the adsorption will be useful. It is
considered desirable to elute t-PA or other proteins by means of
an agent which competes for the binding 6ites on the adsorbant.
For example, t-PA adsorbed to an adsorbant 6ubstrate comprising a
metal chelate 6uch as zinc chelate can be eluted with imidazole,
histidine or zinc, among others. Elution can also be effected by
lo 6uch means as 6alt concentration, pH, or the use of chelating
agents such as sodium ethylenediaminetetraacetic acid (NaEDTA).
The selection of the eluting agent and precise conditions, i.e.,
pH, ionic strength, temperature, are chosen so that the selective
elution of degrated and intact t-PA are achieved thereby.
Plasminogen activator activities characterized by molecular
weights of approximately 50,000 and 32,000, as determined by
zymography (Granelli-Piperino & Reich, supra), using plasminogen-
containing fibrin indicator plates, were eluted by washing the
resin with 2S mM NaCl which contained an agent capable of
disrupting the adsorption of these species, e.g., 100 mM
imidazole. These plasminogen activators could be specifically
inhibited and immunoprecipitated by monoclonal antibodies
directed against t-PA and therefore appear to be degraded t-PA.
u-PA i6 al60 eluted from the resin by this procedure. Since many
tissue culture cells secrete u-PA, this chromatography procedure
1 3~3 1 63
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1 ensures the recovery of t-PA free from this plaminogen activator
which possesses less fibrin-clot 6pecificity.
The decreased ionic 6trength of the a~ueous phase of the
intermediate washes, generally less than the equivalent of 100 mM
NaCl, and desirably below 25 mM NaCl, is an important feature of
embodiments of the present invention employing metal chelate
adsorbant substrates such as Zn++-chelate. The prior art teaches
the use of high ionic strength solutions to minimize non-specific
ionic interactions of proteins with metal chelating resins. We
lo have suprisingly found that the resolution of this resin is
greatly enhanced by use low ionic strength solutions. We have
found that using low ionic strength solutions of less than 100 mM
NaCl or similar salts, degraded t-PA and the bulk of unrelated
proteins adsorbed to the Zn++-chelate resin can be eluted while
retaining most of the intact t-PA adsorbed to the resin. This
allows for the final recovery of t-PA essentially free of
degraded t-PA and for the production of t-PA of greater purity
than i8 possible had the method for chromatography of t-PA on
Zn++-chelate resin in the prior art been used (Rijken et al,
supra; Rijken & Collen, supra).
Complete elution of the adsorbed intact t-PA free of the
degraded t-PA was effected by washing the resin with 1 M NaCl, 50
mM Na EDTA. Alternatively, the eluting buffer can contain 1.0 M
NaCl, 100 mM imidazole or gradually increasing amounts of NaCl
1 3331 63
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1 (0.025 to 1.0 M NaCl) with 100 ~M imidazole. The latter result6
in the cuccessive elution of t-PA ~ubpopulation6, di6tingui6hed
by their differing affinities for the resin under the conditions
of increasing ionic strength. In a preferred embodiment, NaEDTA
effects the highest recovery of t-PA from the adsorbant
substrate.
The intact t-PA recovered from the Zn+l-chelate resin can be
further treated to remove additional, unrelated contaminants.
For example, the intact t-PA recovered from the Zn+l-chelate
resin was then passed though a 6econd column comprising ~n
aminocarboxylic acid (e.g., lysine) linked directly or via
6pacer (e.g. a 8iX carbon aliphatic 6pacer) to a 6upport
substrate (e.g. Sepharo6e*). However, certain benefit~ of the
present invention can be obtained with any compound wherein both
an amino and carboxyl group are free to interact with t-PA.
Such compounds include, e.g., 3-amino-n-proprionic acid, 4-
amino-n-butyric acid, 5-amino-n-heptanoic acid, 6-amino-n-
hexanoic acid, among others. Included also are cyclic compounds
such as tranexamic acid, and other analogs of lysine, such ~s
aminoethylcysteine, lysopine and octopine, which may possess
affinity for t-PA. Such compounds also desirably possess a
reactive side chain, through which the molecule can be coupled to
the 6upport matrix.
When usinq additional adsorbant ~ubstrate in the practice of
the present invention, the benefits of the invention are retained
*Trade mark
1 333 1 63
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1 independent of the order in which the adsorbant substrates are
employed. While the experimental examples necessarily disclose a
certain order, it will be readily understood that no limitation
is expreased or implied thereby.
In those embodiments wherein a ~econd adsorbant substrate
comprising lysine was employed, generally the t-PA solution
containing approximately 1.0 M NaCl obtained from the Zn
chelate resin was diluted ten-fold with 25 mM Tris-HCl (pH 7.5),
0.1% Tween 80 or Pluronic F-68 and 10 KIU aprotinin/ml and passed
lo over L-lysine-Sepharose resin at 4 C at a rate of approximately
two column volumes per hour. In these embodiments, it was
discovered that diluting the t-PA with buffer containing 0.1%
detergent resulted in greater recovery of t-PA than if the
solution had been diluted with buffer containing only 0.01% of
the detergent.
