U.S. patent application number 14/873549 was filed with the patent office on 2016-01-28 for method for preparing a concentrate of factor xi.
This patent application is currently assigned to LABORATOIRE FRANCAIS DU FRACTIONNEMENT ET DES BIOTECHNOLOGIES. The applicant listed for this patent is LABORATOIRE FRANCAIS DU FRACTIONNEMENT ET DES BIOTECHNOLOGIES. Invention is credited to Jean-Francois Martin.
Application Number | 20160024488 14/873549 |
Document ID | / |
Family ID | 46017843 |
Filed Date | 2016-01-28 |
United States Patent
Application |
20160024488 |
Kind Code |
A1 |
Martin; Jean-Francois |
January 28, 2016 |
METHOD FOR PREPARING A CONCENTRATE OF FACTOR XI
Abstract
The invention concerns a concentrate of human Factor XI having
high specific activity prepared using a method comprising a
filtration-adsorption step and a chromatography step on cation
exchange resin. The concentrate obtained is fully adapted for
therapeutic use as substitution therapy in cases of Factor XI
deficiency.
Inventors: |
Martin; Jean-Francois;
(Lille, FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
LABORATOIRE FRANCAIS DU FRACTIONNEMENT ET DES
BIOTECHNOLOGIES |
Les Ulis |
|
FR |
|
|
Assignee: |
LABORATOIRE FRANCAIS DU
FRACTIONNEMENT ET DES BIOTECHNOLOGIES
Les Ulis
FR
|
Family ID: |
46017843 |
Appl. No.: |
14/873549 |
Filed: |
October 2, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14112175 |
Oct 16, 2013 |
9181323 |
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PCT/EP2012/057227 |
Apr 19, 2012 |
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14873549 |
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Current U.S.
Class: |
435/226 |
Current CPC
Class: |
C12N 9/6443 20130101;
C07K 14/745 20130101; C12Y 304/21027 20130101 |
International
Class: |
C12N 9/64 20060101
C12N009/64 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 20, 2011 |
FR |
11 53437 |
Claims
1.-10. (canceled)
11. A Factor XI (FXI) composition obtainable by a method
comprising, a) filtration-adsorption of a supernatant of human
plasma cryoprecipitate using a filter comprising cellulose and
perlites and a charged resin, wherein the filter has a grade
ranging from 0.1 to 0.4 .mu.m, wherein the filtration-adsorption
step comprises passing the supernatant of plasma cryoprecipitate
through the filter to adsorb FXI on the filter, and desorbing or
eluting the adsorbed FXI; and b) chromatography of the product of
step a) on a cation exchange resin to obtain a solution of FXI.
12. The composition according to claim 11 wherein the FXI adsorbed
on the filter is desorbed or eluted using a solution adjusted to a
pH of 5.5 to 6.5 comprising sodium citrate, disodium phosphate,
potassium phosphate, disodium EDTA and sodium chloride, the
concentration of said sodium chloride being higher than 0.5 M.
13. The composition according to claim 12 wherein step a) comprises
washing of the filter prior to eluting the adsorbed FXI, using a
buffer adjusted to a pH of 5.5 to 6.5 comprising sodium citrate,
disodium phosphate, potassium phosphate and sodium chloride, the
concentration of said sodium chloride being equal to lower than 0.5
M.
14. The composition according to claim 11 comprising an additional
step for viral inactivation between step a) and step b), and/or a
virus removal step after step b).
15. The composition according to claim 11 wherein the cation
exchange resin of step b) is equilibrated with a buffer adjusted to
a pH of 5.5 to 6.5 comprising sodium citrate, sodium chloride,
lysine and arginine.
16. The composition according to claim 11 wherein the cation
exchange resin of step b) is loaded with the FXI resulting from
step a), then washed with a buffer adjusted to a pH of 6.1 to 6.9
comprising sodium citrate, disodium phosphate, potassium phosphate,
sodium chloride, lysine and arginine.
17. The composition according to claim 11 wherein the solution of
FXI obtained from step b) is stabilised through the addition of 0.5
to 3 IU Antithrombin III, 0.5 to 4 IU of heparin and 0.5 to 2 IU of
C1-inhibitor per 100 IU of FXI.
18. The composition according to claim 11 further comprising
packaging the stabilised solution of FXI as pharmaceutical
product.
19. The composition according to claim 11 further comprising
lyophilisation of the solution of FXI obtained from step b).
