WO2018069176A1

WO2018069176A1 - Method of preparing formulations of lanthanide metal complexes of macrocyclic chelates

Info

Publication number: WO2018069176A1
Application number: PCT/EP2017/075470
Authority: WO
Inventors: Diederik BUFFEL; Joke NELISSEN; Xavier BOI
Original assignee: T2Pharma Gmbh
Priority date: 2016-10-12
Filing date: 2017-10-06
Publication date: 2018-04-19

Abstract

A method of preparation of a liquid pharmaceutical formulation comprising a metal complex of a lanthanide metal with a macrocyclic chelate, comprising the following steps: (i) mixing a solution of uncomplexed chelate with a lanthanide metal or compound thereof and with a base to obtain complexation of the lanthanide metal with the chelate, in such amounts that not all the chelate is complexed; (ii) removal of the uncomplexed chelate from the solution (i) by contacting the solution one or more times with an anion exchange resin; (iii) separating the solution from the resin; (iv) adding chelate in uncomplexed form to the solution to obtain an amount of uncomplexed chelate in the final pharmaceutical formulation of between 0.002 and 0.4 mol/mol% of the metal complex.

Description

Method of preparing formulations of lanthanide metal complexes of macrocyclic chelates

Description

Technical Field

[0001 ] The present invention relates to a method of preparation of pharmaceutical formulations of lanthanide metal complexes of macrocyclic chelates which further comprise a small excess of uncomplexed chelate. The method uses an anion exchange resin to remove excess uncomplexed chelate, prior to the addition of a defined excess of uncomplexed chelate.

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 ¹H as its signal source which is highly abundant in tissues and it has the highest sensitivity of all the biologically relevant nuclei.

[0003] The 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 7Ϊ , and spin-spin or transverse relaxation, characterised by the time constant Tz.

[0004] Often, when suitable 7Ί- 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. 7Ί contrast agents are based on

paramagnetic metal ion chelates which make the tissue appear brighter on the Τϊ-weighted image (positive contrast) and the majority of these are based on chelates of the gadolinium ion Gd(lll).

[0006] Free ions of lanthanides, and in particular gadolinium, are very toxic for the tissues. Indeed, a pathology known as NSF (nephrologic systemic fibrosis, or fibrogenic dermopathy, with very severe effects on human skin), may be at least partly correlated to the existence of free gadolinium ions, i.e. non- complexed gadolinium, in the body. The level of toxicity depends on the strength of the chelating agent, also known as ligand, chelator or sequestering agent to form a complex with the lanthanide ions. Usually these ligands are organic compounds which form two or more separate coordinate bonds with a single central metal ion, in this case, the lanthanide ion, inactivating it and thus reducing or eliminating its toxic effect in the tissues.

[0007] Polyaminopolycarboxylic acid compounds are the chelate type of choice because they form exceptionally stable complexes with lanthanide metal ions such as Gd(lll) ion. These compounds can be linear (such as pentetic acid or diethylene triamine pentaacetic acid also named as DTPA) or macrocyclic (such as 1 ,4,7,10-tetraazacyclododecane-1 ,4,7,10-tetraacetic acid, DOTA). Complexes of macrocyclic ligands are much more kinetically inert and thus, present an exceptionally high solution stability.

[0008] Formulations of MRI contrast agents based on lanthanide metal ions

comprise an excess of chelate in uncomplexed form or present as a 'weak metal chelate complex' such as with calcium, to complex efficiently any lanthanide ions which may be either liberated or present (US 5,876,695) in the formulation to be administered.

[0009] Table 1 of US 7 385 041 illustrates with LD50 values that free macrocyclic chelates (HP-D03A, D03A, DOTA) are about at least 10 times more toxic than these macrocyclic chelates under the form of a weak metal chelate complex. Hence, if the formulation of a MRI contrast agent based on a lanthanide metal ion comprises uncomplexed macrocyclic chelate, the excess of free macrocyclic chelate should be in a specific and low range.

