U.S. patent application number 11/784706 was filed with the patent office on 2007-09-20 for fibrinogen from transgenic animals.
This patent application is currently assigned to Pharming Intellectual Property BV. Invention is credited to Graham McCreath, Michael N. Udell.
Application Number | 20070219352 11/784706 |
Document ID | / |
Family ID | 27517474 |
Filed Date | 2007-09-20 |
United States Patent
Application |
20070219352 |
Kind Code |
A1 |
McCreath; Graham ; et
al. |
September 20, 2007 |
Fibrinogen from transgenic animals
Abstract
The present invention provides a method for the part
purification of fibrinogen from milk, the method comprising the
transfer of protease enzyme which is present in the milk, into the
whey phase with the removal or partition of fibrinogen into another
phase of the milk. The present invention also provides a method for
obtaining fibrinogen from a fluid, the method comprising: a)
contacting the fluid with a hydrophobic interaction chromatography
resin under conditions where the fibrinogen binds to the resin; and
b) removing the bound protein by means of elution.
Inventors: |
McCreath; Graham;
(Edinburgh, GB) ; Udell; Michael N.; (Edinburgh,
GB) |
Correspondence
Address: |
DRINKER BIDDLE & REATH;ATTN: INTELLECTUAL PROPERTY GROUP
ONE LOGAN SQUARE
18TH AND CHERRY STREETS
PHILADELPHIA
PA
19103-6996
US
|
Assignee: |
Pharming Intellectual Property
BV
P.O. Box 451
Leiden
NL
2300 AL
|
Family ID: |
27517474 |
Appl. No.: |
11/784706 |
Filed: |
April 9, 2007 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
09814371 |
Mar 22, 2001 |
7211650 |
|
|
11784706 |
Apr 9, 2007 |
|
|
|
PCT/GB99/03193 |
Sep 24, 1999 |
|
|
|
09814371 |
Mar 22, 2001 |
|
|
|
60103319 |
Oct 7, 1998 |
|
|
|
60103321 |
Oct 7, 1998 |
|
|
|
Current U.S.
Class: |
530/382 ;
530/418 |
Current CPC
Class: |
A61K 38/00 20130101;
C07K 14/75 20130101; A61L 27/225 20130101; A61L 24/106
20130101 |
Class at
Publication: |
530/382 ;
530/418 |
International
Class: |
C07K 14/75 20060101
C07K014/75 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 24, 1998 |
GB |
9820847.3 |
Sep 24, 1998 |
GB |
9820848.1 |
Sep 24, 1998 |
GB |
9820845.7 |
Sep 24, 1999 |
GB |
PCT/GB99/03193 |
Claims
1. Transgenic fibrinogen having a predetermined F1 fragment to F2
fragment ratio, obtainable from milk, at least partly purified,
having at least one of improved stability or increased integrity of
the fibrinogen alpha chain.
2. The transgenic fibrinogen of claim 1 having a pre-selected A
chain integrity.
3. The transgenic fibrinogen of claim 1, wherein the fibrinogen
comprises at least 80% F1 fibrinogen.
4. The transgenic fibrinogen of any of claims 1 to 3, substantially
free from viral contamination.
5. The transgenic fibrinogen of any of claims 1 to 3, comprising
fibrinogen 1, fibrinogen 2, or a combination thereof.
6. The transgenic fibrinogen of claim 1, obtainable by a method of
part purification of fibrinogen having a high A.alpha.-chain
integrity from milk, the method comprising the following steps: a)
precipitating the fibrinogen from milk; b) separating the
precipitated fibrinogen from protease enzymes contained in whey and
thereby recovering a part-purified fibrinogen, wherein said
part-purified fibrinogen comprises a high and low molecular weight
sub-fractions; c) contacting the part-purified fibrinogen with a
hydrophobic interaction chromatography resin under conditions
wherein the fibrinogen binds to the resin; and d) removing the
bound fibrinogen by means of elution wherein elution results in the
selective removal of said fibrinogen sub-fractions to produce high
A.alpha.-chain integrity fibrinogen.
Description
[0001] This is a divisional of co-pending application Ser. No.
09/814,371, filed Mar. 22, 2001, which is a continuation of
international application PCT/GB99/03193, published in English,
having an international filing date of Sep. 24, 1999, which claims
the benefit under 35 U.S.C. 119(e) of the filing date of
provisional application Ser. No. 60/103,321, filed Oct. 7, 1998,
abandoned, and the filing date of provisional application Ser. No.
60/103,319, filed Oct. 7, 1998, abandoned.
[0002] This disclosure is concerned generally with protein
purification from fluids, in particular, but not exclusively from
milk and specifically with the purification of human fibrinogen
from the milk of transgenic non-human animals.
[0003] Fibrinogen, the main structural protein in the blood
responsible for the formation of clots exists as a dimer of three
polypeptide chains; the A.alpha. (66.5 kD), B.beta. (52 kD) and
.gamma. (46.5 kD) are linked through 29 disulphide bonds. The
addition of asparagine-linked carbohydrates to the B.beta. and
.gamma. chains results in a molecule with a molecular weight of 340
kD. Fibrinogen has a trinodal structure, a central nodule, termed
the E domain, contains the amino-termini of all 6 chains including
the fibrinopeptides (Fp) while the two distal nodules termed D
domains contain the carboxy-termini of the A.alpha., B.beta. and
.gamma. chains. Fibrinogen is proteolytically cleaved at the amino
terminus of the A.alpha. and B.beta. chains releasing
fibrinopeptides A and B (FpA & FpB) and converted to fibrin
monomer by thrombin, a serine protease that is converted from its
inactive form by Factor Xa. The resultant fibrin monomers
non-covalently assemble into protofibrils by DE contacts on
neighbouring fibrin molecules. This imposes a half staggered
overlap mode of building the fibrin polymer chain. Contacts are
also established lengthwise between adjacent D domains (DD
contacts) leading to lateral aggregation. Another serine protease,
Factor XIII is proteolytically cleaved by thrombin in the presence
of Cav into an activated form. This activated Factor XIII (Factor
XIIIa) catalyses crosslinking of the polymerised fibrin by creating
isopeptide bonds between lysine and glutamine side chains. The
first glutamyl-lysyl bonds to form are on the C-terminal of the
.gamma. chains producing D-D crosslinks. Subsequently, multiple
crosslinks form between adjacent A.alpha. chains, the process of
crosslinking imparts on the clot both biological stability
(resistance to fibrinolysis) and mechanical stability [Sienbenlist
and Mosesson, Progressive Cross-Linking of Fibrin .gamma. chains
Increases Resistance to Fibrinolysis, Journal of Biological
Chemistry, 269: 28414-28419, 1994].
[0004] The coagulation process can readily be engineered into a
self sustained adhesive system in vitro by having the fibrinogen
and Factor XIII as one component and thrombin and Ca.sup.++ as the
second component which catalysis the polymerisation process. These
adhesion systems, know in the art as "Fibrin Sealants" or "Fibrin
Tissue Adhesives" have found numerous application in surgical
procedures and as delivery devices for a range of pharmaceutically
active compounds [Sierra, Fibrin Sealant Adhesive Systems: A Review
of Their Chemistry, material Properties and Clinical Applications,
Journal of Biomaterials Applications, 7:309-352, 1993].
[0005] It has been estimated that the annual US clinical need for
fibrin sealants is greatly in excess of the 300 Kg/year that can be
harvested using the current cryoprecipitation methods used by
plasma fractionaters. Alternative sources of fibrinogen, by far the
major component in fibrin sealant, have therefore been explored
with recombinant sources being favored [Butler et al., Current
Progress in the Production of Recombinant Human Fibrinogen in the
Milk of Transgenic animals, Thrombosis and Haemostasis, 78:
537-542, 1997]. It has been shown that mammals are capable of
producing transgenic human fibrinogen at levels of up to 5.0 g/L in
their milk making this a commercially viable method for the
production of human fibrinogen [Prunard et al., High-level
expression of recombinant human fibrinogen in the milk of
transgenic mice, Nature Biotechnology, 14:867-871, 1996; Cottingham
et al., Human fibrinogen from the milk of transgenic sheep. In:
Tissue Sealants: Current Practice, Future Uses. Cambridge
Institute, Newton Upper Falls, Mass., Mar. 30 Apr. 2, 1996
(abstract)].
[0006] Differences have been identified between recombinant human
fibrinogen and fibrinogen which has been purified from human
plasma. Fibrinogen which has been purified from human plasma has
two alternately spliced gamma chains (.gamma. and .gamma.'). In
contrast, recombinant human fibrinogen only has the major form
.gamma.. Further, the glycosylation of the beta and gamma chains
(there is no N-linked glycosylation of the alpha chain) of
recombinant human fibrinogen differs slightly from that on plasma
derived fibrinogen, but is similar to the glycosylation found on
other proteins expressed in the milk of transgenic animals. In
addition, the Ser3 of the alpha chain of recombinant human
fibrinogen is more highly phosphorylated than Ser3 of the alpha
chain of plasma derived fibrinogen, although the difference in
phosphorylation does not result in functional differences. Also,
there are detectable differences in heterogeneity caused by
C-terminal proteolysis of a number of highly protease-sensitive
sites on the alpha chain. Differences of a similar magnitude are
also observed between plasma-derived fibrinogen from different
sources.