The binding efficiency of t-PA to the resin is in part
dependent upon the temperature, pH and salt concentration of the
medium to be contacted. The binding capacity of the resin was
increased with decreasing temperature. The optimal binding of t-
PA to the resin occurs at pH 7 to 8, and when the ionic strength
of the medium is equivalent to approximately 100 mM NaCl.
Dilution, dialysis or gel filtration can be used to modify the
ionic strength of the liquid medium to obtain the optimum
benefits of the present invention. To ensure optimal binding of
( !
f`3331 63
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1 t-PA, approximately 1 liter of resin is used for each 0.2 g of t-
PA. If conditioned ti6sue culture medium i6 directly contacted
with the adsorbant, the optimal dilution is approximately one
part medium to three parts 20 mM Tris-HCl, 0.1% Tween 80.
The lysine-Sepharose with bound t-PA was washed with a
buffer, (20 mM Tris-HCl pH 7.5, 0.01% Tween 80, Pluronic F-68, or
O.OS% Zwittergent 3-12 and 500 mM NaCl) to remove unrelated
proteins, and thereafter the t-PA was eluted with this solution
but at a pH greater than 8.5 or with the same solution
containing an eluting agent ~uch as 10-20 mM 6-amino-n-hexanoic
acid, 20-50 mM L-lysine or 100-300 mM L-arginine.
While it is considered desirable to elute the tissue
plasminogen activator by means of a competitive agent, it will be
readily appreciated that other means can be used to elute the t-
PA from the adsorbant substrate including, for example,
alterations in pH, ionic strength of the buffer, and the addition
of various chaotropic agents. However, it is considered
desirable to use the least disruptive agent for the elution
procedure in order to maximize the recovered plasminogen
activator activity.
It is also considered desirable to use an elution procedure
that will facilitate subsequent formulation of the t-PA for
storage and therapeutic use. The lysine-Sepharose with bound t-
PA is washed with a buffer at pH 7.5 consisting of 10 mM Tris,
500 mM NaCl and 0.01% Pluronic F-68 and followed by a buffer of
:
1 333 1 63
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1 pH 8 (for example, 3 mM Na glutamate containing 160 mM NaCl,
0.01% Pluronic F-68). The bound t-PA can then be eluted by
washing the resin with a buffer of pH 4 (for example 3 m~ Na-
~#'~ glutamate containing 160 mM NaCl and 0.01% Pluronic F-68). Thi6
solution containing the t-PA can be directly concentrated, for
example by pressure dialysis using an Amicon pressure dialysis
cell with a YM30 membrane (Amicon) or with an analogus membrane
in cross-flow apparatus. Using this system at pH 4 it i8
possible to concentrate t-PA to greater than 1 mg/ml. It is
lo important that the pH be maintained relatively acidic to effect
concentration. It has surprisingly been found that t-PA becomes
insoluble at concentrations of 0.1 mg/ml or greater if the pH
exceeds 5.
After concentration 5 mg/ml of mannitol can be added.
This solution can be lyophilized and reconstituted by the
addition of water without any loss of activity. In a buffer
containing 0.1% Pluronic F-68, 160 mM NaCl and 3 mM Na glutamate
(pH 4.0), the t-PA activity is stable for at least 7 days at 23C
and indefinitely when frozen. The t-PA formulated in this manner
was shown to actively mediate the dissolution of blood clots when
administered to rabbits and dogs.
Cation exchange chromatography can also be used to
concentrate the t-PA. The t-PA eluted from the lysine ~olumn at
pH 4 can be directly passed through a column of S-Sepharose-FFtm
~ ¦ra~ <
1 333 1 63
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1 ~Pharmcia, Inc. ~ eguilibrated at ~ with the came ly6ine column
~lution buffer. The t-PA i6 then elutea at pH S.0 (3 ~M Na-
glutamate or 2.5 ~M Na citrate, 0.01% Pluronic F-68 containing
200-500 mM NaCl.)
Non-ion~c detergenta are ordinarily u6ed during cell
extractions and chromatogr~phy to increase t-~A yields ~nd reduce
non-specific adsorption; The use of non-ionic detergent 6uch a6
Iween 80 or Triton X-100 to enhance the recovery of t-PA $s well
known (Ri~ken et al., ~upra). However, since ~ost common non-
ionic detergent6 have critical micellar concentration6 on the
order of 0.001%, they can not be effectively removed by cimple
dialy6i6, and therefore impede the concentration of aolutions.
Zwitterqent 3-12 work6 effectively in ensuring high yielda of t-
PA, ~nd can be used at a concentration of 0.05%, le~s than one-
half of it~ critical micellar concentration. Because of lt6relatively high critical micellar concentration, the detergent
can be removed effectively by dialy6is or gel filtration. The t-
PA can thereby be formulated at the desired concentration with
ppropriate ~urfactants (for example Pluronic F-68) added back,
if desired, at concentrations appropriate for intravenoua u6e.
