PROCESS FOR PURIFYING 1,4,7,10-TETRAAZACYCLODODECANE-1,4,7,10-TETRAACETIC
ACID
Description
Technical field
[0001] The present invention relates to a process for producing and purifying
1,4,7, 0-tetraazacyclododecane-1 ,4,7, 0-tetraacetic acid (DOTA)
including salts and hydrates thereof by using nanofiltration techniques.
The obtained DOTA is highly purified and thus suitable for being used to
produce contrast agents for magnetic resonance imaging. Therefore, the
present invention also relates to a process for obtaining metal ion
complexes thereof and to a process for obtaining pharmaceutical
compositions comprising the metal ion complexes of DOTA.
Background Art
[0002] Magnetic resonance imaging (MRI) is a powerful, non-invasive technique
used to produce detailed two or three-dimensional anatomical images of
tissues in the body. Conventional MRI uses the proton H as its signal
source which is highly abundant in tissues and it has the highest sensitivity
of all the biologically relevant nuclei.
[0003] Contrast, which makes the differentiation of internal structures possible in
the image, arises from how the signal decays and is the difference
between the resulting signals from two tissue regions. The route by which
the protons release the energy they absorbed from the radio-frequency
pulse, thus reducing the transverse magnetisation and causing signal
decay, is known as relaxation. In MRI two independent relaxation
processes occur simultaneously: spin-lattice or longitudinal relaxation
characterised by the time constant 7 , and spin-spin or transverse
relaxation, characterised by the time constant G2.
[0004] Often, when suitable 7 - or ^-weighting sequences are used, the natural
contrast between two tissues is enough to produce a diagnostically-useful
image. However, some conditions do not lead to specific enough changes
in the relaxation times of the affected tissue though and then a contrast
agent is used to locally change the relaxation times of the diseased tissue,
improving the image contrast.
[0005] Most contrast agents work by shortening the relaxation times of the water
protons in the targeted tissue. 7 contrast agents are based on
paramagnetic metal ion chelates which make the tissue appear brighter on
the G -weighted image (positive contrast). T contrast agents are usually
super-paramagnetic iron oxide nanoparticles which create dark spots on
the 72-weighted image (negative contrast). 7 agents are the most widely
used and the majority of these are based on chelates of the gadolinium ion
(Gd3+) .
[0006] To be an effective 7 agent the gadolinium (III) chelate must significantly
increase the proton relaxation rates in water. Gadolinium is the seventh
element in the lanthanide series and, like the other lanthanide elements, it
is most commonly found in the +3 oxidation state, corresponding to the
electronic configuration [Xe]4f 7. This means that Gd3+ has seven unpaired
electrons, making it highly paramagnetic i.e. Gd(lll) ions have large
permanent magnetic moments (due to electron spin angular momentum),
but in the absence of an external magnetic field these are randomly
oriented. Due to its large size, the Gd(lll) ion typically has a coordination
number of nine in its complexes. As free ion, gadolinium is very toxic for
the tissues but is generally regarded as safe when administrated as a
chelated compound.
[0007] The level of toxicity depends on the strength of the chelating agent, also
known as ligand, chelator or sequestering agent.
[0008] Usually these ligands are organic compounds which form two or more
separate coordinate bonds with a single central metal ion, in this case, the
gadolinium ion, inactivating it and thus reducing or eliminating its toxic
effect in the tissues.
[0009] Polyaminopolycarboxylic acid compounds are the ligand type of choice
because they form exceptionally stable complexes with the Gd(lll) ion,
which can be explained by a number of reasons. These compounds can
be linear (such as pentetic acid or diethylene triamine pentaacetic acid
also named as DTPA) or macrocyclic (such as ,4,7,10-
tetraazacyclododecane-1 ,4,7,1 0-tetraacetic acid, DOTA). DOTA is used as
the ligand in the synthesis of the MRI contrast agent gadoterate
meglumine ([Gd(DOTA)(H 2O)](meglumine)).
[0010] Several synthetic routes for the production of DOTA have been proposed,
namely by Stetter, Hermann; Wolfram Frank (1976)- "Complex Formation
with Tetraazacycloalkane-N,N',N",N'";-tetraacetic Acids as a Function of
Ring Size". Angewandte Chemie International Edition in English ( 1 1):
686), by R. Delgado & J.J. Frausto da Silva - Talanta, Vol. 29, pp. 815-
822, Issue 10, 1982, and by J.F. Desreux - Inorg. Chem. 1980, 19, pp.
1319-1324.
[001 ] The preparation of DOTA was first reported in 976 by Stetter & Frank (full
ref. above) through the reaction of 1,4,7,10-tetraazacyclododecane with
chloroacetic acid in aqueous alkali medium to obtain DOTA wherein the
resulting inorganic salts were separated and purified by treatment with an
ion-exchange column Dowex 2x8.
[0012] The method most widely reported in the literature is typified by Delgado et
al. (full ref. above), where cyclen is reacted with chloroacetic acid under
aqueous basic conditions (pH = -10) to form DOTA, which is crystallised
by acidifying the cooled DOTA solution to pH 2 with hydrochloric acid and
placing it in the refrigerator overnight.
[00 3] Desreux (full ref. above) also reported a similar procedure, but specified
sodium hydroxide as being the base used, with a reaction temperature of
80°C, and stated that upon acidification DOTA precipitates out of solution
at pH 2.5.
[0014] E. Clarke & A. Martel (1991) - Inorganica Chimica Acta, 190, pp 27-36),
describes the preparation of DOTA by alkylation of cyclic tetraamine
ligands with bromoacetic acid at a controlled pH between 1.2 to .3
being the resulting product recovered by treatment with a ion-exchange
column as ammonium salts followed by treatment with a potassium cation
solution at pH of 11.5 and vacuum concentration. The resulting ligands
were then reprotonated by addition of HCI and isolated by recrystallization
from hot water. Dota is obtained as a mixture of 1/ .1 mole/mole with KCI.
[0015] WO9905128 discloses a process for producing DOTA compounds by 2
step-alkylation wherein the alkylation agent is preferably bromoacetic acid
but also includes chloroacetic acid, in aqueous solution at a basic pH with
an excess of said alkylation agent, followed by hydrolysis and purification
with ion exchange resins and with an optional recrystallization step in
order to obtain highly purified DOTA compounds. In particular,
WO9905128 discloses a multistep process for the preparation of DOTA
starting from:
a) an alkylation reaction of a 2a,4a,6a,8a-decahydrotetraazacyclo
pentacenaphthylene with an acid in aqueous solution and at a basic pH,
followed by
b) a second alkylation reaction with a different alkylating agent, and by
c) the hydrolysis of any ester groups, and wherein the amount of the first
alkylating agent used in step a) varies between 2 - 2.3 mol of reagent per
mol of substrate and from 2 - 3 mol in step b) and the reaction
temperature varies from room temperature to 80°C, depending on the
reactivity of the alkylating agent.
[0016] To be able to be eventually used as a suitable contrast agent comprising
gadoterate meglumine, the concentrations of process impurities present in
the raw DOTA (both organic and the inorganic) must be removed or
significantly reduced. This is so that the purified DOTA meets the strict
specifications for use in a contrast agent or else it will not be approved for
sale by the relevant medicine regulatory body as it will not be considered
safe enough for human use. Therefore a series of purification steps must
be employed to remove these impurities without introducing too high a
concentration of a new impurity or residual solvent, as these must also
meet the specifications.
[0017] However, the crude DOTA resulting from the above mentioned processes
is still highly contaminated with organic and inorganic impurities, in
particular with chloride and sodium ions, and the conventional purification
steps using ion-exchange resins, as disclosed above, only solves this
problem in some extent.
[0018] In fact, G. Hernandez, M.F. Tweedle and R.G. Bryant, Inorg. Chem., 1990,
29, 5109-51 13, disclose the synthesis of the sodium salt of
[Gd(DOTA)(H 2O)]- (Na[Gd(DOTA)(H 2O)].4H2O). However, this compound
is unsuitable for use as a contrast agent as it contains sodium.
