U.S. patent application number 13/074636 was filed with the patent office on 2011-07-21 for method for obtaining pure monosialoganglioside gm1 for medical use.
This patent application is currently assigned to Laboratoire Medidom S.A.. Invention is credited to Rene-Pierre Bunter, Stefano Carlino.
Application Number | 20110178035 13/074636 |
Document ID | / |
Family ID | 39670910 |
Filed Date | 2011-07-21 |
United States Patent
Application |
20110178035 |
Kind Code |
A1 |
Carlino; Stefano ; et
al. |
July 21, 2011 |
METHOD FOR OBTAINING PURE MONOSIALOGANGLIOSIDE GM1 FOR MEDICAL
USE
Abstract
A process for preparing pure monosialoganglioside GM1 in the
form of its sodium salt. There is provided a process for the
isolation and purification of monosialoganglioside GM1 comprising
(a) separation of GM1 from a lipidic mixture containing the
monosialoganglioside GM1 as the main ganglioside component by ion
exchange column-chromatography using an eluent comprising potassium
or caesium ions, (b) recovery of the solute from the eluted
solution, (c) diafiltration of an aqueous solution of the recovered
solute, and (d) second diafiltration after the addition of 1 M
NaCl, and recovering GM1. The purity level of GM1 obtained is
higher than 99.0%.
Inventors: |
Carlino; Stefano;
(Collombey, CH) ; Bunter; Rene-Pierre; (Venthone,
CH) |
Assignee: |
Laboratoire Medidom S.A.
Geneva
CH
|
Family ID: |
39670910 |
Appl. No.: |
13/074636 |
Filed: |
March 29, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
11812331 |
Jun 18, 2007 |
7943750 |
|
|
13074636 |
|
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Current U.S.
Class: |
514/25 ;
536/17.9 |
Current CPC
Class: |
Y02A 50/30 20180101;
A61P 25/00 20180101; A61K 35/30 20130101; C07H 15/10 20130101; A61K
31/7032 20130101; A61K 31/351 20130101 |
Class at
Publication: |
514/25 ;
536/17.9 |
International
Class: |
A61K 31/7032 20060101
A61K031/7032; C07H 1/06 20060101 C07H001/06; C07H 15/12 20060101
C07H015/12; A61P 25/16 20060101 A61P025/16; A61P 25/28 20060101
A61P025/28; A61P 25/14 20060101 A61P025/14; A61P 25/02 20060101
A61P025/02; A61P 25/00 20060101 A61P025/00 |
Claims
1. (canceled)
2. (canceled)
3. (canceled)
4. (canceled)
5. (canceled)
6. (canceled)
7. (canceled)
8. (canceled)
9. (canceled)
10. (canceled)
11. (canceled)
12. (canceled)
13. A composition, comprising; a monosialoganglioside GM1, wherein
said monosialoganglioside GM1 l is purified by separating said
monosialoganglioside GM1 from a lipidic mixture containing the
monosialoganglioside GM1 as the main ganglioside component, by ion
exchange column-chromatography using an eluent comprising potassium
or caesium ions.
14. The composition according to claim 13, comprising less than
0.1% Fuc-GM1.
15. The composition according to claim 13 further including at
least one pharmaceutically acceptable carrier.
16. (canceled)
17. (canceled)
18. (canceled)
19. The composition according to claim 13, wherein said
monosialoganglioside GM1 is at least 99.0% pure.
Description
[0001] The present invention relates to the monosiaganglioside GM1,
more particularly to processes for the obtaining and purifying the
monosialoganglioside GM1.
BACKGROUND
[0002] Gangliosides are a class of glycosphingolipids, having one
or more sialic acid residues, and are found abundantly in the
cerebral and nervous tissue of humans and animals. The
monosialoganglioside GM1 is known for a number of pharmaceutical
applications, particularly in the repair and treatment of disorders
of the central and peripheral nervous systems.
[0003] Gangliosides are conveniently extracted from bovine or
porcine cerebral tissue according to U.S. Pat. No. 4,849,413. In
order to be suitable for pharmaceutical applications, the
monosialoganglioside GM1 must then be isolated and purified.