We have chosen to use Pluronic F-68 in our final t-PA
formulations. It i6 a more effective detergent at pH 4-5, the
optimal range for concentrating t-PA, than i~ Zwittergent 3-12.
Pluronic F-68 al~o ba~ ~ignific~nt advantages over Tween 80 which
ia widely u6ed to atabilize t-PA (Ri~ken ~ Collen, aupra). lt ia
*Trade mark
1 3351 63
- 2~ -
1 less toxic than Tween 80 and can be lyophilized to a powder,
therefore, making it more compatable in pharmaceutical
formulations.
Com~o~.~s of the present invention, prepared as disclosed
are shown to have the capability of recognizing and binding to
fibrin, which is present in a host's circulatory system at
locations of thromboses. These compounds are also shown to have
fibrinolytic activity and, therefore display thrombolytic
activity as well. Preparations of t-PA produced by the methods
lo of the present invention are an improvement over t-PA prepared by
other procedures in that the enzyme will be consistently and
substantially pure one-chain, substantially free of degradation
products and can be concentrated and formulated in solutions for
therapeutic uses. The methods of the present invention will not
result in the contamination of the product with elements of the
chromatographic resins likely to be antigenic or tumorgenic.
$he absence of degradation products from these preparations
provide a thrombolytic agent having greater specificity and less
systemic activation of plasminogen.
Compounds of the present invention which are shown to have
the above recited physiological effects can find use in numerous
therapeutical applications such as, e.g., dissolving blood clots.
Thus, these compounds can find use as therapeutic agents in the
treatment of various circulatory disorders, such as, for example,
c
1 333 1 63
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1 coronary or pulmonary embolism, ~troke and decreased peripheral
blood flow.
These compounds can be administered to mammals for
veterinary use 6uch a6 with domestic animals, and clinical use in
humans in a manner ~imilar to other therapeutic agent6, that is,
in a physiologically acceptable carrier. In therapy dependent on
t-PA, it may be important to achieve high plasma levels of t-PA
very rapidly by in~ection. In such cases it will be necessary to
have t-PA available in solutions of appropriate concentrations (1
lo to 10 mg/ml or greater). Physiologically acceptable carrier~ or
methods for maint~ ng t-PA in solution at concentrations in
this range have not been known prior to the present invention.
In general the administered dosage will range from about 0.01 to
100 mg/kg, and more usually 0.1 to 10 mg/kg of the host body
weight. Alternatively, dosages within these ranges can be
administered by constant infusion over an extended period of
time, usually exceeding 24 hours, until the desired therapeutic
benefits have been obtained.
These compounds can be administered neat, as mixtures with
2~ other physiologically acceptable active or inactive materials, or
with physiologically suitable carriers such as, for example,
- water or normal 6aline. At the concentrations necessary for
therapeutic admini6tration it may be necessary to maintain t-PA
with an appropriate detergent in the compound to prevent
aggregation of the protein. The compounds can be administered
~ 333 1 63
- 29 -
1 parenterally, for example, by in~ection. In~ection can be
subcutaneou6, ~ntravenous, or by intramuscular in~ection. These
compounds are desirably administered in pharmaceutically
effective amounts and often~as pharmacologically acceptable 6alts
~uch as acid addition 6alts. Such 6alt6 can include, e.g.,
hydrochloride, hydrobromide, phosphate, 6ulphate, acetate,
benzoate, malate, among others.
Com~oul.ds of the present invention can also be used for
preparing anti6era for u6e in immunoa6says employing labelled
lo reagent6, usually antibodies. These compounds and immunologic
reagent6 may be labelled with a variety of labels ~uch as
chromophores, fluorophores 6uch as, fluorescein or rhodamine, or
radioi60topes such 125I, 35S, 14C, or 3H, or magnetized
particles, by means well known in the art. These labelled
compounds and reagents, or labelled reagents capable of
recognizing and 6pecifically binding to them, can find use as
e.g., diagnostic reagents. Samples derived from biological
specimens can be assayed for the presence or amount of substances
having a common antigenic determinant with compounds of the
present invention .
In addition, monoclonal antibodies can be prepared by
methods known in the art, which antibodies can find therapeutic
use, e.g., to neutralize overproduction of immunologically
related compounds in vivo.
1 333 1 63
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1 In addition, t~e t-PA as prepared in this invention when
suitably labelled with radioi~otopes Isuch as 131I, 123I, lllIn
or 99mTc may prove useful for the detection and localization
of thrombi in patients-
The following examples are provided by way of illustration,
rather than implying any limitation of the sub;ect invention.
