U.S. patent application number 14/819001 was filed with the patent office on 2015-11-19 for compositions, methods, and kits for preparing plasminogen; and plasmin prepared therefrom.
This patent application is currently assigned to GRIFOLS THERAPEUTICS INC.. The applicant listed for this patent is Grifols, S.A.. Invention is credited to Anthony Caronna, Jennifer Hunt, Edward Koepf, Myles Lindsay, Charles Miller, James Rebbeor, Rebecca Silverstein, Kenya Stokes, Thomas Zimmerman.
Application Number | 20150329845 14/819001 |
Document ID | / |
Family ID | 42710186 |
Filed Date | 2015-11-19 |
United States Patent
Application |
20150329845 |
Kind Code |
A1 |
Koepf; Edward ; et
al. |
November 19, 2015 |
COMPOSITIONS, METHODS, AND KITS FOR PREPARING PLASMINOGEN; AND
PLASMIN PREPARED THEREFROM
Abstract
Compositions and methods for preparing plasminogen, in
particular recombinant plasminogen, and compositions and methods of
utilizing same for preparing plasmin are provided.
Inventors: |
Koepf; Edward; (Holly
Springs, NC) ; Lindsay; Myles; (Garner, NC) ;
Silverstein; Rebecca; (Cary, NC) ; Hunt;
Jennifer; (Raleigh, NC) ; Rebbeor; James;
(Garner, NC) ; Zimmerman; Thomas; (Raleigh,
NC) ; Miller; Charles; (Apex, NC) ; Caronna;
Anthony; (Apex, NC) ; Stokes; Kenya; (Durham,
NC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Grifols, S.A. |
Barcelona |
|
ES |
|
|
Assignee: |
GRIFOLS THERAPEUTICS INC.
|
Family ID: |
42710186 |
Appl. No.: |
14/819001 |
Filed: |
August 5, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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13147491 |
Sep 23, 2011 |
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PCT/US10/25898 |
Mar 2, 2010 |
|
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14819001 |
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61156990 |
Mar 3, 2009 |
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Current U.S.
Class: |
435/217 |
Current CPC
Class: |
C12Y 304/21007 20130101;
C12N 9/6435 20130101 |
International
Class: |
C12N 9/68 20060101
C12N009/68 |
Claims
1. A method for preparing recombinant plasmin, said method
comprising: (a) contacting a composition comprising a recombinant
plasminogen having a single kringle domain, a peptide linker, an
activation site, and a serine protease domain, wherein said single
kringle domain comprises at least 90% identity to kringle 1 or
kringle 4 of SEQ ID NO:2; said peptide linker sequence between said
single kringle domain and said serine protease domain consists of
residues 91-94 of SEQ ID NO:1 with a cation-exchange medium under a
cation-exchange condition that is sufficient for said
cation-exchange medium to bind said recombinant plasminogen;
wherein said cation-exchange medium comprises a sulfopropyl ligand
coupled to an agarose support; and (b) converting said recombinant
plasminogen to said recombinant plasmin.
2. The method of claim 1, wherein said converting step comprises
contacting said recombinant plasminogen with a plasminogen
activator in an activation solution under an activation condition
sufficient to convert said recombinant plasminogen to a recombinant
plasmin.
3. The method of claim 2 further comprising: contacting said
activation solution with an anion-exchange medium under an
anion-exchange condition to obtain an anion-exchange medium
flow-through comprising said recombinant plasmin, wherein said
anion-exchange condition is such that said anion-exchange medium
preferentially binds said plasminogen activator relative to said
recombinant plasmin.
4. The method of claim 3, wherein said anion-exchange medium flow
through comprises an amount of said plasminogen activator or
fragment thereof that is less than an amount of that which was
present in said activation solution prior to contacting with said
anion-exchange medium, and wherein said anion-exchange medium flow
through comprises an amount of said recombinant plasmin that has a
purity of at least about 50%.
5. The method of claim 3, wherein said anion-exchange medium
comprises a quaternary ammonium or a quaternary aminoethyl.
6. The method of claim 3 further comprising: contacting said
anion-exchange medium flow-through with a second affinity medium
under a second affinity condition sufficient to bind said
recombinant plasmin.
7. The method of claim 3 further comprising: contacting a second
affinity medium eluate comprising said recombinant plasmin with a
hydrophobic interaction chromatography medium under a hydrophobic
interaction condition sufficient such that said hydrophobic
interaction chromatography medium preferentially binds said
plasminogen activator relative to said recombinant plasmin.
8. A method for preparing a recombinant plasmin, said method
comprising: contacting a composition comprising a recombinant
plasmin having a single kringle domain, a peptide linker, an
activation site, and a serine protease domain, wherein said single
kringle domain comprises at least 90% identity to kringle 1 or
kringle 4 of SEQ ID NO:2; said peptide linker sequence between said
single kringle domain and said serine protease domain consists of
residues 91-94 of SEQ ID NO:1 with an anion exchanger whereby, if
present in said composition, a proteinaceous material having an
isoelectric point below that of said recombinant plasmin is
separated from said recombinant plasmin.
9. The method of claim 8, wherein prior to contacting said
composition comprising said recombinant plasmin with said anion
exchanger, a pH of said composition is adjusted to be less than
said isoelectric point of said plasmin but greater than said
isoelectric point of said proteinaceous material to be separated
from said recombinant plasmin.
10. The method of claim 8, wherein said anion exchanger comprises a
quaternary ammonium.
11. The method of claim 8, wherein said proteinaceous material is a
streptokinase or a fragment thereof.
12. The method of claim 8, further comprising an additional
chromatography purification step comprising an affinity
chromatography, an ion exchange chromatography, or a hydrophobic
interaction chromatography.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S. patent
application Ser. No. 13/147,491 having a filing date and a 35
U.S.C. .sctn.371(c) date of Sep. 23, 2011, which is a national
phase application under 35 U.S.C. .sctn.371 of International
Application Serial No. PCT/US2010/25898, filed Mar. 2, 2010, that
claims benefit of priority under 35 U.S.C. .sctn.119 to U.S.
Provisional Application No. 61/156,990, filed Mar. 3, 2009, the
contents of each which are hereby incorporated by reference in
their entirety.
FIELD OF THE INVENTION
[0002] This invention relates to both a method for preparing
plasminogen and a method for preparing plasmin from the
plasminogen, in particular recombinant plasminogen. This invention
also relates to compositions and kits comprising recombinant
plasminogen and/or plasmin prepared therefrom.
BACKGROUND OF THE INVENTION
[0003] The production of large quantities of relatively pure
polypeptides and proteins is important for the manufacture of many
pharmaceutical formulations. For production of many proteins,
recombinant DNA techniques have been employed in part because large
quantities of exogenous proteins can be expressed in host
cells.
[0004] Plasmin, the principle fibrinolytic enzyme in mammals, is a
serine protease with trypsin-like specificity that is derived from
the inactive zymogen precursor plasminogen circulating in plasma.
Plasminogen itself is a 790 amino acid polypeptide having an
N-terminus glutamate residue. Plasminogen activators such as
streptokinase, tissue plasminogen activator (tPA) or urokinase will
cleave the single-chain plasminogen molecule to produce active
plasmin at the Arg560-Val561 peptide bond. The resulting two
polypeptide chains of plasmin are held together by two interchain
disulfide bridges. The light chain of 25 kDa carries the catalytic
center and is homologous to trypsin and other serine proteases. The
heavy chain (60 kDa) consists of five triple-loop kringle
structures with highly similar amino acid sequences. Some of these
kringles contain so-called lysine-binding sites that are
responsible for plasminogen and plasmin interaction with fibrin,
.alpha.2-antiplasmin, or other proteins.
[0005] Plasmin as a potential thrombolytic agent has numerous
technical difficulties. These difficulties include the challenge of
preparing pure plasmin that is relatively free of functional traces
of the plasminogen activator used to generate plasmin from its
inactive precursor, plasminogen. Preparations of plasmin are
typically extensively contaminated by plasminogen activator,
streptokinase or urokinase, and the thrombolytic activity has been,
therefore, attributed to the contaminating plasminogen activators
rather than to plasmin itself. The contaminating plasminogen
activators could also trigger systemic bleeding other than at the
targeted site of thrombosis. Another important technical factor
limiting clinical use of plasmin is that plasmin, as a serine
protease with broad specificity, is highly prone to autodegradation
and loss of activity. This circumstance provides severe challenges
to the production of high-quality plasmin, to the stable
formulation of this active protease for prolonged periods of
storage prior to use, and to safe and effective administration of
plasmin to human patients suffering from occlusive thrombi.
[0006] Preparative isolation of recombinant plasminogen or plasmin
prepared from recombinant plasminogen resulting in pharmaceutical
purity and sufficient yield has eluded the art. Thus, there is need
for compositions and methods for preparing a recombinant
plasminogen and plasmin prepared from recombinant plasminogen
activated by a plasminogen activator.
SUMMARY OF THE INVENTION
[0007] There is now provided compositions and methods for preparing
a plasminogen; and for preparing a plasmin therefrom.
[0008] In one aspect, the present invention provides a method for
preparing plasminogen. The method comprises contacting a
composition comprising a plasminogen with a cation-exchange medium
under a cation-exchange condition that is sufficient for the
cation-exchange medium to bind the plasminogen.
[0009] In another aspect, the present invention provides a method
for preparing plasminogen, the method comprising: [0010] (a)
expressing a recombinant plasminogen using a recombinant expression
system under an expression condition sufficient to produce a
recombinant plasminogen inclusion body; [0011] (b) contacting the
recombinant plasminogen inclusion body with a solubilization buffer
under a solubilization condition sufficient to obtain a solubilized
recombinant plasminogen inclusion body; [0012] (c) contacting the
solubilized recombinant plasminogen inclusion body with a refolding
solution under a refolding condition to obtain a composition
comprising the recombinant plasminogen; [0013] (d) diafiltering the
composition subsequent to step (c); [0014] (e) contacting the
composition with a cation-exchange medium under an ion-exchange
condition that is sufficient for the cation-exchange medium to bind
the recombinant plasminogen; [0015] (f) eluting the recombinant
plasminogen captured by the cation-exchange medium to obtain a
cation-exchange medium eluate comprising the recombinant
plasminogen; [0016] (g) contacting the cation-exchange medium
eluate with a first affinity medium under a first affinity
condition that is sufficient for the first affinity medium to bind
the recombinant plasminogen; and [0017] (h) eluting the recombinant
plasminogen bound by the first affinity medium to obtain a
plasminogen solution comprising the recombinant plasminogen.
[0018] In other aspects, the present invention provides a method
for preparing plasmin, the method comprising: [0019] (a) contacting
a composition comprising a plasminogen with a cation-exchange
medium under a cation-exchange condition that is sufficient for the
cation-exchange medium to bind the plasminogen; [0020] (b)
contacting the plasminogen with a plasminogen activator in an
activation solution under an activation condition sufficient to
convert the plasminogen to a plasmin; and [0021] (c) contacting the
plasmin with an anion-exchange medium under an anion-exchange
condition such that the anion-exchange medium preferentially binds
the plasminogen activator relative to the plasmin.
[0022] In one aspect, the present invention provides a method for
preparing a plasmin. The method comprises: contacting a composition
comprising the plasmin with an anion exchanger whereby, if present
in the composition, a proteinaceous material having an isoelectric
point below that of the plasmin is separated from the plasmin.
[0023] Also provided is a plasminogen and/or a plasmin prepared in
accordance with the methods of the present invention.
[0024] Also provided are kits comprising one or more of the
compositions of the present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] FIG. 1 shows one embodiment of a recombinant plasminogen
amino acid sequence (SEQ ID NO:1).
[0026] FIG. 2 shows the amino acid sequence of a human plasminogen
(SEQ ID NO:2), showing the 19-residue leader sequence numbered as
-19 to -1, and the plasminogen sequence shown as residues 1-791. A
number of features are shown, including the following: one
embodiment of a recombinant plasminogen amino acid sequence (shaded
region corresponds to the amino acid sequence as set forth in SEQ
ID NO:1); kringle domains 1-5 (double underscore); glycosylations
sites Asn289 and Thr346 (in bold); the Arg-Val activation site
(R.sup.561V.sup.562 in bold); and lysine-binding sites in kringle 1
(in underscore and with specific position numbering).
[0027] FIG. 3 shows polypeptide sequence comparisons (i.e., a gap
alignment) between the five kringle domains (1-5) of native human
plasmin(ogen). Amino acid residues that are identical to those of
the same relative position in kringle 1 are shown in
underscore.
[0028] FIG. 4 shows a sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE) gel of purification intermediates.
Samples were run on a 4-12% polyacrylamide gel, under reducing
conditions, and stained with Coomassie Blue R-350 dye. Gel lanes
2-9 were each loaded with 3.5 .mu.g of total protein. Lanes 1 and
10, molecular weight markers; lane 2, solubilized inclusion bodies;
lane 3, refolded recombinant plasminogen; lane 4, SP-SEPHAROSE.TM.
eluate; lane 5, ECH-Lysine SEPHAROSE.TM. eluate; lane 6, plasmin;
lane 7, Benzamidine SEPHAROSE.TM. load; lane 8, Benzamidine
SEPHAROSE.TM. eluate; lane 9, final formulation plasmin. Arrows to
the right of the gel indicate autolysis products of plasmin.
[0029] FIG. 5 shows a size-exclusion analysis of purified
recombinant plasmin. Elution profiles (A.sub.280) for the three
preparations of purified recombinant plasmin are overlaid in the
figure. Monomeric plasmin constituted 98.3.+-.0.1% of the
absorbance eluted from the column during each run. The remainder
(1.7.+-.0.1%) of the eluted absorbance was eluted before the main
peak.
[0030] FIG. 6 is a graph showing the percentage of initial soluble
total protein (A.sub.280) and recombinant plasminogen activity
remaining in solution after addition of solid polyethylene glycol
(PEG) to the indicated concentrations and removal of formed
precipitate by centrifugation.
[0031] FIG. 7 is a graph showing the percentage of initial soluble
total protein (A.sub.280) and recombinant plasminogen activity
remaining in solution after addition of solid ammonium sulfate to
the indicated concentrations and removal of formed precipitate by
centrifugation.
[0032] FIG. 8 is an overlay of chromatograms (A.sub.280 absorbance
traces) of hydrophobic interaction chromatography (HIC) resin
screening experiments demonstrating the direct capture of
recombinant plasminogen (TAL6003Z) from ammonium sulfate
precipitated refolds. The start of the elution phase of each run is
denoted by the black arrow and coincides with the onset of fraction
collection.
[0033] FIG. 9 is a SDS-PAGE.
DETAILED DESCRIPTION OF THE INVENTION
I. Plasminogen
[0034] In one aspect, the present invention provides a method for
preparing plasminogen, the method comprising:
[0035] contacting a composition comprising a plasminogen with a
cation-exchange medium under a cation-exchange condition that is
sufficient for the cation-exchange medium to bind the
plasminogen.
[0036] The amino acid sequence of the plasminogen can correspond to
or be based on any species including, but not limited to, a human,
murine, bovine, ovine, porcine, equine, and avian, in native
sequence or in a genetically engineered form, and from any source,
whether natural, synthetic, or recombinantly produced. In one
embodiment, the plasminogen corresponds to or is based on a human
plasminogen.
[0037] In one embodiment, the plasminogen is recombinant
plasminogen.
A. Cation-Exchange Chromatography
[0038] The cation-exchange medium can be a solid phase that binds
the plasminogen present in the composition. The cation-exchange
chromatography medium can be selected from any of the group of
media commonly described as cation-exchange media, preferably a
strong cation-exchange medium. The medium can possess a chemistry
or a ligand coupled thereto that can allow for selective or
preferential capture of the plasminogen from the composition.
Useful chromatography media comprise a support and one or more
ligand(s) bound thereto that provide(s) the selective or
preferential binding capability for the plasminogen. Useful
supports include, by way of illustrative example, polysaccharides
such as agarose and cellulose, organic polymers such as
polyacrylamide, methylmethacrylate, and polystyrene-divinylbenzene
copolymers. It should be recognized that it is not intended herein
to imply that only organic substrates are suitable for medium
substrate use, since inorganic support materials such as silica and
glasses also can be used.
[0039] In some embodiments, the cation-exchange medium is in the
form of beads, which can be generally spherical, or alternatively
the cation-exchange medium can be usefully provided in particulate
or divided forms having other regular shapes or irregular shapes.
Or, the medium can be in a membrane format. The cation-exchange
medium can be of porous or nonporous character, and the medium can
be compressible or incompressible. Preferred cation-exchange media
will be physically and chemically resilient to the conditions
employed in the plasminogen purification process including pumping
and cross-flow filtration, and temperatures, pH, and other aspects
of the various compositions employed. A wide variety of cation
exchange media, for example, those wherein the coupled ligand is
sulphopropyl or methylsulphate, are known in the art.
[0040] In one embodiment, the cation-exchange medium comprises a
ligand coupled to a support, wherein the ligand is sulfopropyl
(SP), wherein the support is an agarose. For example,
cation-exchange chromatography can be performed in an
SP-SEPHAROSE.TM. column format. SEPHAROSE.TM. is a registered trade
mark for agarose gel in bead form.
[0041] Preferably, the cation-exchange condition is sufficient for
the cation-exchange medium to selectively or preferentially bind
the plasminogen present in the composition relative to one or more
contaminating molecules that may also be present in the
composition. The contaminating molecule can be a substance present
in the composition that is different from the desired plasminogen
molecule and is desirably excluded from the final plasminogen
product. Contaminants can include, but are not limited to, nucleic
acids, polypeptides including, but not limited to, unfolded and
misfolded plasminogens, cell debris, endotoxins, etc.
[0042] For example, the composition can be passed over an SP column
equilibrated with a suitable buffer. The pH of the suitable buffer
can be at least about pH 3.0, illustratively, at least about: pH
3.0, 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0. In some embodiments, the
suitable buffer has an acidic pH, preferably at least about pH 4.0.
In another embodiment, the suitable buffer has an alkaline pH,
preferably a Tris-based buffer at least about pH 8.0. Following the
load step, the column can be washed with the equilibration buffer
or a different buffer so long as a substantial amount of the
recombinant plasminogen remains bound to the medium.
