Development of molecular adsorption processes for the removal of genotoxic impurities from active pharmaceutical ingredients

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1 Development of molecular adsorption processes for the removal of genotoxic impurities from active pharmaceutical ingredients Mariana Duarte de Pina Instituto Superior Técnico, Universidade de Lisboa Avenida Rovisco Pais, Lisboa, Portugal Abstract: Most of the drugs available in the market are synthesized using highly reactive molecules. These molecules may be present in the final API as impurities, that may be genotoxic or carcinogenic. The risk for patient s health caused by these impurities has become an increasing concern of pharmaceutical companies and regulatory authorities. A broad range of unrelated chemicals from very different chemical families have been categorized as genotoxic. These compounds have the ability to react with DNA, preventing its normal replication, resulting in an associated carcinogenic risk. Although it is desirable to avoid the use of GTIs in the manufacture of APIs, this is not always possible, since these compounds are synthetically useful. It is fundamental to produce APIs with low GTI content, controlled below the Threshold of Toxicological Concern (TTC) established by regulatory authorities (1,5 µg/day). So, it is necessary to find simple, robust and economical routes to remove GTIs from APIs. During the development of this thesis, conventional purification techniques (recrystallization, ionic exchange resins and adsorbents), as well as emergent techniques (nanofiltration, molecularly imprinted polymers (MIPs)) were studied. The results achieved suggest that recrystallization is not a cost-effective process. In that sense, it is necessary to find new ways to increase its yield. Using ionic exchange resins and MIPs, it is possible to make recrystallization a viable process for the pharmaceutical industry. Keywords: Genotoxic impurity, purification, recrystallization, molecular imprinting 1. Introduction Carcinogenesis includes three stages: initiation, promotion and progression. Usually, mutational events are involved in the initiation stage; these events are usually corrected almost immediately by DNA repairing mechanisms. Yet, sometimes these mechanisms fail to repair the DNA and the mutated cells start to proliferate promotion state. Then, the cells undergo differentiation, creating new genes. After differentiation, the mutated cells transported by the bloodstream invade healthy tissues; this process in known as metastasis and occurs in the progression stage 1a,1b. Mutagenicity is the capacity to induce transmissible genetic damage, including gene mutations or chromosomal aberrations. The term genotoxicity refers to all genetic damage, including genetic alterations that may result in mutations, which are not transmitted to daughter cells 2. Genotoxic compounds attack the nucleophilic centers of the DNA, which can lead to strand breaks. The nucleophilic centers of DNA are the nitrogen and oxygen atoms of pyrimidine and purine bases and the phosphodiester backbone 1a,2a,3. The stereospecificity of the reaction depends on the chemical nature of the genotoxic compound, steric factors and nucleophilicity; the most nucleophilic sites of the DNA bases are endocyclic nitrogens; on the contrary, exocyclic oxygens are the less nucleophilic 4. Chemical 1

2 mutagens and carcinogens are metabolized by a variety of enzymes; for instance, several forms of human cytochrome P-450 are involved in the oxidative metabolism of chemical carcinogens Regulation The International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceutical for Human Use (ICH) brings together the regulatory authorities from Japan, Europe and United States; it studies the scientific and technical aspects of pharmaceutical product registration 1a. ICH guidelines address impurities in drug substances (Q3A), degradants in drug product (Q3B) and also, residual solvents in drug substance (Q3C). However, these guidelines fail to address a number of important issues, such as the level of impurities in drugs during development and control of GTIs 5. The European Medicines Agency (EMEA), an agency for the evaluation of medicinal products, published a limit guideline for genotoxic impurities in new drug substances that is only used to new applications for manufacturing process changes 5b,5d,6. This guideline recommends the genotoxic impurities division into those interacting directly