U.S. patent application number 13/761676 was filed with the patent office on 2013-06-27 for process for the production of a reversibly inactive acidified plasmin composition.
This patent application is currently assigned to GRIFOLS, S.A.. The applicant listed for this patent is GRIFOLS, S.A.. Invention is credited to George A. Baumbach, Rita T. Bradley, Scott A. Cook, Christopher A. Dadd, Jonathan D. Kent, Marina N. Korneyeva, Valery Novokhatny, Christopher J. Stenland, Tanette B. Villines.
Application Number | 20130164815 13/761676 |
Document ID | / |
Family ID | 23740237 |
Filed Date | 2013-06-27 |
United States Patent
Application |
20130164815 |
Kind Code |
A1 |
Dadd; Christopher A. ; et
al. |
June 27, 2013 |
PROCESS FOR THE PRODUCTION OF A REVERSIBLY INACTIVE ACIDIFIED
PLASMIN COMPOSITION
Abstract
Disclosed is both a process for producing a reversibly inactive
acidified plasmin by activating plasminogen and a process for
producing a purified plasminogen. The produced plasmin is isolated
and stored with a low pH-buffering capacity agent to provide a
substantially stable formulation. The purified plasminogen is
typically purified from a fraction obtained in the separation of
immunoglobulin from Fraction II+III chromatographic process and
eluded at a low pH. The reversibly inactive acidified plasmin may
be used in the administration of a thrombolytic therapy.
Inventors: |
Dadd; Christopher A.; (Holly
Springs, NC) ; Stenland; Christopher J.; (Canyon
Country, CA) ; Kent; Jonathan D.; (Holly Springs,
NC) ; Korneyeva; Marina N.; (Raleigh, NC) ;
Baumbach; George A.; (Knightdale, NC) ; Cook; Scott
A.; (Ballwin, MO) ; Bradley; Rita T.; (Cary,
NC) ; Novokhatny; Valery; (Raleigh, NC) ;
Villines; Tanette B.; (Raleigh, NC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
GRIFOLS, S.A.; |
Barcelona |
|
ES |
|
|
Assignee: |
GRIFOLS, S.A.
Barcelona
ES
|
Family ID: |
23740237 |
Appl. No.: |
13/761676 |
Filed: |
February 7, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10143156 |
May 10, 2002 |
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13761676 |
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PCT/US00/42143 |
Nov 13, 2000 |
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10143156 |
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09438331 |
Nov 13, 1999 |
6355243 |
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PCT/US00/42143 |
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Current U.S.
Class: |
435/184 ;
435/188; 435/217 |
Current CPC
Class: |
A61K 47/12 20130101;
A61K 47/183 20130101; C12N 9/6435 20130101; A61K 47/02 20130101;
C12Y 304/21007 20130101; A61P 7/02 20180101; A61K 38/4833 20130101;
A61K 38/484 20130101; A61K 47/20 20130101; A61K 47/26 20130101;
A61K 45/06 20130101; C12N 9/99 20130101; C12N 9/96 20130101; A61P
9/10 20180101 |
Class at
Publication: |
435/184 ;
435/217; 435/188 |
International
Class: |
C12N 9/99 20060101
C12N009/99; C12N 9/96 20060101 C12N009/96; C12N 9/64 20060101
C12N009/64 |
Claims
1. A method for purifying plasmin comprising: cleaving a
plasminogen in the presence of a plasminogen activator to yield an
active plasmin; substantially removing the plasminogen activator
from the active plasmin to form a plasmin solution; and buffering
the plasmin solution with a low pH-buffering capacity agent to form
a reversibly inactive acidified plasmin.
2. The method of claim 1, wherein the step of substantially
removing the plasminogen activator includes the steps of: binding
the active plasmin to an active plasmin-specific absorbent material
to form a bound plasmin; and eluting the bound plasmin with a low
pH solution to form the plasmin solution.
3. The method of claim 2, wherein the active plasmin-specific
absorbent material comprises benzamidine.
4. The method of claim 2, wherein the plasminogen activator is
further removed by hydrophobic interaction.
5. The method of claim 1, further including cleaving the
plasminogen in the presence of stabilizers comprising omega-amino
acids and glycerol.
6. The method of claim 1, wherein the plasminogen is cleaved using
a catalytic concentration of a plasminogen activator that is
selected from the group consisting of immobilized, soluble and
combinations thereof of plasminogen activators.
7. The method of claim 6, wherein the plasminogen activator is
selected from the group consisting of streptokinase, urokinase, tPA
and combinations thereof.
8. The method of claim 7, wherein the plasminogen activator is
soluble streptokinase.
9. The method of claim 6, wherein the plasminogen activator is
immobilized on a solid support medium comprising SEPHAROSE.
10. The method of claim 1, wherein the low pH-buffering capacity
agent comprises a buffer comprising an amino acid, a derivative of
at least one amino acid, an oligopeptide which includes at least
one amino acid, or a combination thereof.
11. The method of claim 1, wherein the low pH-buffering capacity
agent comprises a buffer selected from acetic acid, citric acid,
hydrochloric acid, carboxylic acid, lactic acid, malic acid,
tartaric acid, benzoic acid, serine, threonine, methionine,
glutamine, alanine, glycine, isoleucine, valine, alanine, aspartic
acid, derivatives thereof, or combinations thereof.
12. The method of claim 1, wherein the buffer is present in the
reversibly inactive acidified plasmin at a concentration at which
the pH of the acidified plasmin is raised to neutral pH by adding
no more than about 5 times the volume of serum to the acidified
plasmin.
13. The method of claim 1, wherein the reversibly inactive
acidified plasmin solution has a pH between about 2.5 to about
4.
14. The method of claim 1, further including stabilizing the
reversibly inactive acidified plasmin by adding a stabilizing agent
selected from a polyhydric alcohol, pharmaceutically acceptable
carbohydrates, salts, glucosamine, thiamine, niacinamide, or
combinations thereof.
15. The method of claim 14, wherein the salts are selected from the
group consisting of sodium chloride, potassium chloride, magnesium
chloride, calcium chloride and combinations thereof.