[0043] In another embodiment, the method further comprises eluting
the recombinant plasminogen that is bound by the cation-exchange
medium to obtain a cation-exchange medium eluate comprising the
recombinant plasminogen. For example, the medium-bound recombinant
plasminogen can be eluted from the cation-exchange medium with an
elution buffer comprising a suitable concentration of a salt. In
one embodiment, a Tris-based buffer comprising at least about 200
mM NaCl is used to elute the recombinant plasminogen from an SP
column.
B. Affinity Chromatography
[0044] The cation-exchange eluate comprising the recombinant
plasminogen can be loaded directly onto a suitable affinity medium
for affinity chromatography. Alternatively, the eluate can be
subjected to further preparation steps prior to affinity
chromatography. In some embodiments, the method for preparing
plasminogen further comprises contacting the cation-exchange medium
eluate with a first affinity medium under a first affinity
condition that is sufficient for the affinity medium to bind the
plasminogen.
[0045] In one embodiment, the first affinity medium comprises a
ligand coupled to a support, wherein the ligand has affinity for
the plasminogen.
[0046] The first affinity medium can be a solid phase that binds
the plasminogen present in the composition. The medium can possess
a chemistry or a ligand coupled thereto that can allow for
selective or preferential capture of the plasminogen from the
composition by way of affinity interactions. Useful affinity
chromatography media comprise a support and one or more ligand(s)
bound thereto that provide(s) the selective or preferential binding
capability for the plasminogen. Useful supports include, by way of
illustrative example, polysaccharides such as agarose and
cellulose, organic polymers such as polyacrylamide,
methylmethacrylate, and polystyrene-divinylbenzene copolymers. It
should be recognized that it is not intended herein to imply that
only organic substrates are suitable for medium substrate use,
since inorganic support materials such as silica and glasses also
can be used.
[0047] In some embodiments, the affinity medium is in the form of
beads, which can be generally spherical, or alternatively the
affinity medium can be usefully provided in particulate or divided
forms having other regular shapes or irregular shapes. The affinity
medium can be of porous or nonporous character, and the medium can
be compressible or incompressible. Preferred affinity media will be
physically and chemically resilient to the conditions employed in
the plasminogen purification process. A wide variety of affinity
media, for example, those wherein the coupled ligand is a lysine,
an antibody, or metal ion, are known in the art.
[0048] In one embodiment, the affinity medium comprises a ligand
coupled to a support. In another embodiment, the ligand is lysine,
wherein the support is an agarose. For example, the first affinity
medium can be performed in an ECH Lysine SEPHAROSE.TM. column
format.
C. Recombinant Plasminogen
[0049] In some embodiments, the plasminogen is a recombinant
plasminogen. For example, a nucleic acid molecule coding for the
plasminogen of the present invention can be prepared from several
sources, for example, through chemical synthesis using the known
DNA sequence or by the use of standard cloning techniques known to
those skilled in the art. cDNA clones carrying the plasminogen
coding sequence can be identified by use of oligonucleotide
hybridization probes specifically designed based on the known
sequence of the plasminogen.
[0050] In one embodiment, the recombinant plasminogen, or a variant
thereof, of the present invention is a recombinant zymogen that is
capable of becoming activated to a functional plasmin enzyme
following an activation event that at least involves proteolytic
cleavage of an Arg-Val peptide bond located between the kringle
domain and the serine protease domain of the zymogen.
[0051] The recombinant plasminogen, or variants, fragments,
derivatives or analogs thereof, can have fibrin- and
antiplasmin-binding as well as activation properties of full-length
native human plasminogen. In various embodiments, the recombinant
plasminogen and/or the plasmin derived therefrom can be
characterized by at least one of the following: [0052] (i) in
particular embodiments, lower molecular weights relative to native
full-length plasmin(ogen) molecules resulting in increased specific
activity (per mg of protein); [0053] (ii) in particular
embodiments, lack of at least two glycosylation sites found in the
native protein, combined with relatively low molecular weights,
facilitates recombinant production of this protein using relatively
inexpensive expression systems; [0054] (iii) in particular
embodiments, the ability of the plasminogen to be activated by the
plasminogen activator streptokinase, urokinase, tPA, and/or
staphylokinase; [0055] (iv) in particular embodiments, the presence
of a single N-terminal kringle domain homologous to a kringle
domain of native human plasminogen, wherein the fibrin-binding
properties of plasmin, which are important for thrombolytic
efficacy, are preserved; [0056] (v) in particular embodiments, the
presence of .alpha.2-antiplasmin-binding sites on the single
N-terminal kringle domain homologous to a kringle domain of native
human plasminogen, which can allow the plasmins to be inhibited
rapidly by this physiological inhibitor of plasmin (a feature which
can prevent bleeding); [0057] (vi) in particular embodiments,
absence of kringle 5, which retains the primary binding site for
intact, undigested fibrin(ogen), can allow use of the plasmin with
reduced depletion of circulating fibrinogen; [0058] (vii) in
particular embodiments, presence of a single N-terminal kringle
domain homologous to a kringle domain of native human plasminogen,
wherein the last four amino acid residues within the kringle domain
are V, P, Q, and C, provides a native-like linkage to the serine
protease domain (i.e., a linkage similar to the naturally occurring
domain juncture between the kringle 5 domain and the serine
protease domain of human plasminogen); and [0059] (viii) in
particular embodiments, following expression of the recombinant
plasminogen, its N-terminus may be cleaved back (e.g., cleaved back
during activation) to provide a native-like N-terminus.
[0060] In other embodiments, the recombinant plasminogen has a
single-kringle-region N-terminal to the activation site and serine
protease domain. In some embodiments, the single kringle region
containing molecules can comprise additional sequences (additional
N-terminal sequences derived from those of native kringle regions
of a native plasminogen) N-terminal to the activation site. The
N-terminal kringle domains can include kringle sequences of
kringles 1 and 4 of native plasmin(ogen) and functional equivalents
thereof. Further, particular embodiments of the recombinant
plasminogens and the plasmins prepared therefrom can exhibit
reduced immunogenicity by virtue of native-like structures. For
example, in some embodiments, the recombinant plasminogen has an
N-terminus identical to that of one of the naturally occurring
forms of human plasma-derived plasminogen, which upon activation by
the plasminogen activator (e.g., streptokinase), produces plasmin
polypeptides comprising native-like N-termini. Additionally, in
other embodiments, the recombinant plasminogen can have a sequence
between the Kringle and Serine protease domains that is similar to
the junction between Kringle 5 and the SP domain in
naturally-occurring human plasmin.
[0061] In another embodiment, the present invention provides a
method for preparing a recombinant plasminogen, wherein the
recombinant plasminogen comprises the sequence shown in SEQ ID NO:1
(FIG. 1). In one embodiment, the recombinant plasminogen
polypeptide is at least 90% or 95%, or 98% identical to the
sequence shown in SEQ ID NO:1. In other embodiments, the
recombinant plasminogen comprises a single kringle domain that is
at least 90% or 95%, or 98% identical to the kringle 1 or kringle 4
domain of native human plasminogen; and the C-terminal domain is at
least 90% or 95%, or 98% identical to the activation site and
serine protease domain of human plasminogen. In some embodiments,
the recombinant plasminogen polypeptide has an amino acid sequence
as shown in SEQ ID NO:1, and conservative substitutions thereof. In
other embodiments, the polypeptide has an arginine residue at a
relative position analogous to that of position 85 of the amino
acid sequence shown in SEQ ID NO:1.
[0062] In other embodiments, the recombinant plasminogens have a
single kringle region N-terminal to the activation site and serine
protease domain, wherein residues at certain positions of the
single N-terminal kringle domain of the plasminogen are conserved
relative to kringle 1 of native human plasminogen. These can be
residues at positions associated with disulfide bridging and lysine
binding, and include Cys84, Cys105, Cys133, Cys145, Cys157, and
Cys162, and Pro136-Pro140, Pro143-Tyr146, and Arg153-Tyr156,
respectively (positions numbered as shown in SEQ ID NO:2 (FIG. 2).
Additionally, particular embodiments of the recombinant plasminogen
can be characterized chemically by contrast to mini-plasmin(ogen)
which has an analogous domain composition (i.e., kringle-serine
protease (K-SP) (see Sottrup-Jensen, L., et al., Progress in
Chemical Fibrinolysis and Thrombolysis, Vol. 3, (Eds: J. F.
Davidson, et al.) Raven Press, New York (1978)) but, inter alia,
lacks an arginine (Arg) at a relative position analogous to that of
position 85 of the amino acid sequence shown in SEQ ID NO:1 (FIG.
1).
[0063] In some embodiments, the recombinant plasminogen of the
invention comprises a single N-terminal kringle domain comprising
an Arg residue at a relative position analogous to that of position
85 of the amino acid sequence shown in SEQ ID NO:1. Non-limiting
examples of a relative position analogous to that of position 85 of
the amino acid sequence shown in SEQ ID NO:1 include Arg(153),
Arg(234), Arg(324), and Arg(426) positions of the amino acid
sequence shown in SEQ ID NO:2 (FIG. 2).
[0064] In other embodiments, the specific positions of the named
residues can vary somewhat while still being present in the
polypeptide at structurally and functionally analogous positions
(i.e., relative to the kringle structure of the N-terminal domain;
see Chang, Y., et al. as discussed above). In some embodiments, the
single N-terminal kringle domain of the plasmin(ogen) polypeptide
has at least one residue greater percent identity with kringle 1 or
kringle 4 of native human plasminogen than with kringle 5 of native
human plasminogen.
[0065] Further, in particular embodiments, the recombinant
plasminogen can be characterized functionally by contrast to
mini-plasmin(ogen). In one embodiment, the plasmin prepared from
the recombinant plasminogen exhibits an increased rate of
inhibition by .alpha.2-antiplasmin, e.g., as much as about one or
two orders of magnitude faster than the rate of inhibition of
mini-plasmin.
[0066] Characterization of the single N-terminal kringle domain of
the plasminogen as "N-terminal" means only that the domain is
present N-terminal to the activation site and does not mean that
additional amino acid residues N-terminal to the domain itself are
not present. Further, the number and identity of residues
interposed between the most C-terminal cysteine residue of the
single N-terminal kringle domain (i.e., the most C-terminal Cys
residue shown in FIG. 3) and the activation site of plasminogen can
be varied without departing from the scope of the present
invention. One of skill in the art will be able to determine these
variations (kringle 1-like binding of .omega. aminocarboxylic
acids, without substantial increase in size of the deletion mutant
or introduction of potentially problematic glycosylation sites)
without undue experimentation based on the disclosure herein and
the references cited herein for guidance regarding kringle 1
function and structure.
[0067] It will further be appreciated that, depending on the
criteria used, the exact "position" or sequence of the kringle,
activation site, and serine protease domains of the recombinant
plasminogen can differ slightly in particular variations within the
scope of the present invention. For example, the exact location of
the kringle domain relative to the activation site can vary
slightly and/or the sequence N-terminal to the kringle domain can
vary in length. Such variants can include, but are to limited to,
deletions, insertions, inversions, repeats, and substitutions.
Guidance concerning which amino acid changes are likely to be
phenotypically silent can be found in Bowie, J. U., et al.,
"Deciphering the Message in Protein Sequences: Tolerance to Amino
Acid Substitutions," Science 247:1306-1310 (1990).
[0068] Thus, variants, fragments, derivatives or analogs of the
polypeptide of SEQ ID NO:1 can be (i) ones in which one or more of
the amino acid residues (e.g., 3, 5, 8, 10, 15 or 20 residues) are
substituted with a conserved or non-conserved amino acid residue
(preferably a conserved amino acid residue). Such substituted amino
acid residues may or may not be one encoded by the genetic code, or
(ii) ones in which one or more of the amino acid residues includes
a substituent group (e.g., 3, 5, 8, 10, 15 or 20), or (iii) ones in
which the mature polypeptide is fused with another compound, such
as a compound to increase the half-life of the polypeptide (for
example, polyethylene glycol), or (iv) ones in which the additional
amino acids are fused to the mature polypeptide, such as an IgG Fc
fusion region peptide or leader or secretory sequence or a sequence
which is employed for purification of the mature polypeptide or a
proprotein sequence. Such fragments, derivatives and analogs are
deemed to be within the scope of those skilled in the art from the
teachings herein.
[0069] As indicated, changes are preferably of a minor nature, such
as conservative amino acid substitutions that do not significantly
affect the folding or activity of the protein. Of course, the
number of amino acid substitutions a skilled artisan would make
depends on many factors, including those described above. Generally
speaking, the number of substitutions for any given plasminogen
polypeptide will not be more than 50, 40, 30, 25, 20, 15, 10, 5 or
3.
[0070] Amino acids in the recombinant plasminogen that are
essential for function can be identified by methods known in the
art, such as site-directed mutagenesis or alanine-scanning
mutagenesis (Cunningham and Wells, Science 244:1081-1085 (1989)).
The latter procedure introduces single alanine mutations at every
residue in the molecule. The resulting mutant molecules are then
tested for biological activity, e.g., as shown in the examples
provided herein. Sites that are critical for ligand binding can
also be determined by structural analysis such as crystallization,
nuclear magnetic resonance or photoaffinity labeling (Smith, et
al., J. Mol. Biol. 224:399-904 (1992) and de Vos, et al. Science
255:306-312 (1992)). Even if deletion of one or more amino acids
from the N-terminus of a protein results in modification or loss of
one or more biological functions of the protein, other biological
activities can still be retained.
[0071] It is also contemplated that recombinant plasminogens can be
produced by solid phase synthetic methods. See Houghten, R. A.,
Proc. Natl. Acad. Sci. USA 82:5131-5135 (1985); and U.S. Pat. No.
4,631,211 to Houghten et al. (1986).
[0072] Polypeptides having an amino acid sequence of an indicated
percent identity to a reference amino acid sequence of SEQ ID NO:1
can be determined using the methods, including computer-assisted
methods, indicated above regarding polynucleotides. Polypeptide
amino acid sequences are examined and compared just as are the
nucleotide sequences in the foregoing discussion. One of skill in
the art will recognize that such concepts as the molecular
endpoints discussed for polynucleotides will have direct analogs
when considering the corresponding use of such methods and programs
for polypeptide analysis. For example, the manual corrections
discussed regarding polynucleotides refer to 5' and 3' endpoints of
nucleic acids, but the same discussion will be recognized as
applicable to N-termini and C-termini of polypeptides.
[0073] The invention also encompasses recombinant plasminogen
polypeptides which are differentially modified during or after
translation, e.g., by glycosylation, acetylation, phosphorylation,
amidation, derivatization by known protecting/blocking groups,
proteolytic cleavage, linkage to an antibody molecule or other
cellular ligand, etc. Any of numerous chemical modifications can be
carried out by known techniques, including but not limited, to
specific chemical cleavage by cyanogen bromide, trypsin,
chymotrypsin, papain, S. aureus V8 protease, NaBH4; acetylation,
deamidation, formylation, methylation, oxidation, reduction;
metabolic synthesis in the presence of tunicamycin; etc.
[0074] Additional post-translational modifications encompassed by
the invention include, for example, e.g., N-linked or O-linked
carbohydrate chains, processing of N-terminal or C-terminal ends,
attachment of chemical moieties to the amino acid backbone,
chemical modifications of N-linked or O-linked carbohydrate chains,
and addition of an N-terminal methionine residue as a result of
vectors and constructs adapted for expression of the recombinant
plasminogen polypeptides, for example for expression in prokaryotic
cultured host cells. In some embodiments, the recombinant
plasminogen also can be modified, for example with an affinity
label (e.g., His-tags, GST-tags) or a detectable label such as an
enzymatic, fluorescent, or isotopic label.
D. Vectors and Host Cells
[0075] In other aspects, the present invention also relates to kits
and vectors that include the recombinant plasminogen molecules of
the present invention; to cultured host cells which are genetically
engineered with the recombinant vectors; and to the recombinant
expression of the plasminogen polypeptides by recombinant
techniques. In one embodiment, the method for preparing plasminogen
or a plasmin prepared therefrom comprises expressing the
recombinant plasminogen using a recombinant expression system.
[0076] The origin of the host cell for protein expression is not to
be limited, for example the host cell can be exemplified by
microorganisms such as bacteria (e.g., those belonging to the genus
Escherichia and those belonging to the genus Bacillus) and yeast
(e.g., the genus Saccharomyces and the genus Pichia). For example,
the genus Escherichia includes, but is not limited to, Escherichia
coli (E. coli) K12DH1, M103, JA221, HB101, X600, XL-1 Blue and
JM109. For example, the genus Bacillus includes, but is not limited
to, Bacillus subtilis MI114 and 207-21. For example, the yeast
includes, but is not limited to, Saccharomyces cerevisiae AH22,
AH22.sup.R-, NA87-11A and DKD-5D and Pichia pastoris. U.S. Pat. No.
6,068,995 is herein incorporated by reference for its teaching of
producing a protein by way of a host cell capable of expressing the
desired protein.
[0077] For example, a molecule having the plasminogen coding
sequence (e.g., SEQ ID NO:1) can be inserted into a cloning vector
appropriate for expression in a host cell. The cloning vector can
be constructed so as to provide the appropriate regulatory
functions required for the efficient transcription, translation,
and processing of the coding sequence. Suitable host cells for
expressing the DNA encoding the plasminogen include prokaryote,
yeast, or higher eukaryotic cells. Suitable prokaryotes include
e.g., bacteria such as archaebacteria and eubacteria. Preferred
bacteria are eubacteria, such as Gram-negative or Gram-positive
organisms, for example, Enterobacteriaceae such as E. coli.
Further, the vector can be, for example, a plasmid, a phage, a
viral or retroviral vector. Retroviral vectors can be replication
competent or replication defective. In the latter case, viral
propagation generally will occur only in complementing cultured
host cells. In one embodiment, the recombinant expression system is
an E. coli-based expression system.
[0078] In addition to prokaryotes, eukaryotic microbes such as
filamentous fungi or yeast also can be suitable expression hosts
for plasminogen-encoding vectors. For example, Saccharomyces
cerevisiae can be used. In another embodiment, the recombinant
expression system is a Pichia-based expression system. However, a
number of other genera, species, and strains are commonly available
and useful herein.
[0079] Suitable host cells appropriate for the expression of the
DNA encoding the plasminogen also can be derived from multicellular
organisms. Examples of invertebrate cells include plant (e.g., from
the duckweed family such as Lemna) and insect cells. Plant cell
cultures of cotton, corn, potato, soybean, petunia, tomato, and
tobacco also can be utilized as hosts. Regulatory and signal
sequences compatible with plant cells are available. U.S. Pat. No.