[0010] Dotarem® is a pharmaceutical formulation for direct administration to

patients which contain a Gd-DOTA complex and an amount of excess free DOTA at a particular low dose range, and which is not under the form of a weak metal chelate complex. The leaflet of DOTAREM^® discloses the amounts of DOTA and Gd₂0₃ , so that, upon simple calculations based on the assumptions that the stated quantities correspond to the Gd-DOTA complex (formed in a quantitative yield), the quantities appear to relate to a formulation containing 0.12 mol/mol% excess free DOTA relative to the amount of complexed DOTA. DOTAREM^® is well established on the market and is known as a very stable and hence safe MRI contrast agent.

[001 1] As an injectable product for diagnostic, it is of very high importance that the final pharmaceutical formulation should be manufactured with extremely precise and delicate industrial scale control of the

concentrations of free macrocyclic ligands. If this is achieved, no additional purification or isolation steps are required anymore.

[0012] WO2009103744 discloses a process for preparing contrast agents

formulations based on a lanthanide chelate, said formulation including an excess of free chelate of 0.002% to 0.4% mol/mol to address the problem of intolerance of lanthanide chelates related to the presence of free lanthanide ions in the formulation to be administered. WO2009103744 stresses that, for an industrial scale pharmaceutical manufacturing processes, the addition of free macrocyclic chelate in this range is difficult to achieve with the required degree of accuracy by weighing alone.

WO2009103744 therefore discloses a method to first carry out the metal complexation with an excess of lanthanide metal ion or an excess of macrocyclic chelate, then secondly to determine accurately the

concentration of excess lanthanide or uncomplexed chelate. That determination is subsequently used to calculate exactly how much additional chelate must be added or removed to achieve the desired excess of macrocyclic chelate in a range between 0.02% and 0.4% mol/mol and in particular between 0.025% and 0.25 % mol/mol of the free macrocyclic chelate. The method requires a very accurate measuring method to assay the very low levels of excess of lanthanide ion or free chelate. To achieve this required accuracy, the measurements include titrations in combination with potentiometry or colorimetric detection. [0013] W016083597 provides a method whereby the lanthanide chelate metal complex is obtained without excess lanthanide ions being present by using a solid phase bound scavenger chelator. Since the process provides an intermediate solution of the lanthanide metal complex without free lanthanide ions, the amount of excess macrocyclic chelator to add to give the desired formulation having a defined excess of free chelator can be calculated readily. The solid phase bound scavenger chelator is a cation exchange resin which will, when complexing the excess of gadolinium ions, release undesirable sodium ions in the pharmaceutical formulation or protons hence further decreasing the pH of the formulation away from the desired value for direct administration. To avoid this unwanted release, W016083597 discloses that the counter cation of the resin has first to be exchanged with meglumine by means of a treatment of the resin with a meglumine solution. This represents an additional step in the process of preparation. The scavenger chelator preferably comprises iminodiacetic acid (IDA) which can be detached from the resin and give undesirable contamination of the pharmaceutical formulation (see Chelex® 100 and Chelex 20 Chelating lonExchange Resin Instruction Manual from Biorad). Furthermore, a cation exchange resin is not compatible with bases present in the solution based on alkali or earth alkali metal hydroxides or meglumine. Moreover, US2016016979A shows that an excess of lanthanide metal, more particularly of Gd can lead to precipitation of Gd- oxides and hydroxides resulting in a loss of raw material and low yields.

[0014] US6951639B discloses the preparation of solutions of lanthanide

complexes of 1 , 4, 7, 10- Tetraazacyclododecane butyltriols wherein after complexation the solution is contacted with an acidic ion exchanger and a basic exchanger. The use of an acidic ion exchanger, which is a cation exchanger, in the preparation process, represents the same

disadvantages as stated above and should be avoided.