Experimental
Example I :Purification of t-PA from
Conditioned Liquid Medium
Liquid medium (1:1 mixture Ham's F-12 and DMEM) containing
0.5% fetal bovine serum, which had been pre-adsorbed with lysine-
Sepharose, and 10 KIU aprotinin per ml was conditioned by
incubation with Bowes melanoma cells (Ri~ken and Collen, supra;
Kluft et al, supra), or alternatively, other plasminogen
15 activator producing cells. This conditioned liquid medium was
clarified by centrifugation at 10,000 x g for 30 minutes at 4C
or by filtration through low-protein binding membranes (e.g.,
Gelman Acrodisc 50A) or filter cartridges (e.g., Sartorius, type
CA or PH). With the cartridge filters it is considered desirable
20 to pretreat the membranes by pre-wetting with a solution of 0.1%
Tween 80 or Pluronic F-68 to decrease the adsorption of t-PA to
the membranes.
Clarified medium was adjusted to approximately pH 7.2 to 7.4
with NaOH, chilled to 4C, and passed through a chelating
25 Sepharose column complexed with Zn+~ as recommended by the
*Trade mark
1 33 3 1 63
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1 manufacturer (Pharmacia, Inc.). The column had been previously
equilibrated with phosphate buffered saline. Up to 200
equivalent column volumes of medium were passed through the resin
at rates up to 50 cm h 1 for a 10 cm bed of Sepharose CL-6B or
300 cm h 1 for an equivalent column of Sepharose-FF; and greater
than 95% of the t-PA activity was bound to the resin. The column
was washed at a rate of 50 cm h 1 with 20 mM Tris-HCl, 1.0 M
NaCl, 0.01% Tween 80, 10 KIU aprotinin per ml until the
absorbance (280 nm) of the eluent buffer was equal to that of the
lo applied buffer. The column was then washed with two to three
column volumes of 20 mM Tris-HCl (pH 7.5), 25 mM NaCl, 0.01%
Tween 80, 10 RIU aprotinin per ml. The t-PA activity associated
with the degraded forms of the enzyme was eluted with 20 mM Tris-
HCl tpH 7.5), 25 mM NaCl, 0.1 M imidazole, 0.01% Tween 80 (termed
eluate "Zn A"). The intact enzyme was recovered by passing 20 mM
Tris-HCl (pH 7.5), 1.0 M NaCl, 50 mM Na EDTA, 0.01% Tween 80
through the resin (termed "Zn B"). The elution profile is ~hown
in Figure lA. Fractions, typically 1/4 column volume, were
collected and aliquots assayed for t-PA activity using
appropriate methods. The t-PA containing fractions of the "Zn B"
elution were collected, diluted ten-fold with cold 20 mM Tris-HCl
(pH 7.5), 0.1% Tween 80, 10 KIU aprotinin per ml and loaded at a
rate of 25 cm per hour onto a 10 cm high bed of lysine-Sepharose.
1 333 1 63
- 32 -
1 A column was c~ocen 6uch that approximately 1 liter of resin was
available for each 0.2 g of t-PA.
The lysine-Sepharose was wa~hed at 4C with one column
volume of 20 mN Tris-HCl (pH 7.5), 100 mM NaCl, 0.1% Tween 80, 10
RIU aprotinin per ml at a rate of about 25 cm per hour. The
column wa~ then washed with 20 ~M Tris-HCl (pH 7.5), 500 ~M NaCl,
0.05% Zwittergent 3-12, 10 KIU aprotinin per ml until the
absor~ance of the eluent buffer was egual to the applied buffer.
Bound plasminogen activator was eluted by washing the column
with 20 ~M Tris-HCl, 500 mM NaCl, 50 mM L-ly~ine, 0.05%
Zwittergent 3-12. Approximately two volume equivalents of
elution buffer were required to complete the recovery (Figure
lB).
Alternatively, t-PA may be eluted from the column by
lowering the pH. A second Zn++-chelate Sepharose was loaded with
serum-free conditioned medium and chromatographed as described
above. The recovered intact t-PA (Zn B) was diluted and loaded
onto a lysine-Sepharose column. This column was washed at 4C
with 10 mM Tris pH 8.0, S00 mM NaCl, 0.01% Pluronic F-68 until the
adsorbance of the eluent buffer was equal to the applied buffer.
The lysine-Sepharose column was washed with 3-4 column volumes
of 3 mM glutamic acid pH 8.0, 160 mN NaCl, 0.01% Pluronic F-68.
Bound plasminogen activator was eluted with 3 mM glutamic
acid pH 4.0 160 mN NaCl, 0.01~ Pluronic F-68. The t-PA
; ~
c
1 3331 63
- 33 -
1 concentration wa6 0.2 - 0.3 mg/ml and the p~ of the eluent was
4.4 + 0.1 (Figure lC).
Thi6 eluted t-PA was concentrated up to 1 mg/ml over an
Amicon YM_30tm membrane by pre~sure dialysis. To avoid
unspecific binding of plasminogen activator, the membrane was
pretreated with 3 mM glutamic acid pH 4.0, 160 mM NaCl, 0.01%
Pluronic F-68.
After concentration, the t-PA solution was brought up to an
Pluronic F-68 concentration of 0.1%. Five mg of ~annitol was
added per ml. This ~olution was lyophilized and reconstituted by
the addition of water without any loss of activity.