6,815,184 is herein incorporated by reference for its teaching of
expressing a polypeptide in duckweed.
[0080] A plasminogen expression vector can be introduced into the
host cells and host cells cultured in conventional nutrient media
modified as appropriate for inducing promoters, selecting
transformants, or amplifying the genes encoding the desired
plasminogen sequences. Numerous methods of transfection are known
to the ordinarily skilled artisan, for example, calcium phosphate
and electroporation. Depending on the host cell used,
transformation is performed using standard techniques appropriate
to such cells. For example, if prokaryotic cells are used to
produce plasminogen, they can be cultured in suitable media in
which the promoter can be constitutively or artificially induced as
described generally, e.g., in Sambrook et al., Molecular Cloning: A
Laboratory Manual (Cold Spring Harbor Laboratory Press, New York
1989). Thus, recombinant constructs can be introduced into cultured
host cells using well-known techniques such as infection,
transduction, transfection, transvection, electroporation and
transformation.
[0081] In some embodiments, polynucleotides coding for the
recombinant plasminogen can be joined to a vector containing a
selectable marker for propagation in a cultured host. Preferred are
vectors comprising cis-acting control regions to the polynucleotide
coding the plasminogen. Appropriate trans-acting factors can be
supplied by the cultured host, supplied by a complementing vector
or supplied by the vector itself upon introduction into the
cultured host. In some embodiments, the vectors provide for
specific expression, which can be inducible and/or cell
type-specific. In one embodiment, among such vectors are those
inducible by environmental factors that are easy to manipulate,
such as temperature and nutrient additives.
[0082] Non-limiting examples of expression vectors useful in the
present invention include chromosomal-, episomal- and virus-derived
vectors, e.g., vectors derived from bacterial plasmids,
bacteriophage, yeast episomes, yeast chromosomal elements, viruses
such as baculoviruses, papova viruses, vaccinia viruses,
adenoviruses, fowl pox viruses, pseudorabies viruses and
retroviruses, and vectors derived from combinations thereof, such
as cosmids and phagemids.
[0083] DNA inserts can be operatively linked to an appropriate
promoter, such as the phage lambda PL promoter, the E. coli lac,
trp and tac promoters, the SV40 early and late promoters and
promoters of retroviral LTRs, to name a few. Other suitable
promoters will be known to the skilled artisan. The expression
constructs will further contain sites for transcription initiation,
termination and, in the transcribed region, a ribosome binding site
for translation. The coding portion of the mature transcripts
expressed by the constructs can include a translation initiating at
the beginning and a termination codon (UAA, UGA or UAG)
appropriately positioned at the end of the polypeptide to be
translated.
[0084] As indicated, the expression vectors can include at least
one selectable marker. Such markers include dihydrofolate reductase
or neomycin resistance for eukaryotic cell culture and tetracycline
or ampicillin resistance genes for culturing in E. coli and other
bacteria. Non-limiting examples of appropriate cultured hosts
include, but are not limited to, bacterial cells, such as E. coli,
Streptomyces and Salmonella typhimurium cells; fungal cells, such
as yeast cells; insect cells such as Drosophila S2 and Spodoptera
Sf9 cells; animal cells such as CHO, COS and Bowes melanoma cells;
and plant cells. Appropriate culture media and conditions for the
above-described cultured host cells are known in the art.
[0085] For example, in other embodiments, the recombinant
expression system is a mammalian-based expression system. Mammalian
cell lines available as hosts for expression are known in the art
including immortalized cell lines available from the American Type
Culture Collection (ATCC). Exemplary mammalian host cells include,
but are not limited to, primate cell lines and rodent cell lines,
including transformed cell lines. Preferably, for stable
integration of the vector DNA, and for subsequent amplification of
the integrated vector DNA, both by conventional methods, Chinese
hamster ovary (CHO) cells are employed as a mammalian host cell of
choice. Other suitable cell lines include, but are not limited to,
HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells
(COS-1), human hepatocellular carcinoma cells (e.g., Hep G2), human
adenovirus transformed 293 cells, mouse L-929 cells, HaK hamster
cell lines, murine 3T3 cells derived from Swiss, Balb-c or NIH mice
and a number of other cell lines. Another suitable mammalian cell
line is the CV-1 cell line. Normal diploid cells, cell strains
derived from in vitro culture of primary tissue, as well as primary
explants, are also suitable. Candidate cells may be genotypically
deficient in the selection gene, or may contain a dominantly acting
selection gene.
[0086] For example, the host cells can be transformed with the one
or more vectors carrying the plasminogen DNA, e.g., by methods
known in the art, and can then be cultured under suitable
conditions if desired, with amplification of one or both introduced
genes. The expressed plasminogen can then be recovered and purified
from the culture medium (or from the cell, for example if expressed
intracellularly) by methods known to one of skill in the art.
[0087] Vectors suitable for replication in mammalian cells can
include viral replicons, or sequences that ensure integration of
the sequence encoding plasminogen into the host genome. Suitable
vectors can include, for example, those derived from simian virus
SV40, retroviruses, bovine papilloma virus, vaccinia virus, and
adenovirus. The components of the vectors, e.g., replicons,
selection genes, enhancers, promoters, and the like, may be
obtained from natural sources or synthesized by known
procedures.
[0088] A suitable vector, for example, can be one derived from
vaccinia viruses. In this case, the heterologous DNA is inserted
into the vaccinia genome. Techniques for the insertion of foreign
DNA into the vaccinia virus genome are known in the art, and
utilize, for example, homologous recombination. The insertion of
the heterologous DNA is generally into a gene which is
non-essential in nature, for example, the thymidine kinase gene
(tk), which also provides a selectable marker.
[0089] Mammalian expression vectors can comprise one or more
eukaryotic transcription units that are capable of expression in
mammalian cells. For example, the transcription unit can comprise
at least a promoter element to mediate transcription of foreign DNA
sequences. Suitable promoters for mammalian cells are known in the
art and include viral promoters such as that from simian virus 40
(SV40), cytomegalovirus (CMV), Rous sarcoma virus (RSV), adenovirus
(ADV), and bovine papilloma virus (BPV).
[0090] The transcription unit also can comprise a termination
sequence and poly(A) addition sequences operably linked to the
plasminogen sequence. The transcription unit also can comprise an
enhancer sequence for increasing the expression of plasminogen.
[0091] Optionally, sequences that allow for amplification of the
gene also can be included, as can sequences encoding selectable
markers. Selectable markers for mammalian cells are known in the
art, and include for example, thymidine kinase, dihydrofolate
reductase (together with methotrexate as a DHFR amplifier),
aminoglycoside phosphotransferase, hygromycin B phosphotransferase,
asparagine synthetase, adenosine deaminase, metallothionien, and
antibiotic resistant genes such as neomycin. Or, for example, the
vector DNA can comprise all or part of the bovine papilloma virus
genome and be carried in cell lines such as C127 mouse cells as a
stable episomal element.
[0092] In one embodiment, the recombinant plasminogen can be
prepared using the PER.C6.RTM. technology (Crucell, Holland, The
Netherlands). Expression of recombinant proteins is disclosed by,
e.g., U.S. Pat. No. 6,855,544, which is herein incorporated by
reference for its teaching of methods and compositions for the
production of recombinant proteins in a human cell line.
[0093] Among vectors preferred for use in bacteria include e.g.,
pET24b or pET22b available from Novagen, Madison, Wis. (pET-24b(+)
and pET-22b(+)=pET Expression System 24b (Cat. No. 69750) and 22b
(Cat. No. 70765), respectively, EMD Biosciences, Inc., Novagen
Brand, Madison, Wis.; see http://www.emdbiosciences.com product
information section regarding pET-24b and pET-22b for details
regarding vector), pQE70, pQE60 and pQE-9, available from Qiagen
Inc., Valencia, Calif.; pBS vectors, PHAGESCRIPT vectors,
BLUESCRIPT vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from
Stratagene, LaJolla, Calif.; and ptrc99a, pKK223-3, pKK233-3,
pDR540, pRIT5 available from Pharmacia (now Pfizer, Inc., New York,
N.Y.). Among preferred eukaryotic vectors are pWLNEO, pSV2CAT,
pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV,
pMSG and pSVL available from Pharmacia. Other suitable vectors will
be readily apparent to the skilled artisan.
[0094] Bacterial promoters suitable for use in the present
invention include the E. coli lad and lacZ promoters, the T3 and T7
promoters, the gpt promoter, the lambda PR and PL promoters, and
the trp promoter. Suitable eukaryotic promoters include the CMV
immediate early promoter, the HSV thymidine kinase promoter, the
early and late SV40 promoters, the promoters of retroviral LTRs,
such as those of the Rous sarcoma virus (RSV), and metallothionein
promoters, such as the mouse metallothionein-I promoter.
[0095] In some embodiments, introduction of a vector construct into
the cultured host cell can be effected by calcium phosphate
transfection, DEAE-dextran mediated transfection, cationic
lipid-mediated transfection, electroporation, transduction,
infection or other methods. Such methods are described in many
standard laboratory manuals, such as Davis et al., Basic Methods In
Molecular Biology, 2nd Edition (1995).
[0096] Transcription of the DNA encoding the plasminogens of the
present invention by higher eukaryotes can be increased by
inserting an enhancer sequence into the vector. Enhancers are
cis-acting elements of DNA, usually about from 10 to 300 bp that
act to increase transcriptional activity of a promoter in a given
cultured host cell-type. Examples of enhancers include the SV40
enhancer, which is located on the late side of the replication
origin at by 100 to 270, the cytomegalovirus early promoter
enhancer, the polyoma enhancer on the late side of the replication
origin, and adenovirus enhancers.
[0097] For secretion of the translated protein into the lumen of
the endoplasmic reticulum, into the periplasmic space or into the
extracellular environment, appropriate secretion signals can be
incorporated into the expressed polypeptide. The signals can be
endogenous to the polypeptide or they can be heterologous
signals.
[0098] In various embodiments, the plasminogens can be expressed in
a modified form, such as a fusion protein, and can include not only
secretion signals, but also additional heterologous functional
regions. For instance, a region of additional amino acids,
particularly charged amino acids, can be added to the N-terminus,
for example, the polypeptide to improve stability and persistence
in the cultured host cell, during purification, or during
subsequent handling and storage. Also, peptide moieties can be
added to the polypeptide to facilitate purification. Such regions
can be removed prior to final preparation of the polypeptide. The
addition of peptide moieties to polypeptides to engender secretion
or excretion, to improve stability and to facilitate purification,
among others, are familiar and routine techniques in the art. A
preferred fusion protein comprises a heterologous region from
immunoglobulin that is useful to solubilize proteins. For example,
EP 0 464 533 A1 (Canadian counterpart, 2,045,869) discloses fusion
proteins comprising various portions of constant region of
immunoglobulin molecules together with another human protein or
part thereof. In many cases, the Fc part in a fusion protein is
thoroughly advantageous for use in therapy and diagnosis and thus
results, for example, in improved pharmacokinetic properties. On
the other hand, for some uses it would be desirable to be able to
delete the Fc part after the fusion protein has been expressed,
detected and purified in the advantageous manner described. This is
the case when Fc portion proves to be a hindrance to use in therapy
and diagnosis, for example when the fusion protein is to be used as
antigen for immunizations. In drug discovery for example, human
proteins have been fused with Fc portions for the purpose of
high-throughput screening assays (such as hIL5-receptor, to
identify antagonists of hIL-5). See, Bennett, D., et al., J.
Molecular Recognition, 8:52-58(1995) and Johanson, K. et al., J.
Biol. Chem., 270(16):9459-9471 (1995).
[0099] In one embodiment, insoluble plasminogen is isolated from
the prokaryotic host cells in a suitable isolation buffer. For
example, the host cells can be exposed to a buffer of suitable
ionic strength to solubilize most host proteins, but in which
aggregated plasminogen is substantially insoluble, and disrupting
the cells so as to release the inclusion bodies and make them
available for recovery by, for example, centrifugation. This
technique is known to one of ordinary skill in the art, and a
variation is described, for example, in U.S. Pat. No. 4,511,503,
which is incorporated by reference herein for its teaching of a
method of solubilizing heterologous protein, produced in an
insoluble refractile form in a recombinant host cell culture. In
one embodiment, the step of expressing the recombinant plasminogen
comprises performing the expression system under an expression
condition sufficient to produce a recombinant plasminogen inclusion
body.
[0100] Without being held to a particular theory, it is believed
that expression of a recombinant protein, in e.g. E. coli,
frequently results in the intracellular deposition of the
recombinant protein in insoluble aggregates called inclusion
bodies. Deposition of recombinant proteins in inclusion bodies can
be advantageous both because the inclusion bodies accumulate highly
purified recombinant protein and because protein sequestered in
inclusion bodies is protected from the action of bacterial
proteases.
[0101] Generally, host cells (e.g., E. coli cells) are harvested
after an appropriate amount of growth and suspended in a suitable
buffer prior to disruption by lysis using techniques such as, for
example, mechanical methods (e.g., sonic oscillator) or by chemical
or enzymatic methods. Examples of chemical or enzymatic methods of
cell disruption include spheroplasting, which comprises the use of
lysozyme to lyse bacterial wall, and osmotic shock, which involves
treatment of viable cells with a solution of high tonicity and with
a cold-water wash of low tonicity to release the polypeptides.
[0102] Following host cell disruption, the suspension is typically
centrifuged to pellet the inclusion bodies. The resulting pellet
contains substantially all of the insoluble polypeptide fraction,
but if the cell disruption process is not complete, it may also
contain intact cells or broken cell fragments. Completeness of cell
disruption can be assayed by resuspending the pellet in a small
amount of the same buffer solution and examining the suspension
with a phase-contrast microscope. The presence of broken cell
fragments or whole cells indicates that additional disruption is
necessary to remove the fragments or cells and the associated
non-refractile polypeptides. After such further disruption, if
required, the suspension can be again centrifuged and the pellet
recovered, resuspended, and analyzed. The process can be repeated
until visual examination reveals the absence of broken cell
fragments in the pelleted material or until further treatment fails
to reduce the size of the resulting pellet. Once obtained from the
solubilized inclusion bodies or at a later stage of purification,
the plasminogen can be suitably refolded in accordance with the
present invention. The degree of any unfolding can be determined by
chromatography including reversed phase-high performance liquid
chromatography (RP-HPLC).
[0103] If the plasminogen is not already in soluble form before it
is to be refolded, it may be solubilized by incubation in a
solubilization buffer comprising chaotropic agent (e.g., urea,
guanidine) and reducing agent (e.g., glutathione, DTT, cysteine) in
amounts necessary to substantially solubilize the plasminogen. This
incubation takes place under conditions of plasminogen
concentration, incubation time, and incubation temperature that
will allow solubilization of the plasminogen to occur. Measurement
of the degree of solubilization of the plasminogen in the buffer
can be carried out by turbidity determination, by analyzing
plasminogen fractionation between the supernatant and pellet after
centrifugation on reduced SDS gels, by protein assay (e.g., the
Bio-Rad protein assay kit), or by high performance liquid
chromatography (HPLC). In one embodiment, the method of preparing
the recombinant plasminogen further comprises contacting the
recombinant plasminogen inclusion body with a solubilization buffer
under a solubilization condition sufficient to obtain a solubilized
recombinant plasminogen inclusion body.
[0104] The pH of the solubilization buffer can be alkaline,
preferably at least about pH 7.5, with the preferred range being
about pH 7.5 to about pH 11. The concentration of plasminogen in
the buffered solution for solubilization must be such that the
plasminogen will be substantially solubilized and partially or
fully reduced and denatured. Alternatively, the plasminogen may be
initially insoluble. The exact amount to employ will depend, e.g.,
on the concentrations and types of other ingredients in the
buffered solution, particularly the type and amount of reducing
agent, the type and amount of chaotropic agent, and the pH of the
buffer. For example, the concentration of plasminogen can be
increased if the concentration of reducing agent, e.g.,
glutathione, is concurrently increased.
[0105] In other embodiments, the method of preparing the
plasminogen further comprises contacting the solubilized
recombinant plasminogen inclusion body with a refolding solution
under a refolding condition to obtain a composition comprising the
recombinant plasminogen. In some embodiments, it is desirable to
produce a more concentrated solubilized protein solution prior to
dilution refolding. For example, in one embodiment, the plasminogen
is diluted with a refolding buffer, preferably at least about five
fold, more preferably at least about ten to about twenty fold. In
other embodiments, the plasminogen is dialyzed against the
refolding buffer.
[0106] The concentration of plasminogen in the refolding buffer can
be such that the ratio of correctly folded to misfolded conformer
recovered will be maximized, as determined by HPLC, RIA, or
bioassay. The refolding incubation is carried out to maximize the
yield of correctly folded plasminogen conformer and the ratio of
correctly folded plasminogen conformer to misfolded plasminogen
conformer recovered, as determined by RIA or HPLC, and to minimize
the yield of multimeric, associated plasminogen as determined by
mass balance, for example.
[0107] In other embodiments, refolding is performed employing a
high-pressure refolding technique. Thus, in some embodiments, the
present invention provides a method for preparing plasminogen,
wherein the method comprises refolding comprising: [0108] (a)
adjusting total protein concentration in a mixture to a first
concentration of at least about 0.01 mg/mL, wherein the mixture
comprises a plasminogen; [0109] (b) subjecting the mixture to a
first pressure of about 0.25 kbar to about 12 kbar for a first time
and a first temperature; [0110] (c) subjecting the mixture to a
second pressure of about 0.25 kbar to about 3.3 kbar for a second
time; and [0111] (d) subjecting the mixture to a third pressure,
whereby the plasminogen in the mixture is disaggregated and
refolded.
[0112] High pressure refolding is disclosed in, e.g., U.S. Pat. No.
7,064,192, which is incorporated by reference herein for its
teaching of a method of refolding protein aggregates and inclusion
bodies.
[0113] In one embodiment, the first concentration is no greater
than 500 mg/mL. In another embodiment, refolding comprises:
contacting the mixture with a chaotropic agent at a second
concentration of no greater than 8 M. In some embodiments, the
second time is about 0.1 hours to about 12 hours. In other
embodiments, the third pressure is about atmospheric pressure. In
particular embodiments, subjecting the mixture to the first
pressure is sufficient to disaggregate the plasminogen. In various
other embodiments, the mixture comprises solubilized inclusion
bodies corresponding to recombinantly prepared plasminogen.