with DNA and those acting through other mechanisms. The first group is of main concern, since there is not enough evidence for a threshold-related mechanism; they can damage DNA at any concentration. In this case, the guideline proposes the application of the ALARP principle (As Low As Reasonably Practicable). This principle is based on a balance between the need to reduce the GTI concentration to the lowest possible level and the possibility of reducing it 6. The guideline proposes the use of a threshold of toxicological concern (TTC) for genotoxic impurities; it refers to a threshold exposure level of compounds that will not pose a significant risk of carcinogenicity or other toxic effects. The draft guideline proposes a TTC of 1,5 μg/day, which corresponds to a 10-5 lifetime risk of cancer 5b,5c ; this risk is justified by the anticipated health benefits for the patient in taking the medicine 5c,7. The Pharmaceutical Research and Manufacturing Association (PhRMA) established a Genotoxic Impurity Task Force, which developed a White Paper. It was proposed that all identified or predicted impurities should be classified into one of five classes 5a-c,7c,8. 1. Impurities known to be genotoxic (mutagenic) and carcinogenic; 2. Impurities known to be genotoxic (mutagenic) but with unknown carcinogenic potential; 3. Impurities containing alerting structures, unrelated to the structure of the API, and of unknown genotoxic (mutagenic) potential; 4. Impurities containing alerting structures, which are related to the API; 5. Impurities with no alerting structures, or where no sufficient evidence exists that genotoxicity is absent. The Center of Drug Evaluation and Research (CDER) of the US FDA is developing guidelines to address genotoxic impurities in pharmaceutical products. Even though genotoxic impurities should be avoided, it is known that complete removal is not always possible. When it is not possible to avoid genotoxic impurities, they should be limited to a level that does not represent a significant risk to patients 5b. 2

3 In 2013 the ICH M7 guideline was published; it provides guidance on analysis of structure activity relationships (SAR) for genotoxicity Genotoxic compounds A wide range of unrelated chemicals, with very different structures and from very different chemical families have been categorized as genotoxic impurities. A genotoxic substance is known as a genotoxin. Sources of genotoxic impurities in the manufacture of APIs include starting materials, reagents, intermediates, side reactions and impurities. Functional groups may also be responsible for starting materials and intermediates genotoxicity. There is a small group of APIs, such as chemotherapeutic agents, for which genotoxic and carcinogenic substances are acceptable; however on the majority of cases, it is desirable to remove the genotoxic impurities, which cannot always be achieved. From a chemical point of view, there are no physical properties or chemical structural elements that provide a definitive categorization of genotoxic. There are some molecules whose genotoxic effect is known, while others are dangerous because they contain reactive groups that may lead to genotoxicity; these reactive groups are molecularly recognized and are cataloged as structural alerts 9. It was estimated that about 20 to 25% of all intermediates used in standard pharmaceutical synthesis contain structural alerts 10. The model compounds selected for this work were Mometasone furoate (Meta) as API, 4-dimethylaminopyridine (DMAP) and methyl p- toluenesulfonate (MPTS) as GTIs. During the synthesis of Meta, sulfonyl chlorides are used in a DMAP base catalyzed sulfonylation reaction (Figure 1). Figure 1 - Model compounds GTI mitigation The first strategy to ease GTIs in the production of APIs is to avoid the use and generation of GTIs by altering the synthetic route. This can be achieved by using different chemical synthesis to obtain the same API or intermediate or by optimizing the existing synthetic route. In many cases, reagents and intermediates are reactive and synthetically useful and cannot be avoided. In these cases, a Quality by Design (QbD) approach can be applied. This approach includes adjustment of parameters such as ph, temperature, reaction time and solvent matrix API purification The API synthesis includes some reaction steps intercalated with purification steps; these purification steps contribute to GTI removal, even though they are not designed for that purpose. For the specific removal of GTIs, the selection of the purification method is dependent on the chemicophysical properties of the compound, such as reactivity, solubility, volatility and ionisability of the GTI 14. It is necessary to guarantee that during the purification steps, the API losses are not significant; another scenario unacceptable at industrial scale. It is important to select a purification process highly selective to a specific impurity, so the API losses are lower and the removal efficiency of the impurity is higher. 3