Summaries of the purification a t-~A from 6erum-supplemented
and serum-free media Are presented in Tables 1 and 2,
respectively. The recovered t-PA, when analyzed by gel
electrophoresi~ under non-reducing conditions (~aemmli, supra)
had an apparent molecular weight of about 66,000 daltons and
represented greater than 95% of the total protein. (Figure 2).
This t-PA exists primarily AS the one-chain form as evidenced by
absence of 32,000 and 34,000 subunits of two-chain seen under
reducing conditions (Figure 2). The enzymatic, physiochemical
and antigenic properties of the recovered protein confirmed that
the material was tir-sue plasminogen activator. Amino acid
sequencing ~Applied Biosystems Model 470A Seguencer) indicated
the presence of two molecular forms with N-terminal ~equences
~hown in Table 3. The ~equences and the N-terminal heterogeneity
i
1 333 1 63
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1 are as reported in the literature. (Wallen et al Eur. J. Biochem
132:681-686 (1983); Pohl et al. Biochemi6try 23:3701-3707
(1984)).
When the procedures described above are used, 5-25% of the
total t-PA i8 in the form of degraded t-PA, ~nd therefore
recovered in the "Zn AH eluate. If liquid medium containing
6erum, which has not been pre-adsorbed with lysine-Sepharose was
u6e, 25 to 100% of tot~l enzyme eluted from the Zn-chelate column
is found in the Zn A eluate. Zymographic analysi6 (Granelli-
Piperno & Reich, ~upra) of typical "Zn A" and "Zn B" pools are6hown in Figure 3 and demonstrated the separation of low
molecular weight forms of t-PA from the bulk of the intact t-PA.
~`` ;
1 333 1 63
- 3~ -
1 Table 1: Recovery of t-PA from 6erum-supplemented
medium conditioned by Bowes melamoma cells.
The lysine-Sepharose column was eluted at
pH 8.0 a8 described in the text.
Volume Total Protein t-PA t-PA Recovered
Step (ml) (mg) (I.U.) (%)
Harvest 5500 1650 983,000 100
Zn++-chelate
Zn A 32 52 37,000 4
ZN B 100 29 681,000 70
Lysine-
Sepharose 28 1.3 739,000 75
Table Legend: Protein was determined relative to bovine
~erum albumin by the BCA method (Pierce Chemical Company.) t-PA
activity was determined using zonal clearing on plasminogen-
enriched fibrin plates (Haverkatet & Brakman, Prog. in. Chem.
Fibrin. Thromb. 1:151-159 (1975)) and was measured relative to a
standardized preparation of t-PA, activity of which had been
previously defined relative to the WHO International t-PA
st~n~A~d.
ACS/CODON/2/PAT13
1 333 1 63
- 36 -
Table 2: Recovery of t-PA from serum-free medium
1 conditioned by Bowes melomona cells. The
lysine-Sepharose column was eluted at pH 4.0
and the final t-PA solution was concentrated
by ultralfilitration as described.
Total
Step Volume Protein t-PA Recovery
(ml) (mg) (I-U) (%)
Harvest 43,400 1,910 9,766,000 100
Zn+ -chelate
Zn A 960 393 1,366,000 14
Zn B 230 46 7,931,000 81
Lysine-Sepharose 103 31 7,800,000 80
t-PA Concentrate 31 31 7,740,000 79
1 333 ~ 63
- 37 -
1 Table 3: Amino-terminal ~equence of t-PA. Purified protein
was subjected to automated sequence analysis on an
Applied Biosystems Model 470A Protein Sequencer.
At each cycle two amino acids present in a ratio
of 3:2 were detected. These data yielded two
sequences which differed by the pregence or
a~sence of three residues. (*) indicates the
reported alternative amino terminus.
Cycle No. Major Peak Minor PeakPredicted Sequence
1 Gly Ser Gly
lo 2 Ala Tyr Ala
3 Arg Gln Arg
4 Ser Val *Ser
S Tyr Ile Tyr
6 Gln Cys Gln
7 Val Arg Val
8 Ile Asp Ile
9 - Glu Cys
Arg Lys Arg
11 Asp Thr Asp
12 Glu Gln Glu
13 Lys Met ~ys
14 Thr Ile Thr
Gln Tyr Gln
16 Met Gln Met
17 Ile _ Ile
18 Tyr His Tyr
19 Gln Gln Gln
- Ser Gln
His
Gln
Ser
Example II: Removal of Degraded t-PA
from Recovered t-PA
t-PA was recovered from conditioned media which had been
~upplemented with O.S% serum. The conditioned medium was applied
to a Zn~-chelate Sepharose and the t-PA recovered using the
(
~ 3331 63
- 38 -
1 protocol taught in the literature ~Riiken et al, 6upra,
Rijken & Collen, ~upra), The recovered t-PA, cont~in~n~ intact
and degraded enzyme, wa~ then chromatographed on ly~ine-
Sepharose as described above. miS preparation of t-PA, which
contained approximately equal amounts of intact and degraded
t-PA, and which was contaminated with other unrelated proteins
was dialyzed against 20 mM Tris-HCl (pH 8.5), 1.0 M NaCl, 0.01%
Tween 80, or other buffers appropriate for the b~n~;~g of t-PA to
Zn-chelating Sepharose resin. This material (Figure 4, ~Load")
lo was applied to a column of the resin, and washed and eluted as
described in Example I. As expected the ma~ority of degraded t-
PA was eluted from the column in the ~Zn A" fraction (Figure 4)
whereas the intact t-PA now substantially free of the 50,000 mw
form was recovered in the "Zn B" fraction (Figure 4).