Nevertheless, the synthetic procedure herein disclosed highlights that high
temperatures (90°C) and long reaction times (6.5 h) are required to
successfully react DOTA and gadolinium oxide (Gd203, an ionic salt which
is the source of the gadolinium ion) together to form the
thermodynamically stable [Gd(DOTA)(H20)]-. This can be accounted for by
the very slow kinetics of formation of the complex.
[0019] Purification of DOTA by using resins has been described in the patent
application EP1 3 152873.9. In this document a process is disclosed that
uses resins combined with specific washes allowing obtaining good yields
of purified DOTA. However, said process is time consuming and uses
chemicals and other consumables, such as resins, ammonia, formic acid
and produces a large volume of waste solvents which have to be removed
in later stages of the process making this process expensive and difficult
to be automated due to the several sequential steps.
[0020] WO20 13/76743 discloses a process for purifying polyaminocarboxylate
compounds without using ion-exchange resins by isolating them under
very acidic conditions and purification of the obtained salts by
recrystallization with water or water solvent mixtures. However, despite the
good quality of the obtained product the yields are rather low.
[0021] Nanofiltration is used to remove monovalent ions from higher valent ions
and from higher molecular weight organic compounds (see Van der
Bruggen, B. and Geens, J. (2008) - Nanofiltration, in Advanced Membrane
Technology and Applications (eds N. N. Li, A. G. Fane, W. S. W. Ho and
T. Matsuura), John Wiley & Sons, Inc., Hoboken, NJ, USA.
doi: 10.1002/9780470276280.ch1 1).
[0022] WO201 1054480 shows how Gadobutrol can be prepared by reacting the
ligand with Gadolinium salts and removing the counterions by
nanofiltration, using a ceramic membrane with a MWCO of 200. This
technique is only suitable for purifying the complex and does not provide a
solution for DOTA. This method is however not suitable for removing high
molecular weight compounds which were formed during the making of the
Butrol ligand.
[0023] In the Journal of Membrane Science 279 (2006) 446 - 452, A. Sorin
describes how polyaminocarboxylic acids such as DOTA can be rejected
by charged nanofiltration membranes. Depending on the pH, the
compound is retained, however as a cation or anion. The anionic or cation
form of DOTA is not suitable for to be used in contrast agents. In neutral
form it is not rejected by the membrane having a MWCO estimated at
2500 and hence this process is not suitable for removing said compounds
from ionic contaminants.
[0024] It is thus desirable to obtain an optimized and efficient process for the
purification of crude DOTA which ensures not only high yields of this
compound, preferably at least 50% relative to the amounts of the starting
reagents used, but also ensures a DOTA of a suitable purity to be used in
the preparation of contrast agents and in a form that was easy to work
with.
[0025] The present invention discloses a process for purifying DOTA including
salts and hydrates thereof by using nanofiltration techniques allowing
obtaining purified DOTA with high yields and in highly purified form.
Summary of invention
[0026] It is an object of the present invention to provide a method for purifying
DOTA, represented by the general formula (I), including salts and hydrates
thereof, for obtaining high yields and a high purity in a simple,
straightforward and reliable process.
Formula 1
[0027] This object is realised by providing a process for purifying DOTA
compounds as defined in claim .
[0028] It is another object of the invention to provide a process for purifying DOTA
without the need for a pH adaptation of the solution to be in the pH range
as specified by the manufacturer of the nanofiltration membrane as
defined in claim 7.
[0029] It is another object of the invention to provide a process for producing
gadolinium complexes comprising DOTA, as defined in claim 0.
[0030] Further advantages and embodiments of the present invention will become
apparent from the following description and the dependent claims.
Brief description of drawings
[0031] Fig. 1: The set-up of the diafiltration process.
(1) Feed tank
(2) Cross-flow cell with nanofiltration membrane
(3) Feed pump
(4) Permeate stream
(5) Diafiltration buffer to keep the volume in the feed tank constant by
adding water or a raw DOTA solution at a flow equal to the permeate flow.
(6)The retentate is recycled into the feed tank
Description of embodiments
[0032] The present invention relates to a process for purifying the compound of
formula , including salts and hydrates thereof, hereafter called DOTA.
The steps of this process are described in §B to C. In §A, preparation
methods of DOTA are described.
A. Preparing DOTA
[0033] There are several ways to prepare DOTA which can be purified according
to the process of the present invention in a preferred embodiment, DOTA
can be produced by reaction of cyclen ( 1,4,7,10-tetra-azacyclododecane)
with a haloacetic acid in basic conditions, at a pH > 10, by addition of a
base. This base (in solid form or as a concentrated solution of preferably
at least 25(wt.)%) is added slowly to the reaction mass by maintaining
preferably the internal temperature at 0°C. An excess of the haloacetic
acid can be used in this step, preferably in an amount of at least 4
equivalents and more preferably between 5 to 6 equivalents with regard to
the initial amount of the cyclen. In this reaction one equivalent
corresponds to one mole per mole ratio. According to the cited prior art,
this step is normally performed at a temperature of approximately 80°C.
However, it was found that by using an excess of haloacetic acid
according to the present invention, lower temperatures may be used.
Therefore, in the scope of the present invention, this step can be
performed at a temperature ranging from 0 to 100°C, preferably from 0 to
65°C and more preferably from 0 to 30°C. In a preferred embodiment, the
haloacetic acid is chloroacetic acid, bromoacetic acid or iodoacetic acid
and more preferably chloroacetic acid.
[0034] As in the prior art, the reaction can be performed at pH values of about 10.
It was surprisingly found that if the reaction was performed at a pH > 13 no
adverse effects were observed and at this pH, the pH has not to be
constantly monitored as described in the prior art. Hence the synthesis
procedure is easier to operate. Therefore, in a preferred embodiment of
the present invention, this step of the DOTA synthesis is performed at a
pH > 13 by addition of the base at the start of the reaction. The base can
be added in excess, of at least 2 times the amount of the haloacetic acid
present in the reaction, namely by using amounts ranging from 8 to 16
equivalents, preferably between 10-12 equivalents with relation to the
cyclene. In the scope of the present invention, preferably a strong base is
used. With a strong base is meant a basic chemical compound that
deprotonates very weak acids in an acid-base reaction and is commonly
recognized as a conjugated base of an acid with pKa of at least 13.
Suitable examples are alkali metal hydroxides, such as KOH, NaOH,
LiOH, RbOH or CsOH and organic non-nucleophilic bases such as
1, 1 ,3,3-tetramethylguanidine. In a most preferred embodiment of the
present invention, NaOH or LiOH is used as the base.
[0035] The reaction mass is then slowly warmed up to 25°C and stirred for at
least 7h, most preferably at least 20h, even more preferably at least 24h.
[0036] Other synthetic routes for preparing DOTA which can be purified by the
process of the present invention, are disclosed in "Complex Formation
with Tetraazacycloalkane-N,N ,,N",N'";-tetraacetic Acids as a Function of
Ring Size", by Stetter, Hermann; Wolfram Frank (1976), Angewandte
Chemie International Edition in English 15 ( ) : 686), Talanta, Vol. 29, pp.
815-822, Issue 10, 1982, by R. Delgado & J.J. Frausto da Silva, Inorg.
Chem. 1980, 19, pp. 1319-1324 by J.F. Desreux , WO9905128 and in
Example 1 of US2014323719.
B. Precipitation and washing of DOTA
[0037] The purification process according to the present invention starts with
preparing an aqueous solution of the DOTA which has to be purified. The
reaction mixture as obtained in §A is such a suitable aqueous solution of
DOTA. The removal of impurities and ions from the aqueous solution of
DOTA by means of filtration over a nanofiltration membrane has been
found not to be successful. The concentration of the ions in the obtained
retentate is much too high in order to be used for the production of
contrast agents. Besides this problem, the yield of DOTA is very low due
to the insufficient retention of the DOTA. After extensive research, it has
been found that prior to the filtration over a nanofiltration membrane,
additional steps including precipitation, filtration and washing the
precipitate are required.