[0007] Milk is well known to contain a number of serine proteases;
of these, the alkaline protease plasmin, which occurs in milk
together with its inactive zymogen plasminogen, is the most
significant protease contributing to proteolytic activity.
Plasmin(ogen) concentration varies with health status of the animal
e.g. mastitic animals exhibit increased proteolytic activity. Also
influencing the proteolytic activity of milk is stage in lactation
i.e. late lactation is associated with higher concentrations of
plasmin [Politis and Ng Kwai Hang, Environmental Factors Affecting
Plasmin Activity in Milk, Journal of Dairy Science, 72:1713-1718,
1989]. In milk, plasmin(ogen) is associated predominantly with the
casein micelles, although it can also be found to a lesser extent
in whey [Politis et al., Distribution of Plasminogen and Plasmin in
Fractions of Bovine Milk, Journal of dairy Science, 75:1402-1410,
1992].
[0008] Plasmin is the serine protease that is predominantly
responsible for the dissolution of fibrin clots in vivo and its
presence is essential for haemostasis. It is very probable that any
fibrinogen degradation product in milk is as a result of the action
of milk proteases. Therefore, the presence of plasmin or other
proteases in milk can be detrimental to the quality of fibrinogen
that is produced by the lactating transgenic animal if steps are
not taken to minimize their effect. Of equal importance is the
removal of any fibrinogen degradation products that may result from
the action of plasmin or other milk proteases. The use of protease
inhibitors to minimize proteolysis is well established in the art
and usually involves the addition of a cocktail of inhibitors of
varying specificity. With transgenic animals the possibility of
proteolytic damage to the recombinant protein has been realized and
suggestions have been put forward to limit degradation (Wilkins and
Velander, Isolation of Recombinant Proteins from Milk, Journal of
Cellular Biochemistry, 49: 33-338, 1992; Velander et al., PCT WO
95/22249). However, increasingly effective methods are constantly a
desideratum.
[0009] In the purification of proteins from milk, one requirement
is the separation of the desired protein from contaminating casein
micelles. For the isolation of transgenic proteins such as AAT, the
first step is precipitation with PEG or other agent, such as
ammonium sulphate. This does not precipitate AAT, but precipitates
casein and is therefore a good way of removing casein from the AAT.
However, when this teaching was applied to transgenic fibrinogen in
milk, it was found that not only did the casein precipitate, but
that the fibrinogen precipitated with it. This was clearly not a
suitable step for removing casein from fibrinogen. Further, the
fibrinogen in the casein/fibrinogen precipitate was unstable and
was very quickly proteolytically damaged, probably due to the
co-precipitation with protease enzymes. The problem was thus how to
separate casein from fibrinogen-like proteins in a milk sample or
fraction thereof. The separation of plasmin(ogen) from casein
micelles can be accomplished by incubation with agents such as
6-aminohexanoic acid (.epsilon.-aminocaproic acid, .epsilon.ACA).
However, 6-aminohexanoic acid also increases the activation of
plasminogen to plasmin which may accelerate proteolysis of any
susceptible desired protein. Furthermore, the separation of plasmin
(or plasminogen) from casein micelles does not assist in the
separation of casein micelles from fibrinogen-like proteins.
[0010] Accordingly, there remains a need to separate desired
proteins from casein micelles without accelerating proteolysis of
the desired protein.
[0011] Human plasma fibrinogen appears heterogeneous by SDS-PAGE
and other methods for separating proteins based on size. A high
molecular weight fraction (HMW Fibrinogen, Fibrinogen 1 or F1) with
a molecular weight of 340,000 daltons contributes approximately
50-70% of total fibrinogen antigen. Low molecular weight fibrinogen
(LMW Fibrinogen, F1 brinogen 2 or F2) with a molecular weight of
approximately 300,000 daltons contributes 20-40%. The residual
amount, designated as low molecular weight' fibrinogen
(LMW'Fibrinogen, Fibrinogen 3, F3 or Fragment X) has a molecular
weight of approximately 280,000 daltons. It has been shown that the
major differences in these fibrinogen molecules results from
proteolytic damage to the carboxy terminus region of the A.alpha.
chains (A.alpha.C-terminal region) resulting in differing lengths
of A.alpha. chain C-terminus. Fibrinogen, purified from
cryoprecipitate by the use of precipitation techniques has been
shown to have partially digested A.alpha. chain [Stroetmann, U.S.
Pat. No. 4,427,650] Although it was first thought that plasmin or
plasmin-like enzymes were responsible for degradation of F1
fibrinogen to F2 and F3 sub-families [Lipinska et al., Fibrinogen
Heterogeneity in Human Plasma Electrophoretic demonstration and
characterization of two major fibrinogen components, Journal of
Laboratory & Clinical Chemistry, 84: 509-516, 1974] it is
apparent that plasmin itself is probably not responsible for the
direct proteolysis of F1 to F2 fibrinogen [Dempfle et al.,
Fibrinogen Heterogeneity in Homozygous Plasminogen Deficiency Type
1: Further evidence that plasmin is not involved in formation of
LMW and LMW'-Fibrinogen, Thrombosis and Haemostasis, 77:879-883,
1997]. It has been suggested that F2 fibrinogen may actually be a
group of degradation products produced by several enzymes including
matrix metalloproteases [Nakashima et al., Human Fibrinogen
Heterogeneity: the COOH-terminal residues of defective A.alpha.
chains of fibrinogen II, Blood Coagulation and Fibrinolysis,
3:361-370, 19921. Recombinant fibrinogen expressed in CHO cells has
also been shown to be heterogeneous comprising of F1 fibrinogen and
a smaller F2-like sub fraction that is also lacking the C-terminal
region of the A.alpha. chain illustrating that the recombinant
fibrinogen is also susceptible to proteolysis [Gorkun et al., The
conversion of fibrinogen to fibrin: Recombinant fibrinogen typifies
plasma fibrinogen, Blood 89:4407-4414]. Similarly, recombinant
human fibrinogen, produced in yeast, has also been shown to possess
an F2-like fraction having partially degraded A.alpha. chains [Roy
et al., Secretion of Biologically Active Fibrinogen by Yeast,
Journal of Biological Chemistry, 270: 23761-23767, 1995],
demonstrating that A.alpha. chain damage may be expected for a
range of expression hosts. As well as the major F2 and F3
fragments, there exist a range smaller fragments generated from
fibrinogen termed Fibrinogen Degradation Products (FDPs). F3 is
also often referred to as a FDP. These FDPs (Fragment Y, D and E)
are well characterized and can be found in small amounts in human
plasma.
[0012] Differences in the rate of clot formation, the structure of
the final clot and the mechanical properties of the final clot have
been observed by various investigators for each of the major
fibrinogen fragments. Also, the presence of FDPs, and their
influence on the clotting progress has been investigated. Gorkan et
al., [Role of the .alpha.C domains of fibrin in clot formation,
Biochemistry 33: 6986-6997, 1994] established that F1 fibrinogen is
95% clottable while F2 fibrinogen is 92% clottable. While total
clottability of these two fractions appears similar, a distinct
difference in clotting time i.e. onset of visible clot formation
following the action of thrombin, was observed with the F2
fibrinogen exhibiting a greater lag time before clot formation.
This has also been observed previously (e.g. Holm et al., 1985,
Purification and Characterization of 3 Fibrinogens with different
molecular weights obtained from Normal Human Plasma, Thrombosis
& Haemostasis, 37: 165-176) where F1 fibrinogen was observed to
have a clotting time of 14s compared to 20s for F2 and 25s for F3.
Evidence therefore suggests that the extent of proteolysis of the
A.alpha. c-terminus influences fibrin polymerization. The
3-dimensional structure of the clot is also influenced by the
degree of degradation of the .alpha.C regions of fibrinogen. Clots
formed from F2 and F3 fibrinogen exhibit a low degree of
protofibril branching with increased porosity. It has been
postulated that partially degraded fibrinogens are more prone to
lateral aggregation of protofibrils. This leads to the formation of
thicker fibers resulting in coarser clots as observed by light
scattering experiments. Further evidence for the importance of the
A.alpha. chain C-terminus in clot formation arises from the fact
that clots formed from Fibrinogen Milano III, a naturally occurring
variant with truncated A.alpha. chains exhibits a reduced degree of
protofibril branching [Furlan et al., Binding of calcium ions and
their effect on clotting of Fibrinogen Milano II, a variant with
truncated A.alpha. chains, Blood Coagulation and Fibrinolysis, 7:
331-335, 1995]. Differences in mechanical properties of clots
formed with different fibrinogen species has also been observed
where clots formed from F2 and F3 appear less resistant to
mechanical disturbances. Thromboelastography (TEG) reveals that
clots made from F1 fibrinogen are more elastic than clots formed
from F2 fibrinogen [Hasegawa and Sasaki, Location of the binding
site "b" for lateral polymerisation of fibrin, Thrombosis Research,
57: 183-195, 1990]. Elasticity is a preferred property for fibrin
sealants whose use may include application in joint or tendon
surgery.