[0004] It is known to treat the extracted lipid mixture by chemical
or enzymatic methods to transform other ganglioside components to
the monosialoganglioside GM1, in order to increase the yield of
monosialoganglioside GM1. Such methods include acid hydrolysis with
a weak acid, such as described in CN 1353112, or enzymatic
hydrolysis using a sialidase, for instance as described in U.S.
Pat. No. 5,296,360.
[0005] Further purification of such GM1 enhanced lipidic mixture by
exclusion chromatography using a chloroform:methanol:water
60:30:4.5 eluent is described in EP 0 150 712. EP 0 489 352
describes purification of a GM1 enhanced lipidic mixture by
ultra-filtration of a solution of the lipidic mixture with
alpha-cyclo-dextrin, followed by extraction of GM1 by solvent
extraction with ethanol. It is reported that GM1 may be obtained
with a purity of 95%.
[0006] Such processes have previously been shown to have drawbacks
with respect to the purity and yield of GM1, and with respect to
cost, efficiency and effectiveness when applied on an industrial
scale.
[0007] For pharmaceutical applications it is required to produce
the ganglioside GM1 at high purity. Accordingly, there remains an
ongoing need for processes for obtaining the ganglioside GM1 at
high-purity.
[0008] The inventors of present application have surprisingly found
that the ganglioside GM1 may be effectively separated from other
gangliosides by a process based on ion-exchange chromatography.
[0009] According to the present invention, it has been found that
the ganglioside GM1 may be prepared at high purity by a process
wherein GM1 is separated from a lipidic mixture containing the
monosialoganglioside GM1 as the main ganglioside component by ion
exchange column-chromatography using an eluent comprising potassium
or caesium ions.
[0010] According to the present invention there is provided a
process for the isolation and purification of the ganglioside GM1
according to claim 1.
[0011] According to a preferred embodiment of the present invention
there is provided a process comprising the general steps of: [0012]
(a) separating GM1 from a lipidic mixture containing the
monosialoganglioside GM1 as the main ganglioside component by ion
exchange column-chromatography using an eluent comprising potassium
or caesium ions, [0013] (b) recovery of the solute from the eluted
solution, [0014] (c) diafiltration of an aqueous solution of the
recovered solute, [0015] (d) addition of a sodium salt and
diafiltration of the resultant solution, and [0016] (e) recovery of
GM1.
[0017] Advantageously the process of the present invention allows
for the preparation of the monoganglioside GM1 at high purity.
According to the present invention the monosialoganglioside GM1 may
be prepared with a purity of higher than 98%, even higher than
99.0% and even 99.9%.
[0018] Further, the process of the present invention advantageously
uses simple steps, is cost-efficient and is suitable for
application on an industrial scale.
[0019] Other objects and advantages of the present invention will
be apparent from the claims and from the following detailed
description and examples.
DETAILED DESCRIPTION
[0020] The present invention provides a process for the
purification of the monoganglioside GM1, wherein GM1 is separated
from a lipidic mixture containing the monosialoganglioside GM1 as
the main ganglioside component by ion exchange
column-chromatography using an eluent comprising potassium or
caesium ions.
[0021] In a preferred embodiment, there is provided a process for
preparing monosialoganglioside GM1 at high purity comprising the
steps of; [0022] (a) separation of GM1 from a lipidic mixture
containing the monosialoganglioside GM1 as the main ganglioside
component by ion exchange column-chromatography, using an eluent
comprising potassium or caesium ions. [0023] (b) recovery of the
solute from the eluted solution of step (a), [0024] (c)
diafiltration of an aqueous solution of the recovered solute of
step (b), in order to eliminate residual potassium or caesium
salts, [0025] (d) addition of sodium ions, preferably in the form
of a suitable sodium salt, in order to displace the potassium or
caesium ions bound to GM1, and diafiltration of the aqueous
solution, in order to eliminate residual sodium salt, and [0026]
(e) recovery of GM1, in the form of its sodium salt.
[0027] The lipidic mixture may be prepared from crude lipid extract
of bovine, ovine, equine or porcine cerebral tissue.
