Note: Descriptions are shown in the official language in which they were submitted.
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DRIED BIOLOGICALLY OR THERAPEUTICALLY ACTIVE PREPARATIONS
FIELD OF THE INVENTION
This invention relates to dried biologically or therapeutically active
preparations,
and in particular it relates to dried or lyophilised Factor VIII preparations.
The present
invention provides in particular for the stabilisation of such Factor VIII
preparations
which are to be subjected to a terminal dry heat treatment step, such as
heating at
80°C for 72 hrs, used as a viral inactivation step.
BACKGROUND OF THE INVENTION
Classic haemophilia, haemophilia A, is an X chromosome-linked disorder of
blood coagulation which causes a decrease in functional levels of a
glycoprotein
known as antihaemophilic factor (AHF) or Factor VIII (FVIII) (Levine 1987).
FVIII is a cofactor for Factor IXa in the activation of Factor X and is
crucial for
activation of the intrinsic coagulation pathway (Foster and Zimmerman, 1989).
FVIII
is present in the plasma of normal individuals at a level of 1 unit/mL (100
ng/mL). It
circulates in plasma in an inactive form bound to another high molecular
weight
glycoprotein, the FVIII-related antigen, von Willebrand Factor (vWF) at a
molar ratio
of approximately 1:100. vWF is deficient or abnormal in patients with von
Willebrand's
disease. Mature vWF consists of 2050 amino acids residues and circulates in
plasma
as a series of disulfide-linked multimers between 1 to 20x1 O6 daltons. The
association
with vWF serves to stabilise the FVIII molecule in plasma as well as localise
the
clotting cascade to the site of vascular damage through vWF adhesion to the
activated
platelet surtace (Weiss 1977).
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The molecular weight of FVIII in plasma ranges between 250,000 to 300,000
daltons. It is composed of heavy and light chains with molecular weights of
approximately 90 and 80 kDa respectively, separated by a heavily glycosyiated
~i-
domain region of approximately 130 kDa (Fulcher and Zimmerman 1982, Rotblat et
al.
1985). FVIII is susceptible to proteolytic cleavage by thrombin and Factor Xa.
This .
serves to remove the ~i-domain, resulting in a conformational change whereby
the
heavy and light chains become linked by calcium ion binding. This cleavage
correlates with an increase in functional activity and represents the
activated form of
the molecule, FVllla (Eaton et al. 1986, Fulcher et al. 1985, Andersson et al.
1986,
Fay et al. 1986, Pittman and Kaufman 1986).
Once FVllla has fulfilled its roles, it is proteolytically cleaved further by
either
thrombin, FACTOR Xa or activated protein C, correlating with loss of
functional
activity. Peptides range from 43 kDa to 80 kDa form, depending on the
participating
enzyme (Eaton et al. 1987}.
The cloning of human FVII1 gene and the expression of active recombinant FVIII
have been reported. The cDNA codes for a single chain FVIII consist of 2332
amino
acid residues with a molecular weight of 264,763 Da prior to glycosylation and
the
glycosylated form of approximately 330 kDa (Toole et al. 1984, Gitschier et
al. 1984,
Rotblat et al. 1985). It has been shown that calcium is necessary for
maintaining the
association of FVlll heavy and light chains, and the removal of calcium from
FVIII
preparations has been demonstrated to result in loss of procoagulant activity
(Fass et
al. 1982, Andersson et al. 1986, Mikaelsson et al. 1983). The reconstitution
of FVIII
activity from isolated heavy and light chains requires the presence of
divalent cations,
with Mn2+ having the strongest effect followed by Ca2'" and Coz+ (Fay 1988).
The incidence of haemophilia A is approximately 1 in 10,000 males, by the
inheritance of an autosomal recessive pattern (Gitschier et al. 1984). Without
treatment, haemophiliacs experience haemorrhage into joints and muscles, are
easily
bruised, and suffer prolonged and potentially fatal postoperative
haemorrhages.
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Restoration of plasma FVIII levels to greater than 1 % of normal with plasma-
derived
commercial concentrates containing FVIII significantly reduces disease
symptoms and
markedly improves the quality of life for afflicted individuals (Aronson 1990,
Esmon et
al. 1990).