[0013] The C-terminal regions of the A.alpha. chains also serve
other purposes distinct from clot formation. They enclose
crosslinking sites for the transglutaminase, Factor XIIIa where
FXIIIa catalyses the formation of isopeptide bond between adjacent
fibrinogen molecules thereby adding strength and stability to the
clot. Crosslinking also increases the clots resistance to
proteolysis and is responsible for localizing other molecules
involved in the clotting process to the surface of the clot most
notably .alpha.2-antiplasmin, which is covalently crosslinked into
the A.alpha. chains by Factor XIIIa further enhancing the stability
of the clot to proteolytic degradation [Rudchenko et al.,
Comparative, Structural and Functional Features of the Human
.alpha.C domain and the Isolated .alpha.C Fragment, Journal of
Biological Chemistry, 271: 2523-2530, 1996]. Fibronectin,
Thrombospondin and von Willibrands Factor are also crosslinked into
this region. The A.alpha. C-regions are also important for
enhancing the activation of plasminogen by tPA on the clot surface
therefore leading to effective fibrinolysis [Matsuka et al., Factor
XIIIa catalysed crosslinking of Recombinant .alpha.C Fragments of
Human Fibrinogen, Biochemistry, 35: 5810-5816, 1996]. It has also
been postulated that the A.alpha. C-terminus of fibrinogen encloses
specific recognition sites for platelet receptors located in
residues A.alpha. 572 through A.alpha.574 [Hawiger, Adhesive ends
of fibrinogen and its adhesive peptides: The end of a saga,
Seminars in Haemotology, 32: 99-109, 1995]. Platelet aggregation is
essential for haemostasis and therefore it may be expected that
fibrinogen molecules having degraded A.alpha. chains would be less
capable of aggregating platelets.
[0014] The importance of A.alpha. C-terminal regions to fibrinogen
properties has inspired the development of techniques whereby
fibrinogen molecules having varying degrees of A.alpha. chain
proteolysis can be separated for study. Various methods have been
described for the separation of the major fibrinogen sub families
and FDPs. For example precipitation techniques have been used to
separate F1 and F2 from purified fibrinogen [Sasaki and Kito,
Simplified determination of fibrinogen sub-fractions by glycine
precipitation, Thrombosis and Haemostasis 42: 440-443, 1979]. Holm
et al., have described a method for the separation of purified
plasma fibrinogen into F1, F2 & F3 subfamilies by using a
series of precipitations with ammonium sulphate. F3, fragments Y, D
and E have been separated based on size using size exclusion
chromatography [Morder and Raphael Shulman, High molecular weight
derivatives of human fibrinogen produced by plasmin, Journal of
Biological Chemistry, 244:2120-2124, 1969]. These authors also
demonstrated that F3 fibrinogen and FDPs Y, D and E actually
possess anticoagulant activity and are inhibitory to clot
formation; a non-desirable feature of a molecule used to prepare a
surgical adhesive. Most attention has been paid to the terminal
degradation products D) and E which have been separated using anion
exchange chromatography [Kemp et al., Plasmic degradation of
fibrinogen: the preparation of a low molecular weight derivative of
fragment D, Thrombosis and Haemostasis, 3:553-564, 1973], cation
exchange chromatography [Rutjven Vermeer et al., A novel method for
the purification of rat and human fibrin(ogen) degradation
products, Hoppe-Seyler's Z. Physiological Chemistry, 360:633-637]
Lysine-SEPHAROSE (cross-linked beaded agarose) chromatography [Rupp
et al., Fractionation of plasmic fibrinogen digest on Lysine
agarose. Isolation of two fragments D, fragment E and simultaneous
removal of plasmin, Thrombosis Research, 27:117-121, 1982] and Zinc
chelated affinity chromatography [Structural features of fibrinogen
associated with binding to chelated zinc, Scully and Kakkar,
Biochim et Biophys. Acta., 700:130-133, 1982]. In none of the above
methods has the simultaneous separation of fibrinogen into
sub-fractions F1, F2 &F3 and FDPs Y, D & E been described
using a single technique.
[0015] As introduced above, plasmin is the serine protease that is
predominantly responsible for the dissolution of fibrin clots in
vivo and its presence is essential for haemostasis. However, as
discussed previously, while the participation of plasmin in
A.alpha. chain degradation of F1 to F2 and F3 is still under
debate, it is very probable that any fibrinogen degradation product
in milk will be as a result of the action of milk proteases.
Therefore, the presence of plasmin or other proteases in milk can
be detrimental to the quality of fibrinogen that is produced by the
lactating transgenic animal if steps are not taken to minimize
their effect. Of equal importance is the removal of any fibrinogen
degradation products that may result from the action of plasmin or
other milk proteases. The use of protease inhibitors to minimize
proteolysis is well established in the art and usually involves the
addition of a cocktail of inhibitors of varying specificity.
[0016] From the above discussion, it is clear that the
incorporation of fibrinogen degradation products (F3 fibrinogen,
fragments Y, D & E) and even F2 fibrinogen into preparations
whose end-use would be either as a heamostasis or sealing agents is
not desirable. Techniques which can be incorporated into a
purification of fibrinogen from the milk of transgenic animals
which reduce fibrinogen degradation products enabling fibrinogen
with a defined A.alpha. chain integrity to be produced for varying
applications would be of considerable use.
[0017] The invention provides an efficient and effective method
whereby a protein produced in the milk of transgenic animals is
recovered and purified.
[0018] This patent application describes techniques whereby
part-purification of fibrinogen may be carried out. Precipitation
techniques are used which include chemical agents capable of
disrupting the interactions between protease enzymes and casein.
Using these techniques it is possible to segregate fibrinogen
product from damaging protease activity in the early stages of
processing and, subsequently, remove protease activity.
Precipitation is carried out in such a manner that enables the
collection of substantially purified (up to 85%) with very little
remaining protease activity. This absence of protease enzymes
renders the fibrinogen more stable during subsequent processing
thus improving product yield and forgoing the necessity for
expensive refrigeration equipment or toxic protease inhibitors
which would have to be removed.
[0019] This patent application describes chromatographic techniques
for the purification of proteins, in particular fibrinogen from
milk and for the removal of fibrinogen degradation products
(Fragments X, Y, D, E & C-terminal A chain fragments). Using
these techniques it is possible to purify fibrinogen to up to and
at least 99% pure. Also, use of these techniques allow for prior
selection of fibrinogen molecules in a product with regard to the
integrity of the A chain. Thus it becomes possible to select a
fibrinogen product which could compose 100% F1 fibrinogen of 80% F1
fibrinogen or any lower concentration of F1. As F1 fibrinogen has
superior functional characteristics with regard to clot elasticity,
rigidity and stability, the ability to predetermine and therefore
select the F1 content enables a range of fibrinogen products to be
produced for varying purposes. The removal of degradation products
also allows for a superior fibrinogen sealant in a controlled and
reproducible manner.
[0020] Accordingly, the first aspect of the present invention
provides a method for the part purification of a desired protein
from milk, the method comprising the transfer of protease enzyme
which is present in the milk, into the whey phase with the removal
or partition of the desired protein into another phase of the
milk.
[0021] The desired proteins according to the present invention are
any of those which may be produced in milk, including naturally
produced milk proteins and transgenic proteins. Preferred proteins
according to the present invention are those having fibrinogen-like
characteristics which result in co-precipitation with casein in the
presence of PEG or ammonium sulphate. Such proteins include, but
are not limited to; fibrinogen, collagen, fibronectin, Factor VIII
and alpha-2-macroglobulin.
[0022] The present invention is preferably in relation to the
isolation of transgenic proteins from milk, that is proteins
produced as a result of transgenic manipulation of an animal. This
accordingly allows for the isolation of proteins, such as
fibrinogen, collagen, fibronectin, Factor VIII and
alpha-2-macroglobulin from animal milk which does not normally
contain such proteins. The present invention is useful for the
production and isolation of individual proteins per se, or proteins
which have been altered in some way to facilitate transgenic
expression, such as by fusion to other proteins.
[0023] In the present text, the term "part purification" means
purification to a level of from 50% free from other contaminants,
preferably 60, 70, 80, 90% free from other contaminants. Preferably
the recovery rates are in the range 50% to about 80%, more
preferably in the range 65% to 85%.
[0024] The present invention is preferably in relation to the
isolation of fibrinogen, in particular transgenic fibrinogen from
milk, that is fibrinogen produced as a result of transgenic
manipulation of an animal. This accordingly allows for the
isolation of fibrinogen from animal milk which milk does not
normally contain such proteins. The present invention is useful for
the production and isolation of fibrinogen protein per se, or
fibrinogen which has been altered in some way to facilitate
transgenic expression, such as by fusion to other proteins.