4 Some of the conventional purification steps include crystallization, precipitation, solvent extraction, column chromatography, treatment with activated carbon, resins as well as distillation. The efficiency of the separation is based on the differences in the properties of the agents to be separated and/or their relative affinities for a selective agent. During the last decade some other techniques, such as membrane separations or molecularly imprinted polymers, have been developed Materials and Methods 2.1. Materials DMAP was purchased from Sigma-Aldrich, MPTS was purchased from Acros Organic and Meta was kindly provided by Hovione. Amberlite resins were purchased from Sigma- Aldrich, AG 50W-X2 resin was purchased from BioRad and activated charcoal was purchased from Merck Recrystallization The recrystallization process was based on the WO patent. 50 ml of a solution having ppm of Meta and 1000 ppm of DMAP in DCM was concentrated under reduced pressure to 5 ml. 10 ml of MeOH were added, the solution was heated to 50ºC and the mixture was concentrated to 5 ml. This procedure was repeated twice and precipitation occurred. The solution was cooled to 20ºC over 1 hour, cooled further to 10ºC and agitated for 2 hours. The Meta was filtered and washed 2 times with MeOH cooled to 10ºC. 0,3 g of charcoal, 10 ml of MeOH and 10 ml of DCM were added to the wet cake. The API was dissolved at 50ºC followed by filtration of the charcoal. The filtration equipment was rinsed twice with 2 ml of DCM. This solution was combined with the API solution and concentrated under reduced pressure to 5 ml. 10 ml of MeOH were added and the solution was once again concentrated under reduced pressure to 5 ml. The mixture was cooled to 23ºC over 1 hour, cooled further to 10ºC and agitated for 2 hours. The Meta was filtered and washed 2 times with MeOH cooled to 10ºC and dried in an oven at 70ºC for 24 hours. This procedure was repeated twice with cooling the solution to 4ºC in the crystallization steps Resins screening Ionic exchange resins (AG 50W-X2, Amberlite CG400, IRA458, IRA68, IRC50, IRC 86, XAD16 and XAD7) and adsorption systems (activated charcoal) may be used to purify the washing solutions from the recrystallization procedure. The influence of ph and temperature in the adsorption process was studied, as well as adsorption isotherms and kinetics. Since the solvent in washing solutions is MeOH and the resins are prepared to be used in aqueous solutions, the following procedures were made using aqueous solutions of DMAP, then using a mixture of water and MeOH (1:1) and finally MeOH. 20 mg of the scavenger resins were put in contact with 4 ml of DMAP solutions with ph values between 12 (DMAP completely deprotonated) and 6 (DMAP completely protonated); these solutions were stirred for 24 hours and then assayed by HPLC-UV. The same was done to study the temperature influence; in this case, the solutions were stirred for 24 hours at 25ºC, 35ºC and 45ºC. To obtain the adsorption isotherms, 20 mg of the selected resins were completely dispersed in 4 ml of DMAP solutions at concentration of 100, 250, 500, 750 and 1000 ppm. Then the solutions 4

5 were continuously agitated for 24 hours at a desired temperature so that the adsorption reached equilibrium. The resin was separated from the solution and the residual content of DMAP was determined by HPLC-UV. The kinetic experiments were identical to the isotherm experiments, while 1 ml of the sample was taken at defined time intervals. The same procedure was followed to study MPTS adsorption onto resins. In this case only two ph values were used (1,18 and 9,67) since a flow of dry nitrogen. The polymerization tubes were sealed and the polymerization occurred at desired temperature. After the polymerization was completed, the polymers were crushed using a pestle and mortar. The template was extracted in a Soxhlet-apparatus with a solution of 0,1 M HCl in MeOH for 48 hours. The remaining acid was washed out with