16. The method of claim 1, further including stabilizing the
reversibly inactive acidified plasmin by adding a sugar or sugar
alcohol selected from glucose, maltose, mannitol, sorbitol,
sucrose, lactose, trehalose, or combinations thereof.
17. A method for purifying plasmin comprising: cleaving a
plasminogen to yield an active plasmin; binding the active plasmin
to an active plasmin-specific absorbent material to form a bound
plasmin; and eluting the bound plasmin with a substantially neutral
amino acid to form a final plasmin solution which is substantially
free of degraded plasmin.
18. The method of claim 17, wherein the plasminogen is cleaved
using a catalytic concentration of a plasminogen activator.
19. The method of claim 17, wherein the activated plasmin solution
is stabilized by the addition of omega-amino acids and sodium
chloride.
20. The method of claim 17, wherein the substantially neutral amino
acid comprises an omega-amino acid.
21. The method of claim 20, wherein the omega-amino acid selected
from the group consisting of lysine, epsilon amino caproic acid,
tranexamic acid, poly lysine, arginine, analogues thereof and
combinations thereof.
22. The method of claim 17, further including filtering out the
substantially neutral amino acid from the final plasmin
solution.
23. The method of claim 17, further including adding a low
pH-buffering capacity agent to the final plasmin solution to form a
reversibly inactive acidified plasmin.
24. The method of claim 23, further including adjusting the pH of
the reversibly inactive acidified plasmin to a pH between about 2.5
to about 4.
25. The method of claim 17, further including adding a stabilizer
to the final plasmin solution.
26. The method of claim 25, wherein the stabilizer is selected from
the group consisting of amino acids, salts or combinations
thereof.
27. A process for the purification of plasminogen from a plasma
source comprising: adding the plasminogen containing solution to a
plasminogen-specific absorbent material; eluting the plasminogen
from the plasminogen-specific absorbent material at a pH of between
about 1 to about 4; and collecting the plasminogen containing
eluate.
28. The process of claim 27, wherein the plasma source is derived
from Fraction II+III of Cohn plasma fractionation process.
29. The process of claim 27, further comprising the steps of:
extracting plasminogen from a plasma paste fraction with a buffer
solution at a pH in a range from about 3.5 to 10.5 and collecting
the plasminogen containing solution; adding polyethylene glycol or
ammonium sulfate to the plasminogen containing buffer solution to
precipitate impurities; separating the precipitated impurities from
the effluent containing plasminogen; and adding the effluent
containing plasminogen to a lysine affinity resin.
30. The process of claim 29, wherein the plasminogen extraction
solution is at a pH in a range from about 7.0 to about 10.5.
31. The process of claim 29 wherein about 1 to about 10% w/w
polyethylene glycol or 80 to 120 g/L ammonium sulfate is added.
32. The process of claim 29, further including adding a plasminogen
solubility enhancer.
33. The process of claim 32, wherein the plasminogen solubility
enhancer is selected from the group of omega-amino acids consisting
of lysine, epsilon amino caproic acid, tranexamic acid, poly
lysine, arginine, combinations thereof and analogues thereof.
34. The process of claim 32, further including removing the
plasminogen solubility enhancer
35. The process of claim 27, wherein the eluted plasminogen is
treated at a pH between about 3 and about 4.
36. The process of stabilizing plasminogen during pH adjustment
from about 3 to neutral by adding omega-amino acids prior to pH
adjustment.
37. The process of claim 27, further including removing
pathogens.
38. The process of claim 37, wherein the removing pathogens
includes inactivating viral pathogens and removing TSE
pathogens.
39. The process of claim 38, wherein viruses are inactivated or
removed by the steps selected from the group consisting of heat
treatment, caprylate addition, solvent detergent addition,
nanofiltration and combinations thereof.
40. The process of claim 38, wherein TSE are removed by the steps
selected from the group consisting of PEG precipitation, depth
filtration and nanofiltration.
41. The process of claim 27, wherein the plasminogen-specific
absorbent material comprises a lysine affinity resin.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 10/143,156 filed on May 10, 2002, which is a
continuation of International Application PCT/US2000/42143 filed on
Nov. 13, 2000 and published in English on May 25, 2001, which is a
continuation-in-part of U.S. patent application Ser. No.
09/438,331, filed Nov. 13, 1999, each of which are incorporated
herein by reference in their entirety.
TECHNICAL FIELD
[0002] The present invention relates generally to a method of
producing plasmin and more particularly to a method of purifying
and isolating the plasmin under conditions, which stabilize against
degradation.
BACKGROUND
[0003] Fibrin is a white insoluble fibrous protein formed from
fibrinogen by the action of thrombin. In the clotting of blood,
fibrin forms the structural scaffold of a thrombus, which is a clot
of blood formed within a blood vessel that remains attached to its
place of origin. Under normal conditions, the blood clotting system
is maintained in equilibrium and the fibrin deposits are dissolved
by the fibrinolytic enzyme system. Unfortunately, events such as
vascular damage, activation/stimulation of platelets, and
activation of the coagulation cascade may disturb the equilibrium,
which can result in thrombosis or the blockage of a blood vessel by
a blood clot.
[0004] Intravascular thrombosis is one of the most frequent
pathological events accounting for greater than 50% of all deaths
as well as a variety of other serious clinical problems. Most
spontaneously developing vascular obstructions are due to the
formation of intravascular blood clots, also known as thrombi.
Small fragments of a clot may detach from the body of the clot and
travel through the circulatory system to lodge in distant organs
and initiate further clot formation. Myocardial infarction,
occlusive stroke, deep venous thrombosis (DVT), and peripheral
arterial disease are well known consequences of thromboembolic
phenomena.
[0005] Plasminogen activators are currently the favored agents
employed in thrombolytic therapy, all of which convert plasminogen
to plasmin and promote fibrinolysis by disrupting the fibrin matrix
(M. A. Creager and V. J. Dzau, Vascular Diseases of the
Extremities, ppgs. 1398-406 in Harrison's Principles of Internal
Medicine, 14.sup.th ed., Fauci et al., editors, McGraw-Hill Co.,
New York, 1998; the contents of which is incorporated herein by
reference in its entirety).