[0015] It is thus desirable to obtain an optimized process for producing a

pharmaceutical liquid formulation suitable for industrial scale comprising a complex of macrocyclic chelate with a lanthanide, which do not require a measurement step, which is compatible with the presence of bases in the reaction mixture and which is straightforward without the need of a further purification or isolation step(s).

Summary of invention

[0016] It is an object of the invention to solve the above stated problems. The invention is a method of preparing a pharmaceutical formulation according to claim 1. The method is based on the removal of uncomplexed chelate by means of an anion exchange resin after mixing of a solution of uncomplexed chelate with a lanthanide metal or compound. The removal step is followed by addition of chelate in uncomplexed form to obtain an amount of uncomplexed chelate in the final pharmaceutical formulation of between 0.002 and 0.4 mol/mol% of the metal complex.

[0017] Preferred embodiments are described by the dependent claims 2 to 10.

[0018] Further advantages and embodiments of the present invention will become apparent from the following description and the dependent claims.

Description of embodiments

[0019] According to the invention the preparation of a liquid pharmaceutical

formulation comprising a metal complex of a lanthanide metal with a macrocyclic chelate and the chelate in uncomplexed form in an amount between 0.002 and 0.4 mol/mol% of the metal complex, in the final pharmaceutical formulation, comprises the following steps:

(i) Mixing a solution of uncomplexed chelate with a lanthanide metal or compound thereof to obtain complexation of the lanthanide metal with the chelate, in such amounts that not all the chelate is complexed.

[0020] The chelate is preferably a derivative of tetraaza macrocycles such as 1 ,4,7,10-tetraazacyclododecane (cyclen), 1 ,4,7,10-tetrazacyclotridecan (homocyclen) and ,4,8,1 1 -tetraazacyclotetradecane (cyclam), preferably DOTA (1 ,4,7,10-tetraazacyclododecane-1 ,4,7,10-tetraacetic acid), NOTA (1 H-1 ,4,7-Triazonine-1 ,4,7-triacetic acid, hexahydro), DOTAGA (1 ,4,7,10- Tetraazacyclododecane-1 ,4,7,10-tetraacetic acid, a-(2-carboxyethyl)), D03A (1 ,4,7,10-Tetraazacyclododecane-1 ,4,7-triacetic acid), D03A-butrol (1 ,4,7,10-Tetraazacyclododecane-1 ,4,7-triacetic acid, 10-[2,3-dihydroxy-1 - (hydroxymethyl)propyl]), HP-D03A (1 ,4,7,10-Tetraazacyclododecane- 1 ,4,7-triacetic acid, 10-(2-hydroxypropyl)) and PCTA (3,6,9,15- Tetraazabicyclo[9.3.1 ]pentadeca-1 (15), 1 1 ,13-triene-3,6,9-triacetic acid), more preferably DOTA, D03A, HP-D03A and even more preferably DOTA.

[0021 ] The chelate in uncomplexed form refers to the 'free chelate', i.e.

substantially without any coordinated metal ions. Hence, the chelate in uncomplexed form does not have any coordinated lanthanide or other metal ions, and is thus fully available for subsequent metal complexation. The chelate in uncomplexed form may contain metal ions in ionic form, such as when present as salts of metal donor group, e.g. of a carboxylic acid.

[0022] Lanthanide metal or compounds thereof comprise the fifteen metallic

chemical elements with atomic numbers 57 through 71 , and their compounds thereof such as oxides, hydroxides, halogenides ... Preferred lanthanides are Gadolinium (Gd), Yttrium (Y) and Terbium (Tb) and most preferred is Gadolinium. The most preferred Gadolinium compound used in the invention is Gd₂O3.

[0023] Suitable solvents for the complexation of step (i) are known in the art [The Chemistry Of Contrast Agents in Medical Magnetic Resonance Imaging, 2" Edition, A.Merbach, L.Helm k E.Toth (Eds), Wiley (2013)] and are preferably aqueous, most preferably water.