[0114] For example, overexpression of recombinant plasminogen in E.
coli can form inclusion bodies that correspond to relatively dense,
insoluble particles of aggregated plasminogen protein. In one
embodiment, once isolated, the inclusion bodies can be solubilized
by a variety of techniques, or a combination of pressure and
chaotropic agent (and optionally, also a reducing agent).
Renaturation to a biologically proper plasminogen conformation can
proceed under conditions of elevated pressure, and optionally in
the presence of a non-denaturing (e.g., at ambient atmospheric
pressures) concentration of chaotropic agent and/or redox reagents
(e.g., dithiothreitol and oxidized glutathione).
[0115] For example, pressure can be generated using high-pressure
nitrogen (e.g., 400 bar) connected to a 10-fold hydraulic
intensifier equipment (High Pressure Equipment Company, Erie, Pa.).
Time to reach the desired pressure can be about 10 min. The
nitrogen input can be connected to a 10-fold hydraulic intensifier,
which can be connected to a 2-liter cloverleaf reactor rated to
about 30,000 psi (2 kbar). For higher pressures, equipment can be
modified for higher ratings. Samples can be prepared in heat-sealed
bulbs of SAMCO.TM. plastic transfer pipettes (Fisher Scientific,
Pittsburgh, Pa.), for example, and placed in a 2 liter clover leaf
reactor rated to 2,000 bar and filled with water. Samples can be
slowly pressurized (over 10 minutes) to the final desired pressure.
The depressurization rate can be about 10 bar per minute.
[0116] Another embodiment of the invention employs
pressure-facilitated refolding of denatured plasminogen. In this
embodiment, denatured plasminogen in solution is provided in the
presence of denaturing amounts of a chaotropic agent. The
plasminogen concentration in solution can be about 0.001 mg/mL to
about 500 mg/mL, preferably about 0.1 mg/mL to about 25 mg/mL, more
preferably about 1 mg/mL to about 10 mg/mL. The chaotropic agent
concentration can be about 2 M to about 8 M. The denatured
plasminogen solution can be incubated at elevated pressure in the
pressure range effective for facilitating renaturation, which, in
some embodiments, can be about 0.25 kbar to about 3.3 kbar,
preferably from about 2 kbar to about 3.3 kbar. While under
pressure, the concentration of chaotropic agent can be reduced by
any suitable means, for example, by dilution or by dialysis.
Incubation can take place for a time sufficient to permit refolding
of the plasminogen. At the end of the pressure incubation period,
the pressure can be reduced to about atmospheric pressure. In other
embodiments, redox agents, stability agents, surfactants and the
like can be added to the solution.
[0117] In one embodiment, the refolding composition can be
subjected to one or more filtering or diafiltering steps following
the step of refolding but prior to further downstream processing to
eliminate or substantially reduce aggregated forms of recombinant
plasminogen following the refolding step. For example, refolding
mixtures can be passed through a depth filter, then diafiltrated,
followed by a subsequent filtration prior to any downstream
chromatography. In some embodiments, recovery of plasminogen
activity through one or more sequential filtration and/or
diafiltration is at least about 70%, preferably at least about 80%,
90%, or more. In one embodiment, the method for preparing the
plasminogen further comprises diafiltering the composition
subsequent to the step of refolding and prior to the step of
contacting with the cation-exchange medium.
[0118] In other embodiments, aggregated recombinant plasminogen is
selectively precipitated by adding an appropriate concentration of
polyethylene glycol (PEG) or a salt of a sulfate to the refolding
mixture. Under the appropriate conditions, the aggregated protein
can be rendered insoluble by the added PEG or salt, and most of the
correctly refolded recombinant protein can be maintained in
solution. Following removal of the precipitated protein aggregates
by either centrifugation or filtration, the resulting
supernatant/filtrate can be further processed. For example, in one
embodiment, at the completion of the refolding step, solid PEG or
salt (e.g., ammonium sulfate or a nonvolatile salt of sulfate such
as sodium or potassium) can be added to an appropriate
concentration, and the mixture mixed to dissolve the PEG or salt
and to allow the resultant precipitation of aggregated protein to
proceed. Following a precipitation time, the precipitate can be
removed by one or more clarification methods (e.g., depth
filtration, centrifugation, microfiltration, etc., and combinations
thereof), preferably by depth filtration. The resulting filtrate
can then be subjected to further processing to prepare recombinant
plasminogen.
[0119] Examples of PEG include, but are not limited to, PEG 200,
PEG 300, PEG 400, PEG 600, PEG 1000, PEG 2000, PEG 3350, PEG 4000,
PEG 4600, PEG 5000, PEG 6000, and PEG 8000. In one embodiment, a
PEG is added to a refolding solution under a precipitation
condition, wherein the refolding solution comprises the plasminogen
and aggregated polypeptides, wherein the plasminogen is refolded
plasminogen, wherein the precipitation condition is sufficient to
precipitate all or a substantial portions of the aggregated
proteins. In other embodiments, the PEG is added to achieve final
concentrations of PEG of at least about 1% (w/v), illustratively,
about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, and 20% (w/v). In some embodiments, a first PEG
precipitation is performed and at least one further PEG
precipitation.
[0120] In other embodiments, ammonium sulfate (e.g., solid ammonium
sulfate) is added to the refolding solution to achieve final
concentrations of ammonium sulfate corresponding to at least about
1% saturation, illustratively, at least about 1, 5, 10, 15, 20, 25,
30, 35, 40, 45, 50, 55, 60, and 70%.
[0121] In one embodiment, the resulting filtrate (following PEG or
salt precipitation) can be contacted with a suitable hydrophobic
interaction chromatography medium under a suitable hydrophobic
interaction condition sufficient such that the hydrophobic
interaction chromatography medium preferentially captures the
plasminogen. The hydrophobic interaction medium can be a solid
phase that binds the plasminogen. The hydrophobic interaction
chromatography medium can be selected from any of the group of
chromatography media commonly described as hydrophobic interaction
media. The medium can possess a chemistry or a ligand coupled
thereto that can allow for selective or preferential capture of the
plasminogen. Useful chromatography media comprise a support and one
or more ligand(s) bound thereto that provide(s) the selective or
preferential binding capability for the plasminogen. Useful
supports include, by way of illustrative example, polysaccharides
such as agarose and cellulose, organic polymers such as
polyacrylamide, methylmethacrylate, and polystyrene-divinylbenzene
copolymers. It should be recognized that it is not intended herein
to imply that only organic substrates are suitable for medium
substrate use, since inorganic support materials such as silica and
glasses also can be used.
[0122] In some embodiments, the hydrophobic interaction medium is
in the form of beads, which can be generally spherical, or
alternatively the second affinity medium can be usefully provided
in particulate or divided forms having other regular shapes or
irregular shapes. In one embodiment, the medium is in the form of a
membrane. The hydrophobic interaction medium can be of porous or
nonporous character, and the medium can be compressible or
incompressible. Preferred hydrophobic interaction media will be
physically and chemically resilient to the conditions employed in
the purification process. A wide variety of hydrophobic interaction
media, for example, those wherein the coupled ligand is a phenyl,
octyl, or butyl moiety, are known in the art.
[0123] In one embodiment, the hydrophobic interaction medium
comprises a ligand coupled to a support, wherein the ligand is a
phenyl moiety, wherein the support is an agarose. For example,
hydrophobic interaction chromatography can be performed in a
phenyl-Sepharose.TM. column format.
[0124] In some embodiments, following removal of the precipitated
plasminogen aggregates by either centrifugation or filtration, the
resulting supernatant/filtrate comprising the correctly refolded
recombinant plasminogen, optionally, is captured and purified
directly by hydrophobic interaction chromatography.
[0125] If desired, the use of selective precipitation and/or
hydrophobic interaction chromatography can, but need not, replace
the one or more filtering/diafiltering steps of the refolding
mixture thereby providing an alternative approach to purification
of the recombinant protein.
[0126] In some embodiments, following the step(s) of subjecting the
refolding mixture to one or more filtering/diafiltering steps
and/or a selective precipitation steps and/or hydrophobic
interaction chromatography, the resulting solution comprising the
recombinant plasminogen can be further purified by contacting the
resulting solution with the cation-exchange medium (e.g.,
SP-SEPHAROSE.TM.) and/or the first affinity medium (e.g.,
ECH-Lysine column).
[0127] Following one or more preparation steps in accordance with
the present invention, the plasminogen thus prepared can be
activated to obtain plasmin or stored at a suitable temperature
(e.g., -20.degree. C., -80.degree. C.) prior to activation of the
plasminogen.
[0128] Thus, in particular embodiments, the present invention
provides a method for preparing plasminogen, the method comprising:
[0129] (a) expressing a recombinant plasminogen using a recombinant
expression system under an expression condition; [0130] (b)
contacting the recombinant plasminogen with a solubilization buffer
under a solubilization condition sufficient to obtain a solubilized
recombinant plasminogen; [0131] (c) contacting the solubilized
recombinant plasminogen with a refolding solution under a refolding
condition to obtain a composition comprising the recombinant
plasminogen; [0132] (d) diafiltering the composition subsequent to
step (c); [0133] (e) contacting the composition with a
cation-exchange medium under an ion-exchange condition that is
sufficient for the cation-exchange medium to bind the recombinant
plasminogen; [0134] (f) eluting the recombinant plasminogen
captured by the cation-exchange medium to obtain a cation-exchange
medium eluate comprising the recombinant plasminogen; [0135] (g)
contacting the cation-exchange medium eluate with a first affinity
medium under a first affinity condition that is sufficient for the
first affinity medium to bind the recombinant plasminogen; and
[0136] (h) eluting the recombinant plasminogen bound by the first
affinity medium to obtain a plasminogen solution comprising the
recombinant plasminogen.
[0137] In one embodiment, the expression condition is sufficient to
provide a recombinant plasminogen inclusion body. In some
embodiments, the recombinant plasminogen inclusion body can be
contacted with a solubilization buffer under a solubilization
condition sufficient to obtain a solubilized recombinant
plasminogen inclusion body. In other embodiments, the solubilized
recombinant plasminogen inclusion body can be contacted with a
refolding solution under a refolding condition to obtain a
composition comprising the recombinant plasminogen.
II. Plasmin
[0138] In other aspects, the present invention provides a method
for preparing plasmin. In one embodiment, the method comprises
contacting a plasmin composition with an affinity medium (e.g.,
benzamidine-SEPHAROSE.TM.), wherein the plasmin composition
comprises plasmin prepared by activating a plasminogen with a
plasminogen activator. In some embodiments, the plasminogen is
prepared in accordance with the present invention. In one
embodiment, the plasminogen is a recombinant plasminogen prepared
in accordance with the present invention.
A. Converting Plasminogen to Plasmin
[0139] In some embodiments, the method for preparing plasmin
comprises contacting the plasminogen with a plasminogen activator
in an activation solution under an activation condition sufficient
to convert the plasminogen to a plasmin.
[0140] Generally, the plasminogen can be activated (i.e., cleaved
to provide plasmin) using a catalytic concentration of a
plasminogen activator (e.g., streptokinase, urokinase, tPA,
trypsin), which can be soluble and/or immobilized. In some
embodiments, the activation of the plasminogen can occur at about
4.degree. C. or more, e.g., about 4, 10, 20, 25, 37 or more degrees
celsius and typically can take at least several minutes or more,
preferably at least about 1, 2, 4, or more hours. The plasminogen
can be cleaved in the presence of one or more reagents including
stabilizers and/or excipients such as omega-amino acids, salts,
sucrose, alcohols (e.g., ethanol, methanol, 1,2-propanediol,
1,3-propanediol, glycerol, ethylene glycol), and combinations
thereof. The omega-amino acids can include lysine, epsilon amino
caproic acid (.epsilon.-ACA), tranexamic acid, poly-lysine,
arginine and combinations or analogues thereof. Stabilizing agents
are described by, e.g., U.S. Patent Publication No. 2003/0012778,
which is herein incorporated by reference in its entirety.
[0141] In one embodiment, the plasminogen activator is a soluble
plasminogen activator. In another embodiment, the method for
preparing plasmin comprises contacting the plasminogen with a
plasminogen activator in an activation solution under an activation
condition sufficient to convert the plasminogen to a plasmin,
wherein the plasminogen activator is an immobilized plasminogen
activator.
[0142] For example, the plasminogen activator can be adsorbed onto
a suitable matrix. For example, it has been reported that
streptokinase is still capable of activating plasminogen to plasmin
when streptokinase is bound tightly to nitrocellulose (Kulisek et
al., Anal. Biochem. 177:78-84 (1989)). Also, adsorption of
streptokinase to a suitable ion-exchange resin can render it
immobilized and still capable of activating plasminogen.
[0143] Immobilized streptokinase also is discussed by Rimon et al.,
Biochem. Biophy. Acta 73:301 (1963) using a diazotized copolymer of
p-aminophenylalanine and leucine. These authors utilized the
immobilized streptokinase to study the mechanism of activation of
plasminogen. Sugitachi et al., Thromb. Haemost. (Stuttg.) 39:426
(1978) discuss the immobilization of the plasminogen activator,
urokinase, on nylon. U.S. Pat. No. 4,305,926, incorporated herein
by reference for its teaching of immobilized plasminogen activator,
proposes immobilization of streptokinase onto a biocompatible
polymer such as a nylon, Dacron, collagen, polyvinylpyrolidine, or
copolymeric p-aminophenylalanine and leucine.
[0144] In one embodiment, the streptokinase is immobilized on a
surface using an affinity tag as described in U.S. Pat. No.
6,406,921, which is incorporated herein by reference for its
teaching of immobilizing streptokinase. The surface can be either
organic or inorganic, biological or non-biological, or any
combination of these materials. In one embodiment, the surface is
transparent or translucent. Numerous materials are suitable for use
as a surface. For example, the surface can comprise a material
selected from a group consisting of silicon, silica, quartz, glass,
controlled pore glass, carbon, alumina, titanium dioxide,
germanium, silicon nitride, zeolites, and gallium arsenide. Many
metals such as gold, platinum, aluminum, copper, titanium, and
their alloys are also options for surfaces. In addition, many
ceramics and polymers can also be used. Polymers which may be used
as surfaces include, but are not limited to, the following:
polystyrene; poly(tetra)fluorethylene; (poly)vinylidenedifluoride;
polycarbonate; polymethylmethacrylate; polyvinylethylene;
polyethyleneimine; poly(etherether)ketone; polyoxymethylene (POM);
polyvinylphenol; polylactides; polymethacrylimide (PMI);
polyalkenesulfone (PAS); polyhydroxyethylmethacrylate;
polydimethylsiloxane; polyacrylamide; polyimide; co-block-polymers;
and Eupergit.TM. Photoresists, polymerized Langmuir-Blodgett films,
and LIGA structures may also serve as surfaces in the present
invention.
[0145] In some embodiments, the activation condition is sufficient
to convert all or a substantial amount of the plasminogen to a
plasmin thereby providing a composition comprising the plasmin.
B. Anion-Exchange Chromatography
[0146] In other aspects, the present invention provides a method
for preparing a plasmin. The method comprises: contacting a
composition comprising the plasmin with an anion exchanger whereby,
if present in the composition, a proteinaceous material having an
isoelectric point below that of the plasmin is separated from the
plasmin.
[0147] Without being held to any particular theory, it is believed
that for any one proteinaceous material there will be a pH at which
the overall number of negative charges equals the number of
positive charges. This is the protein's isoelectric point (or pI),
i.e., the pH at which the protein carries no net charge. Above its
pI, the protein will have a net negative charge and bind to an
anion exchanger.
[0148] In a preferred embodiment, the plasmin that is present in
the composition is a product of a plasminogen having been activated
by a plasminogen activator. For example, a recombinant plasminogen
or a blood-derived plasminogen can be contacted with a plasminogen
activator (e.g., a streptokinase) to provide the plasmin. In one
embodiment, the composition comprising the plasmin is an activation
solution that has been pH-adjusted, if necessary, prior to
contacting with the anion exchanger.
[0149] In another embodiment, the composition comprising the
plasmin is an eluate or a flow-through solution of a chromatography
step (e.g., affinity chromatography using benzamidine), wherein the
eluate or flow-through solution has been pH-adjusted, if necessary,
prior to contacting with the anion exchanger.
[0150] The proteinaceous material to be separated from the plasmin
has a pI below that of the plasmin's pI. In some embodiments, the
proteinaceous material is the plasminogen activator (e.g.,
streptokinase) or a fragment thereof, wherein the composition
comprising the plasmin is contacted with an anion-exchange medium
under an anion-exchange condition sufficient to obtain an
anion-exchanger flow-through comprising the plasmin, wherein the
anion-exchange condition is such that the anion-exchange medium
selectively or preferentially binds the plasminogen activator or a
fragment thereof relative to the plasmin.
[0151] Without being held to any particular theory, it is believed
that proteolysis of streptokinase as a consequence of its
activation of plasminogen occurs following streptokinase contact
with plasminogen. In addition to the formation of plasmin,
streptokinase fragments of varying molecular weights can form upon
activation of the plasminogen by the streptokinase.
[0152] In one embodiment, the proteinaceous material is a
streptokinase fragment having a molecular weight of less than about
45 kD as determined by gel electrophoresis under denaturing
conditions, for example. In another embodiment, the fragment has a
molecular weight of about 40, 25, 15, 10 kD or less. In some
embodiments, the fragment has a molecular weight of about 15
kD.
[0153] In one embodiment, prior to the contacting of the
composition comprising the plasmin with the anion exchanger, the pH
of the composition is adjusted to be less than the pI of the
plasmin but greater than the pI of the proteinaceous material to be
separated from the plasmin. .degree. In some embodiments, the pH of
the composition is adjusted to be about 5.0 to about 10.0,
illustratively, about: 10, 9.9, 9.8, 9.7, 9.6, 9.5, 9.4, 9.3, 9.2,
9.1, 9, 8.9, 8.8, 8.7, 8.6, 8.5, 8.4, 8.3, 8.2, 8.1, 8, 7.9, 7.8,
7.7, 7.6, 7.5, 7.4, 7.3, 7.2, 7.1, 7, 6.9, 6.8, 6.7, 6.6, 6.5, 6.4,
6.3, 6.2, 6.1, 6, 5.9, 5.8, 5.7, 5.6, 5.5, 5.4, 5.3, 5.2, 5.1, and
5. In another embodiment, the pH of the composition is adjusted to
be about 6.5 to about 7. In other embodiments, the pH of the
composition is adjusted to be about 7 to about 8. In still further
embodiments, the pH of the composition is adjusted to be about 6.5
to about 8.