Description
FIELD OF THE INVENTION
[0001] The present invention concerns a method for preparing Factor
XI from a cryosupernatant of plasma precipitate, comprising an
adsorption-filtration step and a chromatography step on cation
exchange resin.
STATE OF THE ART
[0002] Factor XI or the precursor of plasma thromboplastin is a
glycoprotein which is part of the contact pathway in the mechanism
of haemostasis, through its activating action on Factor IX, and is
part of the fibrinolysis pathway through its activating action on
plasminogen.
[0003] Factor XI deficiency is hereditary and is transmitted as a
recessive autosomal character. It is a rare deficiency but
widespread in some populations of Middle-East origin.
[0004] As is the case for other factors whose deficiency is rare
(Factor V, Factor XIII, Factor X), therapeutic products purified
from human plasma are still scarce or inexistent and the sole
substitution therapy uses total plasma or the supernatant fraction
of plasma cryoprecipitate, but this entails the simultaneous
injection of useless quantities of other plasma proteins and hence
a risk of various major secondary reactions after multiple
injections.
[0005] Patent EP 512 883 B1 describes a method for preparing a
concentrate of Factor XI comprising a filtration-adsorption step
and a chromatography step on cation exchange resin. The
filtration-adsorption step of the method described in this patent
is performed on a series of depth filters composed of cellulose and
perlites carrying negative charges and a small quantity of
positively charged resin. These filters have a porosity ranging
from 0.5 to 2 .mu.m and have the property of binding FXI. The yield
of FXI purification at this step is about 50% and the specific
activity of the eluted FXI is about 10-20 U/mg. However, in the
event of a reduced yield, the subsequent chromatography step can be
strongly influenced and hence cause non-conformity of the end
product for faulty FXI concentration.
[0006] There is therefore a true need to develop a method for
purifying FXI allowing the yields of the purification method to be
increased, in particular after the adsorption-filtration step, so
that it is possible to avoid any problem of non-conformity of the
end product.
SUMMARY OF THE INVENTION
[0007] The present invention therefore concerns a method for
preparing a concentrate of Factor XI, comprising a
filtration-adsorption step conducted on a filter composed of
cellulose and perlites and a small quantity of positively charged
resin, the said filter having a grade ranging from 0.1 to 0.4
.mu.m, preferably from 0.2 to 0.4 .mu.m, preferably from 0.25 to
0.35 .mu.m, and preferably 0.3 .mu.m; and a chromatography step on
cation exchange resin.
[0008] At the adsorption-filtration step of the preparation method
of the present invention, the elution of FXI adsorbed on the filter
is performed using a solution adjusted to a pH of 5.5 to 6.5, for
example comprising sodium citrate, disodium phosphate, potassium
phosphate, disodium EDTA and sodium chloride, the concentration of
said sodium chloride being higher than 0.5 M, preferably higher
than or equal to 0.6 M or preferably higher than or equal to 0.75 M
or preferably equal to 1 M.
[0009] The adsorption-filtration step may also comprise washing of
the filter prior to elution of the adsorbed FXI, using a buffer
adjusted to a pH of 5.5 to 6.5 for example comprising sodium
citrate, disodium phosphate, potassium phosphate and sodium
chloride, the concentration of said sodium chloride being equal to
or lower than 0.5 M, preferably equal to or lower than 0.4 M,
preferably equal to 0.3 M.
[0010] Preferably the filtering flow rate of the
adsorption-filtration step lies in the range of 7 to 20
mL/h/cm.sup.2, preferably 10 to 15 mL/h/cm.sup.2, preferably 12 to
14 mL/h/cm.sup.2.
[0011] Preferably the volume load used at the adsorption-filtration
step lies in the range of 11 to 50 mL/cm.sup.2, preferably 20 to 40
mL/cm.sup.2, preferably 25 to 35 mL/cm.sup.2.
[0012] The Factor XI fraction resulting from the
adsorption-filtration step undergoes a diafiltration step before
the purification step by chromatography. The method of the
invention may also comprise additional steps of concentration and
viral inactivation between the adsorption-filtration step and the
purification step by chromatography.