[0025] When the desired protein is fibrinogen, the method for the
part purification thereof is optionally followed by a method step
comprising: [0026] (a) contacting the part purified fibrinogen with
a hydrophobic interaction chromatography resin under conditions
where the fibrinogen binds to the resin; and [0027] (b) removing
the bound protein by means of elution.
[0028] Preferably the part purified fibrinogen, optionally to be
further purified by the steps a) and b) described above, is in a
liquid form, either as a direct result of the first part of the
method, or otherwise.
[0029] As used herein, the term "fibrinogen" refers to the main
structural protein responsible for the formation of clots and
includes the whole glycoprotein form of fibrinogen as well as other
related fibrinogen species, including truncated fibrinogen, amino
acid sequence variants (muteins or polymorphic variants) of
fibrinogen a fibrinogen species which comprises additional residues
and any naturally occurring variants thereof. The same variations
described in relation to fibrinogen also apply to other
fibrinogen-like proteins which can be isolated from milk according
to the present invention.
[0030] As use herein, "milk" is understood to be the fluid secreted
from the mammary glands in animals. Milk according to present
invention includes whole milk, skimmed milk, milk fraction and
colosteral milk. It also includes a milk-derived fluid where the
desired protein, in particular fibrinogen, was originally produced
in milk.
[0031] The present invention enables the part purification of the
desired protein by transferring protease enzymes present in the
milk away from the phase into which the desired protein is
obtained. The protease enzyme is transferred into the whey phase
(whey phase being the phase/portion/fraction of milk which contains
predominantly non-casein proteins) with the removal or partition of
the desired protein into another phase of the milk. The removal or
partition of the desired protein may be simultaneous to the
transfer of the protease enzyme in the whey phase. Alternatively,
it is possible to have a two-step process, whereby the protease
enzyme is transferred first to the whey under conditions which
retard proteolytic damage to the desired protein, followed by the
removal or partition of the desired protein. Such conditions can be
constructed by using protease inhibitors or low temperature. The
transfer of protease enzyme into the whey phase predominately
relates to the transfer of plasmin and/or plasminogen. Other milk
proteases, such as serine proteases (alkaline or acid) may also be
transferred.
[0032] The desired protein is recovered from the milk by the use of
precipitation techniques well known to those in the art, such as by
the use of protein precipitation agents including, but not
exclusively, PEG, sodium sulphate, ammonium sulphate, glycine or
temperature. The precipitation is preferably carried out with
generally low concentrations of the chemical precipitation agents
(e.g. 5-20% w/v sodium and ammonium sulphate, 5-20% w/v glycine or
.beta.-alanine; 2-15% PEG) as this reduces co-precipitation of whey
proteins.
[0033] The transfer of the protease enzyme into the whey phase of
the milk is preferably by the presence of lysine or lysine analogue
such as .epsilon.-aminocaproic acid or other basic amino acids,
such as arginine or histidine. The concentration of lysine or a
lysine analogue according to the invention depends on a number of
factors such as the type of milk from which the desired protein is
being purified, the amount of the desired protein present and the
manner of removal or partition of the desired protein from the whey
phase of the milk. Concentrations typically range from 1 mM-2M,
preferably 10-200 mM.
[0034] Most preferably, the method of the first aspect of the
invention is repeated at least once, and up to approximately four
times. This repetition can greatly increase the purity of the
desired proteins in particular in respect of contaminating
micelles.
[0035] The method according to the first aspect of the invention
increases the stability of the part purified desired protein toward
proteolysis, especially when the desired protein is a transgenic
protein.
[0036] In the optional second step of the first aspect of the
invention, when the desired protein is fibrinogen, a hydrophobic
interaction chromatography resin is used.
[0037] Hydrophobic Interaction Chromatography (HIC) resins are
known in the art and include resins such as Butyl SEPHAROSE
(Amersham Pharmacia Biotechnology), Phenyl SEPHAROSE (low and high
substitution), Octyl SEPHAROSE and Alkyl SEPHAROSE, wherein
SEPHAROSE is cross-linked beaded agarose.
[0038] Conditions under which the fibrinogen is contacted with the
hydrophobic interaction chromatography resin to enable fibrinogen
to bind to the resin include the presence of any "structure
forming" salt (solution), such as ammonium sulphate, sodium
sulphate and other salts as described in: Melander and Horuath,
Salt Effects on Hydrophobic Interactions in Precipitation and
Chromatography: An Interpretation of the Lyotropic Series, Archives
of Biochemistry and Biophysics, 183:200-215, 1977 and Srinivason
and Ruckenstein, Role of Physical Forces in Hydrophobic Interaction
Chromatography, Separation and Purification Methods, 9: 267-370,
1980. Removal of the bound protein is by means of standard elution
techniques known in the art. Such elution can be carried out by
decreasing the concentration of the structure forming salt, such as
decreasing the concentration of ammonium sulphate in the eluting
buffer. Elution may be by gradient elution or more preferably, by a
series of steps to predetermine and thus define the fibrinogen that
is eluted from the column in terms of its sub-fraction ratios and
hence its A.alpha. chain integrity.
[0039] Preferably, the optional method step of the first aspect of
the invention includes a step of washing the resin, to remove
unbound components, between steps (a) and (b). Washing the resin is
usually carried out with a washing buffer which has the same
concentration of salt in it that was used for loading. A higher
concentration of salt in the washing buffer is possible, but not
preferred.
[0040] When the optional second method step of the first aspect of
the invention is used, it preferably achieves at least one of the
following:
(a) increases the purity of the fibrinogen
(b) resolves the fibrinogen into its fractions
(c) enables isolation of higher integrity fibrinogen A.alpha.
chain.
[0041] Since the present invention takes advantage of genetic
manipulation of animals in order to obtain proteins from transgenic
sources ("transgenic fibrinogen"), the source of the fibrinogen can
be carefully selected. Preferably, the fibrinogen is human, bovine
or ovine derived (that is, corresponding essentially to human,
bovine or ovine fibrinogen). For medical purposes, it is preferred
to employ proteins native to the intended patient. Thus human
transgenic fibrinogen is preferred. Where the fibrinogen is
recombinantly encoded, so that fibrinogen from a species other than
the species in which it is being expressed, the glycosylation
pattern may be different from the glycosylation pattern of the
naturally occurring fibrinogen. A transgenic animal closer in
biological taxonomy to the source of the transgenic fibrinogen may
thus be preferred.
[0042] Clearly, any animal which produces milk, and, animals which
can be genetically manipulated to produce transgenic fibrinogen in
their milk, are preferred. In this respect, animals which lactate
and produce suitable milk include sheep, cow, goat, rabbit, water
buffalo, pig or horse. Transgenic animals for the production of a
transgenic protein according to the present invention, do not
include transgenic humans.
[0043] A second aspect of the invention provides a method for the
part purification of a desired protein from milk, the method
comprising precipitation of the desired protein in the presence of
lysine or a lysine analogue. When the desired protein is
fibrinogen, the method is optionally followed by a method step
comprising: [0044] (a) contacting the part purified fibrinogen with
a hydrophobic interaction chromatography resin under conditions
where the fibrinogen binds to the resin; and [0045] (b) removing
the bound protein by means of elution.
[0046] A related second aspect of the invention provides for the
use of lysine or a lysine analogue in the purification of a desired
protein from milk (preferably transgenic protein). The use of the
lysine or lysine analogue in this aspect of the invention is
preferably in combination with the precipitation of the desired
protein.
[0047] All description and details with respect to the first aspect
of the invention, also apply to the second.
[0048] The present invention further provides a useful method
whereby fibrinogen is recovered from a fluid.
[0049] A third aspect of the present invention provides a method
for obtaining fibrinogen from a fluid, the method comprising:
[0050] (a) contacting the fluid with a hydrophobic interaction
chromatography resin under conditions where the fibrinogen binds to
the resin; and [0051] (b) removing the bound protein by means of
elution.
[0052] The fluid-containing fibrinogen may be any. In particular,
it is one or more animal body-fluids such as milk, blood plasma or
urine. It is, in particular a fluid-containing fibrinogen which is
or has been derived from a body fluid of an animal (such as one of
those described above) and/or a fluid which has been used to
solvate the fibrinogen, for example following a previous method
step such as part-purification by precipitation.
[0053] Any animal body fluid can be used according to the method of
the present invention. Preferred body fluids include milk, blood
plasma or urine. Clearly, the natural production of fibrinogen in
some body fluids, such as plasma, can provide an animal body fluid
from which naturally occurring fibrinogen can be isolated. However,
the present invention is preferably in relation to the isolation of
transgenic fibrinogen as a result of transgenic manipulation of an
animal. This accordingly, allows for the isolation of fibrinogen
from animal body fluids which do not naturally contain fibrinogen,
such as milk and urine. The present invention is useful for the
production of fibrinogen per se or fibrinogen which has been
altered in some way to facilitate transgenic expression, such as by
fusion to other proteins.