MeOH using a Soxhlet-apparatus for 24 hours. Then the polymers were crushed again and sieved; the fraction µm was used to evaluate the MPTS pka value is -2,58. binding properties of the polymers. The 2.4. MIPs synthesis polymers were dried in the oven overnight at 40ºC. The same fraction was used for the The polymers composition can be found in Table 1. DMAP was used as template, methacrylic acid (MAA) was used as monomer, ethylene glycol dimethacrylate (EDGMA) was characterization of the scavengers. The nonimprinted polymer was prepared in the same way as described above, but without the template molecule. To study the binding used as cross linking agent and properties of the different scavengers prepared, azobisisobutyronitrile (AIBN) was used as initiator. MAA was dissolved in DCM, which works as porogen. The template was added to the MAA solution and left for 5 minutes. 25 mg and 50 mg of the polymer was added to solutions of 100 and 1000 ppm DMAP in DCM. These solutions were stirred for 24h at 60 rpm. The scavengers were separated from the EGDMA and AIBN were added to the solutions, and the DMAP concentration in polymerization solution, which was purged with solution was determined by HPLC-UV. Table 1 Polymer composition and polymerization conditions. Reaction Template (mmol) Monomer (mmol) Cross-linker (mmol) Initiator (mmol) Polymerization conditions MIP % 65ºC for 24 hours MIP % 40ºC for 12 hours, then the MIP % temperature was increased MIP % (5ºC/30 minutes) to 65ºC for NIP % an additional 3 hours The isotherm adsorption was determined as described above for resins. In this case, 75 mg of the scavenger was added to 1,5 ml of DMAP solutions at concentration of 20, 50, 150 and added to 1,5 ml of a solution of 1000 ppm DMAP and ppm Meta in DCM. After the solution was stirred at 60 rpm for 24 hours, it was assayed by HPLC-UV. 250 ppm. To confirm that Meta was not adsorbed by the polymers, 75 mg of MIP was 5

6 GTI (mg) GTI (mg) API (mg) API (mg) 3. Results and discussion 3.1. Recrystallization GTI limits in APIs are calculated by the TTC divided by the maximum daily dose (g/day) giving the limit in ppm applied to the active substance. Considering a 5 mg/day dose of Meta the GTI is required to be controlled under 300 ppm (0,3 mgdmap/gmeta). Having a poststream reaction containing ppm of Meta and 1000 ppm of DMAP (100 mgdmap/gmeta), it is necessary to remove more than 99,7% of the GTI. The recrystallization shows a high API loss without acceptable compensation in the API purity achieved. This process is commonly used to purify APIs in the pharmaceutical industry due to the fact that it removes unwanted solvent occlusions from the API and allows the control of particle size. The largest fraction of API loss was observed in the charcoal adsorption, representing 53,32% (recrystallization at 10ºC) and 78,86% (recrystallization at 4ºC) of the total API loss over the three steps. When the recrystallization procedure was performed at 10ºC, DMAP was almost all removed in the first recrystallization. When these steps were performed at 4ºC, DMAP removal could not be assigned preferentially to any of the steps. This may be due to the fact that at higher temperatures, the crystallization occurs at a slower rate. It was not possible to obtain a solution with 0,3 mgdmap/gmeta after recrystallization. Meta was mostly lost during charcoal adsorption and through mother liquors. It is necessary to find a way to purify Meta lost during this process Recovered API API loss in washing solutions Another API losses Step Recovered API API lost in washing solutions Another API losses Step Another GTI losses GTI removed in washing solutions Recovered GTI Step Another GTI losses GTI removed in washing solutions Recovered GTI Step Figure 2 API lost and GTI removed during recrystallization procedures (in the left pictures the recrystallization steps were made at 10ºC and in the right pictures at 4ºC) 6