[0013] During the chromatography step, the cation exchange resin is
preferably equilibrated with a buffer adjusted to a pH of 5.5 to
6.5 comprising sodium citrate, sodium chloride, lysine and
arginine. The cation exchange resin is then loaded with the FXI
resulting from the adsorption-filtration step, and washed with a
buffer adjusted to a pH of 6.1 to 6.9 comprising sodium citrate,
disodium phosphate, potassium phosphate, sodium chloride, lysine
and arginine. The Factor XI is finally eluted from the cation
exchange resin with a buffer adjusted to a pH of 7 to 8 comprising
sodium citrate, disodium phosphate, potassium phosphate, sodium
chloride, lysine and arginine in which the concentration of sodium
chloride is between 0.15 and 0.20 M.
[0014] The resulting solution of FXI is preferably stabilised
through the addition of antithrombin III, heparin and C1-inhibitor
and may at this stage be subjected to a viral removal step
preferably by filtering through a grade 15 nm filter. The FXI
solution is then packaged and lyophilised. The stabilised FXI
solution is in particular packaged as a pharmaceutical product.
[0015] The present invention also concerns a composition of FXI
able to he obtained using the method of the invention.
[0016] The applicant has surprisingly discovered that the use of a
filter composed of cellulose and perlites, comprising a charged
resin and having a lower grade than the filters used in the state
of the art and in particular in the method of patent EP 512 883 not
only allows adsorbing of the FXI to a greater extent and hence the
obtaining of a significant increase in FXI yield and in FXI purity,
but also allows a significant increase in volume load, the
filtering flow rate used at this step, compared with the method
described in patent EP 512 883. This filter therefore offers
greater industrial advantages than the filters used in the method
of patent EP 512 883 such as a reduction in the number of filter
cartridges and hence a reduction in occupied space in production
facilities, upgrading to large-scale, simplified installation and
assembly, and a reduction in the overall time of the fabrication
process. Quite unexpectedly the method of the present invention,
which uses filters having a grade ranging from 0.1 to 0.4 .mu.m,
preferably 0.2 to 0.4 .mu.m, preferably 0.25 to 0.35 .mu.m and
preferably of 0.3 .mu.m facilitates the adsorbing of FXI at the
adsorption-filtration step even when the protein load and flow rate
used are significantly increased.
[0017] It therefore appears that the novel method of the invention
allows both an improvement in the yield and specific activity of
the FXI purified at the adsorption-filtration step whilst reducing
the duration of this step. This leads to a reduction in costs and
in the production time of FXI.
DETAILED DESCRIPTION OF EMBODIMENTS
[0018] The present invention concerns a method for preparing a
concentrate of Factor XI comprising a filtration-adsorption step,
selectively retaining Factor XI, and a chromatography step on
cation exchange resin.
[0019] The first step of the purification method corresponds to a
filtration-adsorption step performed on a depth filter. The filter
used is composed of cellulose and perlites, and comprises a charged
resin. The filter used is of a grade ranging from 0.2 to 0.4 .mu.m.
preferably 0.2 to 0.4 .mu.m, preferably 0.25 to 0.35 .mu.m, and
further preferably 0.3 .mu.m. In one preferred embodiment, the
filter used is a Sartoclear.RTM. S5P filter (marketed by Sartorius
stedim biotech GmbH) and having a grade of 0.3 .mu.m. Other
comparable filters that are commercially available may also be
used.
[0020] In one particular embodiment, the method of the invention is
implemented from a supernatant of plasma cryoprecipitate. The
supernatant used in the present invention can be obtained from any
suitable source and preferably corresponds to plasma of human
origin.
[0021] In one preferred embodiment, the purification method of the
present invention applies to the supernatant of human plasma
cryoprecipitate and can be adapted to volumes of at least 1000
litres to 1200 litres. It also advantageously allows the production
of Factor XI for therapeutic use from about 1100 litres of plasma
and within a fairly short time (about 28 hours) including an
optional viral inactivation treatment time (about 8 hours).
[0022] In one preferred embodiment, the method of the invention
comprises an adsorption step of FXI on the depth filter by passing
the supernatant of plasma cryoprecipitate through the filter. The
filter is then advantageously washed with a buffer adjusted to a pH
of 5 to 7, preferably pH 6, comprising sodium citrate, disodium
phosphate, potassium phosphate and sodium chloride. The
concentration of sodium chloride in said buffer is preferably equal
to or less than 0.3 M and is preferably 0.06 M. This filtering step
removes most plasma proteins.