Although quite beneficial to most patients, treatment with some FVlll products
can also have deleterious effects. In addition to the risk of viral
transmission via
plasma-derived FVIII products (Fletcher et al. 1983, Ragni et al. 1983), and
the
production of inhibitory antibodies, capable of neutralising FVIII (Shapiro
and Hutlin
1975), treatment with cryoprecipitate or FVIII concentrates may result in
functional and
phenotypical immune deficiencies (Lederman et al. 1983, Menitove et al. 1983,
Schulman 1991, Allersma et 1996). These immune deficiencies include impaired
monocyte and phagocyte function as well as abnormalities of T cell subsets,
irrespective of HIV infection (Moffat et al. 1985).
FVlll concentrates can be classified on the basis of specific activity
(expressed
in IU/mg total protein in the final product state) as either intermediate (1-5
IUlmg
protein), high (10-200 IUlmg) or very high purity (1000-3000 IUlmg).
Intermediate
purity products are prepared by precipitation reactions and often contain high
levels
of fibrinogen and fibronectin as well as a number of other non-FVIII proteins.
The
yields from these processes are generally high. High purity products have
reduced
yields but significantly lower amounts of non-FVIII proteins, and are prepared
using
conventional chromatography techniques. Very high purity products contain
essentially FVIII alone, and include monoclonal antibody purified concentrates
and
recombinant products. However, albumin is often added back into these products
at
the end of the manufacturing process to stabilise the product and to avoid
Factor VIII
dispersion on the glass wall of the container resulting in a lower specific
activity than
several high purity products. If the albumin content is subtracted from high
or very
high purity Factor VIII concentrates, figures in the range of 10-200 IU/mg or
1000-3000
IU/mg are obtained for specific activity, respectively.
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Fractionation methods have been developed with the use of monoclonal
antibodies directed against the FVIII molecule or von Willebrand Factor
antigen
(vWF:Ag) which successfully removed FVIII from cryoprecipitate during
immunoaffinity
chromatography. The protein content of these concentrates is dramatically
decreased.
Monoclonal antibody purified concentrates show a very high specific activity
and are .
virtually free of extraneous plasma proteins. The achievement of a very low
protein
content seems to be strongly advisable because there is increasing evidence
that
immunodeficiency in multi-transfused haemophiliacs can be related not only to
HIV
infection but also the allogenic protein overload derived from replacement
therapy
(Morfini 1989).
The problem of virus transmission among the haemophilia population has been
recognised since the early 1970s, hepatitis C virus and later HIV being the
major
viruses transmitted by clotting factor concentrates, and more recently
hepatitis A virus
and parvovirus B19. In order to reduce or abolish the virus load a variety of
chemical
and/or physical processes as well as methods of heat treatment under different
experimental conditions (dry, steam, wet heat) have been introduced into the
different
stages of clotting factor concentrate production. The principal methods used
are as
follows:
~ dry heat on freeze dried final product
~ pasteurisation
~ vapour heating under pressure
~ solvent detergent treatment.
Terminal dry heat treatment is a popular methodology introduced in the late
1980s to ensure a high degree of viral safety (Winkelman et al. 1989).
Coagulation
factor concentrates that are heated at 80°C for 72 hours have an
excellent safety
record (Rizza et al. 1993, Cash 1988, Skidmore et al. 1990).
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The use of sugars as stabilisers of biologically active proteins has been
previously disclosed in US patents 2,826,533 (1958) and 4,089,944 (1978).
Since the
FVIII molecule is a heat sensitive protein, FVIII concentrates of intermediate
purity
have been stabilised with different concentrations of various sugars,
lyophilised and
exposed to dry heat regimes of 80°C for 72 hours (Roberts 1995).
The stabilising effect of a commercially available synthetic mixture of 14
synthetic amino acids (Synthamin 17) designed for intravenous use as a
nutrient has
also been extensively documented for the large scale manufacture of
intermediate
to FVIII concentrates. Intermediate purity FVIII concentrate (1 IU/mg) was
stabilised by
the addition of Synthamin 17 when heated at 60°C for 48 hours {Benny et
al. 1988).
Synthamin 17 added in amounts of up to 4mg per unit FVIII, to a
cryoprecipitate FVIII
concentrate, resulted in better solubility and stability on storage. Heating
the
concentrate at 70°C for 16 hours with 1mg per unit maintained
solubility and activity
(Margolis and Eisen 1984).