[0006] The most widely used plasminogen activators include a
recombinant form of tissue-type plasminogen activator (tPA),
urokinase (UK) and streptokinase (SK), as well as a new generation
of plasminogen activators selected for improved pharmacokinetics
and fibrin-binding properties. All of these plasminogen activators,
however, by virtue of their mechanism of action, act indirectly and
require an adequate supply of their common substrate, plasminogen,
at the site of the thrombus to effect lysis.
[0007] UK and tPA convert plasminogen to plasmin directly by
cleaving the Arg560-Va1561 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. SK and staphylokinase
activate plasminogen indirectly by forming a complex with
plasminogen, which subsequently behaves as a plasminogen activator
to activate other plasminogen molecules by cleaving the
arginyl-valine bond.
[0008] Although thrombolytic drugs, such as tissue plasminogen
activator (tPA), streptokinase and urokinase, have been
successfully employed clinically to reduce the extent of a
thrombotic occlusion of a blood vessel, it appears that serious
limitations persist with regard to their use in current
thrombolytic therapy. For example, because the activation of
plasminogen by tPA is fibrin dependent for full proteolytic
activity to be realized (Haber et al. 1989), excessive bleeding may
result as a side effect of its use. Other adverse sequelae
associated with the use of these thrombolytic agents include
myocardial infarction, occlusive stroke, deep venous thrombosis,
and peripheral arterial disease.
[0009] Additionally, the known plasminogen activators currently
used suffer from several limitations that impact their overall
usefulness in the elimination of a thrombus. For example, at best,
the use of current thrombolytic therapy results in restored
vascular blood flow within 90 min in approximately 50% of patients,
while acute coronary re-occlusion occurs in roughly 10% of
patients. Coronary recanalization requires on average 45 minutes or
more, and intracerebral hemorrhage occurs in 0.3% to 0.7% of
patients. Residual mortality is at least 50% of the mortality level
in the absence of thrombolysis treatment.
[0010] A different approach to avoid the problems associated with
the systemic administration of a plasminogen activator to generate
sufficient plasmin at the site of the thrombus, is to directly
administer the plasmin itself to the patient.
[0011] In U.S. Pat. No. 5,288,489, Reich et al., disclose a
fibrinolytic treatment that includes parenterally introducing
plasmin into the body of a patient. The concentration and time of
treatment were selected to be sufficient to allow adequate active
plasmin to attain a concentration at the site of an intravascular
thrombus that is sufficient to lyse the thrombus or to reduce
circulating fibrinogen levels. However, the necessity of generating
the plasmin from plasminogen immediately prior to its introduction
into the body is also disclosed.
[0012] In contrast, U.S. Pat. No. 3,950,513 to Jenson teaches that
plasmin compositions may be stabilized at pH 7.0 by including a
physiological non-toxic amino acid. This method dilutes stock
plasmin solutions stored at low pH with the neutralizing amino acid
immediately prior to administration. There are advantages, however,
in maintaining low pH of the plasmin composition as long as
possible to minimize autodegradation. Ideally, the plasmin will be
retained at a low pH until encountering the target fibrin.
[0013] Yago et al. disclose plasmin compositions useful as a
diagnostic reagent in U.S. Pat. No. 5,879,923. The compositions of
Yago et al. comprise plasmin and an additional component which may
be (1) an oligopeptide comprising at least two amino acids, or (2)
at least two amino acids, or (3) a single amino acid and a
polyhydric alcohol. However, the compositions of Yago et al. are
formulated at a neutral pH to maintain the enzymatic activity of
plasmin.
[0014] Plasmin as a potential thrombolytic agent has numerous
technical difficulties. These difficulties include the challenge of
preparing pure plasmin that is free of all functional traces of the
plasminogen activator used to convert plasmin from its inactive
precursor, plasminogen. Preparations of plasmin are typically
extensively contaminated by plasminogen activator, streptokinase or
urokinase, and the thrombolytic activity was, 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. A
drawback of streptokinase containing plasmin preparations is that
streptokinase can cause adverse immune reactions including fever
and anaphylactic shock.
[0015] One of the more important technical factors 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. Thus, there is need for
a method of producing stable plasmin.
SUMMARY
[0016] The present invention provides for both a process for
producing a reversibly inactive acidified plasmin by activating
plasminogen and a process for producing a purified plasminogen. The
produced plasmin is isolated and stored in a low pH buffering
capacity agent to provide a substantially stable formulation. The
purified plasminogen is typically purified from a fraction obtained
in the separation of immunoglobulin from Fraction II+III by
affinity chromatography with an elution at a low pH. The reversibly
inactive acidified plasmin may be used in the administration of a
thrombolytic therapy.
[0017] Briefly, the method for purifying plasmin comprises cleaving
a plasminogen in the presence of a plasminogen activator to yield
an active plasmin and removing the plasminogen activator from the
active plasmin to form a plasmin solution. A low pH-buffering
capacity agent can then be added to the final plasmin solution to
form a reversibly inactive acidified plasmin. The final plasmin
solution may be buffered to a pH of between about 2.5 to about
4.
[0018] The plasminogen activator can be removed from the active
plasmin by binding the active plasmin to an active plasmin-specific
absorbent material to form a bound plasmin. One such active
plasmin-specific absorbent material can comprise benzamidine. Once
bound, the active plasmin can be eluted with a low pH solution to
form a final plasmin solution. Plasminogen activator may also be
further removed by hydrophobic interaction.
[0019] A further method of purifying plasmin comprises cleaving
plasminogen to yield an active plasmin and binding the active
plasmin to an active plasmin-specific absorbent material to form a
bound plasmin. The bound plasmin can be eluted with a substantially
neutral amino acid to form a final plasmin solution which is
substantially free of degraded plasmin. The substantially neutral
amino acid can comprise an omega-amino acid and is typically
filtered out of the final plasmin. The final plasmin may also be
buffered with a low pH-buffering capacity agent.