[0054] The term "blood plasma" includes whole blood plasma and any
fraction thereof. The term "urine" also refers to whole urine, or
fractions thereof, in particular concentrated urine.
[0055] Preferably the fluid is milk or a milk-derived fluid. In
such situations (where the fluid is milk or milk-derived) the
method according to the third aspect of the invention may be
optionally preceded by a method step comprising the part
purification of fibrinogen from milk, the method comprising the
transfer of protease enzyme which is present in the milk, into the
whey phase with the removal or partition of the fibrinogen into
another phase of the milk.
[0056] The optional preceding method step at least partially
purifies the fibrinogen from milk, thus allowing a better
purification and/or separation of fibrinogen by virtue of the HIC
method step.
[0057] Clearly, any animal which produces a body fluid which may be
used according to the third aspect of the invention is
contemplated. Preferably, animals which can be genetically
manipulated to produce transgenic fibrinogen in their milk, are
preferred. In this respect, animals which lactate and produce
suitable milk include sheep, goat, cow, camel, rabbit, water
buffalo, pig or horse. These animals are also useful for the
production of other body fluids according to the invention.
Transgenic animals for the production of a transgenic protein
according to the present invention, do not include transgenic
humans.
[0058] In order to achieve the maximum result from the method
according to the first aspect of the invention, it may be
preferable to at least partially purify the fibrinogen from the
animal body-fluid. Such a purification will depend on the body
fluid from which the fibrinogen is derived and the nature of
potential contaminates present. The fibrinogen is preferably
purified to a level of from 20 through 40% before undergoing the
method according to the first aspect of the invention. Any
pre-purification method can be used, for example those known in the
art, e.g. precipitation of fibrinogen as described in PCT WO
95/22249. The fact that the fibrinogen may already be part purified
before application of the first aspect of the invention, does not
detract from the fact the fibrinogen may have been originally
produced in the body fluid of an animal.
[0059] All description and details in respect of features of the
first and second aspects of the invention also apply to the third.
The details of the option HIC step in the first and second aspects
apply to the HIC step in the third aspect.
[0060] In accordance with the third aspect of the invention, a
related third aspect provides the use of HIC in one or more of the
following: [0061] (a) increasing the purity of fibrinogen [0062]
(b) resolving fibrinogen into its fractions [0063] (c) selecting of
fibrinogen with high integrity of A.alpha. chains from fibrinogen
in a fluid, preferably a body fluid from an animal.
[0064] The use of HIC in the sixth aspect of the invention is
preferably in combination with a salt solution as described
according to the first aspect of the invention. Relevant preferred
features of aspects one and two also apply to the third. The use of
the HIC in all relevant aspects of the invention includes a batch
format or a column format. In batch format, the liquid may be
contacted with the HIC resin in a well stirred tank. Separation of
the HIC resin from the liquid may then be facilitated by
sedimentation or be centrifugally assisted. In column format, which
is preferred, the liquid is preferably pumped through a column into
which HIC resin has already been added. Column formats are
preferred as they result in greater adsorption efficiency. This
column format could be regarded as either a "Packed" or "Fixed" bed
format. Further, "Expanded bed" or "fluidized bed" contactors may
also be applicable.
[0065] A fourth aspect of the invention provides a method for
obtaining fibrinogen from a fluid, the method comprising: [0066]
(a) contacting the fluid with a hydrophobic interaction
chromatography resin under conditions where the fibrinogen binds to
the resin; and [0067] (b) removing the bound protein by means of
elution.
[0068] Where the fluid is milk or milk-derived (preferable), the
method according to the fourth aspect of the invention is
optionally preceded by a method step comprising the part
purification of the fibrinogen from milk, the method comprising
precipitation of the desired protein in the presence of lysine or a
lysine analogue.
[0069] All description and detail of features of aspects one to
three also apply to the fourth.
[0070] The milk from which the fibrinogen is to be part purified is
preferably derived from animals which can be "farmed" in order to
produce sufficient quantities of milk from which to obtain
pharmaceutical proteins and include sheep, cow, goats, rabbit,
camel, water buffalo, pig or horse. Such animals may clearly be
transgenically modified animals. Preferably, although not
exclusively, the transgenic protein is bovine or human derived.
Human derived proteins are preferable as these, when isolated and
purified for pharmaceutical use from the milk of a transgenic
animal are less likely to cause an unwanted immunological reaction
when administered to a human in need thereof for medicinal
purposes. The present invention does not relate to transgenically
modified humans.
[0071] The plasminogen activation system in milk has been a focus
of interest for a number of years. It is generally accepted that
milk contains the primary enzymes responsible for fibrinolysis in
vivo e.g. plasminogen activator (both tissue type, tPA and
urokinase type, uPA], plasminogen and plasmin. The action of
proteolysis is often observed during storage of milk or milk
products where casein appears to be the milk protein most
susceptible to degradation. It was soon illustrated that in milk,
plasminogen activators, plasminogen and plasmin were associated
mainly with the casein micelles and not in the whey (or serum)
phase. The mechanism by which these molecules associate with casein
has not been categorically determined but it is probable that as
these molecules contain Kringle domians (structured polypeptide
chains with an affinity for basic amino acids) these domains
probably mediate their interaction with casein. Heegaard et al.,
1997 [Plasminogen Activation System in Human Milk, Journal of
Paediatric Gastroenterology and Nutrition, 25: 159-[66] have shown
that casein immobilised on Sepharose is capable of binding tPA and
when casein is present, the tPA catalysed conversion of plasminogen
to plasmin is accelerated. This seems to suggest that the
juxtaposition of casein, plasminogen and tPA results in enhanced
plasminogen activation. The mechanism of enhanced activation is not
clear but may be due to plasminogen undergoing a conformational
change on binding to casein resulting in a molecule more readily
activated with tPA [Markus et al., Casein, A Powerful Enhancer of
the rate of Plasminogen Activation, Fibrinolysis 7: 229-236]. It is
therefore apparent that an agent (such as Lysine or Lysine
analogue) added to milk in sufficient concentration will dissociate
tPA and plasmin(ogen) from casein transferring them to the whey
phase.
[0072] The consequences of this are that active plasmin and
plasminogen are then present in the same phase as the transgenic
protein. In terms of fibrinogen, as discussed above, the result of
this is that proteolysis, especially of the A.alpha. chain will
occur. It is known in the art that .epsilon.ACA is relatively
ineffective at inhibiting primary fibrinolysis i.e Fragment X (F3)
formation from fibrinogen or fibrin and it has been postulated that
initial degradation of fibrin may occur independent of noncovalent
plasmin-fibrin interaction (which is mediated through kringle
domains on plasminogen binding to basic amino acids in the
fibrinogen Acc chain), unlike the later steps which result in the
formation of fragments Y, D and E. Indeed it has been shown
[Francis et al., Structural and Chromatographic Heterogeneity of
Normal Plasma Fibrinogen associated with the Presence of Three
.gamma.-chain types with Distinct Molecular Weights, Biochimica et
Biophysica Acta, 744: 155-164] that A.alpha. chain proteolysis in
commercial fibrinogen preparations proceeds during chromatographic
separation into fibrinogen sub-families even with the inclusion of
20 mM .epsilon.-Aminocaproic acid and Aprotinin (a potent protease
inhibitor) at 10 Kallikrein units/ml. It is therefore apparent that
addition of E-Aminocaproic acid during the purification of human
fibrinogen from milk would have no beneficial, and even negative
effects.
[0073] Paradoxically we have discovered that E-aminocaproic acid is
a useful aid in preventing degradation of fibrinogen during its
purification from milk if it is included during a stage which
partitions the fibrinogen, such as a precipitation stage. The
similarity between fibrinogen and casein in terms of susceptibility
to precipitation; a technique widely used in the purification of
fibrinogen from plasma and cryoprecipitate [e.g. Schwarz et al.,
U.S. Pat. Nos. 4,362,567; 4,377,572 & 4,414,976], and in the
separation of casein from milk [Swaisgood, Developments in dairy
Chemistry-1: Chemistry of Milk Protein, Applied Science Publishers,
NY, [982] leads to the co-precipitation of at least part of the
casein fraction when precipitating fibrinogen from milk using
precipitating agents well known to those in the art (e.g. but not
exclusively Zinc, Copper, sodium and ammonium salts, amino acids
(e.g. glycine, alanine), alcohol (e.g. ethanol) and polymers (e.g.
polyethylene glycol, dextran or hydroxyethyl starch. Even by adding
these precipitants at relatively low concentrations (e.g 5-20% w/v
sodium and ammonium sulphate, 5-20% w/v glycine or .beta.-alanine;
2-15% PEG) sufficient to precipitate fibrinogen or a majority
fraction of it also co-precipitates a fraction of the casein phase
including some whey proteins. This can be reduced if the
precipitation is carried out more than once. The inclusion of
.epsilon.-aminocaproic acid or a similar analogue of lysine during
the precipitation stage (at a concentration of 10-200 mM) results
in the dissociation of kringle containing proteins from casein and
fibrinogen and maintains them in the solution phase while the
fibrinogen is precipitated. The method of protection of the
fibrinogen is therefore one of exclusion. The precipitated
fibrinogen can then be reconstituted in a suitable buffer and is
not only significantly less susceptible to proteolysis but also
significantly more pure. Such a technique would works equally well
if temperature is used as a method of precipitation. The added
advantage of this invention is that not only is the
.epsilon.-aminocaproic acid preventing proteolytic damage to the
fibrinogen, it does not contaminate the precipitated fibrinogen as
it remains in the solution phase.