[0028] Advantageously, the lipidic mixture containing the
monoganglioside GM1 as the prevailing ganglioside component may be
prepared from a lipid extract containing at least 30%, preferably
at least 50% and more preferably at least 70% of a mixture of
gangliosides. The remainder of the lipid extract may generally be
composed of sulfatides, cerebrosides, fatty acids and proteins.
[0029] The lipid extract may advantageously be first subjected to a
diafiltration through a membrane having pore size of 10000 to
100000 Daltons, preferably about 50000 Daltons, in order to desalt
the solution. For the diafiltration any conventional dialysis
membrane may be used. Advantageously filter cassettes, e.g. of the
SARTOCON.RTM. (Sartorius) polysulfone cassettes type may be used,
for example with a cut-off of about 50000 Daltons.
[0030] Preferably the lipid extract is subject to treatment by
hydrolysis to transform other major ganglioside components, such as
GT1b, GD1a, and GD1b, into GM1 in order to increase the GM1
content.
[0031] The hydrolysis may be carried out using conventional
methods. Advantageously the hydrolysis may be carried out using
either of two general methods for hydrolysis of gangliosides known
in the literature, namely acid hydrolysis or enzymatic
hydrolysis.
[0032] Acid hydrolysis may be carried out, for example, using
dilute mineral acid, such as dilute hydrochloric acid, sulphuric
acid and nitric acid. The acid hydrolysis may be effected by
adjusting the pH of an aqueous solution of the lipid extract to a
pH between 3.5 and 5, preferably around pH 4.0, and heating the
solution to a temperature preferably between 90.degree. C. and
100.degree. C. for the time necessary to complete the conversion of
the other major ganglioside components to GM1. The time needed to
hydrolyze the major gangliosides to GM1 depends on the pH and
temperature chosen. In general, the higher the pH the longer the
time required to complete the hydrolysis, and the higher the
reaction temperature the shorter the time required to complete the
hydrolysis. The hydrolysis reaction may generally be carried out
over 2 to 5 hours. For example, where the hydrolysis is carried out
at pH 4.0 and 95.degree. C., the time required to complete the
hydrolysis reaction is about 3 hours.
[0033] Enzymatic hydrolysis may be carried out using any suitable
sialidase. Preferably Arthrobacter ureafaciens sialidase strain S
or Vibrio cholerae sialidase may be used. Advantageously,
Arthrobacter ureafaciens sialidase strain S and Vibrio cholerae
sialidase are active on GT1a, GD1a, GD1b but not on GM1. Enzymatic
hydrolysis may be carried out, for example, by adjusting the pH of
an aqueous solution of the lipid extract to a pH at which the
enzyme used has its optimum activity, for instance between pH 4 and
pH 6, for example about pH 5, by adding a suitable buffer, such as
acetate buffer, adding Ca2+ ions in the case that the sialidase is
a Vibrio cholerae sialidase, and heating the solution at a
temperature at which the enzyme used has its optimum activity, for
example around 37.degree. C., for the time need to complete the
transformation. The hydrolysis may generally be carried out over
12-24 hours, dependant on the enzyme units added.
[0034] Acid hydrolysis is less preferred as it is a non-specific
hydrolysis process and generally provides a lower yield in GM1 due
to conversion to other gangliosides. Further the acid hydrolysis
process leads to the formation of asialo-GM1 impurity.
[0035] The enzymatic hydrolysis methods are preferred as they
generally provide a higher yield in GM1 due to the high specificity
of the chemical transformation. For the enzymatic hydrolysis,
Arthrobacter ureafaciens sialidase is preferred as it does not
require the addition of Ca2+ ions for its activity. Further, due to
its molecular weight of 52,000 Daltons (compared to 82,000 Daltons
for Vibrio cholerae sialidase), it may advantageously be washed out
by diafiltration.
[0036] In order to recover the thus produced lipidic mixture having
an enhanced content of the monosialoganglioside GM1 from the
reaction solution, the reaction solution may advantageously be
diafiltered, e.g. through a membrane having pore size of 10000 to
100000 Daltons, preferably about 50000 Daltons. For the
diafiltration any conventional dialysis membrane may be used.
Advantageously filter cassettes, such as of the SARTOCON.RTM.