[0154] In various embodiments, the proteinaceous material is a
plasminogen activator or a fragment thereof. In one embodiment, the
composition comprising the plasmin is an activation solution in
which a plasminogen is converted by a plasminogen activator to form
the plasmin. In some embodiments, the activation solution
(comprising the plasmin formed therein) is directly contacted with
the anion exchanger, wherein prior to the contacting, the
activation solution is pH-adjusted, if necessary. In another
embodiment, prior to contacting the activation solution with the
exchanger, the activation solution is subjected to one or more
plasmin purification steps such as, but not limited to, filtration,
affinity chromatography, ion exchange chromatography, and/or
hydrophobic interaction chromatography. Accordingly, in some
embodiments, an eluate or flow-through composition obtained from
the one or more plasmin purification steps can be contacted with
the anion exchanger following an appropriate pH adjustment of the
eluate or the flow-through composition, if necessary.
[0155] For example, in one embodiment, wherein the plasmin
comprises a pI of about 9 or greater, wherein the material to be
separated is a streptokinase fragment (e.g., a fragment having a
molecular weight of about 15 kD or less) having a pI of about 5,
the pH of the composition can be adjusted to be about 6 to about 8
to effect binding of the fragment (i.e., the material), but not the
plasmin, to the exchanger.
[0156] By way of another example, in other embodiments, wherein the
plasmin comprises a pI of about 7 to about 8, wherein the material
to be separated is a streptokinase fragment (e.g., a fragment
having a molecular weight of about 15 kD or less) having a pI of
about 5, the pH of the composition can be adjusted to be about 6 to
about 7 to effect binding of the fragment (i.e., the material), but
not the plasmin, to the exchanger.
[0157] In some embodiments, the methods may be carried out in batch
or as continuous processes.
[0158] In other embodiments, the flow-through solution obtained
from the anion exchanger can be subjected to further processing
including further purification of the plasmin contained therein
and/or reduction of any pathogens that may be contaminating the
plasmin. In some embodiments, further purification is effected by
additional filtration steps (e.g., nanofiltration) and/or
chromatography steps including, but not limited to, affinity
chromatography, ion exchange chromatography, and hydrophobic
interaction chromatography.
[0159] In some embodiments, the method for preparing plasmin
comprises contacting the activation solution with an anion-exchange
medium under an anion-exchange condition to obtain an
anion-exchanger flow-through comprising the plasmin, wherein the
anion-exchange condition is such that the anion-exchange medium
selectively or preferentially binds the plasminogen activator
relative to the plasmin.
[0160] The anion-exchange medium can be a solid phase that binds
the plasminogen activator present in the activation solution. The
anion-exchange chromatography medium can be selected from any of
the group of chromatography media commonly described as
anion-exchange media, preferably a strong anion exchanger. The
medium can possess a chemistry or a ligand coupled thereto that can
allow for selective or preferential capture of the plasminogen
activator from the activation solution. Useful chromatography media
comprise a support and one or more ligand(s) bound thereto that
provide(s) the selective or preferential binding capability for the
plasminogen activator. Useful supports include, by way of
illustrative example, polysaccharides such as agarose and
cellulose, organic polymers such as polyacrylamide,
methylmethacrylate, and polystyrene-divinylbenzene copolymers. It
should be recognized that it is not intended herein to imply that
only organic substrates are suitable for medium substrate use,
since inorganic support materials such as silica and glasses also
can be used.
[0161] In some embodiments, the anion-exchange medium is in the
form of beads, which can be generally spherical, or alternatively
the anion-exchange medium can be usefully provided in particulate
or divided forms having other regular shapes or irregular shapes.
In one embodiment, the medium is in the form of a membrane. The
anion-exchange medium can be of porous or nonporous character, and
the medium can be compressible or incompressible. Preferred
anion-exchange media will be physically and chemically resilient to
the conditions employed in the purification process including
pumping and cross-flow filtration, and temperatures, pH, and other
aspects of the various compositions employed. A wide variety of
anion-exchange media, for example, those wherein the coupled ligand
is quaternary_ammonium (Q) or quaternary aminoethyl (QAE), are
known in the art.
[0162] In one embodiment, the anion-exchange medium comprises a
ligand coupled to a support, wherein the ligand is quaternary
ammonium, wherein the support is a membrane. For example,
anion-exchange chromatography can be performed in a Q-membrane or
Q-Sepharose.TM. column format. In one embodiment, the
anion-exchanger is a Q-membrane.
[0163] In other embodiments, the anion-exchange medium comprises a
ligand coupled to a support, wherein the ligand is quaternary amine
or ammonium, wherein the support is a membrane. For example,
anion-exchange chromatography can be performed in a Q-membrane or
Q-Sepharose.TM. column format. In one embodiment, the
anion-exchanger is a Q-membrane. Commercially available anion
exchange membrane adsorbers include Sartorius Sartobind.RTM. Q
(Sarorius, Bohemia, N.Y.), Mustang.RTM. Q Port (Pall Corporation,
Washington, N.Y.), and ChromaSorb.TM. (Millipore, Billerica,
Mass.). In another embodiment, the anion exchanger is a
Sartobind.RTM. MA 5 Q membrane.
[0164] In other embodiments, the flow-through solution obtained
from the anion exchanger comprises an amount of the plasminogen
activator or fragment thereof that is less than the amount of that
which was present in the composition prior to contacting with the
anion exchanger. In one embodiment, the resulting plasmin
composition is substantially free of the proteinaceous material
having a pI less than the pI of the plasmin.
[0165] In some embodiments, the plasmin that is present in the
flow-through solution of the anion exchanger will typically have a
purity of at least about 50% (by weight), illustratively, at least
about: 50%, 60%, 70%, 80%, 90%, 95%, 99%, or greater.
[0166] In other embodiments, the flow-through solution of the anion
exchanger is substantially free of the proteinaceous material,
wherein the substantially free of the proteinaceous material is
characterized as levels of the proteinaceous material that are
below limits of detection by a Western blot.
[0167] In another embodiment, at least about 50%, illustratively,
at least about: 50%, 60%, 70%, 80%, 90%, 95%, 99%, or greater of a
streptokinase fragment is removed from the plasmin-containing
composition that is loaded onto the anion exchanger thereby
providing a resulting plasmin composition substantially free of the
fragment. In some embodiments, the streptokinase fragment has a
molecular weight (e.g., as determined by SDS-PAGE) of about 15 kD
or less).
[0168] In one embodiment, the flow-through from the anion exchanger
can be pH-adjusted, e.g., to an acidic pH (e.g., about 3.4). In
some embodiments, this pH-adjusted flow-through comprising the
plasmin can be subsequently concentrated and/or diafiltered by
ultrafiltration/diafiltration.
[0169] By way of another example, the anion exchange flow-through
can be dialyzed with water and acidified with glacial acetic acid.
In some embodiments, any acid providing a pharmaceutically
acceptable acidified carrier (e.g., having a low buffering capacity
buffer and having a pH between about 2.5 to about 4.0) can be used.
For example, also contemplated within the scope of this invention
is the use of other acids and amino acids such as, but not limited
to, inorganic acids, carboxylic acids, aliphatic acids and amino
acids including, but not limited to, formic acid, acetic acid,
citric acid, lactic acid, malic acid, tartaric acid, benzoic acid,
serine, threonine, valine, glycine, glutamine, isoleucine,
.beta.-alanine and derivatives thereof, either singly or any
combination thereof, that will maintain the pH in the
pharmaceutically acceptable carrier of about 2.5 to about 4.0.
C. Affinity Chromatography
[0170] A plasmin composition comprising plasmin can be purified by
affinity chromatography using an affinity medium that binds the
plasmin contained in the composition. For example, in some
embodiments, the anion-exchange medium flow-through is contacted
with a second affinity medium under a second affinity condition
sufficient to bind the plasmin contained in the flow-through. In
particular embodiments, the second affinity condition is such that
the affinity medium selectively or preferentially binds the plasmin
relative to the plasminogen activator that may be present.
[0171] The second affinity medium can be a solid phase that binds
the plasmin. The second affinity chromatography medium can be
selected from any of the group of chromatography media commonly
described as second affinity media. The medium can possess a
chemistry or a ligand coupled thereto that can allow for selective
or preferential capture of the plasminogen activator from the
activation solution. Useful chromatography media comprise a support
and one or more ligand(s) bound thereto that provide(s) the
selective or preferential binding capability for the plasminogen
activator. Useful supports include, by way of illustrative example,
polysaccharides such as agarose and cellulose, organic polymers
such as polyacrylamide, methylmethacrylate, and
polystyrene-divinylbenzene copolymers. It should be recognized that
it is not intended herein to imply that only organic substrates are
suitable for medium substrate use, since inorganic support
materials such as silica and glasses also can be used.
[0172] In some embodiments, the second affinity medium is in the
form of beads, which can be generally spherical, or alternatively
the second affinity medium can be usefully provided in particulate
or divided forms having other regular shapes or irregular shapes.
In one embodiment, the medium is in the form of a membrane. The
second affinity medium can be of porous or nonporous character, and
the medium can be compressible or incompressible. Preferred second
affinity media will be physically and chemically resilient to the
conditions employed in the purification process including pumping
and cross-flow filtration, and temperatures, pH, and other aspects
of the various compositions employed. A wide variety of second
affinity media, for example, those wherein the coupled ligand is
benzamidine, are known in the art.
[0173] In one embodiment, the second affinity medium comprises a
ligand coupled to a support, wherein the ligand is benzamidine,
wherein the support is agarose. For example, second affinity
chromatography in accordance with the present invention can be
carried out using benzamidine-Sepharose.TM. column format. Because
the plasmin that is formed is a serine protease, in other
embodiments, other affinity-type media having similar properties as
benzamidine (e.g., a serine protease adsorbent material) also can
be used.
[0174] For example, in other embodiments, the plasmin obtained from
the cleaved plasminogen can be contained in a solution comprising
one or more reagents (e.g., amino acids, sodium chloride, glycerol)
that allow for stability of the solution for several days at
neutral pH before the solution is applied to a
benzamidine-SEPHAROSE.TM. column. The flow-through pool can contain
both non-activated plasminogen and inactive auto-degradation
products of plasmin.
[0175] Plasmin bound by an affinity medium can be eluted with an
acid buffer or with a substantially neutral pH excipient solution.
For example, the plasmin bound to benzamidine-SEPHAROSE.RTM. can be
eluted with an acidic buffer such as glycine buffer. When a
substantially neutral pH excipient solution is used to elute the
bound plasmin, the final eluted plasmin solution can be
substantially free of degraded plasmin. Typically, the
substantially neutral pH excipient solution has a pH of value of
between about 6.5 to about 8.5. However, the pH of the solution can
range from about 2.5 to about 9.0. In particular embodiments, the
pH can be from about 3.0 to about 7.5. In other embodiments, the pH
can be about 6.0. Examples of excipients include omega-amino acids,
including lysine, epsilon amino caproic acid, tranexamic acid,
polylysine, arginine, and analogues and combinations thereof, salts
such as sodium chloride, and active site inhibitors such as
bezamidine.
[0176] An appropriate concentration of salt can be represented by a
conductivity from about 5 mS/cm to about 100 mS/cm. Generally, the
salt concentration can be varied somewhat inversely in relation to
acidity, i.e., lower pH solutions can work well with lower salt and
solutions having higher pH (within the ranges discussed above) can
work well with higher salt concentrations. When the salt is sodium
chloride, the concentration can be from about 50 mM to about 1000
mM, or from about 100 mM to about 200 mM. When the solution is at
about pH 6.0, the concentration of sodium chloride can be about 150
mM. Thus, in some embodiments, upon the completion of the
activation of the recombinant plasminogen, the plasmin composition
can be filtered and further stabilized for several days at neutral
pH by the addition of excipients such as omega-amino acids and
sodium chloride prior to benzamidine-SEPHAROSE.TM.
chromatography.
[0177] In some embodiments, eluted plasmin can be buffered with a
low pH, low buffering capacity agent. The low pH, low buffering
capacity agent typically comprises a buffer of either an amino
acid, a derivative of at least one amino acid, an oligopeptide
which includes at least one amino acid, or a combination of the
above. Additionally the low pH, low buffering capacity agent can
comprise a buffer selected from acetic acid, citric acid,
hydrochloric acid, carboxcylic acid, lactic acid, malic acid,
tartaric acid, benzoic acid, serine, threonine, methionine,
glutamine, alanine, glycine, isoleucine, valine, alanine, aspartic
acid, derivatives or combinations thereof. The buffer can be
present at a concentration such that the pH of the acidified
plasmin can be raised to neutral pH by adding serum to the
composition in an amount no more than about 4 to 5 times the volume
of acidified plasmin.
[0178] In other embodiments, the concentration of plasmin in the
buffered solution can range from about 0.01 mg/mL to about 50 mg/mL
of the total solution. The concentration of the buffer can range
from about 1 nM to about 50 mM. Of course, these ranges may be
broadened or narrowed depending upon the buffer chosen, or upon the
addition of other reagents such as additives or stabilizing agents.
The amount of buffer added is typically that which will bring the
acidified plasmin solution to have a pH between about 2.5 to about
4.
[0179] The acidified plasmin solution may be further stabilized by
the addition of a stabilizing agent such as a polyhydric alcohol,
pharmaceutically acceptable carbohydrates, salts, glucosamine,
thiamine, niacinamide, or combinations thereof. The stabilizing
salts can be selected from the group consisting of sodium chloride,
potassium chloride, magnesium chloride, calcium chloride and
combinations thereof. Sugars or sugar alcohols may also be added,
such as glucose, maltose, mannitol, sorbitol, sucrose, lactose,
trehalose, and combinations thereof.
[0180] Concentrations of carbohydrate added to stabilize the
acidified plasmin solution include a range from about 0.2% w/v to
about 20% w/v. Ranges for a salt, glucosamine, thiamine,
niacinamide and their combinations can range from about 0.01 M to
about 1 M.
[0181] Plasmin formulated according to the invention in buffered
acidified water has been found to be extremely stable. It can be
kept in this form for months without substantial loss of activity
or the appearance of degradation products of a proteolytic or
acidic nature. At 4.degree. C., plasmin is stable for at least nine
months. Even at room temperature, plasmin is stable for at least
two months. Long-term stability at room temperature can allow this
formulation to be compatible with long regimens of thrombolytic
administration. For example, 36 hours administration of
thrombolytics such as tissue plasminogen activator or urokinase is
common in treatment of peripheral arterial occlusions.
[0182] In a preferred embodiment, the plasmin contained in the
acidified plasmin solution is a reversibly inactive plasmin. The
ability of a buffered acidified plasmin to become fully active upon
transfer to physiological pH is evidenced by its activity in the
caseinolytic assay and also in the I125-fibrin-labelled clot lysis
assays. Both of these assays can be performed at pH 7.4, and there
can be complete recovery of recombinant plasmin activity during the
change of pH and passing through the isoelectric points (pH9.3 and
9.5). This is because recombinant plasmin is formulated in a low
buffering capacity solvent and when added to a buffered solution
(e.g., PBS, plasma) it can adopt the neutral pH instantly and the
precipitation that usually accompanies the slow passage through the
isoelectric point, does not occur.
D. Hydrophobic Interaction Chromatography
[0183] In some embodiments, the method for preparing plasmin can
further comprise hydrophobic interaction chromatography. In one
embodiment, hydrophobic chromatography is optional. In other
embodiments, the method for preparing plasmin comprises contacting
the second affinity medium eluate comprising the plasmin with a
hydrophobic interaction chromatography medium under a hydrophobic
interaction condition sufficient such that the hydrophobic
interaction chromatography medium preferentially binds the
plasminogen activator relative to the plasmin. In particular
embodiments, the hydrophobic interaction condition is such that the
hydrophobic interaction medium selectively or preferentially binds
the plasminogen activator, if present, relative to the plasmin.
[0184] The hydrophobic interaction medium can be a solid phase that
binds the plasmin. The hydrophobic interaction chromatography
medium can be selected from any of the group of chromatography
media commonly described as hydrophobic interaction media. The
medium can possess a chemistry or a ligand coupled thereto that can
allow for selective or preferential capture of the plasminogen
activator. Useful chromatography media comprise a support and one
or more ligand(s) bound thereto that provide(s) the selective or
preferential binding capability for the plasminogen activator.
Useful supports include, by way of illustrative example,
polysaccharides such as agarose and cellulose, organic polymers
such as polyacrylamide, methylmethacrylate, and
polystyrene-divinylbenzene copolymers. It should be recognized that
it is not intended herein to imply that only organic substrates are
suitable for medium substrate use, since inorganic support
materials such as silica and glasses also can be used.
[0185] In some embodiments, the hydrophobic interaction medium is
in the form of beads, which can be generally spherical, or
alternatively the second affinity medium can be usefully provided
in particulate or divided forms having other regular shapes or
irregular shapes. In one embodiment, the medium is in the form of a
membrane. The hydrophobic interaction medium can be of porous or
nonporous character, and the medium can be compressible or
incompressible. Preferred hydrophobic interaction media will be
physically and chemically resilient to the conditions employed in
the purification process. A wide variety of hydrophobic interaction
media, for example, those wherein the coupled ligand is an octyl,
phenyl, or butyl moiety, are known in the art.
[0186] In one embodiment, the hydrophobic interaction medium
comprises a ligand coupled to a support, wherein the ligand is an
octyl moiety, wherein the support is an agarose. For example,
hydrophobic interaction chromatography can be performed in an
octyl-SEPHAROSE.TM. column format. In particular embodiments, the
composition comprising the plasmin is prepared to about 0.1 M in
ammonium sulfate and subjected to hydrophobic interaction
chromatography, e.g., in a column format using a resin such as
octyl-SEPHAROSE.TM..
[0187] In one embodiment, the octyl-SEPHAROSE.TM. flow-through
comprising plasmin can be subjected to nanofiltration. For example,
the flow-through can be subjected to pre-filtration with a 0.1
micron filter capsule, and then subjected to nanofiltration, e.g.,
using an ASAHI NF (normal flow) 1.0 m.sup.2 15 N membrane (PLANOVA
filters, Asahi Kasei America, Inc., Buffalo Grove, Ill.).