[0023] In one preferred embodiment, the filter is then washed with
a buffer adjusted to a pH of 5 to 7 and preferably pH 6, comprising
sodium citrate, disodium phosphate, potassium phosphate and sodium
chloride. The concentration of sodium chloride in said buffer is
preferably equal to or less than 0.5 M, preferably equal to or less
than 0.4 M, and preferably it is 0.3 M. This washing step removes
the plasma proteins weakly adsorbed on the filter and increases the
purity of the FXI eluted at the following step.
[0024] In one preferred embodiment, the Factor XI which remained
adsorbed on the filter is then desorbed or eluted by increasing the
ionic strength of the wash buffer. The Factor XI is therefore
desorbed using a buffer whose final concentration of sodium
chloride is higher than 0.5 M, preferably higher than or equal to
0.6 M, preferably higher than or equal to 0.75 M, further
preferably it is 1M. In one preferred embodiment the elution buffer
may also comprise a small quantity of protease inhibitors,
advantageously antithrombin III (AT III) to protect the Factor XI
against the action of any proteases and hence its activation.
Heparin and/or C1-inhibitor may advantageously be added to
stabilise the mixture obtained.
[0025] In one preferred embodiment of the present invention, the
adsorption-filtration step of FXI is conducted at a filtering flow
rate ranging from 7 to 20 mL/h/cm.sup.2, preferably 10 to 15
mL/h/cm.sup.2, preferably 12 to 14 mL/h/cm.sup.2. In the context of
the present invention, by <<filtering flow rate>> is
meant the linear flow rate calculated by measuring the volume or
weight filtered per unit of time and per unit surface area. This
flow rate is maintained for every step of the adsorption/filtration
step.
[0026] In one preferred embodiment of the present invention, the
adsorption-filtration step of FXI is conducted with a volume load
ranging from 11 to 50 mL/cm.sup.2 preferably 20 to 40 mL/cm.sup.2,
preferably 25 to 35 mL/cm.sup.2. In the context of the present
invention by <<volume load>> is meant the volume or
weight of solution (for example of precipitation cryosupernatant)
that is passed per unit of filtering surface.
[0027] In one preferred embodiment, the FXI solution obtained after
the adsorption-filtration step is subjected to viral inactivation
treatment. This viral inactivation treatment is preferably
performed using the solvent and/or detergent method of treatment
known to persons skilled in the art. In one preferred embodiment of
the invention, the viral inactivation treatment is conducted in the
presence of Tween 80, preferably at a final concentration of 1%
(w/v) and Tri-n-Butyl Phosphate or TnBP, preferably at a final
concentration of 0.3% (v/v). The viral inactivation treatment is
preferably conducted for a time of at least 8 hours.
[0028] In one preferred embodiment, the FXI solution obtained after
the adsorption-filtration step is dialysed and can be concentrated
and frozen to -80.degree. C. for storage before carrying out a
possible viral inactivation step.
[0029] The FXI solution is then purified by chromatography on
cation exchange resin. This chromatography step additionally allows
the complete removal of the residual products remaining after the
viral inactivation step, if this step is carried out. This
chromatography step notably allows the removal of subsisting
contaminating proteins.
[0030] The fraction containing the FXI desorbed from the filter,
dialysed and optionally concentrated, optionally frozen and virally
inactivated, is injected into a chromatography column comprising a
cation exchange resin. In one preferred embodiment, the cation
exchange resin is more particularly Sulfate-Sepharose Fast Flow
(S-Sepharose FF). S-Sepharose gel unexpectedly has a very high
retaining capacity of Factor XI (from 300 to 450 U/ml of gel) for
example making it possible to avoid a subsequent concentration step
of FXI via an ultrafiltration step which would lead to loss of
yield. In addition, binding to S-Sepharose FF allows the FXI to be
eluted with a buffer having properties comparable to those of a
physiological buffer whereas other resins e.g. containing
sulfylpropyl groups require more drastic eluting conditions
involving the setting up of a diafiltration step to return to
conditions of an injectable product.
[0031] In one preferred embodiment, the cation exchange resin is
equilibrated with a buffer adjusted to a pH of 5.5 to 6.5,
preferably pH 6, composed of sodium citrate, sodium chloride,
lysine and arginine.
[0032] In one preferred embodiment, the cation exchange resin is
loaded with the solution containing the FXI derived from the
adsorption-filtration step which is dialysed and optionally
concentrated and virally inactivated. Washing is then preferably
performed with the equilibrating buffer of pH 5.5 to 6.5 preferably
pH 6, composed of sodium citrate, sodium chloride, lysine and
arginine. A second washing is then preferably performed with a
buffer adjusted to a pH of between 6.1 and 6.9, preferably pH 6.5,
comprising sodium citrate, disodium phosphate, potassium phosphate,
sodium chloride, lysine and arginine.