[0074] A fifth aspect of the present invention provides transgenic
fibrinogen, at least partly purified, having improved stability, in
particular in respect of proteolysis. All preferred features of the
first to fourth aspects of the invention also apply to the fifth,
even though the transgenic protein of the fifth aspect may not
necessarily be required to be produced according to the method of
the first to fourth aspects.
[0075] All individual method steps described in aspects one to four
are considered to increase the stability of the fibrinogen to
proteolysis.
[0076] A sixth aspect of the invention provides fibrinogen,
fibrinogen 1 (F1), fibrinogen 2 (F2), or a combination thereof,
which has high integrity of A.alpha. chains.
[0077] The seventh aspect of the invention provides fibrinogen,
fibrinogen 1 (F1), fibrinogen 2 (F2), or a combination thereof,
obtainable by a method according to the first to fourth aspects of
the invention.
[0078] The fibrinogen, fibrinogen 1 and fibrinogen 2 are obtainable
(having high A.alpha. chains) by virtue of the HIC step.
[0079] The fibrinogen 1 and/or fibrinogen 2, according to the sixth
and seventh aspects of the invention are particularly preferred for
use in fibrinogen adhesives or sealants as described hereinbefore
and hereinafter.
[0080] The fifth, sixth and seventh aspects of the invention
preferably produce fibrinogen which is substantially free from
viral contamination. Such fibrinogen can be more easily produced
from non-blood products, such as those from milk or urine.
[0081] An eighth aspect of the invention provides for purified
fibrinogen obtainable according to any of aspects one to four of
the invention as described above. All description and details for
aspects one to seven, also apply to the eighth.
[0082] The fibrinogen according to the invention may be in any
suitable or convenient state, such as in a lyophilised or soluble
state.
[0083] A ninth aspect of the invention provides a fibrin adhesive
or sealant containing fibrinogen according to the fifth to eighth
aspects of the invention. The fibrin adhesive or sealant according
to the ninth aspect of the invention are, in all respects, with the
exception of the particular fibrinogen used, well known and
standard in the art [Sierra, Fibrin Sealant Adhesive Systems: A
Review of Their Chemistry, Material Properties and Clinical
Applications, Journal of Biomaterials Applications, 7:309-352,
1993; Martinowitz and Spotnitz, Fibrin Tissue Adhesives, Thrombosis
and Haemostasis, 78:661-666, 1997; Radosevich et al., Fibrin
Sealant: Scientific Rationale, Production Methods, Properties and
Current Clinical Use, Vox Sanguinis, 72:133-143, 1997].
[0084] As used herein, the term "fibrin adhesive" or "fibrin
sealant" describes a substance containing fibrinogen which is
capable of forming a biodegradable adhesive or seal by the
formation of polymerised fibrin. Such adhesive/sealant systems are
alternatively called "fibrin tissue adhesives" or "fibrin tissue
glues". The adhesive or seal may act as, inter alia a hemostatic
agent, a barrier to fluid, a space-filling matrix or a
drug-delivery agent. Particular use may be found in neurosurgery,
opthalmic, orthopedic or cardiothoracic surgery, skin grafting and
various other types of surgery.
[0085] Other than fibrinogen, the fibrin adhesive or sealant may
contain substances which encourage the formation of the fibrin
adhesive/seal, such as thrombin, Ca.sup.++ (e.g. CaCl.sub.2) and
Factor XIII (and/or Factor XIIIa [in his text, all references to
Factor XIII are also references to factor XIIIa and vice versa).
While it is recognised that thrombin would be the preferred enzyme
with which to incorporate into any system whereby the formation of
a fibrin clot is desired, it is appreciated that there are other
enzymes capable of proteolytically cleaving fibrinogen resulting in
the formation of a fibrin clot. An example of this would be the
snake venom enzyme Batroxobin [Weisel and Cederholm-Williams,
Fibrinogen and Fibrin: Characterization, Processing and
Applications, Handbook of Biodegradable Polymers (Series: Drug
targeting and Delivery) 7:347-365, 1997]. Other components such as
albumin, fibronectin, solubilisers, bulking agents and/or suitable
carriers or diluents may also be included if desired.
[0086] One advantage of fibrin sealant as a biodegradable polymer
is that there are natural mechanisms in the body for the efficient
removal of clots and thus the fibrin sealant may be a temporary
plug for hemostasis or wound healing. Various proteolytic enzymes
and cells can dissolve fibrin depending on the circumstances, but
the most specific mechanism involves the fibrinolytic system. The
dissolution of fibrin clots under physiological conditions involves
the binding of circulating plasminogen to fibrin, and the
activation of plasminogen to the active protease, plasmin, by
plasminogen activators which may also be, also bound to fibrin.
Plasmin then cleaves fibrin at specific sites.
[0087] Depending on the situation, it may be advantageous to let
the natural process of fibrin breakdown take place after applying a
fibrin adhesive or sealant to a site. Indeed, this breakdown may be
encouraged, for example, by the inclusion of plasminogen.
Alternatively, in some situations it may be advantageous to delay
the process by including antifibrinolytic compounds which can, for
example, block the conversion of plasminogen to plasmin or directly
bind to the active site of plasmin to inhibit fibrinolysis. Such
antifibrinolytics include .alpha..sub.2-macroglobulin, which is a
primary physiological inhibitor of plasmin; aprotinin;
.alpha..sub.2-antiplasmin; and .epsilon.-aminocaproic acid.
[0088] The fibrin/sealant may comprise two components, one
component containing fibrinogen and Factor XIII (and/or Factor
XIIIA) and the other component containing thrombin and Ca.sup.++.
Other substances as described above may be included in one or both
of the components if desired.
[0089] A tenth aspect of the invention provides a kit for a fibrin
adhesive or sealant comprising fibrinogen according to any one of
the fourth to eighth aspects of the invention, and instructions for
use or, may comprise fibrinogen according to any one of the fourth
to eighth aspects of the invention in combination with (but not
necessarily mixed with) one or more of: Factor XIII, Factor XIIIa,
thrombin or Ca.sup.++. Furthermore, the kit may comprise two
components: fibrinogen with (but not necessarily mixed with) Factor
XIII (and/or Factor XIIIa) and thrombin with (but not necessarily
mixed with) Ca.sup.++.
[0090] The components of any fibrinogen sealant, according to the
present invention, including the kit forms, may be used separately,
simultaneously or sequentially.
[0091] All relevant description and details in respect of the first
to ninth aspects of the invention also apply to the tenth.
[0092] An eleventh aspect of the invention provides a method for
producing a fibrin adhesive or sealant according to the ninth
aspect of the invention, comprising admixing fibrinogen with
thrombin or any other enzyme which is capable of proteolytically
modifying fibrinogen and causing it to clot. Factor XIII (and/or
Factor XIIIa) and Ca.sup.2+ may also be mixed with the fibrinogen
and thrombin (or other suitable enzyme) in this aspect of the
invention.
[0093] The method of admixing fibrinogen and thrombin may involve
squirting or spraying the components simultaneously or sequentially
to the repair site with a syringe or a related device. The mixing
may result from two syringes held together along their barrels and
at the plunders with two components mixed either after exiting the
needles or in the hub just prior to exiting. Other devices may be
used to produce an aerosol or to spray in a variable pattern,
depending on the application.
[0094] Although various derivatives of fibrinogen have been used in
clinical applications for some time, there are several safety
issues involved in the clinical use of fibrinogen such as concern
over viral contamination, especially with products containing
fibrinogen or components prepared from human blood especially
pooled human blood. Although improvements in viral cleansing
techniques for blood products have been made since the fear of
transmission of pathogenic viruses was brought to the surface, so
that the risk of disease transmission has been greatly reduced, the
risk has not been totally eliminated. The present invention, which
relates to fibrinogen obtained from milk or urine, can be
substantially free from such a concerns.
[0095] A twelfth aspect of the invention provides fibrinogen,
according to the fourth to eighth aspects of the invention, for use
in medicine. Preferably the fibrinogen is used in human medicine.
However, it may also be used in veterinary medicine such as for
horses, pigs, sheep, cows, cattle, rabbits, mice and rats as well
as for domestic pets such as dogs and cats.