[0038] The precipitation of DOTA is performed by addition of an acid to the
reaction mixture until a pH < 3 is achieved. The obtained mixture of
solution and precipitate is called hereafter slurry. Preferably an inorganic
acid is used to achieve a pH < 3. Suitable inorganic acids are HCI, H2SO4,
HNO3, HBr, HI or HCIO4. Besides inorganic acids, organic acids such as
p-toluenesulfonic acid and methanesulfonic acid may also be used.
[0039] The slurry may be subjected to a heating step and cooling step to obtain a
more compact, more crystalline precipitate that is easier to wash. Hence
an improved yield and purity of the precipitated DOTA are obtained. This is
performed by heating the slurry at a temperature ranging from 50 to
100°C, preferably 50 to 70°C, more preferably 50 to 60°C, for a time period
of at least 5 minutes, in order to dissolve the precipitate and obtain a clear
solution. Then the solution is cooled at a temperature ranging from 5 to
25°C, preferably 5 to 15°C, more preferably 5 to 10°C, for a time period of
at least 5 minutes, to obtain DOTA in the form of a salt depending on the
strong inorganic acid selected for lowering the pH of the solution, such as
DOTA hydrochloride or other salt.
[0040] The final pH of the slurry is < 3 and can be even less than 0.5. At this low
pH values the precipitated DOTA is found in its fully protonated form
(H2L)2+ or (H L)3+ wherein L refers to the ligand DOTA. DOTA in its fully
protonated form has counterions, such as chlorides, introduced by the
reaction with the acid, and which are electrostatically bound to it, so that its
form can be expressed as H2l_(X)2/n, wherein X refers to the counterions
and n refers to the charge of the counterion. In the case of chloride
counterions, the salts of DOTA are denoted hereafter as DOTA bis
hydrochloride or DOTA tris hydrochloride. Apart from the negative
counterions the precipitated DOTA is also contaminated with cations
introduced by the reaction with the base that precipitate out alongside with
the DOTA salt.
[0041] The slurry is filtered and dry sucked. Any filter can be used to filter the
slurry. Suitable examples are filter wool, made of polyethylene
terephthalate, polypropylene or nylon, synthetic sponges or foams, various
ceramics and sintered glass. The filter material is preferably stable at the
pH range of the slurry.
[0042] Following the filtration of the precipitate, a washing step is performed. This
can be done with a mixture of water and a water miscible low boiling
organic solvent in a weight ratio ranging from 1:1 .5 to 1:5, preferably in a
weight ratio ranging from 1:2 to 1:4. Water miscible low boiling organic
solvents are solvents having a boiling temperature preferably lower than
85°C. Suitable examples of water miscible low boiling organic solvents are
acetone, ethanol, methanol, iso-propanol, butanone, methyl acetate, ethyl
acetate, acetonitrile or THF. The type of water miscible low boiling organic
solvent and the ratio in the mixture of water and water miscible low boiling
organic solvent used in the washing step can be selected by optimisation
wherein the dissolution of the precipitate is minimized and the removal of
the contaminating cations and anions is maximised. In a preferred
embodiment of the present invention, acetone or ethanol is used as the
water miscible low boiling organic solvent.
[0043] In this way a DOTA can be obtained that has already a low cation content
(<0.5 (wt.)%), but which is still not pure enough to be a suitable reagent for
producing contrast agents. This DOTA is hereafter called raw DOTA.
C. pH adjustment and nanofiltration
[0044] The raw DOTA as obtained in §B is now purified by using nanofiltration
membranes. Therefore an aqueous solution of the raw DOTA has to be
prepared. A concentration range from 0.1 to 20 (wt.) % is suitable,
preferably from 1 to 5%. If the DOTA used is in a protonated form such as
DOTA hydrochloride, the obtained aqueous solution of the raw DOTA is
acidic. If the pH of the obtained aqueous solution is outside the pH range
specified by the manufacturer of the selected nanofiltration membrane, it
has to be adjusted to a value within this range. This pH range of a
nanofiltration membrane corresponds to the pH range in which the
manufacturer of the membrane guarantees substantially no deterioration
of the membrane during continuous operation. The values of said pH
range of the nanofiltration membrane are specified in the technical sheet
provided by the manufacturer.
[0045] The adjustment of the pH of the aqueous solution of the raw DOTA can be
performed by adding a base, an acid or by diluting the solution with water.
Any base is suitable to increase the pH of the aqueous solution of DOTA
in its protonated form such as e.g. DOTA bis hydrochloride. Limitation of
the concentration of the raw DOTA is also a possibility of achieving a pH of
the aqueous solution within the pH range of the nanofiltration membrane
as specified by the manufacturer. A limited concentration of DOTA in order
to avoid a pH value outside the range specified by the manufacturer of the
nanofiltration membrane has the disadvantage that the starting
concentration is reduced, which reduces the productivity of the filtration
process. To obtain satisfactory results in the nanofiltration process the
washing of the precipitate is preferably optimised so that a cation content
in the aqueous solution after making the solution of the raw DOTA and
after the pH adaptation, is preferably less than 100ppm, preferably less
than 60ppm, more preferably less than 30ppm in weight. This optimisation
is achieved by the selection of the water miscible low boiling organic
solvent, selection of an optimal ratio of water / water miscible low boiling
organic solvent and the number of the washing steps performed.
[0046] Raw DOTA, salts and hydrates thereof as obtained via the precipitation
and washing steps according to the present invention can now be purified
in a very efficient way by using nanofiltration membranes with specific
molecular weight cut-off (MWCO) thus removing the inorganic ions and
other impurities from the larger organic molecule DOTA. Experiments with
membranes having a molecular weight cut-off (MWCO) below the
molecular weight of the DOTA to be purified, show a retention of the
DOTA in the retentate whilst low molecular inorganic ions, such as
chloride, sodium, bromide, potassium, etc., are removed via the permeate.
Suitable nanofiltration membranes for the purification of the raw DOTA
solution are the ones with a MWCO in the range from 150 to 500,
preferably from 200 to 300, such as TFC SR100 and SeIRO MPS-34 from
Koch Membrane Systems, Inc. (USA) or TS40 from TriSep Corporation
(USA) or any other membranes with comparable MWCO.
[0047] Filtration by means of nanofiltration membranes can be done according to
different techniques. A preferable technique for the purification of raw
DOTA according to the invention is the diafiltration technique wherein the
solution of the raw DOTA is pumped from the feed tank tangentially along
the surface of the membrane (also called tangential flow filtration or crossflow
filtration). The retentate is then fed back to the feed tank. The design
of a preferable embodiment of the diafiltration is shown in Fig. . The
diafiltration process for purifying DOTA can be done in i) the concentration
mode (CM), ii) the constant volume mode (CVM) or iii) the variable volume
mode (WM). In the CM, no addition of liquid is done to the feed tank and
the concentration of the DOTA in the retentate increases. In the CVM, the
volume of the feed tank is kept constant mostly by adding water further
washing away the ions from the raw DOTA solution. The third mode
combines the 2 other modes to optimise the filtration process.
[0048] In a tangential flow filtration process, the filtrate flux (=permeate flow rate
normalized for the area of the membrane), is proportional to the pressure
difference over the membrane and is called the Trans Membrane Pressure
(TMP). The optimal TMP has to be determined for each membrane. The
optimum TMP is determined by changing the TMP (bar) at constant feed
and measuring the permeate flow (g/min). The optimum TMP is at the
"knee" of the curve where the filtrate flow increases with increasing TMP
up to a point where it levels off. Working outside the working pressure area
can have an irreversible effect, such as mechanical damage, on the
membrane behaviour. Suitable pressures in function of the membrane
properties used according to known methods, are in the range from 3 to 60
atm. Under these conditions a filtrate flow is created containing inorganic
ions and only a very small portion of DOTA is found in the permeate,
showing that a good yield is possible.