[0033] The Factor XI is then eluted from the cation exchange resin
using a buffer comparable to the above-mentioned wash buffer but
having a pH of between 7 and 8, preferably pH 7.5 and whose sodium
chloride concentration is between 0.15 and 0.20 M, preferably 0.17
M.
[0034] In one preferred embodiment, the Factor XI is stabilised
after its elution from the cation exchange resin, through the
addition of small amounts of protease inhibitors e.g. Antithrombin
III, heparin and C1-inhibitor. The Factor XI solution is stabilised
with the addition of 0.5 to 3 IU, preferably 1 to 3 IU, preferably
2 to 3 IU of Antithrombin III, 0.5 to 4 IU, preferably 1 to 4 IU,
preferably 2 to 4 IU of heparin and 0.5 to 2 IU, preferably 1 to 2
IU, preferably 1.5 to 2 IU of C1-inhibitor per 100 IU of Factor
XI.
[0035] The resulting stabilised FXI solution may optionally undergo
a viral removal step preferably via filtration on a 15 nm grade
filter and/or is then sterilised e.g. by filtration, packaged and
lyophilised.
[0036] In one preferred embodiment, the stabilised and optionally
virally filtered solution of Factor XI is packaged as a
pharmaceutical product. This packaging may in particular be
performed by adding pharmaceutically acceptable excipients.
[0037] A further subject of the invention comprises a composition
of Factor XI able to be obtained using the method of the
invention.
[0038] Advantageously the purification factor of the present method
is at least 10000 relative to the starting plasma.
[0039] The Factor XI obtained with the method of the present
invention has specific activity ranging from 80 to 120 U/mg,
preferably at least equal to 100 U/mg of proteins.
[0040] The high purity of the Factor XI obtained is evidenced by
polyacrylamide gel electrophoresis under denaturing conditions (in
the presence of SDS) and biochemical analyses, and its
innocuousness is evidenced by biological tests on animals.
[0041] The concentrate of Factor XI obtained with the method of the
present invention is therefore particularly well adapted to
therapeutic use, in particular as substitution therapy in cases of
congenital or acquired deficiency of Factor XI.
[0042] The present invention is illustrated by the examples and
embodiments described and illustrated, but it is no way limited
thereto insofar as it may have numerous variants accessible to
persons skilled in the art.
EXAMPLE 1
Composition of Factor XI
[0043] Starting material:
[0044] Each batch of Factor XI was prepared from a volume of about
1000 litres of supernatant of human plasma cryoprecipitate. The
cryoprecipitation supernatant was first clarified on a 1 .mu.m
grade filter such as the Profile.RTM. II (Pall) filter.
[0045] First Purification Step
[0046] The cryoprecipitate supernatant was passed through a
Sartoclear.RTM. S5P (Sartorius) filter of grade 0.3 .mu.m at a flow
rate of 13 mL/h/cm.sup.2 (i.e. about 14.3 L/h). The volume load
used corresponded to 30 mL of cryoprecipitate supernatant/cm.sup.2
of filtering surface area, and all filtering was conducted at a
temperature of 2 to 8.degree. C.
[0047] After removing the filtrate containing most of the proteins
of the cryoprecipitate supernatant, the filter was washed with
buffer comprising 5 mM sodium citrate, 5 mM disodium phosphate. 5
mM potassium phosphate, 0.065 M sodium chloride and adjusted to pH
6 with citric acid
[0048] At this filtering stage the FXI is adsorbed on the
filter.
[0049] A more stringent wash step was performed with a buffer
comparable to the wash buffer but containing 0.3 M NaCl.
[0050] The Factor XI was eluted from the filter by increasing the
ionic strength of the wash buffer. The eluting buffer used, to
which disodium EDTA was added. also contained 1M NaCl. To the
resulting FXI solution, 0.2 U/ml antithrombin III were added to
protect the Factor XI against the action of residual plasma
proteases. The disodium EDTA in the buffer also takes part in this
protective action.