(Sartorius) polysulfone cassettes type, may be used, preferably
with a cut-off of 50000 Daltons. The permeate may then be dried to
obtain a powder of the lipidic mixture containing the
monosialoganglioside GM1 as the main ganglioside component. Drying
may be carried out by conventional methods. Advantageously the
drying may be carried out by spray drying or vacuum drying.
[0037] The lipidic mixture may generally have a concentration of
GM1 of 10 to 200 g/lt and preferably of at least 100 g/lt.
[0038] According to the process of the present invention, the
ganglioside GM1 is separated from other gangliosides in the lipidic
mixture using ion-exchange chromatography.
[0039] It has surprisingly been found that where an eluent
containing caesium or potassium ions is used it is possible to
successfully separate out GM1 from other monosialogangliosides.
[0040] Conventionally used ion exchange techniques have generally
been found not to allow effective separation of GM1 from other
monosialogangliosides. Of particular note is the
monosialoganglioside Fucosyl-GM1, which is present as a major
ganglioside impurity in the porcine lipidic mixture produced by the
known hydrolysis treatments.
[0041] The two molecules GM1 and Fucosyl-GM1 have very similar
physical properties. Both have a single negative charge, provided
by the carboxyl group of the sialic acid, and have similar
molecular weights; 1558 and 1704 respectively. Accordingly, when
loaded onto the pre-equilibrated ion-exchanger column, the binding
strength of the two gangliosides with the resin is the same.
[0042] It has been observed that when sodium acetate is added to
the eluent in order to increase the ionic strength of the eluent,
and provide the conditions for displacement of the gangliosides,
both of GM1 and Fucosyl-GM1 are eluted at the same time with the
same ionic strength. No separation of the two monosialogangliosides
can be achieved.
[0043] Whereas present inventors have surprisingly found that where
cesium or potassium ions are used, for instance by the addition of
methanolic potassium or cesium acetate, GM1 and Fucosyl-GM1 are
eluted separately, with Fucosyl-GM1 being eluted first. The
separation is complete, and each of the gangliosides GM1 and
Fucosyl-GM1 can be isolated.
[0044] Whilst not wishing to be bound by any theory, it is
considered by the inventors of the present invention, that the
observed separation may be attributed to the fact that, contrary to
the conventional ion-exchange theory, the solutes GM1 and
Fucosyl-GM1 are not only released from the column in order of their
strength in binding with the resin, but also in order of their
affinity for the counter-ion to which they must bind in order to be
detached from the gel. Accordingly, following this theory, if one
of the two solutes has a different affinity for the counter-ion,
then the solutes will be released with two different ionic
strengths, and purification can occur.
[0045] It has surprisingly been found by the present inventors that
the monosialogangliosides GM1 and Fuc-GM1 have the same affinity
for sodium but do not have not the same affinity for potassium or
caesium, despite the fact that all three metals belong to the same
group. It has been found by the present inventors that Fuc-GM1 has
a higher affinity for potassium and caesium than GM1, and is eluted
first.
[0046] The ion-exchange chromatography method of the present
invention advantageously enables effective separation of GM1 from
Fucosyl-GM1. Further, the method of the present invention
advantageously also allows the separation of GM1 from corresponding
fatty acids. The fatty acids having the same charge as the
ganglioside, but a higher affinity for caesium or potassium
ions.
[0047] For the ion exchange chromatography, any suitable resin may
be used. Advantageously a resin having a quaternary amino group may
be used, for example, FRACTOGEL.RTM. EMD TMAE (S) or Sepharose
resins e.g. Q-Sepharose HP resins.
[0048] In a first stage, the resin is equilibrated with a suitable
solvent. Suitable solvents include ethanol, methanol or a mixture
of methanol and chloroform. Preferably the solvent is methanol,
because it is a solvent in which gangliosides and potassium and
caesium salts are soluble.
[0049] The column may then be loaded with a solution of the lipidic
mixture in a suitable elution solvent. The elution solvent should
contain the same solvent components as the solvent used for
equilibration of the resin. Preferably the solvent chosen is
methanol. Preliminary elution with the elution solvent enables
elution of unbound substances, e.g. cerebrosides.