[0049] To monitor online the filtration process according to the invention, two
conductivity meters can be used. A conductivity measuring probe is set in
the retentate tank, which will represent a decrease in conductivity and
consequently a decrease in concentration of the ions. The second
conductivity measuring probe is placed on the permeate flow, where the
same decrease is monitored. If the conductivity is decreased to a desired
level, the filtration is stopped. Other online or even offline analytical
techniques can be used to monitor the level of ions and other impurities in
the retentate such as ion-selective electrodes and titration.
[0050] When filtering a raw DOTA-solution which is neutralized with a base, for
example NaOH or LiOH, in order to obtain a solution with a pH value
within the allowable pH range of the selected nanofiltration membrane
range as specified by the manufacturer, it is observed that the DOTA is
less well retained by the membrane and can be found in the permeate.
Although this effect is not completely understood, it is beneficial to keep
the concentration of the ions, more specifically the cations in the raw
DOTA solution as low as possible by limiting the addition of inorganic
bases. Preferably the concentration of the cations in the solution has to be
lower than 100ppm (wt.), preferably lower than 60ppm (wt), more
preferably lower than 30ppm (wt.). Achieving a pH value within the pH
range specified by the manufacturer of the nanofiltration membrane can
therefore be done by further dilution of the solution with water of the raw
DOTA or limiting the concentration of the raw DOTA, especially if the raw
DOTA is in its fully protonated form such as e.g. DOTA bis hydrochloride
and/or DOTA tris hydrochloride. The disadvantage of diluting is a lower
efficiency of the purification process because the concentration of the
DOTA has to be increased again later on, or by a longer diafiltration time
(preferably in the CM) or by the evaporation of water.
[0051] Preferably, the nanofiltration is performed by addition of water as
diafiltration buffer to the retentate and continuing the diafiltration keeping
the volume constant or even concentrating the solution. In a preferred
embodiment of the invention, the solution of the raw DOTA is diafiltered, in
a first phase with extra feed of a raw DOTA solution as diafiltration buffer
and in a second phase with pure water. As the pH of the retentate
increases during the diafiltration process of a raw DOTA solution with
DOTA in its fully protonated form such as DOTA bis hydrochloride and/or
DOTA tris hydrochloride, a more concentrated raw DOTA solution than the
raw DOTA solution at the start, can be added as diafiltration buffer, hence
increasing the efficiency of the whole filtration process. This increase in
efficiency of the filtration process can compensate the loss in efficiency
due to the dilution of the raw DOTA solution in order to achieve a pH value
compatible with the membrane. Alternatively, a solution of an electrolyte or
acid such as formic acid in water can be used prior to the diafiltration with
pure water to improve removal of one of the ions.
[0052] Although the filtration process over a nanofiltration membrane can be
performed at room temperature, a higher temperature is beneficial
because it increases the permeate flow, hence increasing the productivity
of the process. Preferably the temperature of the nanofiltration is between
10 and 90°C, more preferably between 20 and 70°C.
[0053] When the concentration of the ions and impurities has reached a required
level in the retentate, the filtration process is ended. In the obtained
solution of purified DOTA, the concentration of the DOTA can be
increased via diafiltration in CM or via evaporation of the water to a
concentration between 5 to 20 (wt.)%, more preferably between 5 to 15
(wt.)%. After increasing the concentration, the DOTA can be precipitated
by adding a water miscible low boiling organic solvent to the concentrated
solution. Water miscible low boiling organic solvents are solvents having a
boiling temperature preferably lower than 85°C. Suitable examples of
water miscible low boiling organic solvents are acetone, ethanol,
methanol, iso-propanol, butanone, methyl acetate, ethyl acetate,
acetonitrile or THF. Preferably acetone, ethanol, methanol or iso-propanol
is used as low boiling organic solvent. The addition of the low boiling
organic solvent to the DOTA solution can be done while stirring the
solution. Speed of the addition and temperature is not critical in obtaining
the precipitate. The precipitated DOTA can be filtered and washed with a
low boiling organic solvent and dried. The drying can take place in a
vacuum dryer or ventilation dryer.
D. Making of a Gadolinium complex.
[0054] The purified compound of formula 1 can be used to make a gadolinium
complex by adding a salt or oxide of gadolinium to an aqueous solution of
the purified compound of formula 1 so as to obtain complexation of the
gadolinium by the purified compound of formula .
[0055] The DOTA of high purity obtained as described above can be used as the
ligand in the formation of the contrast agent gadoterate meglumine,
[Gd(DOTA)(H2O)](meglumine). In a first step, the DOTA-Gd complex has
to be made.
[0056] For this purpose, a gadolinium compound, preferably Gd2O 3 is added to
an aqueous solution of purified DOTA obtained according to the process
of the present invention so as to obtain complexation of the gadolinium by
the purified DOTA. Preferably an excess of DOTA, most preferably in a
molar ratio slightly over 2:1 is used to form an aqueous solution of a
complex DOTA-Gd. The temperature of the reaction solution required to
form the complex is in the range from 80 to 120°C, preferably from 90 to
100°C, more preferably at a temperature of approximately 95°C. As the
kinetics of formation of the complex are very slow, the reaction typically
takes 2 to 8h, preferably from 3 to 6h, more preferably approximately 4h.
[0057] During this time the pH of the reaction solution typically decreases from ~3
to ~ .5-1 .6. In order to complex the Gd(lll) ion by the DOTA, the DOTA
must become fully deprotonated, which releases hydrogen ions into the
solution.
E. Making a pharmaceutical composition based on the contrast agent
gadoterate meglumine(2nd step).
[0058] After allowing the solution obtained in § D. to cool to between 40 and 50
°C, N-methyl-D-glucamine (meglumine) is added to balance the negative
charge of the complex. Meglumine is added until the pH of the solution is
between 6.9 - 7.8, to meet the pH range required to allow the solution to
be safely injected as contrast agent. Meglumine is used as an excipient in
many drugs and can even be present in the final solution in excess
because it can be well tolerated by the body. After stirring for about half an
hour, to ensure the reaction has gone to completion, the reaction solution
is allowed to cool to room temperature and is filtered.
[0059] The obtained permeate was analysed by combined liquid chromatography
with mass spectrometry (HPLC-MS) and was found to contain gadoterate
meglumine, showing that the quality of DOTA being synthesised and
purified according to the present invention can successfully be used to
synthesise a solution of the contrast agent. The DOTA-Gd complex can be
easily identified on the electron spray ionisation (ESI) mass spectrum from
the collection of peaks 1 m/z (particle mass(amu) per charge) value apart,
centred at m/z 560. There are a number of [M + H]+ peaks corresponding
to the dehydrated complex because gadolinium has six stable isotopes,
five (155Gd, 156Gd, 157Gd, 158Gd and 160Gd) of which all have relative
abundances greater than 14 %. Meglumine is also evident on the mass
spectrum with a [M + H]+ peak at m/z 196.
[0060] Specific embodiments will now be described in detail. The examples are
intended to be illustrative and the claims are not limited to the materials,
conditions or parameters set forth in the examples;
EXAMPLES
[0061] The following methods and materials are used in the examples described
below.
1. Methods
A. Purity and Assay by HPLC
[0062] The content of , 4 , 7 , 0-tetraaza-cyclododecane and DOTA was
determined by reversed phase HPLC (High Performance
Chromatography) with a gradient program and a DAD (Diode Array
Detection). The purity of DOTA is expressed as the ratio of the area of the
peaks, with regard to total peak area in %. The assay of DOTA is
expressed in (wt.)% and is measured by using a standard sample of
DOTA (obtained according to the International Conference on
Harmonisation (ICH guidelines)).