[0020] The process for the purification of plasminogen from a
plasma source includes the steps of adding the plasminogen
containing solution to a plasminogen-specific absorbent material
and then eluting the plasminogen from the plasminogen-specific
absorbent material at a pH of between about 1 to about 4. The
purified plasminogen is then collected as an eluate. Additionally,
the process may include methods for the purification of micro- or
mini-plasmin(ogen) or other truncated or modified forms of
plasmin(ogen).
[0021] Thus, a process is now provided that successfully addresses
the shortcomings of existing processes and provides distinct
advantages over such processes. Additional objects, features, and
advantages of the invention will become more apparent upon review
of the detailed description set fourth below when taken in
conjunction with the accompanying drawing figures, which are
briefly described as follows.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] In the Drawings:
[0023] FIG. 1 graphically depicts the effect of on plasminogen
recovery and lipid removal from CCI filtrate I through PEG
precipitation/depth filtration.
[0024] FIG. 2 graphically depicts nephelometry data for CCI extract
and the subsequent filtrates I and II.
[0025] FIG. 3 depicts a gel of coomassie stained reduced SDS-PAGE
(10-20% Tris-Glycine) of CCI extract, filtrates, and UF/DF
retentate.
[0026] FIG. 4 depicts a coomassie stained reduced SDS-PAGE (10-20%
Tris-Glycine) of lysineSEPHAROSE 4B affinity purification of
Pmg.
[0027] FIG. 5 graphically depicts a lysineSEPHAROSE 4B chromatogram
for the affinity purification of Pmg.
[0028] FIG. 6 depicts a coomassie stained reduced SDS-PAGE (10-20%
Tris-Glycine) of pH adjustment of the lysineSEPHAROSE 4B eluate
(Pmg) with and without epsilon amino caproic acid present.
[0029] FIG. 7 graphically represents streptokinase activation
solution stability following 0.5 M NaCl, 0.25 M .epsilon.-ACA
stop.
[0030] FIG. 8 graphically represents benzamidine SEPHAROSE 6B
chromatogram for the affinity purification of SK activated Pm.
[0031] FIG. 9 depicts a coomassie stained reduced SDS-PAGE (10-20%
Tris-Glycine) of benzamidine SEPHAROSE 6B purified Pm.
[0032] FIG. 10 graphically depicts the hydrophobic interaction
chromatography (OctylSEPHAROSE 4 FF) chromatogram for the removal
of streptokinase.
[0033] FIG. 11 depicts a non-reduced SDS PAGE and anti-SK Western
Blot.
DETAILED DESCRIPTION
[0034] The present invention comprises both a method for producing
a reversibly inactive acidified plasmin in combination with a low
pH-buffering capacity agent and a method for the purification of
plasminogen from a plasma source. The inactive acidified plasmin
solution may also include a stabilizer in addition to being
inactivated in buffered solution. The process for purifying
plasminogen provides for both inactivation and removal of pathogens
and the elution of the plasminogen at a low pH. The inactive
acidified plasmin preparation can be used in the administration of
a thrombolytic therapy.
Purification of Plasminogen
[0035] The present invention includes both a process for the
purification of plasminogen and plasmin and concurrently, methods
for the inactivation and removal of viral and Transmissible
Spongiform Encephalopathies (TSE) contaminants during these
processes. The starting material, plasminogen, can be purified from
Cohn Fraction II+III paste by affinity chromatography on
Lys-SEPHAROSE as described by Deutsch & Mertz (1970). SEPHAROSE
is a trade name of Pharmacia, Inc. of New Jersey for a high
molecular weight substance for the separation by gel filtration of
macromolecules. The process may be performed on any plasma source,
recombinant source, cell culture source, or transgenic source. For
example, plasma from a waste fraction derived from the purification
of immunoglobulin from a chromatographic process can be used as
described in commonly owned U.S. patent application Ser. No.
09/448,771, filed Nov. 24, 1999, which is incorporated by reference
herein.
[0036] Plasminogen was extracted from this waste fraction (referred
to herein as the "caprylate cake I" (CCI)) over a wide range of pH.
Conditions of extraction can be varied from a pH of about 3.5 to
about 10.5 using a variety of buffers capable of providing a pH in
this range, including citrate, acetate, tris, imidazole, histidine,
HEPES and/or phosphate buffers. The extraction can occur at
temperatures from about 4.degree. C. to 37.degree. C. and can be
run for 1 to 24 hours without deleterious effect. In addition, the
ionic strength can be varied by the addition of about 0.2 Molar
sodium chloride without deleterious effect on the extraction of
plasminogen. Following the extraction of plasminogen, lipid and
protein impurities and TSE were reduced by precipitation with the
addition polyethylene glycol (PEG), in a range of about 1 to about
10% weight/volume or the addition of about 80 to about 120 g/L
ammonium sulfate. The PEG or ammonium sulfate precipitate was
removed by depth filtration and the resulting solution placed on a
lysine affinity resin column.
[0037] If desired, the solubility of plasminogen may be enhanced by
the addition of omega-amino acids (lysine, arginine, tranexamic
acid, or epsilon amino caproic acid, or combinations or analogues
thereof) after extraction of the caprylate cake I. Solubility
enhancement may be accomplished with from about 0.02 M to about 1 M
omega-amino acid, preferably about 0.1 M lysine appears to be
sufficient. If added, the lysine is preferably removed after the
PEG or ammonium sulfate precipitation and depth filtration by
diafiltration and the resulting solution placed on a lysine
affinity resin column. The phrase "lysine affinity resin" is used
generally for affinity resins containing lysine or its derivatives
or epsilon caproic acids as the ligand. The column can be eluted
with a low pH solution of approximately 1 to 4.
[0038] The protein obtained after elution from the affinity column
is generally at least 80% plasminogen. The purified plasminogen is
then stored at low pH in the presence of simple buffers such as
glycine and lysine or omega-amino acids. Storage at low pH also
provides an opportunity for viral inactivation and removal and TSE
removal as determined by spiking methods. Our studies suggest that
plasmin meets the most stringent requirements for 6 log clearance
of non-enveloped viruses including one 4 log removal step, and 10
log clearance for enveloped viruses including two independent 4 log
elimination steps. In addition to sufficient virus clearance,
plasmin has greater than 6 logs of TSE infectivity removal for
added safety.