[0024] The base to be added is required to increase the pH as the lanthanide metal and / or macrocyclic chelate in the solvents give solutions of low pH values. Most macrocyclic chelates comprise acidic groups and the complexation reaction comprises the exchange from protons by lanthanide metal which is rather slow. This is particularly the case with DOTA and Gadolinium. The exchange rate therefore increases when the macrocyclic chelate has lost at least one proton, hence with increasing pH. For acidic macrocyclic chelates, the pH value is preferably equal to or higher than 2.5, more preferably equal to or higher than 2.5, to have complexation reaction times which are suitable in an industrial production process.

Suitable bases are inorganic bases as hydroxides, carbonates,

bicarbonates of for example sodium, potassium, lithium, magnesium or calcium and/or organic bases as primary, secondary and tertiary amines, such as ethanolamine, morpholine, glucamine, N-methyl and N,N dimethylglucamine, as well as basic amino acids, such as lysine, arginine and ornithine or of amides of originally neutral or acidic amino acids. L- lysine and N-methyl-D-glucamine also known as meglumine are preferred because these compounds do not have to be removed during the preparation of the pharmaceutical formulation due to its high acceptance by the body when administered. The base can also be introduced in the method of the invention as a salt of the macrocyclic chelate, e.g.

meglumine-DOTA.

[0025] The complexation of lanthanides by macrocyclic chelates (e.g. DOTA) is a multistep process that involves a somewhat stable initial complex that slowly matures to give the final, thermodynamically stable metal complex. In step (i), it is preferred to ensure that said stable lanthanide complex has been generated completely, as is known in the art [Moreau et al, Chem. Eur. J., 10(20), 5218—32 (2004)] , e.g. by heating, prolonged reaction times, raising the pH, or combinations thereof, before proceeding to step (ii). The raising of the pH is preferably performed by adding lysine or N- methyl-D-glucamine. The heating takes place in a temperature range, preferably between 70°C and 100°C, more preferably between 90°C and 100°C, most preferably between 95°C and 100°C.

[0026] To achieve the condition in step i) that not all the chelate is complexed, it is required that the amount of chelate with respect to the lanthanide is in stoichiometric excess. The amount of uncomplexed chelate, also called excess of the chelate in step (i) can be obtained by calculation of the molar ratios knowing the stoichiometry of the lanthanide macrocyclic chelate metal complex. That is typically a 1 :1 complex. Such information is given in The Chemistry of Contrast Agents in Medical Magnetic Resonance

Imaging, 2" Edition, A.Merbach, L.Helm k E.Toth (Eds), Wiley (2013); for gadolinium complexes by Port et al [Biometals, 21 , 469-490 (2008)]; and for DOTA complexes by Viola-Villegas et al. [Coord. Chem. Rev. , 253, 1906-1925 (2009)]. Molar ratios of chelate over lanthanide are hence to be above 1.0, preferably less than or equal to 1 .4, advantageously between 1.001 and 1 .3, particularly advantageously between 1 .005 and 1.2, and in particular between 1.005 and 1 .02. The less the ratio is above 1 .0, the less resin is to be consumed (see §(ii)).

[0027] In order to calculate the stoichiometric amounts, the chemical purity of both the lanthanide used and the chelate in question can also be taken into account. More specifically, the water content of the chelate is preferably to be taken into account. Therefore a measurement of the moisture content is preferably performed prior to the calculation. Methods for measuring the moisture content are the Karl Fisher method and the determination of the solid content by heating the chelate at 120 to 130 °C. Another preferable method is measuring the content of the macrocyclic ligand by a suitable analytical technique, e.g. HPLC or titration and calculate the amount of macrocyclic chelate. In order to compensate for the additional amount of moisture which will be taken up by the

macrocyclic ligand after the measurement of the moisture content and before the formation of the complex, it is preferred to use an extrapolated moisture content. The extrapolated moisture content can be obtained using a linear or non-linear extrapolation.