[0063] The chemicals and reagents used are:
• Acetonitrile: HPLC grade
• Water: HPLC grade or Milli-Q-water
• Orthophosphoric acid: HPLC grade
Potassium dihydrogen phosphate: AR grade
[0064] The apparatus used is an Agilent 1100/1200 series HPLC system with UV
DAD detector, or equivalent.
[0065] The chromatographic parameters were:
• Column: Prevail Organic Acid, (250 x 3.0)mm,5.0 miti
• Column Temperature: 30°C
Detector Wavelength: 195 nm
Pump Configuration: Gradient
Flow rate: 0.44mL/min
Injection Volume: 5m
Run Time: 40 min
• Mobile phase A: 20mM KH2PO4 in water at pH 2.5 using Diluent (see
below)
Mobile phase B: Acetonitrile : Mobile phase A (volume ratio of 60:40)
• Mobile phase C: Acetonitrile : Water (volume ratio of 60:40)
• Mobile phase D: Acetonitrile : Water (volume ratio of 90:10)
Diluent: 0.1 (wt.) % Orthophosphoric acid in water
[0066] The gradient used, is summarised in Table 1:
Table 1
[0067] Retention times were for DOTA: 4.6 min, for 1,4,7,1 0-tetraazacyclododecane:
2.7 min. and for chloroacetic acid: 6.9 min.
B. Determination of chloride content in DOTA by potentiometric titration.
[0068] The solution of the sample, obtained by dissolving 50 mg of solid DOTA in
a mixture of 20 ml water and 80 mL acetic acid, is titrated with 0.001M
AgNO3 using a Mettler DL 25 potentiometric auto titrator. The chloride
content is expressed as ppm (wt.) with respect to the weight of DOTA,
unless otherwise specified.
C. Determination of sodium and lithium content in DOTA by ICP-OES
(Inductively Coupled Plasma - Optical Emission Spectrophotometry).
[0069] The sodium and lithium content is expressed as ppm (wt.) or as (wt.)%
with respect to the weight of DOTA. The lithium content in the aqueous
solution of the raw DOTA is also determined by means of ICP-OES.
D. Determination of sodium content by measurement with an Ion Selective
Electrode.
[0070] The sodium content in the aqueous solution of the raw DOTA is measured
with a Metrohm 781 pH/ion meter connected to a sodium glass electrode
and an Ag/AgCI reference-electrode. The sample preparation is performed
as prescribed in the Manual Ion-selective electrodes (ISE), Metrohm AG ,
Herisau, Switzerland, 2010. The results are given in ppm (wt.) or as (wt.)
%.
E. Determination of the moisture content of DOTA.
[0071] Water content of DOTA is determined with a modified Karl-Fischer titration
by heating a sample at 200°C in a drying oven, absorbing the removed
water in dry methanol. The water absorbed in the methanol is titrated with
Hydranal Composite 5 KF reagent, using a Metrohm Titrando 835 with a
774 Oven Sample Processor.
F. Determination of 1,1,3,3-tetramethyl guanidin (TMG) in DOTA
[0072] The 1,1 ,3,3-tetramethyl guanidin (TMG) content in DOTA is determined via
H-NMR.
G. Determination of the optimal TMP for each membrane.
[0073] The optimum TMP was practically determined by changing the TMP (bar)
at a constant feed (= 35 g/min) and measuring the permeate flow (g/min).
The optimum TMP is at the "knee" of the curve where the filtrate flow
increases with increasing TMP up to a point where it levels off. The
optimum TMP was determined with deionised water.
2. Materials
[0074] All reagents used to prepare DOTA were obtained commercially and used
as received:
,4,7,10-tetraaza-cyclododecane (cyclen) from IS Chemical
technology.
• Chloroacetic acid from S.r. Drugs & Intermediates Pvt.Ltd
Gd2O3 from Rhodia.
• N-methyl-D-glucamine (meglumine) from Merck.
• Acetone from Rekha Chemical Corporation.
• Lithiumhydroxide monohydrate from Merck
1, 1 ,3,3-tetramethyl guanidin (TMG) from Acros Chemicals
[0075] For the nanofiltration experiments, deionised water was used with a
conductivity of less then 5 S/cm. All nanofiltration membranes used were
obtained commercially and used as received. The pH range disclosed
hereafter is specified by the manufacturer of the membranes.
• SeIRO MPS-34, from Koch Membrane Systems, Inc., pH range:0-14;
• TS 40, from TriSep Corporation, pH range: 2-1 1.
• Koch TFC-SR1 00, pH range: 4- 0
The pressure ranges of the membranes as specified by the
manufacturer are:
• Trisep TS40: 3 - 14 atm.;
• Koch TFC-SR100: 14 - 41atm.;
• Koch SeIRO MPS-34: 15 - 35 atm.;
[0076] The nanofiltration cell used, is a teflon CF042 cell obtained from Sterlitech.
The filtration area is 42 cm2 and the maximum pressure for this cell is 29
atm.
[0077] An aqueous solution of NaOH (29(wt.)%) was obtained from M.R. Fine
Chem., an aqueous solution of HCI (36 (wt.)%), was obtained from RFCL
Limited and diluted using deionised water as required.
[0078] In Process Control (IPC) is using HPLC according to the method of § 1.
The values are expressed as the ratio (in %) of the area of the peaks, with
regard to the total peak area, to report values for cyclen as starting
material and DOTA as product.
[0079] Conductivity is measured using a conductometer from Metrohm using a
standard conductivity cell; the result is expressed in S/c .
[0080] The yield of the raw DOTA is reported as the number of moles of isolated
product (DOTA bis hydrochloride, without correcting for assay) per number
of moles of cyclene starting material. The yield of DOTA in the purification
step is reported as the weight of isolated pure DOTA per weight of raw
DOTA input corrected for the assay.
3. Preparation of DOTA to be purified
[0081] Seven batches of DOTA were prepared. Batch B-01 was prepared by
adding chloroacetic acid (54.86 Kg, 580.48 mol) to a solution of 1,4,7,10-
tetraaza cyclododecane (20 Kg, 116.1 mol) in water (120 L) and the
reaction mixture was cooled to 5±5°C. A solution of sodium hydroxide
(48.77 Kg in 120 L water, 219 mol) was added slowly to the reaction
mass by maintaining the internal temperature at 0±5°C. The reaction
mass was slowly warmed to 25±5°C and stirred for 20 h. The obtained
DOTA batch B-01 , according to IPC has a DOTA content of 75.53
(area)%, contains also an intermediate compound (at retention time of
0.84 from the retention time of DOTA) of 1.2 (area)% and a cyclene
content of 0.0%.
[0082] Batch B-02 was prepared by adding chloroacetic acid (197.48 g, 2090
mmol) to a solution of 1,4,7,10-tetraaza cyclododecane (72.0 g, 418 mmol
mol) in water (438 g) and to cool the reaction mixture to 10°C. A solution
of NaOH (29 (wt.)% in water, 599.5 g) was added while keeping the
temperature between 8 and 3 °C. The reaction mass was stirred, heated
to 30 °C and kept at this temperature during 22 h.
[0083] Batch B-03 was prepared by adding chloroacetic acid (23.16 g, 0.245
moles) to a solution of cyclen (8.44 g, 49 mmole) in 5 1 ml_ water. The
reaction mixture was cooled at 10 °C and 2.2 g of LiOH powder (0.51
mole) was added at once via a funnel that was rinsed afterwards with 5 g
water. A slightly turbid solution was obtained with a pH = 9. Then the
reaction mixture was heated to 30 °C and stirred for 24 h, during which
time the pH did not change.