[0039] The plasminogen in solution was then activated to plasmin by
the addition of a plasminogen activator, which may be accomplished
in a number of ways including but not limited to streptokinase,
urokinase, or the use of urokinase immobilized on resin and use of
streptokinase immobilized on resin. The preferred plasminogen
activator is soluble streptokinase. The addition of stabilizers
such as glycerol, and omega-amino acids such as lysine, poly
lysine, arginine, epsilon amino caproic acid and tranexamic acid
were shown to enhance the yield of plasmin.
Purifying Plasmin
[0040] Plasmin was purified from unactivated plasminogen by
affinity chromatography on resin with benzamidine as the ligand and
elution with a neutral omega-amino acid solution or low pH
solution. This step can remove essentially all degraded plasmin as
well as the majority of the streptokinase.
[0041] As a polishing step for the removal of remaining
streptokinase, hydrophobic interaction chromatography at low pH is
performed. Following the HIC step, the plasmin is formulated as a
sterile protein solution by ultrafiltration and diafiltration and
0.22 .mu.m filtration.
[0042] The present method additionally includes the steps of
activating plasminogen to plasmin using a plasminogen activator and
then capturing the formed active plasmin on an active plasmin
specific absorbent material. The bound plasmin is then eluted with
a low pH buffer. The eluted plasmin is buffered with a low
pH-buffering capacity agent such as an acid. Typically, the eluted
plasmin is buffered to a pH of between about 2.5 to about 4.
[0043] The low buffering capacity of the acidic buffer aids in
enabling the reversibly inactivated acidified plasmin to be brought
up to physiological pH quickly and then activated when administered
as a thrombolytic agent. Typically, the buffer is added in a
concentration at which the pH of the acidified plasmin is raised to
neutral pH by adding no more than about 5 times the volume of serum
to the acidified plasmin.
Cleaving the Plasminogen to Yield an Active Plasmin
[0044] Plasminogen can be cleaved to plasmin by using a catalytic
concentration of an immobilized or soluble plasminogen activator.
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
soluble streptokinase, tissue plasminogen activator (tPA) or
urokinase will cleave the single-chain plasminogen molecule to
produce active plasmin at the Arg560-Va1561 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,
alpha2-antiplasmin, or other proteins.
[0045] The activation of plasminogen can occur at about 4.degree.
C. to about 37.degree. C. and typically takes between about 2 to 24
hours. The plasminogen can be cleaved in the presence of
stabilisers such as omega-amino acids and glycerol. The omega-amino
acids can include lysine, epsilon amino caproic acid, tranexamic
acid, poly lysine, arginine and combinations or analogues
thereof.
[0046] Upon the completion of the activation, the plasmin solution
can be filtered and further stabilised for several days at neutral
pH by the addition of omega-amino acids and sodium chloride and
applied to benzamidine-SEPHAROSE.
Removing Plasminogen Activator and Impurities
[0047] The active plasmin formed from the cleaving of the
plasminogen can then be bound to an active plasmin specific
absorbent to substantially remove the plasminogen activator. Since
the protein of interest is an active serine protease with
trypsin-like specificity, benzamidine may be used as an active
plasmin specific absorbent that allows for the capture of the
active plasmin. Other active plasmin specific absorbents having
similar properties as benzamidine may also be used. The benzamidine
can be immobilized in a solid support medium. The solid support
medium can be a resin or SEPHAROSE. Additionally, hydrophobic
interaction may be used to further remove the plasminogen
activator.
[0048] More specifically, the cleaved plasminogen is typically
contained in a solution of amino acids, sodium chloride and
glycerol, which allows for stability of the solution for several
days at neutral pH before it is applied to a benzamidine-SEPHAROSE
column equilibrated with about 0.05 M Tris, pH 8.5, 0.5 M NaCl. The
column is typically run at 4.degree. C. The front portion of the
non-bound peak contains high-molecular weight impurities, with the
rest of the non-bound peak being represented by residual
non-activated plasminogen and by inactive autodegradation products
of plasmin.
[0049] The bound plasmin can then be eluted with an acid buffer or
with a substantially neutral omega-amino acid. The plasmin bound to
benzamidine-SEPHAROSE can be eluted with an acidic buffer such as
glycine buffer. When a substantially neutral pH omega-amino acid is
used to elute the bound plasmin, the final eluted plasmin solution
can be substantially free of degraded plasmin. Typically, the
substantially neutral pH amino acid has a pH of value of between
about 6.5 to about 8.5. Examples of neutral omega-amino acids
include lysine, epsilon amino caproic acid, tranexamic acid, poly
lysine, arginine, and analogues and combinations thereof.
Buffering the Plasmin Solution with a Low pH-Buffering Capacity
Agent
[0050] The eluted plasmin can be buffered with a low pH-buffering
capacity agent. The low pH-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-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 in the reversibly inactive
acidified plasmin at a concentration at which the pH of the
acidified plasmin is raised to neutral pH by adding no more than
about 4 to 5 times the volume of serum to the composition.
[0051] 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 mM
to about 50 mM. Of course, these ranges may be broadened or
narrowed depending upon the buffer chosen, or upon the addition of
other ingredients such as additives or stabilizing agents. The
amount of buffer added is typically that which will bring the
reversibly inactive acidified plasmin solution to have a pH between
about 2.5 to about 4.
Further Stabilizing the Inactive Acidified Plasmin Solution
[0052] The reversibly inactive 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.
[0053] Concentrations of carbohydrate added to stabilize the
reversibly inactive 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.
[0054] The plasmin being formulated in a buffered acidified water
has been found to be extremely stable. It can be kept in this form
for months without any 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 is important because it
would make this formulation 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.