[0096] While the main use of fibrinogen is thought to be for the
preparation of adhesive or sealing agents as hereinbefore
described, fibrinogen has other applications in the field of
medicine, for example as a coating for polymeric articles as
disclosed in U.S. Pat. No. 5,272,074. A particular use of
lyophilised fibrinogen of the present invention is within or part
of a gauze or bandage (preferably made from polylacetic acid
compounds used in surgical stitches). Such a wound dressing can be
supplied (also incorporating the other components required for the
formation of a clot (described above), optionally in a package or
kit form, for application direct to the skin or to an internal
organ. All details and features of previously discussed aspects,
also apply to the twelfth.
[0097] A thirteenth aspect of the invention provides a fibrin
adhesive or sealant, according to the ninth aspect of the
invention, for use in medicine.
[0098] The use in medicine may be any of those described herein.
All details and features of aspects one to eleven, also apply to
the thirteenth.
[0099] A fourteenth aspect of the present invention provides a
method of surgery or therapy comprising placing fibrinogen
according to the fourth to eighth aspects of the invention, on or
within a animal or a body part of an animal. The animal in question
is preferably in need thereof. Preferably the animal is a human.
The fibrinogen may be mixed with one or more of thrombin, Factor
XIII, Factor XIIIa or Ca.sup.2+ separately, sequentially or
simultaneously with the fibrinogen. The fibrinogen may thus be in
the form of a sealant according to the ninth aspect of the
invention. The fibrinogen may be applied by squirting using a
syringe or a related device. It may be applied very precisely in a
localised area or broadly over a wide area to any tissue. All
details and preferred features of aspects one to thirteenth also
apply to the fourteenth.
[0100] A fifteenth aspect of the invention provides the use of
fibrinogen, according to the fourth to eighth aspects of the
invention in the manufacture of a fibrin adhesive or sealant.
[0101] In this invention, purification of fibrinogen is achieved or
a preferred optional step by the use of Hydrophobic Interaction
Chromatography (HIC) which is carried out in such a way that
enables not only the separation of milk proteins, leading to a
substantially pure product, but also the simultaneous fractionation
of fibrinogen into F1, F2 and degradation products. In general,
fibrinogen, preferably partially purified by precipitation, is
contacted with a HIC resin (e.g. Butyl SEPHAROSE (cross-linked
beaded agarose)) under conditions where the fibrinogen is retained
(e.g. 0.2-0.8M, preferably 0.3 to 0.6M, ammonium sulphate). The
resin is then washed, either in batch fashion by centrifugation or
by inclusion in a chromatography column. Elution of bound material
is facilitated by decreasing the concentration of salt (e.g.
ammonium sulphate in decreasing concentration 0.5 to 0M) in the
mobile phase so that resolution of fibrinogen from non-fibrinogen
components is achieved. By careful selection of salt concentration,
the fibrinogen is not only separated from the majority of milk
components but can also be fractionated into subfamilies. Elution
can either be carried out using a decreasing gradient whereby the
slope of the gradient determines the resolution or, more
conveniently, by use of a series of decreasing steps of
concentration. The use of HIC enables the fibrinogen product to be
defined with respect to its A.alpha. C-terminal region.
[0102] The plasminogen activation system in milk has been a focus
of interest for a number of years. It is generally accepted that
milk contains the primary enzymes responsible for fibrinolysis in
vivo e.g. plasminogen activator (both tissue type, tPA and
urokinase type, uPA), plasminogen and plasmin. The action of
proteolysis is often observed during storage of milk or milk
products where casein appears to be the milk protein most
susceptible to degradation. It was soon illustrated that in milk,
plasminogen activators, plasminogen and plasmin were associated
mainly with the casein micelles and not in the whey (or serum)
phase. The mechanism by which these molecules associate with casein
has not been categorically determined but it is probable that as
these molecules contain Kringle domians (structured polypeptide
chains with an affinity for basic amino acids) these domains
probably mediate their interaction with casein.
[0103] It is realized that proteolysis of the human protein may
occur within the mammary gland or udder of the lactating transgenic
animal. The incubation period of the transgenic protein in the
mammary gland or udder can be approximated to the time period
between milking of the animal. Therefore it is apparent that
increasing the frequency of milking minimizes this time period.
However, increasing the frequency of milking to above 3 or 4
milkings per day not only creates a measure of discontinuity for
the animal but involves a cost addition to Dairy expenses. It is
accepted therefore that a measure of degradation of the human
fibrinogen will occur. As discussed in the Prior Art, the presence
of fibrinogen degradation products in a fibrin tissue adhesive
compromises the usefulness of the product and therefore any
degradation products must be removed. This invention discloses how
the inventors have discovered an extremely efficient way of
achieving this which also allows the ratio of F1 and F2 fibrinogen
in the final product to be selected and defined.
[0104] Techniques for the separation of plasma fibrinogen into its
various sub-fractions, as described in the prior art, generally
fall into two categories. Those which rely on the differential
solubility of subfractions in high concentration of salts (e.g.
ammonium sulphate and glycine), often refereed to as selective
precipitation techniques [Holm et al., Purification and
Characterisation of 3 Fibrinogens with different molecular weights
obtained from normal human plasma, Thrombosis Research, 37:
165-176, 1985], and those which take advantage of the fact that
degradation products have a different molecular size and can
therefore be separated using size exclusion chromatography.
[0105] The two categories of techniques described above are quite
contrasting in their ability and ease of use, at industrially
enabling scales, for subfractionating fibrinogen. While
precipitation techniques are relatively easy to operate and scale,
their inherent mode of separation does not allow for the extremely
high levels of resolution that would be required to ensure that the
fibrinogen produced could be accurately defined with respect to its
F1:F2 ratio and hence A.alpha.. chain integrity. Indeed, advocates
of this technique at the laboratory scale often report
contamination of subfractions with each other [Lipinska et al.,
Fibrinogen Heterogeneity in Human Plasma: Electrophoretic
demonstration and characterization of two major fibrinogen
components, Journal of Laboratory & Clinical Chemistry, 84:
509-516, 1974] and low yields.
[0106] In contrast to techniques based on differential solubility,
size exclusion chromatography can potentially result in very good
resolution of fibrinogen sub-fractions in high yield. The main
drawbacks of this technique are expense and scale. Although F1
& F2 fibrinogen and F2 & F3 differ by some 35-40 Kdal, the
size of the molecule itself (340 Kdal) is near the limit of the
fractionation range of most size exclusion matrices. This results
in poor resolution if expensive resins are not used. Another
limitation is scale, as SEC is not a chromatographic technique
favored at process scale when subtle separations have to be carried
out. Also, SEC is usually a very expensive technique as only a
small fraction of a column volume of material could be loaded while
maintaining resolution.
[0107] Hydrophobic Interaction Chromatography (HIC) is a
separations technique which exploits the binding of proteins to
mildly hydrophobic resins in the presence of low concentrations of
salts which expose hydrophobic patches on the surface of proteins.
In the presence of these so-called "structure forming" salts,
selective interactions can be initiated between different proteins
and the matrix. The technique is most often used to discriminate
between different proteins in a heterogeneous mixture. The
inventors have discovered that not only is HIC a very good
fractionation technique for the recovery of fibrinogen from a
partially purified extract, it is also a surprisingly powerful
technique for resolving fibrinogen sub-fractions i.e. F1, F2, F3
(Fragment X), Fragment Y and Fragments D & E.
[0108] Transgenic human fibrinogen, partially purified from milk is
bound to HIC resins (e.g. but-not exclusively Butyl SEPHAROSE
(cross-linked beaded agarose) 4FF Amersham Pharmacia Biotechnology)
in the presence of ammonium sulphate or other "structure forming"
salt at a concentration enabling fibrinogen to bind e.g. a range
0.2-1.0M (preferably 0.3-0.6M) is used. By decreasing the
concentration of ammonium sulphate in the irrigation buffer, the
bound material elutes from the column in the order milk components
(0.485-0.37M ammonium sulphate), F1 fibrinogen (0.37-0.2M ammonium
sulphate), F2 fibrinogen (0.2-0.14M ammonium sulphate) and F3
fibrinogen and degradation products (0.10-0.0M ammonium sulphate).
The range of concentrations of ammonium sulphate over which the
bound components elute is determined, in part, by the operating
conditions and those skilled in the art would be able to adjust
either the temperature or the pH or both to change the
concentrations of ammonium sulphate over which the fractions elute.
Using this technique it is possible by means of gradient elution or
more preferably by a series of steps to predetermine and thus
define the fibrinogen that it is eluted from the column in terms of
its F1 to F2 ratio and hence its A.alpha. chain integrity.
[0109] This text refers to the accompanying figures of which:
[0110] FIG. 1 is SDS-PAGE of part purified fibrinogen from example
A, in the absence or presence of .epsilon.ACA.
[0111] FIG. 2 is a chromatogram illustrating the various fractions
generated from the HIC column in example 1. The chromatogram was
generated using UV at 280 nm.
[0112] FIG. 3 is SDS-PAGE of transgenic human fibrinogen elution
from an HIC (Butyl SEPHAROSE (cross-linked beaded agarose) 4FF)
column in example 1.
[0113] FIG. 4 is SDS-PAGE of transgenic human fibrinogen elution
from an HIC (Butyl SEPHAROSE (cross-linked beaded agarose) 4FF)
column in example 2.