[0028] The obtained excess of uncompiexed macrocyclic chelate can be checked by measuring the concentration of uncompiexed chelate by a suitable method such as HPLC or titration. Instead of calculating the amount of uncompiexed chelate to be obtained in step (i), the amount can also be measured by means of a suitable method such as HPLC or titration.

[0029] The presence of an excess of chelate can also be checked by means of a measurement of the free lanthanide concentration, i.e. the concentration of uncompiexed lanthanide. This last measurement gives also an indication that the complexation reaction is completed.

(ii) removal of the uncompiexed chelate from the solution obtained in step (i) by contacting the solution with an anion exchange resin

[0030] Step (ii) is carried out such that the solution obtained in step (i) is exposed to the resin. This can be carried out by two principal methods, or combinations thereof. The first option is to mix the resin with the solution. Alternatively, the resin can be provided as a column and the solution is eluted through the column. For either option, the solution can be contacted to the resin one or more times.

[0031] Anion-exchange resins contain contain functional groups which are

suitable to bind anions from a solution. Different types exist: strong basic anion exchange resin and weak basic anion exchange resins.

[0032] The strongly basic anion exchange resin can be selected from any

commercially available type I (namely with trimethylammonium functional groups) or type II (namely with dimethyl-2-hydroxyethylammonium functional groups), gel, porous, macroporous and macroreticular type. Suitable commercial resins are for example the Amberlite series IRA402, IRA400, IR410, IRA900, IRA910, Amberlyst A26, Dowex series SBR, SAR, MSA-1 C, all from Dow. Other suitable strong anion exchange resins are the Trilite series SAR10 and SAR 1 from SamyangCorp, the Dianion sesies SA10A, SA20A, PA412 from Mitsubishi, SBG1 , SBG2 from

ResinTech, the Purolite series A400, A600, A200, A300 from Purolite and the Lewatit monomplus series M500, M600, MP600 from Lanxess.

Preferably, Amberlyst 26 is to be used.

[0033] Weak basic anion exchange resins include acrylic and styrene resins with secondary amine or with a tertiary amine such as the Dianion series WA10, WA20, WA30 from Mitsubishi, WBMP from ResinTech, the

Amberlite series IRA60, IRA67 from Dow and MP62 from Lanxess.

[0034] When available, small particle size grades are preferred, as they allow a faster exchange: for example, "fb" grade is the preferred grade for Dianion 3A or 3AS. The resin can be present in the method of the invention as particles, fibres and membranes. Preferably the resin is present as particles or beads. Suitable fibre shaped anion exchange resins are Smopex-103 and Smopex-105 from Johnson Matthey. Suitable anion- exchange membranes are Sartobind Q membranes from Sartorius Stedim Biotech GmbH.

[0035] Anion exchange resins are further characterised by their capacity

expressed as the number of sites available for exchange (also called equivalents) / L of resin. The number of sites available for exchange is determined by the number of functional groups of the resin: for example quaternary amino groups, such as, trimethylammonium groups for strongly basic anion exchange resins and primary, secondary, and/or tertiary amino groups for weak basic anion exchange resins. The total capacity of an amount of resin, expressed as equivalents or total number of sites available for exchange, is then obtained by multiplying the capacity with the amount of the resin.

[0036] The strong basic anionic exchange resin, prior to use is preferably brought into its hydroxyl-ion containing form. Resins in this form can be purchased as such or have to be regenerated by means of contacting the resin with an alkaline solution. It is preferable to replace all exchangeable anions bound to the resin by hydroxyl ions prior to its use, to limit the

contamination of the lanthanide metal complex containing solution by anions such as chlorides. In this case, the number or equivalents of sites available for exchange of the resin is equal to the equivalents of hydroxyl ions. Chloride anions, if its concentration is too high, have to be removed in an additional purification step.

[0037] The reaction time of the ion exchange may vary and can be for example 1 to 10 hours, or may be performed for 2 to 5 hours. The ion-exchange step, can be performed in a temperature range between 0 and 70°C, preferably between 20°C and 30°C.