[0055] 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
.sup.125I-fibrin-labelled clot lysis assays. Both of these assays
are performed at pH 7.4, and there was complete recovery of plasmin
activity during the change of pH and passing through the iso-pI
point (pH 5-5.5). This is because plasmin is formulated in a
non-buffered solvent and when added to a buffered solution (either
PBS of plasma) it adopts the neutral pH instantly and the
precipitation that usually accompanies the slow passage through the
iso-pI point, does not occur.
[0056] A feature of the active plasmin as used in the present
invention is the maintenance of the plasmin in an acidic buffer and
its formulation in acidified water, providing a pure and stable
active plasmin. Its efficacy was demonstrated in in vitro assays
and in an in vivo rabbit jugular vein thrombolysis model unified,
substantially purified or partially purified enzyme such as, but
not limited to, plasmin or any composition containing plasmin that
is within the scope of the present invention.
[0057] A description of a method of treating thrombolysis and
related ailments employing aspects of the claimed invention is
disclosed in U.S. patent application Ser. No. 10/143,157, which
issued as U.S. Pat. No. 6,964,764, entitled "Method of Thrombolysis
by Local Delivery of Reversibly Inactivated Acidified Plasmin,"
which is incorporated herein by reference in its entirety.
[0058] Additionally, compositions made in accordance with the
claimed invention are disclosed in U.S. patent application Ser. No.
10/143,112, entitled "Reversibly Inactivated Acidified Plasmin,"
and which published as U.S. Patent Publication No. US 2003/0012778
A1, which is incorporated herein by reference in its entirety.
[0059] 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 process is
only limited in scope by the appended claims.
EXAMPLES
Example 1
Caprylate Cake I (CCI) Extraction and Lipid Reduction by PEG
Precipitation and Filtration
[0060] Caprylate cake I (CCI) is a fraction resulting from a pH 5
caprylate precipitation of resuspended Fraction II+III in the
IGIV-C process. Plasminogen (Pmg) is extracted from the CCI by
solubilizing at a cake:buffer ratio of about 1:10 for 2 to 3 hours
at 4.degree. C. with mixing. While several extraction solutions
were investigated, the current method was performed with 100 mM
Tris pH 10.5 to maintain the pH at or above neutral; a condition
favorable to Pmg solubilization from the CCI. Table 1 depicts the
extraction solutions investigated along with their final extract pH
and Pmg potency.
TABLE-US-00001 TABLE 1 CCI Extraction Solutions and Their Resulting
Final Extract pHs and Pmg Activities Extraction Solution Final
Extract pH Pmg (IU/mL) 0.1M Tris pH 10.5 9.2-9.5 1.77 0.2M Tris pH
7.5 7.5 2.06 0.05M Citrate, 0.2M .epsilon.-ACA, 6.0 1.49 0.4M NaCl
pH 6.5 0.15M Citrate pH 8.3 6.7 1.21 0.4% Acetic Acid pH 3.5 3.5
0.05
[0061] Following 2 to 3 hours of extraction, the temperature of the
extract is adjusted to 20.degree. C. and the pH to 7.5. Table 2
shows the Pmg yield, based on nephelometry, from Clarified Plasma
Pool through Fraction II+III and CCI Extract.
TABLE-US-00002 TABLE 2 Step and Process Yields for Pmg from
Clarified Plasma Pool to CCI Extract. % Pmg % Pmg Process Cohn
Fraction mg Pmg/g (SD), n Step Yield Yield Clarified Plasma Pool
0.124 (0.013), 33 -- -- Fraction II + III 0.143 (0.024), 30 65.6 --
CCI Extract (post L- 0.145 (0.01), 7 101 66.3 lysine)
[0062] Only about 66% of the Pmg in plasma tracks to Fraction
II+III while virtually all of the Pmg found in the resuspended
Fraction II+III precipitates to and is extracted from CCI.
Extraction of CCI in Tris pH 10.5, final CCI Extract pH of 9.2-9.5,
solubilizes all of the Pmg found in the CCI.
[0063] The addition of lysine derivatives (100 mM L-lysine, 50 mM
epsilon amino caproic acid (EACA)) increases the solubility of Pmg
in the CCI Extract resulting in increased recoveries during
subsequent PEG precipitation and filtration steps as illustrated in
FIG. 1.
[0064] Reduction of lipid is achieved through precipitation by the
addition of PEG 3350 to 3%-4% w/w. As mentioned previously, the
addition of L-lysine to 100 mM prior to PEG addition is necessary
to maintain high Pmg recovery in the PEG filtrate, or about 90%.
Without the addition of lysine, only about 25% of the Pmg is
recovered in the PEG filtrate (FIG. 1). The PEG precipitation
proceeds for 1 to 2 hours at 20.degree. C. with mixing. Filter aid
is added to 4% w/w and mixed prior to depth filtration through a
CUNO 30SP followed by further clarification with 0.5 micron and
0.22 micron filters.
[0065] FIG. 1 shows the lipid content, determined by cholesterol
and triglycerides concentration, is reduced by 60-70% following PEG
precipitation and filtration (CCI Filtrate I). The CCI Filtrate I
is diluted 1:1 with phosphate buffered saline pH 7.5 and held at
20.degree. C. for 1 to 2 hours as precipitation often continues
following filtration. The CCI Filtrate I is filtered through 0.5
.mu.m and 0.22 .mu.m filters to remove any additional precipitate;
CCI Filtrate II. Nephelometry data for CCI Extract and CCI
Filtrates I and II are illustrated in FIG. 2. Note that fibrinogen
and apolipoprotein A-1 concentrations are reduced following PEG
precipitation.
[0066] The CCI Filtrate II is diafiltered by tangential flow
filtration (TFF) against phosphate buffered saline pH 7.5 to reduce
the L-lysine concentration such that it will not act as a
competitive inhibitor for Pmg binding to the lysine affinity resin.
Experiments were performed to illustrate the necessity of lysine
removal. Loading the CCI Filtrate II directly onto a lysine
affinity resin without reduction in soluble lysine concentration,
results in the capture and release of about 4% of the Pmg activity.
Diluting the CCI Filtrate II 1:1 with TBS (10 mM Tris, 150 mM NaCl
pH 7.5) still resulted in capture and release of only about 5% of
the Pmg activity. Following 5 volumes of diafiltration to reduce
the lysine concentration, about 22% of the Pmg activity was
captured and released from the lysine affinity resin (in
retrospect, the column was overloaded by about 50%).