The synthetic amino acid mixture was used for the large scale manufacture of
an intermediate FVlll concentrate (1 IU/mg) using controlled pore glass
adsorption
chromatography. Heat stability of the freeze dried material was tested and no
loss of
FVIII activity was shown after 30 hours at 70°C compared with other
intermediate
purity concentrate preparations tested which exhibited approximately 25% of
activity
after only hours at 70°C and solubility was significantly impaired
{Margolis et al. 1984,
Austen 1979). Similar FVIII preparations have been stabilised by addition of
the
synthetic amino acid mixture (1.6% w/v) in the final product. The lyophilised
material
heated at 60°C for 72 hours reduced the FVIII loss to less than 10%
compared with
a non-amino acid stabilised concentrate, which resulted in the loss of 30% of
FVIII:C
activity (McGrath et al. 1985).
The clinical experience reported has been favourable. Patients infused with
heat-treated material had a similar mean ex vivo recovery not significantly
different
from that observed with the non heated concentrates. The mean half life of
both
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heated and on heated material were reported to be identical. Stabilisation
with
Synthamin 17 and heat treatment does not influence the in vivo recovery and
haemostasis was uniformly effective, with no adverse side effects being
reported
(Ockelford et al. 1987).
Established FVIII products are indeed true concentrates and unlike pure plasma
products such as albumin or immunoglobulin, FVIII is only a small fraction of
the
protein contained in the concentrates (i.e. <1%), the majority being
fibrinogen,
immunoglobulin, fibronectin and many others. The presence of such proteins has
also
been shown to help stabilise the FVIII molecule over the terminal dry heat
stage.
Concentrate purity is now a very important prerequisite to be taken into
account
when developing new FVtll concentrates. The current trend is towards
increasing the
purity of FVIII preparations, purer products will have less
fibrinogenlfibronectin.
However purity itself further complicates the product stability over the
terminal dry heat
step. The protein content of high purity concentrates is dramatically
decreased.
Monoclonal antibody purified concentrates show a very high specific activity
and are
virtually free of extraneous plasma proteins. However, as noted above albumin
is
often added at the end of the manufacturing process to stabilise the product.
It is well
known that albumin has a low risk for viral transmission and is possibly less
antigenic
than other proteins due to its low molecular weight. Whether albumin can be
considered as an essential component of the protein overload and thereby of
the
immune depression observed in heavily-infused patients is still a matter of
controversy. Cohn Fractionation Albumin solutions used to stabilise high
purity FVllt
concentrates are unlikely to be more than 95% pure and so trace amounts of
other
proteins may therefore be added back into the FVIII concentrates. However,
this
practice largely defeats the purpose of purification.
The first documented report of a high purity FVIII concentrate which was shown
to be stable at ambient temperatures and at pasteurisation temperatures
(60°C) in the
absence of human serum albumin was recently disclosed in International Patent
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Publication WO 96122107. The addition of trehalose at 0.15 to 2.5mg per unit
FVIII
to a recombinant FVIII preparation showed excellent stability of FVIII
activity when
stored at 60°C for an extended period of time in the complete absence
of human
serum albumin. Trehalose is known to be a highly effective stabilising agent
for
delicate proteins as disclosed in US Patent No. 4,891,319 and Australian
Patent No. .
591160, enabling proteins to be dried at temperatures above freezing.
The stability of FVIII concentrates has been well established using sodium
citrate as an anticoagulant in plasma fractionation. Early studies with
fibrinogen
preparations as disclosed in US Patent No. 2,826,533 illustrated the
anticoagulant
and solubilising properties of sodium citrate. Citrate was also reported to
have a
marked stabilising effect on early preparations of chromatographically
purified FVIII
concentrates (Hynes et al. 1969) which helped establish the use of 20mM
trisodium
citrate in commercial FVIII concentrates (Newman et al. 1971 ). FVlil activity
shows
a biphasic deterioration in the presence of citrate (Preston 1967). This loss
of activity
stems from the chelation by citrate groups of calcium ions which are essential
for
maintaining the normal structure of the FVIII molecule. Formulating during
processing
by adding calcium chloride to maintain physiological levels of ionised calcium
stabilises FVIII activity (Fay 1988).
The present invention provides improved stabilising compositions which are
particularly, but not exclusively, directed to the stabilisation of high and
very high
purity Factor VIII concentrates as discussed above. The present invention also
provides stabilising compositions for these concentrates which avoid the
necessity for
adding back albumin, although albumin may still be added if desired and
acceptable.
SUMMARY OF THE INVENTION
According to a first aspect of the present invention, there is provided a
dried,
heat-treated product comprising a heat labile, biologically or therapeutically
active
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protein or peptide preparation, and a stabilising effective amount of a
composition
comprising sucrose, trehalose and at least one amino acid.