[0038] The amount of resin, expressed as equivalents of the resin is preferably between 1 and 50 times the calculated amount of uncomplexed

macrocyclic chelate in moles, more preferably between 1 and 20. The amount of strong basic anion exchange resin, expressed as equivalents of the resin, is preferably between 3 and 10, more preferably between 4 and 9 times the calculated amount of uncomplexed macrocyclic chelate in moles. An amount of strong basic anion exchange resin above 10 times the amount of uncomplexed chelate, leads to considerable loss in metal- chelate complex due to the removal of the metal-chelate complex by the resin. This leads to a reduced yield in the production of the pharmaceutical formulation. [0039] On an industrial scale, the required amount of resin is preferably added based on a volume measurement. This has the advantage that it takes into account the amount of water absorbed by the resin.

[0040] The method of the invention provides the additional advantage that, as part of the process, many impurities present in the chelate are removed from the solution. These impurities can be e.g. triacetic acid derivatives of the chelate, more particularly, 1 ,4,7,10-tetraazacyclododecane-1 ,4,7- triacetic acid but also contaminants such as endotoxines which can be generated due to the presence of bacteria present in the used raw materials and which should be avoided or removed because these endotoxines can induce fever when injected into the blood or

cerebrospinal fluid.

[0041] In order to check if the concentration of the uncomplexed macrocyclic chelate is sufficiently removed such as that the concentration is zero, a measurement of the concentration of the chelate can be performed. The term zero concentration of chelate shall be understood as meaning that the concentration is zero or substantially zero, typically less than 10"⁴ M and advantageously less than 10 ⁵ M or 10 ⁶ M, the possible presence in solution of an extremely small amount of chelate not being able to be totally excluded. The reason for this is that concentrations less than 10"⁴ M cannot be measured sufficiently reliably by the current analytical methods such as HPLC or titration. If the concentration of the uncomplexed chelate is not substantially zero, the contact time with the resin can be prolonged or the solution can be eluted one or more times through the column.

(iii) separation of the solution obtained in step (ii) from the resin

[0042] The separation of step (iii) is achieved by e.g. filtration or decantation of the solution to remove the resin and resin bound to chelate, by collecting the eluate from the column elution or passing the solution through a membrane. After the separation of the solution from the resin, a

measurement of the uncomplexed macrocyclic chelate can be performed as described in § (ii). (iv) addition of chelate in uncomplexed form to the solution obtained in step (iii) to obtain an amount of uncomplexed chelate in the final

pharmaceutical formulation of between 0.002 and 0.4 mol/mol% of the metal complex

[0043] Contrary to the methods of the prior art such as WO 2009103744 the

addition of step (iv) may be carried out without a prior, in-process measurement of the concentration/amount of uncomplexed chelate in either the solutions obtained in step (i), (ii) or (iii). That is because such a step is unnecessary for the present method. The removal and separation in steps (ii) and (iii) leads to a solution with a substantial zero

concentration of uncomplexed chelate. Because the concentration of uncomplexed chelate is substantial zero, the amount of uncomplexed chelate to be added to obtain a concentration between certain specs of free chelate in the pharmaceutical solution can be calculated without the need of a measuring step before the addition. Not only the presence of free lanthanide ions but also a too high amount of free chelate in a pharmaceutical solution can be toxic to the patient. The method according to the invention guarantees a free chelate concentration between wanted values without a measuring step.

[0044] The addition of step (iv) can be carried out on the basis of a calculated amount of chelate in uncomplexed form, based on an assumed 100% conversion in the complexation reaction of step (i). The free chelate can be added either as a solid, or as a solution and preferably as a solution.

When a solution of the macrocyclic chelate is prepared in order to carry out step (i), then a most preferred method is to remove a suitable volume fraction from that solution prior to the addition of the lanthanide (e.g.

removing ca. 1 L from a 1000L reaction volume or equivalent). This volume fraction is then conveniently used for the addition of step (iv). This approach obviates the need to make up multiple solutions, and/or to carry out multiple calculations to correct for purity or water content of the macrocyclic chelate. [0045] The adjustment step (iv) can further comprise at the end a step of adjustment of the pH advantageously with a base such as meglumine for DOTA and/or of the volume.