[0084] Batch B-04 was prepared by adding chloroacetic acid (23.16 g, 0.245
moles) to a solution of cyclen (8.44 g, 49 mmole) in 5 1 m water. The
reaction mixture was cooled at 10 °C and 58.7 g of TMG (1,1 ,3,3-
tetramethyl guanidine, 0.51 mole) was added at once via a funnel that was
rinsed afterwards with 5 g water. A slightly turbid solution was obtained
(pH=9). Then the reaction mixture was heated to 30 °C and stirred for 24
h, during which time the pH did not change.
[0085] Batch B-05 was prepared by dissolving 49 mmoles of the cyclen
hydrochloride (15.6 g) into 5 1.4 mL of water and by adding 23.1 g of
chloroacetic acid (245 mol) at a temperature of 10°C. Then 94.6 g of a
NaOH solution (29 %, 0.686 mol) where added to the reaction mixture and
the temperature was raised to 30°C so as to obtain a clear solution. The
reaction mixture was kept at 30°C during 24 h.
[0086] Batch B-06 was prepared by dissolving 49 mmoles of the cyclen
hydrochloride (15.6 g) into 5 1.4 mL of water and by adding 23.1 g of
chloroacetic acid (245 mole) at a temperature of 10°C. Then 12.2 g of
LiOH powder (0.51 mole) was added at once via a funnel that was rinsed
afterwards with 5 g water. A slightly turbid solution was obtained with a pH
= 9. Then the reaction mixture was heated to 30 °C and stirred for 24 h,
during which time the pH did not change.
[0087] Batch B-07 was prepared by dissolving 40 mmoles of the cyclen
hydrochloride ( 5.6 g) into 5 .4 mL of water and by adding 23.1 g of
chloroacetic acid (245 mole) at a temperature of 10°C. Then, 58.7 g of
TMG ( 1 ,1 ,3,3-tetramethyl guanidine, 0.51 mole) was added at once via a
funnel that was rinsed afterwards with 5 g water. A slightly turbid solution
was obtained (pH=9). Then the reaction mixture was heated to 30 °C and
stirred for 24 h, during which time the pH did not change.
4. Addition of an acid to an aqueous solution of DOTA (step a)) and
filtration (step b)).
[0088] From the batch B-01 of the reaction mixture obtained in §3. a portion of 50
g. was taken. This part is denoted as batch B-01 A. 24.6 g of an HCI
solution (36 (wt.)%) is added to batch B-01 A having a temperature of
5°C.The obtained slurry was filtered on a sintered glass filter. The
precipitate on the filter had a high volume and was difficult to wash with
water/acetone mixtures due to the slow passage of the liquids.
[0089] The remaining part of batch B-01 was cooled to 5±5°C, acidified with 150
L of an HCI solution of 36 (wt.)% and stirred at 5±5°C for 0-15 min. A
slurry comprising a white solid was obtained. Batch B-01 was further
slowly warmed to 25±5°C and heated to 65±5°C. A clear solution was
obtained and the solution was stirred for 10 min at the same temperature.
The solution was then cooled to 5±5°C over a period of 4-5 h. and stirred
for 10 min. Again, a slurry was obtained. The slurry was filtered using a
polypropylene filter cloth with a mesh size of 40 m and suck dried for 10
min. The reactor was rinsed with a mixture of water (62.2 L) and acetone
(157.2 L). The wet precipitate was mixed with the mixture of water and
acetone used for rinsing the reactor while stirring. The solid was suck dried
for 30 min and dried in a ventilated tray dryer at 67±3°C for 6 h. The
obtained raw DOTA batch, RB-01 had a moisture content of 4.8 (wt.)%
(after 6 h.) according to IPC. The amount of obtained raw DOTA is 41.6
Kg. The purity by HPLC is 89.74%; the Na-content is 188 ppm; the Clcontent
is 9.0 (wt.)%. The assay by HPLC of DOTA is 78.9 (wt.)%.
[0090] Batch B-02 was cooled to 5 °C, a portion of 200 mL was taken and the pH
of this portion was adjusted to 8.1 with an HCI solution (36 (wt.) % in
water). This portion is denoted as batch B-02A. The remainder batch B-02
was acidified with 531 .1 g of HCI (36 (wt.)% solution in water) while stirring
and keeping the temperature at 5°C. At pH=3 precipitation occurred. After
completion of the addition, the obtained slurry was stirred for 30 more
minutes at 5 °C. Then the mixture was heated to 68°C for 30 minutes to
obtain a clear solution. This solution was cooled to 10°C and stirred at that
temperature during 3 h. The obtained slurry was then filtered using a
sintered glass filter with a mesh size of 40mhi and the precipitate was
mixed and washed 3 times on the filter with in total 675 g of a 2:1 mixture
of acetone : deionised water (w/w) while stirring. The moist crystals (134 g)
were dried in a ventilated oven at 40 °C until a constant weight is
achieved; 7 g of raw DOTA as dry crystals were obtained. The obtained
raw DOTA batch, RB-02 had a Na-content of 809 ppm, a Cl-content of
12.7 (wt.)%, a moisture content of 13.4 (wt.)%, and an assay by HPLC of
76.8 (wt.)%.
[0091] Batches B-03 and B-04 were cooled to 5°C and then acidified with 75 g of
36% (w/w) HCI solution while keeping the temperature at 5°C. A slurry
with a pH = 3 was obtained. This slurry was heated to 70 °C and a clear
solution was obtained and after 30 minutes cooled to 10 °C with a cooling
gradient of 1°C/min. The white crystals were filtered on a P3 glass filter
and suspended during 1h in a mixture of water (26 g) and acetone (53 g).
The crystals where filtered on a P3 glass filter and washed on the filter
with a water/acetone mixture containing 26 g of water and 52 g of acetone.
From batch B-03 17.3 g of a raw DOTA as a white powder was obtained
(HPLC content of 90.5 (wt.)%, Cl-content of 3(wt.)%, a Li-content lower
than 50 ppm measured by ICP-OES, the yield of DOTA is 79 (wt.)%) after
drying in a ventilated tray dryer. From batch B-04, 19.05 g of Raw DOTA
was isolated (HPLC content 88.4%, Cl-content measured by titration is
15%, the TMG content measured by NMR is lower than 0.4 (wt.)%, the
yield of DOTA is 85 (wt.)%).
[0092] Batches B-05, B-06 and B-07 were cooled to 5°C and acidified with 94.3 g
of a 36% HCI solution (0.931 moles), yielding a slurry. This slurry was
redissolved by heating to 70 °C and kept at that temperature for 30 min.
The solution was cooled again to 10 °C with a gradient of 1 °C/min and
stirred during 1 h. The precipitate was filtered on a P3 glass filter and
stirred during 1 h in a mixture of 26 g water and 53 g of acetone. From
batch B-05, 17.506 g of DOTA as white crystals where obtained (HPLC
content 88.6%, Cl-content is 16%, Na-content is 3887 ppm, the yield = 78
(wt.)%) after drying in a ventilated dryer. From batch B-06, 3.74 g of raw
DOTA where obtained (HPLC content 86.5%, Cl-content is 19%, Licontent
is lower than 50ppm, the yield is 60 (wt.)%) after drying in a
ventilated dryer. From batch B-07, 16.24 g of raw DOTA is obtained
(HPLC content 87.5%, Cl-content of 16.3 (wt.)%, TMG-content is lower
than 0.1 (wt.)%, and a yield of 88 (wt.)%) after drying in a ventilated dryer.
5. Dissolving the precipitate (step c)) and pH adjustment of the aqueous
solution if required.
[0093] Aqueous solutions of raw DOTA (S-02 to S-06, see Table 2) were
prepared by dissolving parts of the raw DOTA batches RB-01 and RB-02,
to obtain concentrations of DOTA and amounts as mentioned in Table 2.