Example III: Recovery of t-PA
from-E. coli Extracts
A guanidine-HCl extract of E. coli expressing pre-pro-t-PA
was prepared as described previously (Pennica et al., Nature
301:214-221 (1983)). The extract was diluted to a concentration
of 1 M in guanidine-HCl with 20 mM Tris-HCl (pH 7.~), 0.01 M
NaCl, 0.01% Tween 80 and loaded onto the Zn++-chelate Sepharose
column. Chromatography on the Zn++-chelate and lysine Sepharose
columns proceeded as described in Example I with the intact
E. coli t-PA activity eluting as expected for intact mammalian
1 333 1 63
- 3g -
1 cell enzyme.
- Example IV: Effect of pH on the solubility
of t-P~
Aliguots of a ~olution of intact t-PA at a concentration of
approximately 0.1 mg/ml were dialyzed to equilibrium again6t 10
mM buffers of several pH values containing 160 mM NaCl and 0.1%
Tween 80. Each ~ample was transferred to a centrifuge tube,
mixed thoroughly and an aliquot was assayed on a plasminogen
enriched fibrin plate as described in Table 1. The sample was
centrifuged at 16,000 g to sediment insoluble material. The t-PA
activity remaining in the supernatant fractions was assayed on
fibrin plates. Prior to centrifugation each sample was ~hown to
contain the same amount of t-PA; however, in those sample6 with
pH values greater than pH 5 and up to at least pH 10.5 a
substantial fraction of the t-PA was contained in aggregates
which could be removed by centrifugation (Table 4).
Table 4: Solubility of t-PA at various pH value.
The t-PA remaining in solution after
centrifugation of the samples was determined
on fibrin plates. All samples prior to
centrifugation contained approximately
46,000 units/ml.
Soluble t-PA
pH (I.U./ml)
4.0 46,000
5.0 46,000
6.0 34,000
7.8 8,500
:
f `
C~
1 333 1 63
-- 41~ --
1 9.3 15,500
10.5 34,000
This experiment demonstrates that at neutral pH values t-PA
ayy~eyates even in relativeiy dilute solutions. Therefore, to
concentrate t-P~ for u~e in a phar~aceutical formulAtion weakly
buffered solutions of acidic pH ~hould be employed.
Example V: The use of cation eyçhA~ge
chromatography for the
concentration of t-PA.
The experiments described in Example IV demonstrate that t-
PA is maximally ~olu~le at acidic pH. The isoelectric point of
t-PA is approximately pH 7.5 to 8, therefore in acidic solutions
t-PA should possess a net positive charge and bind to cation
exchange resins such as SP-Sepharo6e or S-Sepharose-FF. These
cation exchangers typically will reversibly bind 10 to 100 mg of
protein per ml of resin, and thus provide a matrix for the
concentration of t-PA.
One ml of S-Sepharose Fast Flowtm was eguilibrated with 0.01
M sodium acetate, lSO mM NaCl, 0.01% Tween 80, 0.02% sodium azide
at pH 4.5 and then packed into a 0.5 cm (i.d.) column. Five mg
of t-PA in 150 ml of 3 mM glutamic acid, 160 mM NaCl, 0.01%
Pluronic F-68, pH 4.0 was applied to the resin at approximately
50 ml h-l.
The ~o~u~n was washed at 12 ml h 1 with 2.5 ~M sodium
citrate, ~00 mM NaCl, 0.1% Pluronic F-68, pH 5.0 until the
1 3 3 3 1 63
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1 adsorbancy at 280 nm of the effluent egualed that of the solution
applied to the column. The column was eluted at 12 ml hrh 1 with
2.5 mM 60dium citrate, 1 M NaCl, 0.07% Pluronic F-68 at pH 5Ø
~ractions, typically 1/4 column volume were collected and
aliquots were assayed for t-PA activity (Table 5).
Table 5: Recovery of t-PA from S-Sepharose
Fast Flow. t-PA activity was assayed
~s described Table 1.
Sample Volume t-PA Recovery
(ml) (I.U.) (%)
t-PA load 150 3,000,000 100
S-Sepharose FF
Flow through 5 500 0
Peak fractions 8.5 3,500,000 115
The product was concentrated by a factor of 20 to a final
concentration of 0.65 mg/ml with full recovery of activity. This
t-PA solution was dialyzed to eguilbrium without loss of activity
against a solution containing 3 mM glutamic acid, 160 mM NaCl and
0.01~ Pluronic F-68 (pH 4.0). This ~olution is suitable for
further concentration by ultrafiltration or direct formulation in
a pharmaceutical preparation.