In yet another aspect, the present invention provides a method of preparing a
dried, heat-treated product comprising a heat labile, biologically or
therapeutically .
active protein or peptide preparation, which comprises (i) adding a
stabilising effective
amount of a composition comprising sucrose, trehalose and at least one amino
acid
to said protein or peptide preparation; (ii) drying said admixture; and (iii)
heat-treating
said dried product.
In yet another aspect, the invention provides a composition for use in the
stabilisation of a heat labile, biologically or therapeutically active protein
or peptide
preparation, comprising sucrose, trehalose and at feast one amino acid.
The terms "drying" and "dried" are used herein in the broad sense to refer to
removal of water from an aqueous product and products obtained thereby, and
include
in particular the freeze drying process (also known as lyophilisation).
The terms "heat-treating" and "heat-treated" are used herein to refer to
methods
of heat treatment in order to reduce or abolish a virus load in a biologically
or
therapeutically active product and products obtained thereby, and include dry
heat
treatment at greater than 75°C for up to 72 hours, more particularly
dry heat treatment
at 80°C for 72 hours.
In one particular embodiment, the present invention comprises a dried Factor
Vlll preparation comprising a Factor VIII concentrate, and a stabilising
effective
amount of a composition comprising sucrose, trehalose and at least one amino
acid.
Preferably, the Factor VIII concentrate is a high purity (10 - 200 IUlmg) or
very
high purity (1000-3000 IU/mg) product.
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References herein to a "high purity" or "very high purity" Factor VI II
concentrate
should be understood as referencing to the purity of the concentrate prior to
the
addition of a protein stabiliser.
Alternatively, the protein or peptide preparation may be a von Willebrand
Factor
concentrate.
Optionally, the stabilising composition in accordance with the present
invention
may also comprise albumin, particularly human serum albumin.
Throughout this specification and the claims which follow, unless the context
requires otherwise, the word "comprise", or variations such as "comprises" or
"comprising", will be understood to imply the inclusion of a stated integer or
group of
integers but not the exclusion of any other integer or group of integers."
IS
DETAILED DESCRIPTION OF THE INVENTION
The formulations of the present invention have the potential to provide
another
alternative to the adding back of protein such as human serum albumin (HSA) as
a
stabilising agent for high purity FVIII concentrates. Whilst the use of
albumin in the
composition of this invention is optional, the addition of albumin is not an
ideal option
because it adds a complex component to the product formulation. The
formulations
detailed in this invention relate to defined chemical additives containing
specific
components designed to prevent adsorption, and to maintain the stability and
solubility
of high purity FVIII concentrates exposed to heat treatment regimes, in
particular the
dry heat treatment of lyophilised product heated at 80°C for 72 hours.
The technology
of the current invention offers many advantages over the practice of adding
back
albumin to stabilise high purity FVIII concentrates and the use of trehalose
as
disclosed in International Patent Publication WO 96122107. The major
advantages are
highlighted below:
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(i) Coagulation factor concentrates that are heated at 80°C for 72
hours have an
excellent viral safety record. It has not been thought possible to prepare a
high
purity dried blood FVIII concentrate which is stable over this severe dry heat
treatment step without the addition of albumin.
(ii) Trehalose as disclosed in WO 96/22107 may be used to stabilise a dried
blood
factor composition which undergoes heat treatment at pasteurisation
temperatures (60°C). The amount of trehalose used in some of the
formulations in the present invention when used alone is insufficient to
produce
a stabilising effect at temperatures greater than 60°C (i.e.
80°C for 72 hours).
However, trehalose in combination with other chemical additives, sugars and
amino acids as disclosed in this invention provides significant stability to
high
purity FVIII concentrates undergoing severe dry heat treatment.
(iii) The disclosed formulations provide a method of preparing aqueous
solutions
containing FVIII, which may be freeze dried at temperatures greater than
25°C.
(iv) The chemical compounds, sugars and amino acids used in the disclosed
formulations can be chemically produced in large quantities under GMP
conditions. By contrast, human serum albumin derived from plasma introduces
considerable problems of purification since it is essential that the protein
is free
of viral contamination. Using recombinant HSA to overcome these problems
is expensive but it is also derived from animals cells or bacterial culture,
and
therefore the risk of contamination by infectious agents or unknown materials
is possible.
In the stabilising composition of the present invention, sucrose is preferably
present in amounts of from 0.5 to 10% by weight, more preferably from 2.5 to
5% by
weight. Similarly, trehalose is preferably present in amounts of from 0.5 to
5% by
weight, more preferably from 1.25 to 2.5% by weight.