Depending on the type of the nanofiltration membrane and the
concentration of the raw DOTA, the pH of the solutions had to be adjusted
towards a value within the pH range as specified by the manufacturer of
the membrane. This pH value was obtained by adding a NaOH solution
(29 (wt.) %) to the solution S-02 or by limiting the concentration of the raw
DOTA, which behaves acidic because the raw DOTA was present as
DOTA bis hydrochloride. Besides the solutions S-02 to S-06, an aqueous
solution S-07, in an amount of 1134 g of 2.3 (wt.)% of DOTA was
prepared which was to be used as diafiltration buffer. The pH of this
solution was .6 and was not adjusted.
[0094] As a comparative, solution S-01 was prepared by adjusting the pH of the
reaction mixture B-02A to 8.1 with 20 g of the HCI solution (36(wt.)%), and
by diluting 97.5 g of this solution with 236.8 g of deionised water to obtain
334 g of an aqueous solution having a concentration of DOTA of 3 (wt.)%.
The measured pH of this solution was 8.2, and is within the pH range as
specified by the manufacturer of the nanofiltration membrane. The
prepared solutions are summarised in Table 2.
Table 2
* : calculated ion concentration in S-01 at the start of the diafiltration process.
6. Filtration of aqueous solutions of raw DOTA over a nanofiltration
membrane (step d)).
[0095] The solutions S-01 to S-06 were put in the feed tank before the start of
each of the filtration processes. From solution S-03, 375 g solution was put
in the feed tank. The diafiltration processes were performed as set-up in
fig. , with a feed rate of 35 g/min. and a pressure of 12.5 atm. for the
TS40 membrane (using the Prominent Gamma L 1602 pump) or 20 atm.
for the SeIRO membrane (using the Waters Delta 600 pump). The
solutions and installation were kept at room temperature during the
filtration process. The volume in the feed tank of the solutions S-01 , S-02
and S-06 was kept constant by adding water as diafiltration buffer. In case
of the solutions S-04 to S-05, in a first phase, the volume in the feed tank
is kept constant by adding the raw DOTA solution of 2.3 (wt.)% DOTA
from §5. as the diafiltration buffer (=CVM) to the feed tank. The total
weight of this solution added as diafiltration buffer is displayed in Table 3.
In case of the solution S-03, in a first phase, the volume of the feed tank is
kept constant by adding the remaining part of the solution S-03 as a
diafiltration buffer to the feed tank. After complete addition of the raw
DOTA solutions during the diafiltration of S-03 to S-05, deionised water
was added as diafiltration buffer so as to keep further the volume in the
feed tank constant. The filtration experiment was continued until the
permeate conductivity was less then 250 pS/cm, except for S-01 and S-05.
The filtration with S-05 was stopped after 55,5 h. The total permeate of S-
02 to S-06 was collected and weighed. The weights are mentioned in
Table 3. The filtration loop was rinsed with 200 g of deionised water. 500 g
of the obtained retentate from P-02 to P-06, including the rinsing liquid of
the reactor was concentrated with a rotary evaporator to yield a moist
residue of 8.64g. This residue was redissolved in water to obtain a solution
of 90 g. and precipitated with 180 g of acetone. The precipitate was
filtered, first washed with 75 g of acetone : water (2:1 w/w) and then
washed with 45 g of acetone and dried in a ventilation drier at 60 °C until a
constant weight is achieved. The content of Na, C in the solid purified
DOTA (P-02 to P-06) and the yields are included in Table 3.
Table 3
P-01 P-02 P-03 P-04 P-05 P-06
(COMP) (INV) (INV) (INV) (INV) (INV)
Weight of raw DOTA solution - - 375 567 567 -
as diafiltration buffer (g) S-03 S-07 S-07
Concentration of Na-ions in 7(wt.)% 0.28 2.4 8.1 8.1 2 1
the feed tank at the start of (wt) % ppm ppm ppm ppm
filtration process
Weight of permeate (kg.) 2.71 5.46 4.18 6.73 4.44 5.87
End value of the conductivity 2800 38 195 110 500 250
(MS/cm)
Na content (ppm) 1700 8234 37 109 <50 629
C content (ppm) 148000 3 1 226 <30 490 1870
Yield of DOTA (%) 63 35 59 66 85 92
[0096] Purified DOTA from solution S-01 was obtained differently than from
solutions S-02 to S-06. The solution S-01 did not show a significant
decrease in conductivity during the diafiltration. After 72h., the diafiltration
was stopped. The DOTA could not be isolated from the aqueous solution
by the process of concentrating, redissolving and precipitation as it could
with the retentate of S-02 to S-06. To determine the yield of the purified
DOTA and the content of Na and CI ions in the DOTA of the comparative
example, the retentate of S-01 after attempting isolation as described
above, was concentrated and redissolved in 47 g of water, acidified with
29g of HCI solution (36 (wt.)%), and the obtained slurry was filtered. The
precipitate was suck dried and dried in a ventilation oven to obtain 7.1 g of
DOTA (P-01).
[0097] It can be concluded from the above results that the purification of DOTA
based on filtration over a nanofiltration membrane can only significantly
reduce the concentration of Na and CI ions if the DOTA, as obtained by
the reaction of ,4,7,10-tetra-azacyclododecane with a halo-acetic acid
with a base at a pH > 10, is subjected to preceding purification steps.
These purification steps comprise a precipitation by adding an acid to the
reaction mixture (step a)), filtrating and washing of the precipitate (step b))
and redissolving the obtained raw DOTA to make an aqueous solution of
this raw DOTA (step c)). Indeed, Cl-ion and Na-ion concentrations in
purified DOTA, P-01 without these purification steps, are both very high
with respect to the other batches.
[0098] It is also clear from the results that, if an adjustment of the pH of the
aqueous solution of the raw DOTA is required, this adjustment is
preferably done by limiting the DOTA bis hydrochlorate concentration prior
to the filtration process over a nanofiltration membrane so as to avoid
extra cations into the solution introduced by the use of NaOH as the base
to adjust the pH. The Na-ion concentration in the purified DOTA batches,
P-03 to P-06 where the aqueous solutions of raw DOTA were not
subjected to a pH adjustment with a base, is lower than the Na-ion
concentration in the DOTA batch P-02.
7. Determination of the retention factors for DOTA and for the ionic
contaminants.
[0099] In order to determine retention factions, solutions of DOTA were prepared
based on purified DOTA. The purified DOTA was obtained from raw DOTA
and purified with ion exchange resins as described in WO2014/1 14664.
From the raw DOTA batch RB-01 , 36 kg was dissolved in 540 L of
demineralised water, 2 6 kg of Amberlite IR120(H+) resin (freshly washed
with water until the pH of the decantate is higher than 4) were added; the
suspension was stirred for 16 h at room temperature; the supernatant was
removed and the resin was rinsed with water until the pH of the washing
solution is higher than 4 (in portions of 540 L). The resin was stirred with
720 L portions of ammonia solution (3(wt.)% in water) at room temperature
until no more DOTA (less then 2(wt.)% of the input quantity) showed in the
supernatant (Thin Layer Chromatography: water/methanol 8/2, staining
with KMnO4) . The combined ammonia wash solutions were concentrated
by evaporation under vacuum to a 10 (wt.)% solution of DOTA in water.
Water (540 L) was added and the solution was concentrated by
evaporation in vacuum. This was repeated 3 times.
[00100] Subsequently, the DOTA solution (10 (wt.)%) was stirred with Amberlyst
A26(OH-) resin (183 kg, freshly washed with 460 L portions of water until
the pH was < 10) during 6 h at room temperature. The supernatant was
removed by decanting and the resin was washed with 460 L portions of
demineralised water until the pH of the washing solution was lower than
0. Then, the resin was washed twice with 6 0 L of 0.02 (wt.)% formic acid
in water, the washing solutions were discarded. Next, the resin was
washed with 615 L portions of a 1(wt.)% formic acid solution in water until
the DOTA concentration in the washing solution was lower than 0.05
(wt.)%, determined as above. The combined 1(wt.)% formic acid washes
were concentrated in vacuum until a 10(wt.)% solution of DOTA in water
was obtained, 460 L water was added and the solution was concentrated
to 10 (wt.)%. This last action was repeated 5 times.