Implementing nanofiltration further downstream in the process,
after octyl hydrophobic interaction chromatography, can improve
throughput and membrane flux properties due to a more pure
feedstream. In some embodiment, the step of nanofiltration
subsequent to hydrophobic interaction chromatography is
optional.
III. Therapeutics and Kits
[0188] In other aspects, the plasminogens and/or the plasmins
prepared therefrom can be formulated for therapeutic use, for
example in accordance with the methods described in U.S. Pat. No.
6,964,764; and Novokhatny, V., et al., J. Thromb. Haemost.
1(5):1034-41 (2003), both incorporated herein by reference. For
example, a low-pH (from about 2.5 to about 4), low-buffering
capacity buffer can be used for formulation of plasmin prepared in
accordance with the present invention. In some embodiments, the
plasminogen and/or the plasmin prepared therefrom can be used to
treat a variety of thrombotic diseases or conditions, for example,
according to the methods as described in U.S. Pat. Nos. 6,355,243
and 6,969,515, each incorporated herein by reference for its
teaching of treatment methods. Additionally, other methods and
formulations known to those of skill in the art, as practiced with
plasmin, mini-plasmin, and/or micro-plasmin, can be used to
formulate the plasminogen and/or the plasmin prepared therefrom of
the present invention for therapeutic administration.
[0189] In still further aspects, the present invention provides
kits comprising the recombinant plasminogens and/or the plasmins
prepared therefrom described herein. Such kits can generally
comprise, in one or more separate compartments, a pharmaceutically
acceptable formulation of the plasminogen and/or the plasmin
prepared therefrom. The kits also can further comprise other
pharmaceutically acceptable formulations. The kits can have a
single container, or they may have distinct container for each
desired component. Kits comprising reagents necessary for preparing
the recombinant plasminogens and/or the plasmins derived therefrom
also are contemplated, for example reagents such as, but not
limited to, expression vectors, recombinant host cells comprising
the expression vectors, and plasminogen activators. Further,
wherein the components of the kit are provided in one or more
liquid solutions, the liquid solution is an aqueous solution, with
a sterile aqueous solution being particularly preferred. However,
the components of the kit may be provided as dried powder(s). When
reagents or components are provided as a dry powder, the powder can
be reconstituted by the addition of a suitable solvent. It is
envisioned that the solvent may also be provided.
[0190] The following examples are given only to illustrate the
present process and are not given to limit the invention. One
skilled in the art will appreciate that the examples given only
illustrate that which is claimed and that the present invention is
only limited in scope by the appended claims.
EXAMPLES
Example 1
Preparing Recombinant Plasminogen
[0191] An expression vector comprising the DNA encoding the
recombinant plasminogen polypeptide shown in SEQ ID NO:1 (see also
shaded amino acid sequence shown in FIG. 2) was transformed into a
variety of cells including BL21(DE3) RIL (Stratagene, La Jolla,
Calif.), BL21(DE3) (genotype: F-ompT hsdSB (rB-mB-) gal dcm (DE3))
(EMB Biosciences, Inc., San Diego, Calif.), and BLR(DE3) (genotype:
F-ompT hsdSB (rB-mB-) gal dcm (DE3)
.DELTA.(srl-recA)306::Tn10(TetR)), and protein over-expression
following induction by 1 mM IPTG
(isopropyl-beta-D-thiogalactopyranoside) was analyzed by SDS-PAGE.
Expression estimates were at least about 250 mg/L cell culture in
shaker flasks.
[0192] Cell type BL21(DE3) RIL is engineered to express rare E.
coli tRNAs coding for Arg, Ile, and Leu. Further, both BL21(DE3)
and BLR(DE3) are B strain E. coli that is classified as
non-pathogenic to humans and animals based on the absence of
virulence and colonization factors. BLR(DE3) cells lack the recA
gene for DNA recombination, and induction of lamba phage has not
been reported with these cells. A research cell bank of the
recombinant plasminogen construct in BLR(DE3) cells was produced
and tested for purity, identity, and induction of bacteriophage at
Charles River Laboratories (Malvern, Pa.). The testing confirmed
the identity and purity of the research cell bank and the cells
passed the phage induction test with no phage observed (data not
shown).
[0193] Production of recombinant plasminogen (i.e., based on SEQ ID
NO:1) was confirmed in larger scale expression in which cells were
lysed and both soluble protein and purified inclusion bodies were
examined by SDS-PAGE.
[0194] The following typical protocol has been used for expression
of recombinant plasminogen: A single colony of E. coli cells (e.g.,
BL21(DE3) RIL, BL21(DE3), or BLR(DE3) containing the recombinant
plasminogen vector was used to inoculate 5 mL of LB/kanamycin (30
.mu.g/mL) and was incubated for 8 hours at 37.degree. C. on a
shaker. After that, a 50 .mu.L-aliquot was taken form the cultured
bacterial suspension for further growth in fresh media. The
procedure was repeated after 16 hours with 6 mL of bacterial
culture and 250 mL of the media. Cultures were grown at 37.degree.
C. with shaking to an OD.sub.600 nm of .about.1.0, and IPTG was
added to 1 mM final concentration. Cultures were grown for an
additional 5 hours. Cells were harvested by centrifugation at
5,000.times.g and cell pellets were dissolved in 20 mM Tris pH 8.0
containing 20 mM EDTA and frozen at -80.degree. C.
[0195] To purify recombinant plasminogen, cell pellets were thawed
and buffer added until the solution volume was approximately 1/20th
that of the original cell culture volume. After that, lysozyme was
added to a final concentration of 0.5 mg/mL and the cells were
stirred rapidly at 4.degree. C. for 10-15 minute. Then, Triton
X-100 was added to 1% final concentration and stirring continued
for another 10 min. DNAse I (0.05 mg/mL) and MgCl.sub.2 (2.5 mM)
were added and stirring was continued at 4.degree. C. for 30
minutes or until the solution was no longer viscous. The final
solution was centrifuged at 4.degree. C. for 30 min at
15,000.times.g and the supernatant was discarded. The cell pellet
was washed three times with wash solution (50 mM Tris-HCl, pH 7.4
containing 10 mM EDTA, 1% Triton X-100, and 0.5 M urea).
[0196] The recombinant plasminogen comprises the amino acid
sequence shown in FIG. 1. The primary structure of the recombinant
plasminogen begins at Met69 and has the linker sequence VPQ in
place of ILE at positions 160-162 (human plasminogen numbering
system); the latter change was incorporated to make the linker
region joining the kringle to the serine protease domain identical
to that of the native kringle 5-serine protease sequence. The
N-terminal sequence of the recombinant plasminogen yields N-termini
of Lys78 and Val79 after activation by SK and cleavage of the
pre-activation peptide. The mean yield of inclusion bodies isolated
from 100 L of culture was 2.17.+-.0.63 kg (n=3), which contained
approximately 20% recombinant plasminogen protein by weight.
Example 2
Solubilizing and Refolding of Plasminogen
[0197] Crushed, frozen inclusion bodies (26 g) were added to 480 mL
of cold solubilization buffer (7 M urea, 10 mM reduced glutathione,
10 mM Tris, 0.25 M arginine, 2 mM EDTA, pH 7.5), and this
suspension was stirred vigorously at 6.degree. C. for 4 hr. This
solution of solubilized inclusion bodies was then diluted 1:20 into
cold refolding buffer [0.5 M urea, 1.0 mM each of reduced and
oxidized glutathione, 0.5 M arginine, 1.0 mM EDTA, 5.0 mM
.epsilon.-ACA, 50 mM Tris, pH 8.0] and stirred for 17 hr in the
cold. The recombinant plasminogen concentration in this refolding
milieu was approximately 0.5 mg/mL.
[0198] Solubilized inclusion bodies were analyzed by reducing
SDS-PAGE; based upon densitometric analysis of Coomassie
Blue-stained gels, approximately 70% of the total protein was
estimated to be recombinant plasminogen (FIG. 4, Lane 2). Thus,
deposit of expressed recombinant plasminogen in inclusion bodies
provided relatively pure target protein at the beginning of this
process.
[0199] The refolding procedure was carried out at protein
concentration of about 0.5 g/L. Solubilized/reduced recombinant
plasminogen from inclusion bodies was oxidatively refolded, by
dilution refolding, to catalytically-active protein (determined in
the presence of stoichiometric SK) having a specific activity of
0.34; this corresponds to an estimated yield of 34% for the
refolding step (relative to total protein). If the refolding yield
is normalized based only upon the content of recombinant
plasminogen protein present in inclusion bodies, it increases to
approximately 48%.
Example 3
Filtration/Diafiltration
[0200] The refolding mixture in example 2 was clarified by passing
through a 0.054 m2 Millipore Millistak Pod+AlHC depth filter. This
filtrate was further filtered through a 0.01 m2 Millipore Express
SHC Opticap XL 150 filter (0.5/0.2 m). Diafiltration of this latter
filtrate was performed with two 0.11 m2 30 kDa GE Healthcare Kvick
cassettes. The diafiltration buffer was 10 mM Tris, 1.0 mM EDTA,
5.0 mM-ACA, 50 mM urea, pH 9.0. Between 37 and 40 L of buffer
exchange was required to reach the target conductivity of 1-2
mS/cm.
[0201] Refolding mixtures were first passed through a depth filter,
to remove particulate aggregates of protein, and were then
diafiltered. A subsequent filtration prior to chromatography was
done to safeguard against fouling of the down-stream
cation-exchange chromatography. Recovery of recombinant plasminogen
activity through these three sequential
filtration/diafiltration/filtration steps was approximately
81%.
Example 4
Polyethylene (PEG) Precipitation
[0202] Solid polyethylene glycol (PEG) was added to a refolding
composition (an undialyzed refolding composition comprising 0.5 M
arginine and 0.85 M urea) of recombinant plasminogen to precipitate
aggregated protein that may be present following oxidative
refolding.
[0203] Amounts of solid PEG were added to cold (5.degree. C.) 50-mL
portions of refolding mixture, with vigorous stirring, to achieve
final concentrations of PEG of 5, 10, 15 or 20% (w/v). Stirring was
continued for 60 minutes to ensure complete solubilization of the
PEG. These samples, as well as a sample of the original refolding
mixture to which no PEG had been added, were centrifuged at 16,000
rpm for 30 minutes to pellet any precipitate that had formed. The
clear supernatants were collected and assessed for total protein
content (by measurement of A.sub.280) and for content of active
recombinant plasminogen (by SK-activation assay, DiaPharma, West
Chester, Ohio) and were analyzed by size-exclusion high-performance
liquid chromatography (SEC-HPLC). The results are summarized in
Table 1 and FIG. 6 and show that addition of PEG resulted in: (i.)
relatively selective precipitation of protein that was not active
recombinant plasminogen; (ii.) a resultant increase in the specific
activity of the recombinant plasminogen that remained soluble; and
(iii.) elimination of aggregated protein. These results demonstrate
that addition of PEG to undialyzed refolding mixture caused
precipitation of some protein (that was not active recombinant
plasminogen) and preserved most of the active-SK-active recombinant
plasminogen in solution. Moreover, SEC-HPLC demonstrated that
addition of PEG to the undialyzed refolding mixture resulted in a
progressive reduction in aggregated protein and in a corresponding
increase in monomeric recombinant plasminogen in the PEG
supernatants. The results indicate that PEG somewhat selectively
precipitated incorrectly refolded or aggregated recombinant
plasminogen and, thereby, enriched correctly refolded recombinant
plasminogen in the PEG supernatant. The specific activity of
SK-active recombinant plasminogen in this undialyzed refolding
mixture increased from 0.33 to 0.53 in the 20% PEG supernatant. The
results indicated that PEG precipitation is a viable method for
selectively eliminating at least a portion of the SK-inactive
recombinant plasminogen present in refolding mixtures prior to a
chromatography or an SK-activation step.
TABLE-US-00001 TABLE 1 PEG Precipitation Analysis Sample Col. 1
Col. 2 Col. 3 Col. 4 Col. 5 Col. 6 Col. 7 Col. 8 0% PEG 1.349 0.812
0.266 0.328 `100` `100` 41.5 52.5 Supernatant 0.259 0.319 5% PEG
1.305 0.786 0.255, 0.324, 96.7 97.5 60.6 39.5 Supernatant 0.257
0.327 10% PEG 0.987 0.595 0.241, 0.405, 73.2 92.0 86.9 13.1
Supernatant 0.242 0.407 15% PEG 0.789 0.475 0.227, 0.478, 58.5 86.9
98.6 1.4 Supernatant 0.229 0.482 20% PEG 0.691 0.416 0.219, 0.526,
51.2 82.9 99.8 0.2 Supernatant 0.216 0.519 In Table 1: Column 1,
A.sub.280 of PEG supernatant; Column 2, concentration of total
recombinant plasminogen present in PEG supernatant, calculated by
dividing the A.sub.280 value by the extinction coefficient of 1.66
for a 1.0-mg/mL solution of recombinant plasminogen; Column 3,
concentration of SK-active recombinant plasminogen present in PEG
supernatant (duplicate assay values); Column 4, specific activity
of SK-active recombinant plasminogen, calculated by dividing values
in Column 3 by corresponding values in Column 2 (duplicate values
based upon duplicate assays); Column 5, percent of original
A.sub.280 value remaining in PEG supernatant; Column 6, percent of
original SK-active plasminogen remaining in PEG supernantant;
Column 7, percentage of total recombinant plasminogen present in
the PEG supernatant as monomeric recombinant plasminogen (based
upon SEC-HPLC); Column 8, percentage of total recombinant
plasminogen present in PEG supernatant as aggregated protein (based
upon SEC-HPLC).
Example 5
Ammonium Sulfate Precipitation
[0204] Solid ammonium sulfate was added to a refolding composition
(an undialyzed refolding composition comprising 0.5 M arginine and
0.85 M urea) of recombinant plasminogen to precipitate aggregated
protein that may be present following oxidative refolding.
[0205] Solid ammonium sulfate was added to cold (5.degree. C.)
50-mL portions of refolding mixture, with vigorous stirring, to
achieve final concentrations of ammonium sulfate corresponding to
10, 20, 30 or 40% saturation, respectively. Stirring was continued
for 60 minutes to ensure complete solubilization of the ammonium
sulfate. These samples, as well as a sample of the original
refolding mixture to which no ammonium sulfate had been added, were
centrifuged at 16,000 rpm for 30 minutes to pellet any precipitate
that had formed. The clear supernatants were collected and assessed
for total protein content (by measurement of A.sub.280) and for
content of active recombinant plasminogen (by SK-activation assay,
DiaPharma, West Chester, Ohio), and were analyzed by size-exclusion
high-performance liquid chromatography (SEC-HPLC). The results are
summarized in Table 2 and FIG. 7 and show that addition of ammonium
sulfate resulted in: (i.) relatively selective precipitation of
protein that was not active recombinant plasminogen; (ii.) a
resultant increase in the specific activity of the recombinant
plasminogen that remained soluble; and (iii.) elimination of
aggregated protein. These results demonstrate that addition of
ammonium to undialyzed refolding mixture caused precipitation of
some protein (that was not active recombinant plasminogen) and
preserved most of the active-SK-active recombinant plasminogen in
solution. Moreover, size-exclusion high-performance liquid
chromatography (SEC-HPLC) demonstrated that addition of ammonium to
the undialyzed refolding mixture resulted in a progressive
reduction in aggregated protein and in a corresponding increase in
monomeric recombinant plasminogen in the ammonium sulfate
supernatants. The results indicate that ammonium sulfate somewhat
selectively precipitated incorrectly refolded or aggregated
recombinant plasminogen and, thereby, enriched correctly refolded
recombinant plasminogen in the ammonium sulfate supernatant. The
specific activity of SK-active recombinant plasminogen in this
undialyzed refolding mixture increased from 0.33 to 0.48 in the 30%
saturated ammonium sulfate supernatant; addition of a higher amount
of ammonium sulfate had the apparent effect of causing significant
precipitation of SK-active recombinant plasminogen. The results
indicated that ammonium sulfate precipitation is a viable method
for selectively eliminating at least a portion of the SK-inactive
recombinant plasminogen present in refolding mixtures prior to a
chromatography or an SK-activation step.
TABLE-US-00002 TABLE 2 Ammonium Sulfate (AmSO.sub.4) Precipitation
Analysis Sample Col. 1 Col. 2 Col. 3 Col. 4 Col. 5 Col. 6 Col. 7
Col. 8 0% AmSO.sub.4 1.349 0.812 0.270, 0.333, `100` `100` 46.1
44.5 Supernatant 0.272 0.335 10% AmSO.sub.4 1.316 0.793 0.274,
0.346, 97.6 100.4 49.1 48.2 Supernatant 0.270 0.340 20% AmSO.sub.4
1.109 0.668 0.256, 0.383, 82.2 94.5 94.6 5.4 Supernatant 0.256
0.383 30% AmSO.sub.4 0.824 0.496 0.236, 0.476, 61.1 88 99.1 0.9
Supernatant 0.241 0.486 40% AmSO.sub.4 0.669 0.403 0.170, 0.422,
49.6 62.7 100 0 Supernatant 0.170 0.422 In Table 2: Column 1,
A.sub.280 of ammonium sulfate supernatant; Column 2, concentration
of total recombinant plasminogen present in ammonium sulfate
supernatant, calculated by dividing the A.sub.280 value by the
extinction coefficient of 1.66 for a 1.0-mg/mL solution of
recombinant plasminogen; Column 3, concentration of SK-active
recombinant plasminogen present in ammonium sulfate supernatant
(duplicate assay values); Column 4, specific activity of SK-active
recombinant plasminogen, calculated by dividing values in Column 3
by corresponding values in Column 2 (duplicate values based upon
duplicate assays); Column 5, percent of original A.sub.280 value
remaining in ammonium sulfate supernatant; Column 6, percent of
original SK-active plasminogen remaining in ammonium sulfate
supernantant; Column 7, percentage of total recombinant plasminogen
present in the ammonium sulfate supernatant as monomeric
recombinant plasminogen (based upon SEC-HPLC); Column 8, percentage
of total recombinant plasminogen present in ammonium sulfate
supernatant as aggregated protein (based upon SEC-HPLC).