[0051] The FXI solution thus recovered was concentrated about 30
times and dialysed to remove the EDTA using a Pall ultrafiltration
system having a cut-off threshold at 10 kD. The expressed flow rate
is a linear flow rate. It can be calculated by measuring the volume
or weight filtered per unit of time and per unit surface area. This
flow rate was maintained for all the steps of the
adsorption/filtration step. The dialysis step also allows the FXI
fraction to be placed under the osmolality and chemical composition
conditions of the equilibrating buffer in the S-Sepharose FF
column, to guarantee FXI binding in the column.
[0052] The dialysis buffer was composed of 5 mM sodium citrate, 140
mM sodium chloride, 5.5 mM L-lysine and 20 mM arginine, and
adjusted to pH 6.
[0053] The dialysed solution was passed through a KA2NLP 0.45 .mu.m
filter (Pall)+0.22 .mu.m K2NFP1 (Pall) filter to clarify the
solution and optionally remove any bacterial contaminants
present.
[0054] Viral Inactivation Treatment
[0055] The solution containing Factor XI was subjected to treatment
with a solvent-detergent known for its efficacy against lipid
enveloped viruses (Horowitz et al 1985, Transfusion 25, 516-522)
and which comprised 8-hour incubation at 25.degree. C. in the
presence of 0.3% tri-n-butyl-phosphate (TnBP) and 1% Tween 80.
[0056] Second Purification Step
[0057] The objective of this step was to increase the purity of
Factor XI (essentially by removing Factor II, Factor VII, Factor X,
high molecular weight kininogens, the Prekallikrein Activator, M
immunoglobulins, G immunoglobulins, fibronectin and fibrinogen,
whilst removing from the solution the solvent-detergent used for
viral inactivation treatment.
[0058] A chromatography column was used containing a cation
exchange resin, more particularly Sulfate-Sepharose Fast Flow gel
(S Sepharose FF, manufactured and distributed by Pharmacia,
Uppsala, Sweden).
[0059] The column was equilibrated with the above-described
dialysis buffer.
[0060] After loading the protein solution in the column, the column
was rinsed with at least 20 volumes of the above-described dialysis
buffer pH 6.0 to remove the proteins non-adsorbed on the gel and
the viral inactivation agents. The gel was then washed with a
buffer solution comprising 10 mM sodium citrate, 5 mM disodium
phosphate, 5 mM potassium phosphate, 92 mM sodium chloride, 27 mM
lysine and 11.5 mM arginine, pH 6.5, to elute the proteins weakly
adsorbed on the gel.
[0061] The Factor XI was eluted from the column by increasing the
pH of the buffer to 7.5 and increasing the NaCl concentration to
170 mM.
[0062] The linear flow rate to pass the equilibrating buffer, wash
and eluting solutions was 50 cm/h.
[0063] To the eluted FXI solution there were added three
stabilisers:
[0064] Antithrombin III in an amount of about 3 IU Antithrombin per
100 IU of FXI;
[0065] C1-inhibitor in an amount of about 2 U of C1-inhibitor per
100 IU of FXI; and
[0066] heparin in an amount of about 4 IU heparin per 100 IU of
FXI.
[0067] The stabilised solution was filtered through 0.22 .mu.m
Millipak 20, dispensed (10 ml per bottle) and lyophilised. It may
be filtered through a grade 15 nm filter before 0.22 .mu.m
filtering and dispensing into bottles.
[0068] Biochemical and Biological Analyses of the Factor XI
Concentrate
[0069] The purity yields (%) of FXI (IU/mg) and <<activation
levels>> measured using the ratio of activated Factor
XI/Factor XI (FXIa/FXI) obtained at the different unit steps of the
method are illustrated in Table I below:
TABLE-US-00001 TABLE 1 Steps Mean of 3 batches Cryosupernatant FXI
(IU/mL) 0.74 FXI (Total IU) 23660 (100%) Specific activity (IU/mg)
0.01 FXIa/FXI (.mu.g/IU) 0.236 Sartoclear eluate FXI (IU/mL) 4.1
FXI (Total IU) 17763 (82%) Specific activity (IU/mg) 31.3 FXIa/FXI
(.mu.g/IU) 3.1 After Ultrafiltration FXI (IU/mL) 85.1 FXI (Total
IU) 15630 (74%) Specific activity (IU/mg) 23.3 FXIa/FXI (.mu.g/IU)
ND S-Sepharose eluate FXI (IU/mL) 121 FXI (Total IU) 12126 (55%)
Specific activity (IU/mg) 292 FXIa/FXI (.mu.g/U) 0.46 Stabilised
FXI (IU/mL) 102.7 Specific activity (IU/mg) 95.7 FXIa/FXI (.mu.g/U)
<0.001
[0070] With the method of the invention, it is possible to obtain a
filtration/adsorption yield of FXI of about 80% and FXI purity of
about 20 to 40 IU/mg. By way of comparison, the method described in
patent EP 512 883 allows a filtration/adsorption yield to be
obtained of about 50% and FXI purity of about 10 to 20 IU/mg.