[00101] Then 185 L of ethyl alcohol was added. The mixture was concentrated by
evaporation under vacuum to a 10 (wt.)% solution. Acetone (245 L) was
added and the suspension was cooled to 15°C. The precipitate was
centrifuged, the cake in the centrifuge was washed with 6 1 L of acetone
and then dried in a vacuum tray dryer at 67°C and 550 mm Hg till the
water content was lower than 10 (wt.)%. The dried DOTA was dissolved in
110 L of water at 45 °C, 330 L ethylalcohol were added slowly at 45 °C,
the mixture was cooled to room temperature and centrifuged; the cake
was rinsed with 220 L of ethylalcohol and dried in a vacuum tray drier at
550 mm Hg, first at 25°C and then at 67°C until the water content was
lower than 10 (wt.)%. After homogenising, 27 kg of purified DOTA were
obtained, the Na-content is 5 ppm, the Cl-content is 76 ppm, the formiatecontent
is 495 ppm, the ammonia-content is 46 ppm. The HPLC purity is
99.97 %, the assay is 91.5 (wt.)%.
[00 02] Solution S-07 was prepared by dissolving 15 g (37.1 mmole) of purified
DOTA in 480 g of water and 20 g of a 30 (wt.) % NaOH solution in water
was added. Solution S-08 was prepared according to the same way as S-
07. Solution S-09 was prepared by dissolving 15 g (37.1 mmole) of purified
DOTA in 480 g of water and 3.63 g of LiOH (148.4 mmole) was added.
Solution S-10 was prepared according to the same way as S-09.
[00103] To determine the retention factors for DOTA and for the ionic
contaminants (Na+ or Li+ and CI-) a different set-up was used: a solution
of DOTA and ions was circulated over the membrane and both retentate
and permeate were recycled to the feed tank. After 1 h, samples were
taken from the feed tank, the retentate and the permeate stream and
analysed for DOTA, cationic and chloride content.
[00104] The retention factor (in %) for species I is calculated according to formula
2:
R f = 00 X ( 1 - [l]permeate/[l]retentate ) (formula 2)
wherein:
[l]permeate : concentration of the species I in the permeate
[l]retentate: concentration of the species I in the retentate
[00105] Solutions S-07 and S-09 were circulated over a SeIRO MP34 membrane
using a Prominent Hydro type 2 2506 pump at 20 atm and a feed flow of
35 g / min. After 1 h samples were taken from feed, permeate and
retentate streams and analysed for DOTA, Na and CI content. In the feed
tank, also pH and conductivity were measured. The results of the
measurements are listed in Table 4 . Then 3.65 g of a 37% HCI in water
was added (37.1 mmol, 1 equivalent with regard to DOTA) and the
obtained solution was again re-circulated over the membrane as above.
After 1 hour, samples were taken as described before and analysed. This
procedure (adding an equivalent aliquot of HCI) was repeated 4 times (in
total 5 equivalents of HCI are added). The results are presented in Table
4.
Table 4 .
Solution HCI pH Conduc¬ Permeate R f R f R f
equival. tivity flow (g/min) DOTA Na / Li CI
S-07 0 12 13270 0.9 99.8% 92.8% -
S-07 1 9.3 14270 0.9 99.6% 88.3% 43.8%
S-07 2 8.7 7350 0.9 99.6% 70.1 % 49.1 %
S-07 3 4.6 20200 0.9 98.0% 69.8% 55.9%
S-07 4 3.9 22900 0.7 97.3% 70.8% 69.3%
S-07 5 2.1 30700 0.6 98.8% 72.5% 75.6%
S-09 0 11.3 8690 0.8 99.8% 98.2% -
S-09 1 9.1 10800 1 99.6% 9 1.8% 62.7%
S-09 2 7.2 15600 1. 1 99.7% 66.8% 28.8%
S-09 3 4.1 18000 1 99.6% 76.6% 66.0%
S-09 4 3.2 2 500 0.6 99.8% 78.0% 69.7%
S-09 5 2.7 26100 0.5 99.6% 8 .3% 77.9%
[00106] The solutions S-08 and S-10 were circulated over a Trisep TS40
membrane using a Prominent gamma L 1602 pump at 12.5 atm and a
feed flow of 35 g / min. After 1 h samples were taken from feed, permeate
and retentate streams and analysed for DOTA, Na-, Li- and Cl-content. In
the feed tank, also pH and conductivity were measured. The results of the
measurements are listed in Table 5. Then 3.65 g of a 37% HC in water
was added (37.1 mmol, 1 equivalent with regard to DOTA) and the
obtained solution was again re-circulated over the membrane as above.
After 1 hour, samples were taken as described before and analysed. This
procedure (adding an equivalent aliquot of HCI) was repeated 3 and 2
times (in total 4 and 3 equivalents of HCI were added). The results are
presented in Table 5.
Table 5.
[00107] From these results it is clear that the retention for DOTA is better with the
SeIRO membrane and that in the presence of Li ions the DOTA retention
is even higher. The experiments at lower pH show a higher concentration
of chloride ions then metal ions in the permeate.
Claims
1. A process for purifying a compound of formula ,
HOOC~ "^COOH
N N
N N
HOOC-^/ V--COOH
Formula 1
comprising the following steps:
a) adding an acid to an aqueous solution of the compound of formula ,
including salts thereof so as to obtain a pH < 3 whereby a slurry is obtained;
and
b) filtering the slurry and at least one time washing the obtained precipitate
with a liquid comprising water; and
c) dissolving the precipitate obtained in step b) in water to obtain an aqueous
solution; and
d) filtering of the solution obtained in step c) over a nanofiltration membrane
having a Molecular Weight Cut Off in the range from 150 to 500 and wherein
optionally, between step c) and step d) the pH of the aqueous solution is
adjusted to a pH value in the pH range as specified by the manufacturer of the
nanofiltration membrane.
2. The process according to claim 1 wherein a heating step and a cooling step is
performed on the slurry.
3. The process according to claims 1 or 2 wherein the liquid in step b) is a
mixture of water and a water miscible low boiling organic solvent, the mixture
having a water : water miscible low boiling organic solvent weight ratio of 1 :
.5 to 1 : 5.
4. The process according to claim 3 wherein the water miscible low boiling
organic solvent is selected from the group of acetone, ethanol, methanol, isopropanol,
butanone, methyl acetate, ethyl acetate, acetonitrile and THF.
5. The process according to claims 2 to 5 wherein the heating step is performed
at a temperature in the range from 50°C to 100°C, for at least 5 minutes, and
the cooling step is performed at a temperature in the range from 5°C to 25°C,
for at least 5 minutes.
6. The process according to any of the preceding claims, wherein the aqueous
solution of the compound of formula 1 in step a) is obtained by reacting
,4,7,10-tetra-azacyclododecane and a halo-acetic acid with a base at a pH >
10.
7. The process according to any of the preceding claims, wherein step d) is
performed by a diafiltration process.
8. The process according to claim 7, wherein a part of the solution obtained in
step c) is added as a diafiltration buffer to a feed tank.
9. The process according to any of the preceding claims, further comprising the
following steps:
e) increasing the concentration of the compound of formula 1 in the solution
obtained in step d) via diafiltration or evaporation of water so as to obtain a
concentration from 5 to 20 % by weight; and
f) precipitating the compound of formula 1 by adding a water miscible low
boiling organic solvent to the solution obtained in step e); and
g) filtering and drying the precipitate.
10. A process for preparing a gadolinium complex of the compound of formula 1
comprising the steps of:
h) purifying the compound of formula 1 as defined in claims 1 to 9; and
i) adding a salt or oxide of gadolinium to an aqueous solution of the purified
compound of formula 1 so as to obtain compiexation of the gadolinium by the
purified compound of formula 1.