Example 6
Hydrophobic Interaction Chromatography Following Ammonium Sulfate
Precipitation
[0206] Solid (NH.sub.4).sub.2SO.sub.4 was added to a recombinant
plasminogen-containing refolding composition (pH 8.0) to 1 M. The
precipitate formed was removed by filtration (0.45 .mu.m) and the
post-filtration solution was contacted with the following
hydrophobic interaction chromatography media:
[0207] Run 1. HiTrap.TM. Phenyl Sepharose FF: Load: 25 mL
(A.sub.280: 0.839); Flow-through (FT)/Wash: 49.4 g (A.sub.280:
0.266); Eluate: 96 well plate with fractions.
[0208] Run 2. HiTrap.TM. Octyl Sepharose FF: Load: 25 mL
(A.sub.280: 0.839); Flow-through (FT)/Wash: 49.6 g (A.sub.280:
0.407); Eluate: 96 well plate with fractions. This run did not show
any real eluate peak.
[0209] Run 3. HiTrap.TM. Butyl Sepharose FF: Load: 25 mL
(A.sub.280: 0.839); Flow-through (FT)/Wash: 49.05 g (A.sub.280:
0.215); Eluate: 96 well plate with fractions.
[0210] Run 4. HiTrap.TM. Phenyl Sepharose HP: Load: 25 mL
(A.sub.280: 0.839); Flow-through (FT)/Wash: 49.01 g (A.sub.280:
0.183); Eluate: 96 well plate with fractions.
[0211] Chromatography: Buffer A: 25 mM Tris-HCl, pH 8.0, 1 mM EDTA,
and 1 M (NH.sub.4).sub.2SO.sub.4; and Buffer B: 25 mM Tris-HCl, pH
8.0, and 1 mM EDTA (100-0% reverse gradient in 40 CV at 1 mL/min).
Fractions were collected at the 1 mL scale.
[0212] The following fractions from the runs were subjected to
dialysis for at least two days against 10 mM Tris-HCl, pH 9.0, and
1 mM EDTA at 4.degree. C.: Fraction #1--Phenyl Sepharose FF
(B1-B11); Fraction #2--Phenyl Sepharose FF (B12-D5); Fraction
#3--Phenyl Sepharose FF (D6-D11); Fraction #4--Phenyl Sepharose FF
flow-through; Fraction #5-Octyl Sepharose flow-through; Fraction
#6--Column load; Fraction #7--Butyl Sepharose (A6-B12); Fraction
#8--Butyl Sepharose (C1-C7); Fraction #9--Butyl Sepharose (C8-D12);
Fraction #10--Butyl Sepharose flow-through; Fraction #11--Phenyl
Sepharose HP (A12-B12); Fraction #12--Phenyl Sepharose HP (C1-D1);
Fraction #13--Phenyl Sepharose HP flow-through; and Fraction
#14--Column load.
[0213] The A.sub.280 absorbance traces of the four hydrophobic
interaction chromatography runs are shown in FIG. 8. Potency
analysis of selected fractions showed that the Phenyl Sepharose FF
(B12-D5) pooled Fraction #2 had a specific activity of 0.91, while
the flow-through pool Fraction #4 had a specific of 0.003.
Similarly, the specific activity of the Phenyl Sepharose HP
(A12-B12) pooled Fraction #11 was 1.01, while the specific activity
of the flow-through Fraction #13 for this run was negligible.
Fraction #7, Butyl Sepharose (A6-B12), had a specific activity of
0.61, Fraction #8, Butyl Sepharose (C1-C7) a specific activity of
0.21, and Fraction #10, Butyl Sepharose flow-through a specific
activity of 0.05. No capture peak was achieved with the Octyl
Sepharose resin, and the flow-through Fraction #5 from this run had
a specific activity of 0.42.
[0214] The result show that the supernatant/filtrate from the
ammonium sulfate precipitation step is amenable to direct
application to hydrophobic interaction chromatography. Phenyl
Sepharose FF, Phenyl Sepharose HP, and Butyl Sepharose FF are able
to selectively capture most of the active recPlasminogen protein
from the refold mixture, evident by a high specific activity in the
eluate fractions and a low specific activity in the flow-through
pools. Thus, for example, following ammonium sulfate precipitation,
the precipitate can be clarified by one or more methods (e.g.,
depth filtration, centrifugation, microfiltration, etc., and/or
combinations thereof) that remove all or a substantial amount of
any precipitate, and the clarified filtrate comprising plasminogen
can be subjected to hydrophobic interaction chromatography to
capture the recombinant plasminogen contained therein.
Example 7
Cation-Exchange Chromatography
[0215] Refolded, diafiltered recombinant plasminogen was captured
on a 206-mL column of SP Sepharose FF (GE Healthcare, Pittsburgh,
Pa.) at room temperature. The column equilibration buffer was 25 mM
Tris, 1.0 mM EDTA, pH 8.0. After the column was washed with 10
column volumes of equilibration buffer, the recombinant plasminogen
was eluted with 25 mM Tris, 200 mM NaCl, 1.0 mM EDTA, pH 8.0.
[0216] The SP Sepharose column eluate provided nearly homogeneous
recombinant plasminogen, having a specific activity of 0.98, with a
recovery of approximately 89% of the recombinant plasminogen
activity. This chromatography step was highly effective in reducing
contamination of the recombinant plasminogen by host-cell protein
as shown in Table 3.
TABLE-US-00003 TABLE 3 Purification of recombinant plasminogen from
E. coli inclusion bodies Protein Total Total Activity Host Cell
Purification Concentration Protein Activity Recovered Specific
Protein Step (mg/mL) .sup.a (mg) .sup.a,b (mg) .sup.a,b (%)
.sup.a,b activity .sup.c (ng/mg) Solubilized 8.88 .+-. 0.72 4.424
.+-. 356 DNDd -- -- DND inclusion bodies Refolded 0.51 .+-. 0.05
5.133 .+-. 454 1.720 .+-. 46 33.6 .+-. 2.2 0.34 .+-. 0.02 DND
plasminogen (Post-refold 0.38 .+-. 0.02 4.039 .+-. 274 1.604 .+-.
185 93.2 .+-. 9.9 0.40 .+-. 0.02 DND filtrate plasminogen)
(Diafiltration 0.34 .+-. 0.03 3.386 .+-. 282 1.604 .+-. 252 99.8
.+-. 9.2 0.47 .+-. 0.04 DND retentate plasminogen) (Pre-SP 0.32
.+-. 0.02 3.313 .+-. 132 1.379 .+-. 5 87.6 .+-. 15.0 0.42 .+-. 0.02
2.464 .+-. 897 Sepharose filtrate plasminogen) SP 2.29 .+-. 0.13
1.255 .+-. 25 1.225 .+-. 26 88.8 .+-. 1.9 0.98 .+-. 0.02 31.9 .+-.
20.5 Sepharose eluate plasminogen ECH Lysine 5.96 .+-. 0.56 1.137
.+-. 104 1.194 .+-. 34 97.4 .+-. 0.7 1.05 .+-. 0.08 1.60 .+-. 1.4
eluate plasminogen .sup.a Values represent the mean .+-. the
standard deviation from the mean for three different punfication
runs, each starting with a different batch of inclusion bodies.
.sup.b Values not corrected for volume of sample withdrawn for
bioanalytical assays. .sup.c Specific activity, mg of active
recombinant plasminogen per mg of total protein. .sup.d Analysis
was not conducted on these samples.
Example 8
Affinity Chromatography
[0217] Recombinant plasminogen eluted from the SP Sepharose column
was subsequently bound to, and eluted from, an ECH-Lysine Sepharose
column. This latter affinity chromatography step also served to
confirm that all or substantially all of the protein processed
further downstream contains a correctly refolded lysine-binding
site on the kringle domain.
[0218] The eluate from the SP Sepharose column was loaded on to a
295-mL column of ECH-Lysine Sepharose 4 FF (GE Healthcare,
Pittsburgh, Pa.) at room temperature equilibrated with 50 mM Tris,
200 mM NaCl, 1.0 mM EDTA, pH 8.0. After the column was washed with
4 column volumes of equilibration buffer and then with 3 column
volumes of wash 2 buffer (50 mM Tris, pH 8.0, and 1 mM EDTA),
plasminogen was eluted with 50 mM Tris, 20 mM .epsilon.-ACA, 1.0 mM
EDTA, pH 8.0. This column eluate was stored frozen at -80.degree.
C. until the next step of the purification process was
initiated.
[0219] Almost quantitative recovery of the recombinant plasminogen
was achieved from the ECH-Lysine Sepharose chromatography step,
with a specific activity of unity. This affinity column was also
effective in further reducing the content of host-cell protein.
Example 9
Activation of Plasminogen to Plasmin Using Soluble
Streptokinase
[0220] Thawed recombinant plasminogen was activated with
streptokinase (SK) for 4 hr at room temperature under the following
solution conditions: 2.5 mg of plasminogen/mL; 12.5% (v/v)
1,2-propanediol; 200 mM .epsilon.-ACA; 87.5% (v/v) ECH-Lysine
Sepharose elution buffer; 25 .mu.g of SK/mL; pH 7.0.
[0221] SK was used to activate the recombinant plasminogen to
plasmin in free solution. The conditions for activation were
selected carefully to maximize activation of the recombinant
plasminogen and to minimize autolysis. The specific activity of
plasmin at the end of the 4-hr activation period was 0.77,
indicating an approximately 80% yield at this step.
Example 10
Activation of Plasminogen to Plasmin Using Immobilized Recombinant
Polyhistidine-Tagged Streptokinase
[0222] Recombinant polyhistidine-tagged streptokinase (100 .mu.g)
in 10 mM Tris-HCl (pH 8.0) and 100 mM NaCl is added to 100 .mu.l of
immobilized metal ion affinity chromatography (IMAC) matrix. After
incubation at 22.degree. C. for 5 min, the slurry is applied to a
Spin-X microcentrifuge spin column (Costar, Cambridge, Mass.)
fitted with a 0.45-.mu.m cellulose acetate filter. The matrix is
pelleted by centrifugation at 2,000.times.g for 3 min and is
subsequently washed several times with 20 mM Tris-HCl, pH 7.4. The
matrix is removed from the Spin-X unit, placed in a microcentrifuge
tube, and resuspended in 200 mL of 50 mM Tris-HCl buffer, pH
7.4.
[0223] An equimolar amount of the recombinant plasminogen is added
to the immobilized streptokinase in an activating solution: 50 mM
Tris-HCl buffer, pH 7.4. Samples are incubated at 22.degree. C. and
placed on a rotating platform to keep the matrix in suspension.
Upon completion of activation, the activation solution is filtered
from streptokinase-SEPHAROSE.TM. on a glass filter and immediately
applied on Q-membrane or benzamidine-SEPHAROSE.TM. column. To
monitor the progress of plasminogen activation, at different
intervals a sample is selected and the reaction is terminated by
the addition of 0.1 volumes of 10.times. stop buffer (1.0 M
NaHCO.sub.3, 1.0 M .epsilon.-aminocaproic acid [pH 9.4]). The
sample is transferred to a Spin-X microcentrifuge tube and pelleted
by centrifugation at 2,000.times.g for 3 min. Immobilized reactants
are eluted by addition of 25 .mu.l of 100 mM EDTA, followed by
centrifugation at 5,000.times.g for 10 min. Reduced SDS-PAGE was
carried out by conventional methodology.
Example 11
Anion Exchange Chromatography
[0224] The SK-activation mixture of Example 9 was diluted 1:1 with
200 mM .epsilon.-ACA/12.5% 1,2-propanediol and pH-adjusted to 9.0.
This solution was passed through a Sartobind SingleSep Q Nano 1-mL
membrane (Sartorius) at room temperature.
[0225] The activation solution was passed through a Q membrane to
remove SK, with 91% recovery of plasmin; this separation is made
possible by the disparate pI values for the recombinant plasmin
(9.3 and 9.5) and SK (5.2). Based upon Western blot analysis of the
load material and flow-through from this Q membrane, SK was reduced
to below the level of quantification by this step.
Example 12
Affinity Chromatography
[0226] The Q membrane flow-through of Example 11 was supplemented
with NaCl (to 0.5 M) and pH-adjusted to 7.0 prior to being loaded
on to a 200-mL column of Benzamidine Sepharose 4 FF (high sub) (GE
Healthcare, Pittsburgh, Pa.) equilibrated with 25 mM Tris, 500 mM
NaCl, 250 mM .epsilon.-ACA, pH 7.0 at room temperature. After the
column was washed with 5-column volumes of equilibration buffer,
the plasmin was eluted with 200 mM sodium citrate, 200 mM
.epsilon.-ACA, 300 mM NaCl, pH 3.0. This column eluate was
collected in a vessel containing one column volume of elution
buffer, for the purpose of rapidly lowering the pH (to 3.0) of
plasmin present in the peak front.
[0227] The Q membrane flow-through was loaded on to a Benzamidine
Sepharose column to bind active plasmin and to eliminate unreacted
recombinant plasminogen and autolyzed plasmin. Plasmin activity was
recovered with a yield of approximately 95% during this step.
Example 13
Ultrafiltration/Diafiltration
[0228] Plasmin present in the eluate from the Benzamidine Sepharose
column was concentrated approximately 5-fold (to 5 mg/mL) and
diafiltered against 5.0 mM sodium citrate (pH 3.3), using a 5 kDa
Millipore Pellicon XL (0.005 m2) membrane. Following diafiltration,
this acidic plasmin solution was stored at -80.degree. C.
Example 14
Plasmin Concentration, Activity and Purity
[0229] Total protein was quantified in upstream purification
intermediates by the pyrogallol red method using a plasminogen
standard that had been calibrated based upon its absorbance at 280
nm. The extinction coefficient for pure recombinant plasmin was
calculated to be 1.66 mg.sup.-1mL based upon amino acid
composition; absorbance at 280 nm was used to quantify plasmin
protein in downstream purification intermediates. Plasminogen
activity was assayed using the COAMATIC.RTM. Plasminogen kit
(DiaPharma Group, Inc.). The latter kit was also used to assay
plasmin activity but without addition of SK to the assay mixtures;
these assays were calibrated and validated to measure the
concentration of catalytically-active zymogen. Conventional
SDS-PAGE was performed under reducing conditions. Size-exclusion
HPLC was performed with a 7.8 mm.times.30 cm TSK-GEL G2000SW.sub.XL
column (Tosoh BioScience) with 10 mM acetic acid, 100 mM NaCl, pH
3.4, as mobile phase. E. coli BL21 host-cell proteins were assayed
with an ELISA kit from Cygnus Technologies. SK was estimated with a
semi-quantitative Western blot using custom-prepared,
affinity-purified rabbit anti-SK sera.
[0230] The primary criterion for purity of plasminogen and plasmin
is the specific activity of the preparations, with a specific
activity of 1.0 representing 100% pure protein. As shown in Tables
3 and 4, the specific activities of the plasminogen intermediate
and the plasmin final product were both close to 1.0.
TABLE-US-00004 TABLE 4 Purification of plasmin from purified
plasminogen Col. 5 Col. 6 Col. 7 Col. 1 Col. 4 Col. 3 Col. 4
Specific (ng/mg (.mu.g/mg Purification step (mg/mL) (mg).sup.a,b
(mg).sup.a,b (%).sup.a,b activity.sup.c Protein) Protein)
Streptokinase-activated 2.47 .+-. 0.01 636 .+-. 49 489 .+-. 47 82.9
.+-. 3.6 0.77 .+-. 0.03 DND.sup.d 4.2 .+-. 1.5 plasmin Q membrane
flow- 1.00 .+-. 0.01 574 .+-. 46 425 .+-. 30 91.1 .+-. 0.3 0.74
.+-. 0.01 DND <QL.sup.e through Benzamidine Sepharose 1.09 .+-.
0.08 472 .+-. 43 409 .+-. 37 96.1 .+-. 4.1 0.87 .+-. 0.03 <12
<QL eluate plasmin Ultrafiltered/diafiltered 6.19 .+-. 0.28 459
.+-. 29 414 .+-. 11 101.7 .+-. 6.5 0.90 .+-. 0.05 <0.9 <QL
plasmin Streptokinase-activated 2.47 .+-. 0.01 636 .+-. 49 489 .+-.
47 82.9 .+-. 3.6 0.77 .+-. 0.03 DND.sup.d 4.2 .+-. 1.5 plasmin In
Table 4: Column 1: Protein Concentration; Column 2: Total Protein;
Column 3: Total Activity; Column 4: Activity recovered in process
step; Column 5: Specific activity; Column 6: Host cell protein;
Column 7: SK Concentration. .sup.aValues not corrected for volume
of sample withdrawn for bioanalytical assays. .sup.bValues
represent the mean .+-. the standard deviation from the mean for
three different purification runs. .sup.cSpecific activity, mg of
active recombinant plasmin per mg of total protein. .sup.dAnalysis
was not conducted on these samples. .sup.eBelow the level of
quantification (0.15 .mu.g/mL).
[0231] Corroborating these estimations of purity are the SDS-PAGE
results presented in FIG. 4, which illustrate the progressive
purification of plasminogen, conversion of single-chain plasminogen
into two-chain plasmin by SK, and purification of plasmin. A small
percentage of autolysis products was observed in the
ultrafiltered/diafiltered plasmin at the end of the purification
process (FIG. 4, lane 9). Size-exclusion HPLC analyses of the three
samples of final product are overlaid in FIG. 5; this analysis
demonstrated that 98% of the protein was eluted in the peak
corresponding to monomeric plasmin. Host-cell protein was reduced
to <0.8 ng/mg of purified plasmin. SK was reduced to below the
level of quantification (<0.02 .mu.g/mg of plasmin).
[0232] Recombinant plasminogen protein present in solubilized
inclusion bodies was refolded into active protein with a yield of
approximately 48%. The overall recovery of activity after
ECH-Lysine Sepharose chromatography, based upon active, refolded
zymogen, was 70%. The yield of plasmin activity, based upon
starting zymogen activity, was 65%. Thus, the calculated overall
yield of plasmin, based upon active, refolded zymogen, was 46% for
the three purification runs presented in Tables 3 and 4.
Example 15
Anion Exchange Chromatography of Plasmin Prepared from Recombinant
Plasminogen
[0233] A Q membrane chromatography was implemented immediately
after activation of recombinant plasminogen to plasmin using SK.
The SK activation mixture was pH-adjusted from about 7 to about 8,
loaded onto the Q membrane equilibrated in pH 8 Tris buffer, and
the flow-through containing plasmin was collected. Bench scale
studies were performed and membrane loading conditions conducive to
SK binding and plasmin recovery were identified. Samples from bench
scale experiments were assayed for SK content and the sample purity
was confirmed.