[0050] Potassium or cesium ions are then added to the elution
solvent. The potassium or cesium ions are preferably provided in
the form of a methanolic solution of potassium or cesium acetate,
formate, proprionate or as a salt of other organic acid. A
methanolic solution of sodium or potassium acetate is preferred.
Advantageously potassium or cesium acetate may be present in the
eluent in an amount sufficient to impair a conductivity of
1100-1400 .mu.S/cm, preferably a conductivity of 1200-1300 .mu.S/cm
on the eluent. This sodium or potassium salt containing eluent
solution may be passed through the column isocratically at any
suitable flow rate, for instance at a flow rate between 100 ml/h to
140 ml/h.
[0051] Where the separation is carried out using the ion exchange
chromatography process according to the present invention, fatty
acids and Fuc-GM1 are eluted before GM1, whilst sulfatides remain
bound to the column may be eluted during column washing.
[0052] The GM1 containing eluate is collected and, advantageously,
the elution solvent eluted from the column may be recovered by
distillation.
[0053] The GM1 containing solute may be recovered by drying the
eluate solution to produce a powder containing the GM1. Drying may
be carried out using conventional methods, for instance
spray-drying or vacuum drying.
[0054] The GM1 containing solute may then be purified by
diafiltration of an aqueous solution thereof through a membrane
having pore size of 10000 to 100000 Daltons, preferably about 50000
Daltons, in order to eliminate residual potassium or caesium salts.
Optionally, a mineral acid, such as aqueous sulphuric acid, nitric
acid, or preferably hydrochloric acid, may be added to the solution
to adjust to a pH between pH 6 to 8, preferably about pH 7 before
diafiltration.
[0055] Sodium ions, suitably in the form of an aqueous solution of
a sodium salt, preferably NaCl, may then be added to the solution,
in order to displace the potassium or caesium ions linked to GM1,
and obtain GM1 in the form of the physiological sodium salt. The
solution may then be subject to a second diafiltration in order to
eliminate residual salt (e.g. NaCl), using a membrane having pore
size of 10000 to 100000 Daltons, preferably about 50000 Daltons.
Advantageously filter cassettes, such as cassettes of the
SARTOCON.RTM. (Sartorius) polysulfone type, may be used, preferably
with a cut-off of 50000 Daltons.
[0056] The solution may then be dried to recover the GM1 in the
form of a powder. Drying may be carried out by conventional
methods. Advantageously the drying may be carried out by spray
drying or vacuum drying.
[0057] GM1 obtained according to the present invention has a degree
of purity of 98% or more, generally of about 99.0 to 99.9%. The GM1
obtained according to the process of the present invention contains
less than 0.1% Fucosyl-GM1 impurity.
[0058] The process of the present invention advantageously enables
efficient separation of GM1 from other monosialogangliosides.
Particularly, the process of the present invention allows the
separation of GM1 from Fucosyl-GM1 impurity.
[0059] Advantageously the process of the present invention allows
the preparation of the monosialoganglioside GM1 with a good yield
and a high level of purity. Accordingly, the invention includes the
purified monosialoganglioside GM1 in a pharmaceutically acceptable
carrier, which pharmaceutical composition is prepared by techniques
known to those skilled in the art. In one embodiment, the
composition is sterile.
[0060] The purified GM1 according to the process of the present
invention may be used in the treatment of human, mammal or animal
subjects. Particularly, the purified GM1 according to the present
invention is envisaged for the treatment of humans or mammals,
particularly for the repair and treatment of disorders and diseases
of the central and peripheral nervous systems, including cerebral
stroke, Parkinson's disease, spinal cord injury, Alzheimer's
disease, Tardive Dyskenisia, Amyotrophic Lateral Schlerosis,
peripheral neuropathies and autonomic neuropathy. Preferably, a
therapeutically effective amount of a pharmaceutical composition
comprising the purified monosialoganglioside GM1 in a
pharmaceutically acceptable carrier is administered to a patient,
i.e., a human, mammal or animal in need of treatment.
[0061] The present invention is further illustrated by the
following examples.