Examples

1. Measuring methods

1.1. Concentration measurement of DOTA in uncomplexed form (free DOTA) in solution.

[0046] Solution A: 50 g of sodium acetate was dissolved in 10 ml of glacial acetic acid and the volume was adjusted to 1000.0 ml with water free from carbon dioxide. The obtained solution was adjusted to pH (5 ± 0.05) with 0.1 M sodium hydroxide solution or glacial acetic acid.

[0047] Solution B: 50.8 mg of Xylenol orange was dissolved in water free from carbon dioxide, the volume was adjusted the volume to 100.0 ml with the same solvent. Freshly prepared solutions were used.

[0048] Solution C: 3 ml of solution B was added to 30 ml of solution A, the volume of the solution was added to 200.0 ml with water free from carbon dioxide. 0.005 M gadolinium sulphate solution was prepared as follows: 3.735 g of gadolinium sulphate octahydrate was dissolved in water free from carbon dioxide. The volume of solution was adjusted to 000 ml with the same solvent.

[0049] A test solution was prepared as follows: 4.88 ± 0.5 g of meglumine and 50 ± 1 g of hot (70 - 90 °C) sample of the solution comprising the gadolinium- DOTA complex, were transferred into a 100 ml conical flask, mixed for 5 to 10 minutes at 70° C to 90° C and cooled to room temperature. To 2 ml of this solution, 20 ml of water free from carbon dioxide and 10 ml of solution C were added and mixed. The resultant solution was adjusted to pH 5 ± 0.05 with 0.1 M sodium hydroxide solution or glacial acetic acid. The yellow coloration indicates the presence of free DOTA. The solution was titrated with the 0.005 M gadolinium sulphate solution until colour alters to reddish-pink.1 ml of 0.005 M gadolinium sulphate solution corresponds to 4.044 mg of DOTA.

1 .2. Concentration measurement of the Gd-DOTA complex

[0050] The concentration of the Gd-DOTA complex was determined by reversed phase HPLC (High Performance Chromatography) with a gradient program and a DAD (Diode Array Detection). The content of the Gd-DOTA complex in the sample is expressed as the ratio of the area of the peak of the sample over the area of the peak of the standard at the same amount injected for both standard and sample. The concentration of Gd-DOTA is expressed in (wt.)%. As a standard, DOTAREM® was used which contains 279.32 mg Gd-DOTA. The standard was obtained by weighing 0.5 ml DOTAREM in a flask of 25 ml and dissolving it in distilled water and dilute until the marker with distilled water. The obtained calibration curve is linear in the range between 5 and 40 ug Gd-DOTA / injected volume.

[0051] The method and apparatus used is the same as described in [0062-0066] of WO2015/1 1791 1A1. Retention times are 10.2 min for the Gd-DOTA complex.

2. Materials

[0052] All reagents used in the following examples were readily available from commercial sources unless otherwise specified:

Gadolinium oxide : Gd2O₃ from Rhodia

Meglumine: N-methyl-D-glucamine from Merck KGaA, Darmstadt DOTA: was obtained as described in the patent application WO2014/1 14664.