[0067] Constant volume diafiltration was performed by tangential
flow filtration (TFF) against 5 volumes phosphate buffered saline
pH 7.5 using a 30 kDa molecular weight cutoff membrane. Following
diafiltration, the protein solution was concentrated by
ultrafiltration to 4 to 5 A.sub.280/mL. Pmg recoveries in the UF/DF
retentate, by nephelometry, averaged 84% (+1, n=3). FIG. 3 shows
reduced SDS PAGE for each of the process intermediates discussed
thus far. The data in FIGS. 2 and 3 illustrate the complexity and
heterogeneity of the CCI Extract and subsequent Filtrates.
Example 2
Purification of Pmg by Lysine Affinity Chromatography:
[0068] The purpose of lysine affinity chromatography is to purify
Pmg, which represents from about 3 to 5% of the total protein in
the diafiltered CCI Filtrate II. The DF CCI Filtrate II was applied
to a Lysine-SEPHAROSE 4B (Amersham Pharmacia #17-0690-01) column
equilibrated with 0.01 M NaH2PO4, 0.15 M NaCl pH 7.5, at 3.5-4.0
A.sub.280/mL resin. Unbound proteins were washed through the column
with the equilibration buffer and the resin was then washed with
0.01 M NaH2PO4, 0.5 M NaCl pH 7.5 to remove non-specifically bound
protein; no protein was removed. Bound protein, Pmg, was eluted
with 0.1 M Glycine, 0.03 M Lysine pH 3.0 and collected with mixing
to maintain low pH. FIGS. 4 and 5 show SDS PAGE analysis and the
chromatogram of the lysine affinity purification of Pmg,
respectively. The resin was cleaned sequentially with 0.1 N NaOH
and 2.0 M NaCl, 0.1% Triton X-100 and stored in 20% ethanol. Table
3 shows Pmg step yield by nephelometry and purity by reduced SDS
PAGE.
TABLE-US-00003 TABLE 3 Lysine Affinity Chromatography Pmg Step
Yield and Purity Process Intermediate Step Yield % Pmg Purity %
Lysine-SEPHAROSE 4B 75.7 85.9 Eluate
Example 3
Viral Inactivation and Removal and TSE Removal
Nanofiltration
[0069] The optimal placement of a nanofiltration step during the
Plasmin process, along with determining the optimal conditions for
pathogens removal from Pmg lysine affinity eluate (Pmg) for a
particular nanofiltration scheme was tested. Pmg was spiked with
PPV or BVDV and filtered through a PALL DV20 filter membrane. All
runs were performed with 50 mL starting material (0.3 mg/mL Pmg),
30 psi constant pressure, pH 3.4 and room temperature. The
challenge solution was pre-filtered through 0.22 .mu.m prior to
nanofiltration. The determining factors for the optimal conditions
for removal of different pathogens by nanofiltration deal mainly
with the attainment of a minimum of 4 log infectivity removal of
known pathogens, percent product recovery, percent potency
remaining, product concentration and product pH. We detected, that
PPV and BVDV clearance was >4 log 10 TCID.sub.50. The
nanofiltration step has also the capability of removing greater
than 4 log of TSE. All product recoveries obtained in the study
were >95% with no substantial change in Pmg activity.
Caprylate Viral Inactivation.
[0070] Since caprylate inactivation is very much pH dependent and
more efficacious under acidic pH conditions, it was logical to
study virus inactivation by caprylate at the low pH lysine affinity
chromatography elution step. We used BVDV as a model enveloped
virus to study caprylate virucidal activity in lysine affinity
eluate. Complete BVDV inactivation, resulting in >4.4 log 10
reduction, was detected at the lysine affinity column eluate with 3
mM caprylate at pH 3.4 during 30 min of incubation at room
temperature in the presence of 1.5 mg/ml Pmg. In the absence of
product, complete BVDV inactivation (>4.7 log 10 reduction) was
also achieved with 3 mM caprylate after 30 minutes at pH 3.4. No
visible precipitation was observed during the caprylate treatment
suggesting that the product and virus spike remain soluble and are
not being precipitated by the caprylate. The impact of the added
caprylate on product recovery or potency following lysine affinity
column chromatography was minimal.
PEG Precipitation
[0071] We have investigated the effect of PEG on TSE removal. The
clarification and removal of lipids achieved by depth filtration
and 3% PEG precipitation of the Caprylate Cake I Extract resulted
in greater than 2 log 10 of TSE removal.
TABLE-US-00004 TABLE 4 Total Virus/TSE clearance across Plasmin
process Step BVDV PPV TSE Nanofiltration >4 log 4 log 4 log 3 mM
Caprylate >4 log <1 log <1 log Lysine Affinity 3.3 log 2.5
log pending PEG precipitation <1 <1 2-3 logs Total clearance
>12 >6 >6
Example 4
Streptokinase (SK) Activation of Pmg to Pm (Pm)
[0072] The addition of SK to the purified Pmg solution effects the
conversion of Pmg to Pm. The lysine affinity column eluate pH 3.4
is concentrated by TFF to 2 mg/mL through a 30 kD molecular weight
cutoff membrane. The Pmg solution temperature is ramped down to
4.degree. C. and a Pmg stabilizer, EACA, is added to a final
concentration of 20 mM to protect Pmg against damage during pH
adjustment from 3.4 to 7.5. Without the addition of EACA, a 67 kDa
species appears following the pH swing. The presence of EACA during
pH adjustment results in decreased Pmg degradation as compared to
pH adjustment without EACA (FIG. 6). Once the pH is adjusted to
7.5, the Pmg solution is diluted 1:1 with 20% glycerol, 4.degree.
C., to achieve a final condition of 1 mg Pmg/mL 0.05 M glycine,
0.015 M L-lysine, 0.01 M EACA, 10% glycerol pH 7.5. These
conditions have been optimized for minimizing Pm autodegradation.