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The amino acid which is incorporated into the stabilising composition is
preferably lysine, and the amino acid is preferably incorporated in a molar
amount of
from 0.05 to 1 M, more preferably 0.1 to 0.5M. Alternatively, however, the
amino acid
may be incorporated as a mixture of amino acids, particularly the mixture
available
commercially as Synthamin 17, or one or more of the amino acids present in .
Synthamin 17. Synthamin 17 is a formulation of amino acids as follows:
Synthamin 17
Essential Amino Nonessential
Acids (mg1100mL) Amino Acids
(mglmL)
L-Isoleucine (600) L-Alanine (2070)
L-Leucine (730) L-Arginine (1150)
L-Lysine (580) L-Histidine (480)
L-Methionine (400) L-Proline (680)
L-Phenylalanine (560) L-Serine (500)
L-Threonine (420) Glycine (1030)
L-Tryptophan (180)
L-Valine (580)
Such a mixture of amino acids is preferably used in the stabilising
composition
of the present invention in amounts of from 0.5 to 5% by weight, more
preferably from
2 to 3% by weight.
As previously described, albumin (such as human serum albumin) may
optionally be incorporated in the stabilising composition of the present
invention.
When incorporated, albumin may be present in an amount of from 0.1 to 10 mg/mL
of
the Factor VIII concentrate prior to freeze drying.
Whilst the present invention is principally directed towards the stabilisation
of
FVIII concentrates, particularly on drying high and very high purity FVIII
concentrates,
the compositions of the present invention may also be used in the
stabilisation of other
biologically or therapeutically active preparations, particularly those which
are to be
dried or lyophilised and subsequently treated to dry heat treatment as a viral
inactivation step. Such other biologically or therapeutic preparations
include, for
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example, other blood, plasma or serum fractions, enzymes, antibodies,
antigens,
vaccine components and the like.
Further features of the present invention are more fully described in the
following Example(s). It is to be understood, however, that this detailed
description
is included solely for the purposes of exemplifying the present invention, and
should
not be understood in any way as a restriction on the broad description of the
invention
as set out above.
EXAMPLE
This Example relates to a stabilised, dried Factor VIII preparation comprising
a high purity Factor VIII concentrate.
Method of Manufacture
Fresh frozen plasma (FFP) is thawed at temperatures below 5°C and the
FVlll-
rich cryoprecipitate is collected by centrifugation. The FVIII is extracted
with Tris
(hydroxymethyl) methylamine (Tris) buffer. Levels of unwanted proteins,
principally
fibrinogen, fibronectin, immunoglobulin and albumin, are reduced by
precipitation with
heparin followed by reprecipitation of FVIII with sodium chloridelglycine
buffer. The
purified FVIII is redissolved in a sodium chloride-Tris-citrate buffer
containing sucrose
and a low level of calcium chloride. The dissolved precipitate is filtered,
treated with
solventldetergent and incubated. The mixture is then filtered and
chromatographed
on a Sephacryl S400 column pre-equilibrated in the same buffer. The FVIII-rich
eluate
(z50 IU/mg total protein) is then concentrated by ultrafiltration against the
same buffer
and chemical stabilisers added to the retentate. The bulk formulated
concentrate is
sterile filtered, dispensed, freeze dried and heat treated at 80°C for
72 hours.
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Freeze DryingIDry Heat Treatment
The freeze drying cycle proceeds under conditions of programmed
temperature/vacuum/timing for approximately 100 hours. The formulated product
is
loaded into a freeze dryer and the shelves cooled to -50°C. The vacuum
is applied .
and the temperature vamped up to 50°C. The finished lyophilised product
is then
heated in a hot air oven at 80°C for 72 hours.
FVIII Procoagulant Activity
FVIII procoagulant activity was measured throughout the process using a
chromogenic assay kit (Chromogenix, Sweden).
Samples from each batch were taken at various stages throughout the process
(formulated bulk, freeze dried, and dry heated product) and tested for FVIII
procoagulant activity. The results are shown in the following Table in
comparison with
an intermediate purity (4-5 IUlmg) product, AHF(HP), and the high purity
concentrate
formulated with human serum albumin (used as a stabiliser in a number of
commercial
high purity concentrates).