[0114] FIG. 5 is chromatogram illustrating the various fractions
generated from the HIC column in example 2. The chromatogram was
generated using UV.
[0115] FIGS. 6, 7, 8 and 9 are RP-HPLC chromatograms for fibrinogen
and fibrinogen fractions eluted from the HIC column using
conditions outlined in examples 1 and 2. The chromatograms were
generated using a wavelength of 214 nm.
[0116] The following non limiting examples help to illustrate this
invention.
EXAMPLE A
[0117] Milk from a transgenic ewe was thawed from a frozen state in
a water bath at 37.degree. C. and then delipidated by low speed
centrifugation (2000 rpm) for 10 minutes. The skimmed milk was than
aliquoted into 2.times.40 ml fractions and processed as follows. To
one of the fractions was added 40 ml of 27.6% (w/v) ammonium
sulphate in 25 mM citrate, 100 mM .epsilon.ACA, pH 8.0. The tube
was mixed for 20 minutes at room temperature followed by high speed
centifugation in a Beckman J2-21 centrifuge (15.degree. C.). The
supernatant generated was removed and the pellet dissolved in 25 mM
citrate, pH 8.0. Once dissolved up to 40 ml, the precipitation and
resolubilisation was repeated as above. A final precipitation and
resolubilisation step was then carried out, essentially as above
except that .epsilon.ACA was omitted from the salt solution. The
same process as above was then repeated on the second 40 ml aliquot
of skimmed milk except that .epsilon.ACA was not used.
[0118] The Sodium Dodecyl Sulphate PolyAcrylamide Gel
Electrophoresis (SDS-PAGE, 8-16%, Novex) shown as FIG. 1
illustrates the stability of part purified fibrinogen. Lane 1
represents fibrinogen part purified in the presence of .epsilon.ACA
and stored at 4.degree. C. overnight. It can be seen that the
fibrinogen is predominantly F1: F2. The degradation product.
Fragment X(F3) is also present as a faster migrating band under the
F1: F2 bands. Lane 2 represents material stored at 4.degree. C.
purified as in Lane 1 except that .epsilon.ACA was absent during
the precipitation stage. From Lane 1 and Lane 2, it is evident that
some F1 fibrinogen has been proteolytically cleaved even during
storage at 4.degree. C. Lane 3 represents material as in lane 1
except that storage was at 18.degree. C. overnight. As can be seen
this material appears to be more stable than that shown in Lane 2
and in fact is very similar to that shown in Lane 1. Lane 4
represents material purified in the absence of .epsilon.ACA after
overnight storage at 18.degree. C. It is evident that this material
has been severely damaged and is almost lacking in F1 fibrinogen.
This example serves to illustrate that fibrinogen, part purified
from milk by precipitation, is unstable to milk protease action.
This protease action may be diminished by incubation at 4.degree.
C. but is abolished if the precipitation is carried out in the
presence of .epsilon.ACA which prevents milk protease contamination
of the precipitated fibrinogen.
EXAMPLE 1
[0119] Transgenic fibrinogen was, partially purified from the milk
of a transgenic ewe by precipitation with ammonium sulphate, in a
similar manner as described in example A. 2 ml was made to 0.485M
ammonium sulphate by the addition of 1.45M ammonium sulphate in 5
mM citrate, pH 7.5 (1 ml). After mixing, the solution was pumped
onto a HiTrap Butyl SEPHAROSE (cross-linked beaded agarose) 4FF
column (previously equilibrated with 0.485M ammonium sulphate, in
25 mM citrate buffer, pH 7.5) at 0.1 ml/min. The column was washed
with 2 column volumes of 0.485M ammonium sulphate in 5 mM citrate,
pH 7.5 after which elution was carried out in 3 steps 1) 0.40M
ammonium sulphate in 5 mM citrate, pH 8.0, 2) 0.15M ammonium
sulphate in 5 mM citrate buffer, pH 8.0, 3) 5 mM citrate, pH 8.0.
The chromatogram presented below as FIG. 2 shows that 4 major peaks
were obtained from this experiment. The first peak represents
material that does not bind to the column under these adsorption
conditions and is mainly sheep milk proteins. The second peak
represents material that did bind to the column and was eluted with
0.40M ammonium sulphate. The third peak represents fibrinogen and
fractions taken across this are shown on a SDS-PAGE as FIG. 3. The
clear distinction between F1 (High molecular weight fibrinogen) and
F2 (Low molecular weight fibrinogen) and the resolution obtained on
the chromatography can be clearly seen (Lanes 1-5). Lane 6
represents the pooled peak while lanes 7 & 8 represent peak 4
from the chromatogram which can be seen to be F3 (Fragment X)
fibrinogen. Thus it is evident that by changing the concentration
of ammonium sulphate used for elution it is possible to define
eluted fibrinogen with respect to its A.alpha. chain integrity.
EXAMPLE 2
[0120] In another example which illustrates the scale-up potential
of this technique, a procedure equivalent to example 1 above was
scaled up by a factor of 400. Thus 0.9 g (790 ml) of transgenic
human fibrinogen was partially purified by precipitation in the
presence of 50 mM .epsilon.-aminocaproic acid. It was then made to
0.5M ammonium sulphate by the addition of 790 ml of 1M ammonium
sulphate in 5 mM citrate buffer pH 7.5. This material was loaded
onto a column 5 cm.times.21 cm (400 ml) of Butyl SEPHAROSE
(cross-linked beaded agarose) 4FF at a flow rate of 20 ml/min.
After loading, the column washed with 400 ml of 0.5M ammonium
sulphate in 5 mM citrate buffer, pH 7.5. Bound material was eluted
from the column by irrigation with three buffers 1) 0.4M ammonium
sulphate in 5 mM citrate, pH 7.5, 800 ml 2) 0.15M ammonium sulphate
in 5 mM citrate buffer, pH 7.5, 800 ml, and 3) 5 mM citrate, pH
7.5, 800 ml.
[0121] The SDS-PAGE and chromatogram shown as FIGS. 4 and 5
respectively, show results for this experiment. As can be seen from
the SDS-PAGE, F1:F2 fibrinogen was eluted from the column by 0.15M
ammonium sulphate (Lanes 34, FIG. 4) while F3 fibrinogen was eluted
using a step change to 5 mM citrate, pH 7.5 containing no ammonium
sulphate (Lane 8, FIG. 4). Reducing SDS-PAGE is a convenient way of
determining A.alpha. chain integrity as loss of A.alpha. C-terminal
regions results in a decrease in the A.alpha. chain molecular
weight. This decrease is readily qualitatively assessed. In FIG. 4,
Lane 6 shows a reduced F1:F2 fibrinogen with 10 mM dithiotheritol
as the reducing agent. When this is compared to F3 fibrinogen (Lane
8), the loss of A.alpha. chain is clearly seen.
[0122] Quantitative information on A.alpha. chain integrity can be
obtained by the use of Reversed-Phase High Performance Liquid
Chromatography (RP-HPLC) on reduced fibrinogen according to Raut et
al., [Ultra-rapid preparation of milligram quantities of the
purified polypeptide chains of human fibrinogen, Journal of
Chromatography B, 660:390-394, 1994] which allows for integration
of peak areas. FIG. 6 shows a RP-HPLC chromatogram for purified F1
fibrinogen; the three fibrinogen chains elute from the column in
the order A.alpha., BP and y respectively; as can be seen, there
exists a single peak for each chain. Integration of the A.alpha.
chain results in a peak area which is used as a standard against
which fibrinogen with degraded A.alpha. chains can be normalized.
In FIG. 7, a RP-HPLC chromatogram, run under identical conditions
to that in FIG. 6, is shown for F2 fibrinogen where it is evident
that the A.alpha. peak has been separated into two peaks, the
former being intact A.alpha. chain and the latter being A.alpha.
chain being proteolytically cleaved at the C-terminus. Using
on-line integration it can be calculated that the A.alpha. chain
exist as 73% intact, the remaining 27% being degraded A.alpha.
chain. In FIG. 8 a RP-HPLC chromatogram is shown for F3 fibrinogen.
In this chromatogram it is evident that amount of degraded Au
greatly outweighs the amount of non-degraded A.alpha. chain as is
illustrated by the much reduced non-degraded A.alpha. chain peak.
It can be calculated that degraded A.alpha. chain represent 62% of
total A.alpha. chain present.
[0123] It is evident therefore that using the technique of RP-HPLC,
as an analytical tool following Hydrophobic Interaction
Chromatography, allows conditions for the Hydrophobic Interaction
Chromatography to be selected to prepare fibrinogen with a defined
A.alpha. chain integrity. An example of this is given in FIG. 9
which represents elution from the Butyl SEPHAROSE (cross-linked
beaded agarose) 4 FF column using conditions outlined in Example 2
above. From the chromatogram in FIG. 9 it is evident that mainly F1
fibrinogen is selected as the A.alpha. chain is 87% intact.
* * * * *