7 % binding 3.2. MIPs It was not possible to achieve a ratio of 0,3 mgdmap/gmeta using recrystallization. Since most of Meta is being lost during charcoal adsorption, this adsorbent may be replaced with MIPs. MIPs are highly specific for a given molecule. The DMAP binding percentage was determined for all the prepared MIPs. 100 Figure 3 DMAP adsorbed by MIPs prepared. The best results were obtained when using 50 mg of polymer in contact with a 100 ppm DMAP solution. 0 MIP4 MIP3 MIP2 MIP1 100 ppm 50 mg 100 ppm 25 mg 1000 ppm 50 mg 1000 ppm 25 mg The amount of template and cross-linker added to the polymerization reaction affects the binding properties. Increasing the amount of cross-linker, the binding increases from 71% to 93%. This agent fixes the functional groups of MAA around the imprinted molecule and forms a highly cross-linked rigid polymer. When the template is removed, the polymer has some cavities complementary to the target molecule. If the cross-linker content is low, the cross-linking degree is smaller and, consequently, the polymer cannot maintain a stable cavity configuration. The template:monomer ratio also affects the amount of GTI adsorbed. For a ratio 1:4, 93% of DMAP is removed whilst for a ratio 4:4 a lower amount of GTI is adsorbed (66%). This ratio influences the number of binding sites available. The polymerization conditions also influence the binding properties. When the polymerization occurs entirely at 65ºC the quantity of DMAP adsorbed is lower (MIP1) than when a temperature gradient is used. In the latter case, the polymeric chains were formed conveniently, creating binding cavities for molecular recognition. MIP4 revealed the best performance for GTI removal. NIP4, which was prepared like MIP4 but without the template, removed 79% of DMAP. The Freundlich isotherm is the best fit for the adsorption isotherm determined for MIP4, which suggests a multilayer adsorption. The charcoal adsorption stage is very critical, since a great amount of API is lost during this procedure. Since MIPs show good stability in DCM and are effective in GTI removal, they can be used to replace charcoal. Starting with 1,5 ml of a solution of 100 ppm DMAP and ppm Meta and adding 75 mg of MIP4, it is possible to remove 98% while losing 9,65% of Meta. Using data from the isotherm adsorption, it is possible to conclude that MIPs allow to lower the ratio from 74,24 mgdmap/gmeta to 4,79 mgdmap/gmeta Ionic exchange and adsorbent resins During recrystallization at 10ºC, it is possible to remove more than 97% of DMAP, but there s a great amount of Meta that is lost within mother liquors from recrystallization. Ionic exchange resins and adsorbents may be used to remove DMAP from mother liquors DMAP in water DMAP was successfully removed from the solution using cationic exchange resins and activated charcoal. The resins AG 50W-X2 (98%), Amberlite IRC50 (93%), Amberlite IRC86 (98%) and activated charcoal (91%) were capable of removing DMAP from the solution. DMAP was more efficiently removed when it was fully deprotonated (higher ph values). The temperature did not influence the adsorption process. After studying the ph and temperature effect, the adsorption isotherms were obtained. Freundlich isotherm was 7

8 % of GTI removal and API losses the best model to describe the adsorption by activated charcoal. This model is based on multilayer adsorption on heterogeneous surfaces. The ionic exchange resins follow the Sips model, which is a combination of the Langmuir and Freundlich isotherms. The kinetic studies were performed with AG 50W-X2 resin. The adsorption capacity of the resin increases rapidly with increasing of time until the equilibrium. 5 minutes is enough to reach equilibrium DMAP in water and MeOH (1:1) When MeOH is added to the solution, the binding capacity of the resins lowers, especially with activated charcoal (60%). AG 50W-X2, Amberlite IRC50 and Amberlite IRC86 were able to remove 97%, 92% and 95% of DMAP, respectively. Activated charcoal is able to adsorb MeOH, which explains the decrease of the amount of DMAP adsorbed by charcoal. It was also observed that the amount of DMAP adsorbed when the ph of solution is high (12,94) decreases abruptly. Possibly, ionic species are formed and these species compete with DMAP to bind to resins. The temperature influenced the adsorption process, which may be due to the fact that the temperature changes the equilibrium constants. After determining the adsorption isotherms, it was observed that the Sips model was the best fit. The kinetic studies were performed with AG 50W-X2. The adsorption capacity of the resin increases rapidly with increasing of time until the equilibrium. 