Example 16
Anion Exchange Chromatography of Plasmin Prepared from
Plasma-Derived Plasminogen
[0234] Blood-derived plasminogens and/or plasmins prepared
therefrom, are disclosed by, e.g., U.S. Pat. Nos. 6,355,243,
6,964,764, 6,969,515, and 7,544,500; U.S. Patent Publication Nos.
2002/0192794 and 2003/0012778; and Deutsch et al., Science,
170:1095-6 (1970), each of which is herein incorporated by
reference in its entirety. Accordingly, plasminogen is prepared
from blood (e.g., plasma, serum) in order to provide a composition
comprising the plasmin.
[0235] For example, plasminogen is prepared from Cohn Fraction
II+III paste by affinity chromatography on Lys-SEPHAROSE as
described by Deutsch et al. supra. For example, 200 g of a Cohn
Fraction II+III paste is resuspended in 2 liter of 0.15 M sodium
citrate buffer, pH 7.8. The suspension is incubated overnight at
37.degree. C., centrifuged at 14,000 rpm, filtered through
fiberglass and mixed with 500 mL of Lys-SEPHAROSE 4B (Pharmacia).
Binding of plasminogen is performed at room temperature for 2
hours. The Lys-SEPHAROSE is then be transferred onto a 2-liter
glass filter, and washed several times with 0.15 M sodium citrate
containing 0.3 M NaCl until the absorbance at 280 nm drops below
0.05. Bound plasminogen is eluted with three 200-mL portions of 0.2
M .epsilon.-aminocaproic acid. Eluted plasminogen is precipitated
with 0.4 g solid ammonium sulfate/mL of plasminogen solution. The
precipitate of crude (80-85% pure) plasminogen can be stored at
4.degree. C.
[0236] The ammonium sulfate precipitate of crude plasminogen is
centrifuged at 14,000 rpm and resuspended in a minimal volume using
40 mM Tris, containing 10 mM lysine, 80 mM NaCl at pH 9.0 to
achieve a final protein concentration of 10-15 mg/mL. The
plasminogen solution is dialyzed overnight against the same buffer
to remove ammonium sulfate. The dialyzed plasminogen solution
(10-20 mL) is diluted with an equal volume of 100% glycerol and
combined with an appropriate amount of a plasminogen activator,
preferably streptokinase. The use of 50% glycerol can reduce
autodegradation of plasmin formed during activation by the
activator.
[0237] The plasminogen activation by the activator (e.g.,
streptokinase) is at room temperature for about 2 hours to about 24
hours or more. SDS-PAGE is performed under reducing conditions to
monitor the progress of plasminogen activation. Upon completion of
the activation, the activation solution comprising plasmin is
filtered with a glass filter, if desired, and applied to an
affinity adsorbent such as benzamidine-SEPHAROSE. Since the plasmin
is a serine protease with trypsin-like specificity,
benzamidine-Sepharose is an affinity absorbent that can allow
capture of the active plasmin. For example, a solution in 50%
glycerol is applied to the 50 mL benzamidine-Sepharose column
equilibrated with 0.05 M Tris, pH 8.0, containing 0.5 M NaCl with a
flow rate of 3 mL/min. The column is run at 3 mL/min at 3-7.degree.
C. The front portion of the non-bound peak contains high-molecular
weight impurities. The rest of the non-bound peak is represented by
residual non-activated plasminogen and by inactive autodegradation
products of plasmin.
[0238] To protect plasmin from inactivation at neutral pH
conditions, acidic elution conditions are selected. The plasmin
bound to benzamidine-Sepharose is eluted with, e.g., 0.2 M glycine
buffer, pH 3.0 containing 0.5 M NaCl.
[0239] An eluate collected from a benzamidine Sepharose column was
adjusted to pH about 6.5 to 7.0 and diluted 4-fold with water for
injection (WFI). A Q membrane (Sartorius Sartobind MA 5 Q membrane)
was equilibrated with equilibration buffer (62.5 mM ECAC, 37.5 mM
NaCl, pH 6.5 to 7.0) and the diluted benzamidine eluate was loaded
onto the Q membrane. The flow-through and rinse were collected, and
the pH was immediately adjusted to pH 3.4 with 1 N HCl. This
pH-adjusted flow-through was subsequently concentrated and
diafiltered by UF/DF.
[0240] SDS-PAGE analysis confirmed that very little (.about.10% or
less) of plasmin was bound to the Q membrane with the remainder
being captured in the flow-through and rinse fractions (FIG. 9).
And, Western blot analysis confirmed that the SK fragments present
in the Q load were not present in the flow-through and rinse, but
were present in the strip fraction when the Q membrane was treated
with 62.5 mM .epsilon.-ACA, 500 mM NaCl, pH 6.5. The Western blot
was analyzed densitometrically and the results are shown in Table
5.
TABLE-US-00005 TABLE 5 Densitometric analysis of Western Blot
lanes. Lane Sample ID (samples tested neat) 15 kD Conc. .mu.g/mL 1
1.0 .mu.g/mL 15 kD standard 1.000 2 0.5 .mu.g/mL 15 kD standard
0.500 3 0.15 .mu.g/mL 15 kD standard 0.150 4 PBC 050 221
Benzamidine Load >1.0 5 PBC 060 221 Benzamidine Flow-through
>1.0 6 PBC 070 221 Benzamidine Eluate <0.15 7 AJC 007
Benzamidine Load 0.25 8 AJC 008 Q Membrane Load, pH 6.5 <0.15 9
AJC 009 Q Membrane Flow-through, pH 6.5 <0.15 10 AJC 010 Q
Membrane Wash, pH 6.5 <0.15 11 AJC 011 Q membrane Eluate, pH 6.5
0.46
Sequence CWU 1
1
71344PRTHomo sapiens 1Met Arg Asp Val Val Leu Phe Glu Lys Lys Val
Tyr Leu Ser Glu Cys 1 5 10 15 Lys Thr Gly Asn Gly Lys Asn Tyr Arg
Gly Thr Met Ser Lys Thr Lys 20 25 30 Asn Gly Ile Thr Cys Gln Lys
Trp Ser Ser Thr Ser Pro His Arg Pro 35 40 45 Arg Phe Ser Pro Ala
Thr His Pro Ser Glu Gly Leu Glu Glu Asn Tyr 50 55 60 Cys Arg Asn
Pro Asp Asn Asp Pro Gln Gly Pro Trp Cys Tyr Thr Thr 65 70 75 80 Asp
Pro Glu Lys Arg Tyr Asp Tyr Cys Asp Val Pro Gln Cys Ala Ala 85 90
95 Pro Ser Phe Asp Cys Gly Lys Pro Gln Val Glu Pro Lys Lys Cys Pro
100 105 110 Gly Arg Val Val Gly Gly Cys Val Ala His Pro His Ser Trp
Pro Trp 115 120 125 Gln Val Ser Leu Arg Thr Arg Phe Gly Met His Phe
Cys Gly Gly Thr 130 135 140 Leu Ile Ser Pro Glu Trp Val Leu Thr Ala
Ala His Cys Leu Glu Lys 145 150 155 160 Ser Pro Arg Pro Ser Ser Tyr
Lys Val Ile Leu Gly Ala His Gln Glu 165 170 175 Val Asn Leu Glu Pro
His Val Gln Glu Ile Glu Val Ser Arg Leu Phe 180 185 190 Leu Glu Pro
Thr Arg Lys Asp Ile Ala Leu Leu Lys Leu Ser Ser Pro 195 200 205 Ala
Val Ile Thr Asp Lys Val Ile Pro Ala Cys Leu Pro Ser Pro Asn 210 215
220 Tyr Val Val Ala Asp Arg Thr Glu Cys Phe Ile Thr Gly Trp Gly Glu
225 230 235 240 Thr Gln Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala
Gln Leu Pro 245 250 255 Val Ile Glu Asn Lys Val Cys Asn Arg Tyr Glu
Phe Leu Asn Gly Arg 260 265 270 Val Gln Ser Thr Glu Leu Cys Ala Gly
His Leu Ala Gly Gly Thr Asp 275 280 285 Ser Cys Gln Gly Asp Ser Gly
Gly Pro Leu Val Cys Phe Glu Lys Asp 290 295 300 Lys Tyr Ile Leu Gln
Gly Val Thr Ser Trp Gly Leu Gly Cys Ala Arg 305 310 315 320 Pro Asn
Lys Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val Thr Trp 325 330 335
Ile Glu Gly Val Met Arg Asn Asn 340 2810PRTHomo sapiens 2Met Glu
His Lys Glu Val Val Leu Leu Leu Leu Leu Phe Leu Lys Ser 1 5 10 15
Gly Gln Gly Glu Pro Leu Asp Asp Tyr Val Asn Thr Gln Gly Ala Ser 20
25 30 Leu Phe Ser Val Thr Lys Lys Gln Leu Gly Ala Gly Ser Ile Glu
Glu 35 40 45 Cys Ala Ala Lys Cys Glu Glu Asp Glu Glu Phe Thr Cys
Arg Ala Phe 50 55 60 Gln Tyr His Ser Lys Glu Gln Gln Cys Val Ile
Met Ala Glu Asn Arg 65 70 75 80 Lys Ser Ser Ile Ile Ile Arg Met Arg
Asp Val Val Leu Phe Glu Lys 85 90 95 Lys Val Tyr Leu Ser Glu Cys
Lys Thr Gly Asn Gly Lys Asn Tyr Arg 100 105 110 Gly Thr Met Ser Lys
Thr Lys Asn Gly Ile Thr Cys Gln Lys Trp Ser 115 120 125 Ser Thr Ser
Pro His Arg Pro Arg Phe Ser Pro Ala Thr His Pro Ser 130 135 140 Glu
Gly Leu Glu Glu Asn Tyr Cys Arg Asn Pro Asp Asn Asp Pro Gln 145 150
155 160 Gly Pro Trp Cys Tyr Thr Thr Asp Pro Glu Lys Arg Tyr Asp Tyr
Cys 165 170 175 Asp Ile Leu Glu Cys Glu Glu Glu Cys Met His Cys Ser
Gly Glu Asn 180 185 190 Tyr Asp Gly Lys Ile Ser Lys Thr Met Ser Gly
Leu Glu Cys Gln Ala 195 200 205 Trp Asp Ser Gln Ser Pro His Ala His
Gly Tyr Ile Pro Ser Lys Phe 210 215 220 Pro Asn Lys Asn Leu Lys Lys
Asn Tyr Cys Arg Asn Pro Asp Arg Glu 225 230 235 240 Leu Arg Pro Trp
Cys Phe Thr Thr Asp Pro Asn Lys Arg Trp Glu Leu 245 250 255 Cys Asp
Ile Pro Arg Cys Thr Thr Pro Pro Pro Ser Ser Gly Pro Thr 260 265 270
Tyr Gln Cys Leu Lys Gly Thr Gly Glu Asn Tyr Arg Gly Asn Val Ala 275
280 285 Val Thr Val Ser Gly His Thr Cys Gln His Trp Ser Ala Gln Thr
Pro 290 295 300 His Thr His Asn Arg Thr Pro Glu Asn Phe Pro Cys Lys
Asn Leu Asp 305 310 315 320 Glu Asn Tyr Cys Arg Asn Pro Asp Gly Lys
Arg Ala Pro Trp Cys His 325 330 335 Thr Thr Asn Ser Gln Val Arg Trp
Glu Tyr Cys Lys Ile Pro Ser Cys 340 345 350 Asp Ser Ser Pro Val Ser
Thr Glu Gln Leu Ala Pro Thr Ala Pro Pro 355 360 365 Glu Leu Thr Pro
Val Val Gln Asp Cys Tyr His Gly Asp Gly Gln Ser 370 375 380 Tyr Arg
Gly Thr Ser Ser Thr Thr Thr Thr Gly Lys Lys Cys Gln Ser 385 390 395
400 Trp Ser Ser Met Thr Pro His Arg His Gln Lys Thr Pro Glu Asn Tyr
405 410 415 Pro Asn Ala Gly Leu Thr Met Asn Tyr Cys Arg Asn Pro Asp
Ala Asp 420 425 430 Lys Gly Pro Trp Cys Phe Thr Thr Asp Pro Ser Val
Arg Trp Glu Tyr 435 440 445 Cys Asn Leu Lys Lys Cys Ser Gly Thr Glu
Ala Ser Val Val Ala Pro 450 455 460 Pro Pro Val Val Leu Leu Pro Asp
Val Glu Thr Pro Ser Glu Glu Asp 465 470 475 480 Cys Met Phe Gly Asn
Gly Lys Gly Tyr Arg Gly Lys Arg Ala Thr Thr 485 490 495 Val Thr Gly
Thr Pro Cys Gln Asp Trp Ala Ala Gln Glu Pro His Arg 500 505 510 His
Ser Ile Phe Thr Pro Glu Thr Asn Pro Arg Ala Gly Leu Glu Lys 515 520
525 Asn Tyr Cys Arg Asn Pro Asp Gly Asp Val Gly Gly Pro Trp Cys Tyr
530 535 540 Thr Thr Asn Pro Arg Lys Leu Tyr Asp Tyr Cys Asp Val Pro
Gln Cys 545 550 555 560 Ala Ala Pro Ser Phe Asp Cys Gly Lys Pro Gln
Val Glu Pro Lys Lys 565 570 575 Cys Pro Gly Arg Val Val Gly Gly Cys
Val Ala His Pro His Ser Trp 580 585 590 Pro Trp Gln Val Ser Leu Arg
Thr Arg Phe Gly Met His Phe Cys Gly 595 600 605 Gly Thr Leu Ile Ser
Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu 610 615 620 Glu Lys Ser
Pro Arg Pro Ser Ser Tyr Lys Val Ile Leu Gly Ala His 625 630 635 640
Gln Glu Val Asn Leu Glu Pro His Val Gln Glu Ile Glu Val Ser Arg 645
650 655 Leu Phe Leu Glu Pro Thr Arg Lys Asp Ile Ala Leu Leu Lys Leu
Ser 660 665 670 Ser Pro Ala Val Ile Thr Asp Lys Val Ile Pro Ala Cys
Leu Pro Ser 675 680 685 Pro Asn Tyr Val Val Ala Asp Arg Thr Glu Cys
Phe Ile Thr Gly Trp 690 695 700 Gly Glu Thr Gln Gly Thr Phe Gly Ala
Gly Leu Leu Lys Glu Ala Gln 705 710 715 720 Leu Pro Val Ile Glu Asn
Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn 725 730 735 Gly Arg Val Gln
Ser Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly 740 745 750 Thr Asp
Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu 755 760 765
Lys Asp Lys Tyr Ile Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys 770
775 780 Ala Arg Pro Asn Lys Pro Gly Val Tyr Val Arg Val Ser Arg Phe
Val 785 790 795 800 Thr Trp Ile Glu Gly Val Met Arg Asn Asn 805 810
379PRTHomo sapiens 3Cys Lys Thr Gly Asn Gly Lys Asn Tyr Arg Gly Thr
Met Ser Lys Thr 1 5 10 15 Lys Asn Gly Ile Thr Cys Gln Lys Trp Ser
Ser Thr Ser Pro His Arg 20 25 30 Pro Arg Phe Ser Pro Ala Thr His
Pro Ser Glu Gly Leu Glu Glu Asn 35 40 45 Tyr Cys Arg Asn Pro Asp
Asn Asp Pro Gln Gly Pro Trp Cys Tyr Thr 50 55 60 Thr Asp Pro Glu
Lys Arg Tyr Asp Tyr Cys Asp Ile Leu Glu Cys 65 70 75 478PRTHomo
sapiens 4Cys Met His Cys Ser Gly Glu Asn Tyr Asp Gly Lys Ile Ser
Lys Thr 1 5 10 15 Met Ser Gly Leu Glu Cys Gln Ala Trp Asp Ser Gln
Ser Pro His Ala 20 25 30 His Gly Tyr Ile Pro Ser Lys Phe Pro Asn
Lys Asn Leu Lys Lys Asn 35 40 45 Tyr Cys Arg Asn Pro Asp Arg Glu
Leu Arg Pro Trp Cys Phe Thr Thr 50 55 60 Asp Pro Asn Lys Arg Trp
Glu Leu Cys Asp Ile Pro Arg Cys 65 70 75 578PRTHomo sapiens 5Cys
Leu Lys Gly Thr Gly Glu Asn Tyr Arg Gly Asn Val Ala Val Thr 1 5 10
15 Val Ser Gly His Thr Cys Gln His Trp Ser Ala Gln Thr Pro His Thr
20 25 30 His Asn Arg Thr Pro Glu Asn Phe Pro Cys Lys Asn Leu Asp
Glu Asn 35 40 45 Tyr Cys Arg Asn Pro Asp Gly Lys Arg Ala Pro Trp
Cys His Thr Thr 50 55 60 Asn Ser Gln Val Arg Trp Glu Tyr Cys Lys
Ile Pro Ser Cys 65 70 75 678PRTHomo sapiens 6Cys Tyr His Gly Asp
Gly Gln Ser Tyr Arg Gly Thr Ser Ser Thr Thr 1 5 10 15 Thr Thr Gly
Lys Lys Cys Gln Ser Trp Ser Ser Met Thr Pro His Arg 20 25 30 His
Gln Lys Thr Pro Glu Asn Tyr Pro Asn Ala Gly Leu Thr Met Asn 35 40
45 Tyr Cys Arg Asn Pro Asp Ala Asp Lys Gly Pro Trp Cys Phe Thr Thr
50 55 60 Asp Pro Ser Val Arg Trp Glu Tyr Cys Asn Leu Lys Lys Cys 65
70 75 780PRTHomo sapiens 7Cys Met Phe Gly Asn Gly Lys Gly Tyr Arg
Gly Lys Arg Ala Thr Thr 1 5 10 15 Val Thr Gly Thr Pro Cys Gln Asp
Trp Ala Ala Gln Glu Pro His Arg 20 25 30 His Ser Ile Phe Thr Pro
Glu Thr Asn Pro Arg Ala Gly Leu Glu Lys 35 40 45 Asn Tyr Cys Arg
Asn Pro Asp Gly Asp Val Gly Gly Pro Trp Cys Tyr 50 55 60 Thr Thr
Asn Pro Arg Lys Leu Tyr Asp Tyr Cys Asp Val Pro Gln Cys 65 70 75
80
* * * * *
References