SK is added to this solution at a 100:1 Pmg:SK molar ratio. The SK
reaction mixture is mixed at 4.degree. C. for 16 hours to allow
activation of Pmg to Pm. The average relative percent purity, as
determined by reduced SDS PAGE, of each of 4 groups of protein
species (Pmg, Pm HC, Pm LC and impurities/clipped Pm) from 14 SK
activation reactions are listed in Table 5.
TABLE-US-00005 TABLE 5 Relative Average % of Pmg, Pm (HC, LC) and
Impurities/Clipped Pm by Reduced SDS PAGE Following SK Activation;
n = 14. Protein Average % Purity SD Pmg 20.3 5.3 Pm 68.5 4.4 Pm
Heavy Chain 49.0 2.9 Pm Light Chain 19.4 1.5 Impurities/Clipped Pm
11.3 1.8
[0073] The data shows that the SK activation is reproducible and
results in only about 11% clipped Pm/impurities while activation of
Pmg to Pm is about 80%. To stop the activation and Pm
autodegradation reactions, NaCl and EACA are added to final
concentrations of 0.5 M and 0.25 M, respectively. This solution is
stable with respect to Pm integrity, for at least 4 days at
4.degree. C. FIG. 7 illustrates that there is no change in the Pm
purity or Pm autodegradation (Other) over this time period.
Example 5
Purification of Pm by Benzamidine Affinity Chromatography:
[0074] The purpose of benzamidine affinity purification is the
separation of unactivated Pmg and impurities, including Pm
degradation products, from active Pm. The stable SK activation
solution, pH adjusted to 8.5 in 0.05 M glycine, 0.015 M L-lysine,
0.25 M EACA, 0.5 M NaCl, 10% glycerol, is applied to a
Benzamidine-SEPHAROSE 6B (Amersham Pharmacia #17-0568-01) column
equilibrated with 50 mM Tris, 500 mM NaCl, pH 8.5. The Pm, both
clipped and intact, is captured by the affinity resin while the
aforementioned impurities flow through the column. The column is
washed with the equilibration buffer until the absorbance at 280 nm
reaches baseline. The bound Pm is then eluted in either one of two
ways: (1) removing the resin and eluting in batch format with 0.1 M
Glycine, 0.03 M Lysine pH 3.4; (2) eluting in a column format with
1 M EACA pH 7.5. Elution with EACA pH 7.5 removes only the intact
Pm while damaged Pm remains bound to the resin. A step to strip all
remaining protein. FIG. 8 shows a typical column format, EACA
elution profile. The batch elution profile consists only of the
unbound protein peak as the resin is then removed from the column
for Pm elution. The Pm captured and eluted from the affinity resin
is 87-91% intact (non-autodegraded) as illustrated in FIG. 9 and
>99% total Pm. The elution of Pm from the benzamidine resin with
EACA was unexpected as lysine derivatives such as EACA interact
with the heavy chain of Pm while benzamidine interacts with the
light chain.
Example 6
Removal of the Pmg Activator SK
[0075] The purpose of these steps is to remove the Pmg activator SK
such that the only remaining fibrin clot dissolution activity is
that of Pm. The benzamidine affinity step removes >99% of the SK
from the Pm as is illustrated in Table 6.
TABLE-US-00006 TABLE 6 SK removal, as determined by ELISA, by
benzamidine affinity chromatography and hydrophobic interaction
chromatography. Plasmin Process Step Streptokinase (ng/mL) SK
activation 1930.1 Benzamidine-SEPHAROSE unbound 1549.5
Benzamidine-SEPHAROSE eluted Pm 1.9 HIC Unbound Pm 0.7 HIC NaOH
strip (SK) 1.3 Final Formulation Pm <0.5
[0076] The hydrophobic interaction step using Octyl SEPHAROSE 4 FF
(Amersham Pharmacia #17-0946-02) acts as a polishing step to remove
essentially any remaining SK. The final sterile Pm product has no
detectable SK by ELISA. The 1 M EACA eluate pH 7.5, from the
benzamidine affinity column, is adjusted to pH 3.4 and (NH4)2SO4 is
added to a final concentration of 0.1 M. This acts as the protein
load for the Octyl-SEPHAROSE 4 FF column. The column is
equilibrated with 0.1 M (NH.sub.4).sub.2SO.sub.4, 0.1 M Glycine, 30
mM Lysine pH 3.4. Pm flows through the column while SK binds to the
column and is separated from Pm. The captured SK is removed from
the resin along with 0.1 to 1.0 N NaOH. FIG. 9 is an
Octyl-SEPHAROSE 4 FF chromatogram from a proof of principle
experiment. Pmg and SK were mixed at a 2:1 Pmg:SK molar ratio and
subjected to Octyl-SEPHAROSE 4 FF chromatography. The high levels
of SK were used so it could be tracked throughout the
chromatographic cycle using an anti-SK western blot. FIG. 10
illustrates the removal of SK from the Pm by SDS PAGE and anti-SK
western blot. The SK standard (panels A and B; lane 1) migrates
true to its molecular weight of 47 kDa. Once mixed with Pmg, the SK
is modified and migrates faster and as several species. There is no
detectable SK in the unbound protein fraction, which contains the
bulk of the Pm, by anti-SK western blot (panel B; lane 3).
[0077] Results for final sterile preparations of Pm purified by
benzamidine affinity and HIC chromatographies, as described above,
are listed in Table 7.
TABLE-US-00007 TABLE 7 Relative Average % Purity of Pm (HC, LC) by
Reduced SDS PAGE Following HIC; n = 2. Protein Average % Purity Pmg
0.0 Pm 95.5 Pm Heavy Chain 66.5 Pm Light Chain 29.0
Impurities/Clipped Pm 4.5
[0078] While specific embodiments have been set forth as
illustrated and described above, it is recognized that variations
may be made with respect to disclosed embodiments. Therefore, while
the invention has been disclosed in various forms only, it will be
obvious to those skilled in the art that many additions, deletions,
and modifications can be made without departing from the spirit and
scope of this invention, and no undue limits should be imposed
except as set forth in the following claims.
* * * * *