1 minute is enough to reach equilibrium DMAP in MeOH When MeOH is the only solvent, the quantity of DMAP adsorbed lowers. AG 50W-X2, Amberlite IRC50 and Amberlite IRC86 removed 90%, 66% and 64% of DMAP, respectively. Activated charcoal only removed 17% of DMAP; as stated before, this may be due to the fact that this adsorbent is able to adsorb MeOH. Once again, it was observed that at highest ph value (12,05), the DMAP adsorbed decreases substantially, which suggests the formation of ionic species competing with DMAP to bind to the resins. It was also observed that temperature does not affect the adsorption process. The Freundlich isotherm is the best fit for the adsorption isotherm obtained for AG 50W-X2 and Amberlite IRC86, while Sips isotherm describes more properly the adsorption of Amberlite IRC50. Once again, the adsorption capacity of AG 50W-X2 increases rapidly over time, but in this case, 2 hours were necessary to reach equilibrium. Using AG 50W-X2, it is possible to purify the mother liquor from recrystallization 1; a solution with 29,28 mgdmap/gmeta was obtained. Since this ratio is lower than 100, this solution could be fed again to the recrystallization process OSN This study was based on a theoretical model. The data used was based on information available from the membrane GMT-oNF-2, which shows a good stability in DCM. This membrane retains Meta effectively (99,1%), while DMAP can cross the membrane easily (16,5%). 100,00 95% GTI removal 80,00 60,00 40,00 20,00 20% API losses 0, Dilution ratio Remoção de GTI Perdas de API Figure 4 GTI removal and API losses using OSN. 8

9 OSN is very effective in the removal of DMAP. GTI removals superior to 95% can be achieved at the cost of only 2,69% Meta loss at diavolume 3. It is possible to lower the ratio from 100 mgdmap/gmeta to 0,16 mgdmap/gmeta at diavolume 6. However, the higher the number of diavolumes, the higher the API loss and solvent consumption and operation time. It is possible to use a lower number of diavolumes and then feed the mother liquor back to the process and use MIPs to purify the API retained in the membrane. For that purpose, it is possible to dissolve the retained compounds in DCM and put the solution in contact with MIP4 in order to remove DMAP from the solution. Using this approach it is possible to lower the ratio from 100 mgdmap/gmeta to 0,33 mgdmap/gmeta at diavolume 2 if an additional step of MIPs adsorption is performed. process shows a high API loss without compensation in the API purity achieved, therefore this process is not cost effective. Since this is the purification process approved in the manufacture of APIs, it is necessary to find alternatives to increase the recrystallization yield. Ionic exchange resins may be used to purify Meta lost in the mother liquors, while MIPs are a good alternative to replace charcoal in the adsorption step. OSN is another purification process that can be used instead of recrystallization. OSN requires 6 diavolumes to remove 99,85% of the GTI with acceptable API losses (5,28%). Adding a step of MIPs adsorption, only two diavolumes are required to obtain a solution with 0,33 mgdmap/gmeta. MPTS can be removed from solution using ionic exchange resins and adsorbents. Amberlite IRA68 is very efficient in the removal of MPTS MPTS mitigation 5. Acknowledgements MPTS in MeOH The same studies as described above for DMAP were performed with solutions of MPTS in MeOH. Only Amberlite IRA68, whose functional group is a tertiary amine, was able to remove MPTS (96%). The amount of MPTS adsorbed increases slightly with the increase of temperature, but it is not significant. The adsorption isotherm may be described by the Langmuir isotherm, which describes the formation of monolayers. The adsorption capacity of Amberlite IRA68 increases slowly with time being necessary 24 hours to reach equilibrium. 4. Conclusions It was not possible to reduce the GTI in API post reaction streams to levels below the recommend TTC value using recrystallization. This FCT Fundação para a Ciência e Tecnologia for funding through the project PTDC/QEQ- PRS/2757/2012, Removal of Genotoxic Impurities from Active Pharmaceutical Ingredients, and Hovione for supply of API used. To IST, FFUL and FCT-UNL team members that participated in this project. 6. References 1. (a) Székely, G. S., Miriam; Ferreira, Frederico C.; Gil, Marco; Heggie, William, Sources of genotoxic impurities in drug synthesis: a systematic review. p 115. To be submitted; (b) Woo, Y.-t. L., David Y., (Q)Sar Analysis Of Genotoxic And Nongenotoxic Carcinogens: A State- Of-The-Art Overview. In Cancer Risk Assessment: Chemical Carcinogenesis, Hazard Evaluation, and Risk Quantification, Wiley, Ed. 2010; p (a) Benigni, R.; Bossa, C., Chemical reviews 2011, 111 (4), ; (b) Zeiger, E., Environmental and Molecular Mutagenesis 2004, 44 (5),

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