EXAMPLES
Example 1
Preparation of A Lipidic Mixture Containing the
Monosialoganglioside GM1 As The Principal Ganglioside
[0062] Lipid extract containing a mixture of gangliosides having a
purity of 70% is dissolved in purified water at a concentration of
about 25 g/l. This solution is then diafiltered through
SARTOCON.RTM. (Sartorius) polysulfone filter cassettes having a
cut-off of 50 000 Daltons.
[0063] 200 l of the solution are then equilibrated to pH 5.5 by
addition of 50 mM acetate buffer and 4 mM of Calcium chloride.
30000 U of Vibrio cholerae sialidase are added and the solution
heated to 37.degree. C. for 12 h to complete transformation of
other major gangliosides (GT1b, GD1a, GD1b) to GM1. The resulting
solution has a GM1 concentration of 14-15 g/l.
[0064] After the enzymatic hydrolysis, the solution is diafiltered
through filter cassettes having a cut-off of 50 000 Daltons. 1 M
NaCl is then added to the retentate and the solution subjected to a
second diafiltration. After the second diafiltration the retentate
is concentrated to a concentration of GM1 of 100 g/l by letting the
permeate flow without any water replacement. Then, the solution is
dried under vacuum to obtain about 3200 g of a powder having a GM1
concentration of 85% measured by HPLC.
Purification of GM1 From Lipidic Mixture Containing the
Monosialoganglioside GM1 As the Principal Ganglioside
[0065] A methanolic solution at a concentration of 10 g/l is
prepared using the powder obtained in the previous step, and the
solution is filtered through a 0.22 .mu.m Sartopore cartridge
filter (manufactured by Sartorius AG).
[0066] For each cycle, 7 litres of the filtered solution are then
loaded on an FPLC column containing 20 l of Fractogel.RTM. EMD TMAE
(S) resin equilibrated in methanol. The column is then eluted with
methanol: potassium acetate methanolic solution having a
conductivity of 1200-1300 .mu.S/cm, at a flow rate of 120 l/h. The
cycles repeated until the end of the GM1 solution.
[0067] The eluate (about 60-70 l) is continuously concentrated by
distillation, and is then dried to obtain a powder, and methanol is
recovered. The thus-obtained powder is a mixture of pure GM1 and
potassium acetate.
[0068] The thus-obtained powder is dissolved in purified water to a
concentration of 25g/l, and equilibrated to pH 7 by the addition of
18% HCl. The solution is diafiltered through filter cassettes
having a cut-off of 50,000 Daltons. 1M NaCl is then added and the
solution is again diafiltered through filter cassettes having a
cut-off of 50,000 Daltons. After this second diafiltration the
retentate is concentrated up to 100-120 g/l by letting the permeate
flow without any water replacement.
[0069] The concentrated solution is then filtered through a 0.22
.mu.m filter and dried by spray drying to provide about 2700 g of a
white to white-beige powder of GM1, identified by TLC, this GM1
powder having a purity of 99.8% measured by HPLC. The resultant GM1
powder has a Fuc-GM1 content of less than 0.1%, measured by
HPLC.
Comparative Example
[0070] The process for preparing and purifying GM1 was carried out
as in Example 1 above, except that in the column chromatography,
the methanol: potassium acetate methanolic solution was replaced by
a methanol: sodium acetate methanolic solution.
[0071] 3100 g of a powder of GM1 was obtained containing 91% GM1
and 8% Fuc-GM1, measured by HPLC.
[0072] From the above examples it can be seen that a much lower
purity of GM1 is obtained where sodium acetate is used for the
elution of GM1. This may be attributed to the fact that the sodium
counter-ion does not make any difference between GM1 and Fuc-GM1 in
the elution process, compared with potassium or caesium
counter-ions, which on the contrary completely separate both
peaks.
[0073] All publications, patents, and patent applications
identified above are incorporated herein by reference in their
entirety.
[0074] Although this invention has been described in relation to
certain preferred embodiments thereof, and many details have been
set forth for purposes of illustration, it will be apparent to
those skilled in the art that the invention is susceptible to
additional embodiments and that certain of the details described
herein may be varied considerably without departing from the basic
principles of the invention.
* * * * *