1 3331 63
- 42 -
1 Example Vl: Comparison of Adsorbant
Substrates
Chromatographic resins were 6ynthesized by dissolving
approximately one millimole of each of several diaminocarboxylic
acids in one ml of 0.1 M sodium bicarbonate. Each acid was added
to 3 ml of a 66% slurry of CNBr-activated Sepharose or activated
CH-Sepharose in water. Solutions were mixed with gentle
agitation for 20 minutes at 4C.
The coupling reactions were terminated by the addition of
200 ml of triethanolamine. After an additional 30 minutes of
agitation at 4C, the substrates were washed as suggested by the
manufacturer of the Sepharose.
One-half of each packed resin was transferred to a small
column and washed with 5 ml of 20 mM Tris-HCl, pH 8.0, 0.1~ Tween
80. -t-PA 6amples containing 2000 units in 5 ml of the same
buffer were passed over each column. Each adsorbant substrate
was washed with 5 ml of the same buffer. Thereafter, t-PA was
eluted with 20 mM Tris-HCl (pH 8.0), 0.25 M NaCl, 0.2 M e-
aminocaprioic aci*, 0.1% Tween 80. The enzyme activity rec~vered
thereby was measured on plasminogen enriched fibrin plates
(Haverkatet & Brakman, supra) to calculate the fraction of enzyme
bound by the adsorbant. The results, as 6hown in Table 6 below,
demonstrated that L-lysine provides the best chromatographic
ligand and that a six carbon spacer between the ~olid support and
the ligand improved the efficency of t-PA binding.
*Trade mark
1 ~33~ ~
- 43 -
1 Table 6: 8;~ng of t-PA to Immobilized
D~aminocarboxylic Acids
t-~A Units Bound
Immobilized Ligand C~-Sepharose Sepharose
2,3-diaminopropionic acid 11 4
D,L-orinithine 16 4
D-lysine 360 64
L-lysine 1040 780
2,4-diaminobutyric acid 80 56
diaminopimelic acid 180 4
Example VII: The Effect of Aprotinin on
Yield of One-Chain t-PA
from various tissue culture
media
Aprotinin is known to inhibit the conversion of one-chain t-
PA into two-chain t-PA. (Riiken & Collen, supra) The
concentration of aprotinin necessary to optimize recovery of one-
chain t-PA relative to two-chain degraded forms of t-PA was
determined. A genetically engineered strain of Bowes melanoma
cells was grown to confluency in a 24 well plate in a medium
composed of a 1:1 mixture of Ham's F-12 and DMEM (F-12/DMEM)
supplemented with 5~ heat-inactivated fetal bovine serum. The
growth medium was removed, and the cells were washed with serum-free
F-12/DMEM. Serum-free medium, medium supplemented with 0.5%
heat-inactivated fetal bovine 6erum which had been pre-treated
with lysine-Sepharose, or medium supplemented with 0.5% heat-
i
(: `;
1 333 ~ 63
- ~4 -
1 activated fetal bo~ine cerum wa~ added to the cells. Aprotinin
was added to each of the media 80 that lndividual well~ in the
tissue culture plates cont~ne~ 0, 1, 5, 10, 50 or 100 RIU of
aprotinin/ml. The plates wére incubated at 37 C for 48 hours.
The media were harvested, clarified by centrifugation and
a~sayed for t-PA activities. The total t-PA in each ~ample was
determined from the diameter of the zone of clearing effected by
5 ul ~ample placed into a well formed in a plasminogen enriched
fibrin plate tHaverkatet and Brakman, ~upra). Neither the choice
of medium nor concentration of aprotinin had any effect on total
t-PA production. Each sample contained approximately 900 I.U. t-
PA per milliliter.
The effect of aprotinin and medium on the conversion of one-
chain t-PA to the two-chain form was determined by Western blot
analysis (Burnette, Anal. Biochim. 112:195 (1981)). The protein
from one ml of each sample of the conditioned media was recovered
by precipitation with trichloroacetic acid (lOt final
concentration). The pellet of protein obtained by centrifuging
the samples for 10 minutes at 15,000 g was resolubilized in 20 ul
of sample buffer (Laemmli, supra). The ~amples, which contained
10 mM dithiothreitol, were boiled for 7 minutes; then loaded onto
a 8.75% polyacrylamide gel. After running the dye front to the
bottom, the proteins were electroblotted onto nitrocellulose.