The FVIII concentrates used in the experiments detailed in this invention were
formulated in a sodium chloridelTrislcitrate buffer containing sucrose and a
low level
of calcium chloride. The stability of FVIII concentrate using this buffering
system has
been well established. However, this formulation alone provided little
protection of the
FVIII molecule in the high purity product over the terminal dry heat treatment
step.
For the high purity product, a 33% loss of FVlli activity was reported,
compared with
the intermediate purity product in the same buffer where only 13% of the FVIII
activity
was lost over the same step, highlighting the protective effect of the high
levels of
protein contained in this concentrate. Similarly, the high purity product
stabilised with
added albumin only lost 21 % FVI II activity. The post dry heated results also
showed
that additional sugars such as sorbitol, maltose and sucrose alone produced
little or
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no stabilisation over the dry heat treatment step. Significant losses of FVIII
activity
were observed from the formulated bulk concentrate to post terminal dry heat
treatment. Similarly, the post drying results showed that it was not possible
to dry heat
the purity concentrate successfully when trehalose (45% loss of FVIII
activity) or
Synthamin 17 {25% loss of FVIII activity) were also alone.
Trehalose in combination with sucrose also produced significant losses in
FVIII
activity (61 %). However, with a composition of trehalose in combination with
sucrose
and lysine, and with a composition of sucrose, trehalose, lysine in
combination with
other amino acids (Synthamin 17), it was possible to dry heat FVIII
successfully in the
absence of human serum albumin.
These formulations all produced results similar to the high purity concentrate
stabilised with human serum albumin, and from the results disclosed it can be
concluded that a high purity FVIII concentrate can indeed be successfully
freeze dried
and dry heated in a chemical based formulation in the absence of HSA.
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Table 1 Percentage of FVII1 activity loss from formulation to dry heat
treatment
with various stabilisers.
Formulation No. of % FVIII
Activity
Loss
Composition Batches
Dry Formulation
Heat to Dry
Heat
Buffer alone 5 33.2 44.1
Intermediate purity product 4 12.6 14.8
AHF (HP)
Albumin (10 mglmL) 7 20.9 26.6
5% sorbitol 1 98.3 98.8
5% sucrose 2 46.6 78.4
10% sucrose 1 39.9 44.8
2.5% maltose 1 89.3 91.3
2.5% trehalose 1 43.3 45.1
2.5% sucrose + 1.25% trehalose1 60.6 63.3
2% Synthamin 17 + electrolytes1 25.1 41.0
2.5% sucrose, 1.25% trehalose,7 8.8 29.4
0.5M lysine
2.5% sucrose, 1.25% trehalose,
0.1 M lysine, 3 15.8 25.5
2% Synthamin 17 + electrolytes
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chronic
factor VIII substitution on immune parameters in HIV seronegative
haemophiliacs: a
comparison between factor VIII cryoprecipitate and factor VIII concentrate.
Thromb.
Haemostas. (1996); 75(2}:261-6.
Andersson LO, Forsman N, Huang K, Lundin A, Pavlu B, Sadberg H, Sewerin K,
Smart
J. Isolation and characterisation of human factor VIII: molecular forms in
commercial
factor Vlll concentrate , cryoprecipitate and plasma. Proc.Natl. Acad. Sci.
(1986);
83:2979-83.
Aronson D. The development of the technology and capacity for the production
of
factor VIII for the treatment of haemophilia A. Transfusion (1990}; 30:8:748-
58.
Austen D. The chromatographic separation of factor VIII on aminohexyl
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Br. J. Haematol. (1979); 43(4):669-74.
Benny A, Ockelford P, John A, Scott R, Woodfield D, Berry E. Influence of heat
treatment on FVIII:C recovery from freeze dried cryoprecipitate. J. Clin.
Pathol.
{1988): 41:945-7.
Cash J. Coagulation factor VIII concentrates and the market place. Lancet
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ii:814-6.
Eaton D, Rodrigez H, Vehar GA. Proteolytic processing of human factor Vlll.
Correlation of specific cleavages by thrombin, factor Xa, and activated
protein C with
activation and inactivation of factor VIII coagulant activity. Biochemistry
(1986);
25:505-12.
Eaton DL, Hass PE, Riddle L, Mather J, Wieve M, Gregory T, Vehan G.
Characterisation of recombinant Human Factor VIII. J. Biol. Chem. (1987);
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3285-90.
Esmon PC, Kuo HS, Fournel MA. Characterization of recombinant factor VIII and
a
recombinant factor VIII deletion mutant using a rabbit immunogenicity model
system.
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