Resin: Amberlyst A26. The resin was purchased in its hydroxyl-ion containing form. The exchange capacity of the resin is 0.8 milliequivalents / ml, which corresponds to 0.0016 equivalents per gram. 3. Method of preparing a pharmaceutical formulation

[0053] A reactor of 2 L was filled with 90.64 g Gd₂0₃ followed by adding 233.07 g DOTA using a teflon funnel. The added Gd203 had a purity of 99.99 wt.%, the added DOTA contained 5.3 wt.% of water. The molar ratio of DOTA / Gd was: 1.043. Hereafter 768.1g of water was added. The mixture was agitated by means of an impeller stirrer, and heated up to a temperature of 97°C. The mixture was stirred for 180 minutes at 97°C. The obtained solution had a pH value of 1 .75. This solution was divided in aliquots having a mass of 50.0 g. To some aliquots 4.6 g of Meglumine was added such as to obtain a pH between 2.5 and 3.5. To some aliquots, different amounts of resin were added. The amount of resins, expressed as equivalents, is between 6.9 and 10 times the amount of uncomplexed DOTA in the solution, the amount of DOTA is expressed as moles. The obtained mixtures were stirred for 3 hours. After this time, the resin was filtered from the solutions and the amount of DOTA with respect to the amount of Gd-DOTA complex was determined in the solutions S-1 to S-4. The results are listed in Table 1.

Table 1

Solution: S-1 S-2 S-3 S-4 mass of aliquot 50.0 50.0 50.0 50.0

Mass of meglumine added (g) 0.0 4.6 4.6 4.6 pH before addition of resin 1 .75 3.4 3.4 3.4

Mass of resin added (g) 6.0 0.0 4.0 5.0

Ratio meq resin/ mmol uncomplexed 10.0 0.0 6.9 8.63 DOTA (step(ii))

Amount of uncomplexed DOTA after 16.95 4.75 0.00 0.00 filtration of resin (mol/mol%)

Comparative / Invention COMP COMP INV INV [0054] From the results it can be seen that, from a certain amount of resin added, the concentration of DOTA is substantially zero. i.e. below the detection limit. To the obtained solutions all having a mass of approximately 45 g and having an amount of Gd-DOTA complex in the range between 20 and 23 wt.%, an amount of DOTA of 15 mg is added such as to obtain an excess of uncomplexed DOTA in the formulation of 0.2 mol/mol% of the metal complex. This was confirmed by concentration measurements of the uncomplexed DOTA in the pharmaceutical formulation.

[0055] The obtained formulation does not require further purification or isolation steps and is suitable to be directly administered to a patient after a heat- sterilisation step and a filtration step using 0.2 to 0.45 μιη filters.

Claims

Claim 1 . A method of preparation of a liquid pharmaceutical formulation

comprising a metal complex of a lanthanide metal with a macrocyclic chelate, the method comprising the following steps:

(i) mixing a solution of uncomplexed chelate with a lanthanide metal or compound thereof and with a base to obtain complexation of the lanthanide metal with the chelate, in such amounts that not all the chelate is complexed; and

(ii) removal of the uncomplexed chelate from the solution (i) by contacting the solution one or more times with an anion exchange resin; and

(iii) separating the solution from the resin ; and

(iv) adding chelate in uncomplexed form to the solution to obtain an amount of uncomplexed chelate in the final pharmaceutical formulation of between 0.002 and 0.4 mol/mol% of the metal complex.

Claim 2. The method according to claim 1 wherein the solution obtained in step (i) has a pH value equal to 2.0 or higher.

Claim 3. The method according to claim 1 or 2 wherein the base is L-lysine or meglumine.

Claim 4. The method according to any of the preceding claims wherein the lanthanide metal is gadolinium, the compound thereof preferably Gd203.

Claim 5. The method according to any of the preceding claims wherein the macrocyclic chelate is DOTA.

Claim 6. The method according to any of the preceding claims wherein the temperature at which the complexation in step (i) takes place is between 60 °C and 100°C.

Claim 7. The method according to claims 1 to 5 wherein the temperature of the solution in step (i) is increased to a value between 60 °C and 100°C before the base is added to the solution.

Claim 8. The method according to any of the preceding claims wherein step

(ii) is performed by mixing particles of the anion exchange resin with the solution.

Claim 9. The method according to claims 1 to 7 wherein during step (ii) the anion exchange resin is provided in a column and the solution is eluted through the column.

Claim 10. The method according to any of the preceding claims wherein the resin is a strong basic anion exchange resin.