The nitrocellulose was incubated in S% BSA in 10 mM Tris-
HCl pH 7.5, 0.9% NaCl for 30 minutes at room temperature, ~nd then
incubated with rabbit anti-t-PA (antiserum to denatured human t-
PA) (10 microliters ~erum in 10 ml of 10 mM Tris-HCl pH 7.5, 0.9%
~ :`
1 3331 63
- 45 -
1 The nitrocellulose was ~n~h~ted in 5% BSA in 10 mM Tris-
HCl pH 7.5, 0.9~ ~a~l for 30 minutes at room temperature, and then
incubated with ra~bit anti-t-PA (anti6erum to denatured human t-
PA) (10 microliter6 6erum in 10 ml of 10 mM Tris-HCl pH 7.5,~0.9%
NaCl 3% ~SA, 0.05% Tween 20) overnight at 4C. The b;n~;n~ of
the rab~it ~nti-t-PA was detected using the Yectastain ABCtm
(avidin-biotin-horseradish peroxidase complex) kit and 4-chloro-1-
naptithol as the substrate for the peroxidase (Figure 5). On the
blots, one-chain t-PA i6 seen a6 a band at approximately 66,000
mw, while the subunits of the two-chain enzyme are detected as
bands at 32,000 and 34,000 mw. In those experiments wherein
serum was used no single-chain t-PA can be visualized. In
control experiments this was 6hown to be the result of the large
amount of albumin in the 6ample which both distorts the single-
chain t-PA band during the gel electrophoresis and further
inhibits the complete binding of proteins in this molecular
weight range to the nitrocellulose. However, the presence of
6erum had no effect on the migration of transfer of the two-
chain bands.
Complete inhibition of the conversion of one-chain t-PA to
two-chain was observed at 5-10 KIU/ml aprotinin for either ~erum-
free medium (Figure 5) or medium containing 0.5% "scrubbed serum"
(Figure 5C). When "non-scrubbed" serum was used even 100 XIU
aprotinin per ml was not adeguate to completely inhibit formation ,
1 333 1 63
- ~6 -
1 In media cont~ n~ serum the conversion of one-chain t-PA
to two-chain t-PA is a result of proteolytic activities ~nvolving
t-PA as ~ sub~trate. These activities will additionally cause
degradation of t-PA. Eliminating or blocking the activity which
causes degradation of t-PA is an important step in maintaining
the integrity of t-PA in its medium.
Example VIII: The stimulation of "intact~ and
"degraded" t-PA by fibrinogen
fragments.
lo A striking difference between tissue plasminogen activators
and uro~ 6e is that the former adsorb to fibrin (Thorsen et al
supra), which results in a marked enhancement of the activation
of plasminogen (Wallen, Prog. Chem. Fibrinolysis Thrombolysis
3:167-181 (1978)). Fragments generated from a CNBr cleavage of
fibrinogen (Niewenhuizen et al, Biochim. Biophys. Acta. 755:531-
533 (1983)) also timulate the process of plasminogen activation
by t-PA.
Twenty 1 of t-PA solutions each containing 0.2 units of
one-chain, two-chain or degraded (50,000 mw) t-PA as measured by
fibrin plate assay, was added to 180 1 of solution containing
plasminogen, the chromogenic substrate S-22Sl (Kabi), and with or
without fibrinogen fragments (Niewenhuizen et al, 6upra). In this
assay (Wiman et al, Biochim. Biophys. Acta 579:142-154 (1979)) t-
PA cleaves plasminogen to form active plasmin. The resulting
plasmin activity is assayed using the chromogenic substrate S-
2251, which yields a yellow color ~A405nm), upon hydrolysis by
plasmin. The mixture (0.2 units t-PA, 0.2 mM S-2251, 20 ~g/ml
( :
1 333 1 63
- 47 -
1 CNBr-fragment6 of human fibrinogen) was incubated at 37 and the
absorbance change at 40S nm was read at 15 minute intervals.
Activity i6 determined from a plot of adsorbace V6 (time)2 which
is linear (Drapier et al, Biochimie 61:463-571 (1979)~.
As is 6hown is Table 7 equivalent amounts of t-PA, ~s
defined by egual zones of clearing on a fibrin plate, exhibited
very different activities in the S-2251 assay. In the absence of ~
fibrinogen fragments two-chain t-PA was much more active than
either one-chain or degraded t-PA. The addition of fibrinogen
fragments greatly stimulated the activity of the one-chain enzyme
and to a lesser extent that of the two-chain form ~uch that the
resulting activities were equivalent. The degraded (50,000 mw)
t-PA was much less stimulated by the fibrinogen fragments.
These data suggest that the best form of t-PA for
therapeutic applications i~ the one-chain enzyme a6 its activity
is much more fibrin-specific than that of the two-chain form.
Both forms of the intact enzyme are much more fibrin-specific
than the degraded t-PA.
Table 7: Stimulation of t-PA by fibrinogen fragments(FF)
Enzyme Activity( A405/min2 x 105)Fold Stimulation
-FF ~FF
One-cha~n 0.0614.8 250
Two-chain 0.3416.3 48
Degraded 0.08l.S 19
(50,000 mw)
f
1 333 1 63
- 48 -
1 Although the foregoing invention has been described in some
detail ~y way of illustration and example for purpo~es of clarity
of underst~~g, it will be obvious that certain changes and
modifications may be practiced within the scope of the appended
claims. However, it must be stressed that the production of
intact t-PA which is suitable for subseguent formulation in
pharmaceutical compositions requires following the essential
steps described in the foregoing invention.