[0071] The yield and purity obtained in the present invention
therefore prove to be significantly higher than obtained when
implementing the method of the prior art. In addition, the method
of the invention was performed under FXI binding and eluting
conditions in which the protein load and filter flow rate were
respectively increased by a factor of 3 and a factor of 2 compared
with the conditions applied in the method of patent EP 512 883.
[0072] As a result, the method of the invention allows a
significant reduction in the cost and production time of FXI,
whilst providing a Factor XI having a purity level higher than that
described in the state of the art for the filtration/adsorption
step.
[0073] The removal of the proteins present in the cryoprecipitate
supernatant during the purification method is illustrated in
following Table 2. The results were compared with the data reported
for the method in patent EP 512 883.
[0074] Removal of Proteins During FXI Purification
TABLE-US-00002 Depth filter eluate of the invention FXI solution
obtained FXI solution obtained with the method of the with the
method of invention (mean of 3 EP 0 512 883 batches) (mean of 3
batches) Proteins (g/L) 3.8 8.2 High molecular weight .gtoreq.300
418 kininogens (.mu.g/mL) Factor II (IU/mL) 1.04 8.8 G
Immunoglobulins 0.28 0.9 (mg/mL) M Immunoglobulins 0.21 0.26
(mg/mL) Fibronectin (mg/mL) 0.018 0.09 C3 component of 0.041 0.1
complement (mg/mL) C1-Inhibitor (mg/mL) 0.011 0.029 .alpha.-2
Macroglobulin 0.039 0.17 (mg/mL) S-Sepharose FF eluate Proteins
(g/L) 0.42 0.64 High molecular weight <10 123 kininogens
(.mu.g/mL) Factor II (IU/mL) <0.1 <0.126 G Immunoglobulins
0.003 0.02 (mg/mL) M Immunoglobulins 0.011 0.022 (mg/mL)
Fibronectin (mg/mL) <0.003 0.03 C3 component of <0.008
<0.03 complement (mg/mL) C1-Inhibitor (mg/mL) 0.01 0.044
.alpha.-2 Macroglobulin <0.003 <0.01 (mg/mL) Stabilised
S-Sepharose FF eluate Proteins (g/L) 1.1 0.73 High molecular weight
<10 49 kininogens (.mu.g/mL) Factor II (IU/mL) <0.1 <0.126
G Immunoglobulins 0.004 0.018 (mg/mL) M Immunoglobulins 0.01 0.01
(mg/mL) Fibronectin (mg/mL) <0.004 0.01 C3 component of
<0.008 <0.03 complement (mg/mL) C1-Inhibitor (mg/mL) 0.52*
0.53* .alpha.-2 Macroglobulin <0.003 <0.01 (mg/mL) *after
addition for stabilisation
[0075] The absence of residual contamination by coagulation factors
and constituents of the kinin system (kininogens) was carefully
controlled using conventional methods.
[0076] After reconstituting the lyophilised product, conventional
tolerance tests on animals were performed:
[0077] thrombogenicity test in rabbits;
[0078] hypotension test in rats;
[0079] toxicity test in mice.
[0080] The tests on rabbits showed that the composition of the
invention is not thrombogenic since ED50 (effective dose for 50%)
is higher than 1000 U FXI/kg whereas this same value is 40 to 60
U/kg for a concentrate of PPSB (coagulating fraction composed of
proconvertin, prothrombin, Stuart factor, anti-haemophilic factor
B) which is therefore much more thrombogenic and may effectively
lead to phenomena of thrombosis and disseminated intravascular
coagulation in man at a high dose.
[0081] The FXI composition of the invention does not cause
phenomena of hypotension when injected via intravenous route into
rats at a dose of 50 U FXI/kg. This animal model is highly
sensitive to the presence of plasma components having vasoactive
properties and showed the absence of these components in the Factor
XI composition obtained using the described method.
[0082] When injected via intravenous route into mice at high dose
(2500 U/kg) it does not cause any lethality or disturbed behaviour
over a 7-day period.
* * * * *