A Generic Industry Approach To Demonstrate ... - ACS Publications

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Organic Process Research & Development

A generic industry approach to demonstrate efficient purification of potential mutagenic impurities in the synthesis of drug substances Nevenka Lapanja,*,† Borut Zupančič,† Renata Toplak Časar,† Damir Orkič,† Matjaž Uštar,† Astrid Satler,† Sabina Jurca† and Bojan Doljak ‡ †

Lek Pharmaceuticals d.d., Verovškova 57, 1526 Ljubljana, Slovenia

‡

Faculty of Pharmacy, Aškerčeva 7, 1000 Ljubljana, Slovenia

Corresponding Author *E-mail: [email protected] Address: Lek Pharmaceuticals d.d., Verovškova 57, 1526 Ljubljana, Slovenia Phone number: +386 1 5803443

ACS Paragon Plus Environment

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TABLE OF CONTENTS GRAPHIC

Cl

N H HCl

Cl

Cl NO2

S N NH HCl


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ABSTRACT: Determination of theoretical purge factors for the evaluation of risk of carryover of potential mutagenic impurities (MIs) into the final active pharmaceutical ingredient (API) has been discussed as a possible approach to demonstrate efficient purification of potential MIs (Substances I, II, III and IV) in the synthesis of the vortioxetine drug substance. Theoretical purge factors for the four potential MIs were determined based on the physicochemical properties of an MI in relation to processing conditions. Compared to depletion studies of I and III, the calculated purge factors were very conservative in predicting impurities reduction. However, even a conservatively calculated purge factor correctly predicted high purging capability of the process to eliminate substance I. This novel approach could help pharmaceutical companies to focus on those impurities that are more likely to be carried over into the final API thus obviating the use of analytical testing where not necessary.

KEYWORDS: drug substance, impurities, mutagenicity, purge factors


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INTRODUCTION: Increased concern has been expressed over the last decade about mutagenic impurities (MIs) and their possible presence in pharmaceuticals. Because of their DNA reactivity they can induce genetic mutations and have the potential to cause cancer. To limit potential carcinogenic risk, such impurities have to be properly controlled. By using Threshold of Toxicological Concern (TTC) approach which defines a limit of 1.5 µg/day that is associated with an acceptable risk, industry can avoid unnecessary toxicological testing and safety evaluations.1 However, to prove that potential MIs are controlled in line with the limits, analytical data have to be presented in most cases. Since the limits for MIs are very low, developing a reliable analytical method with such a low quantification limit (1 ppm or less) is often a major issue.2 Moreover, the efforts of the pharmaceutical industry to limit such impurities are associated with additional financial cost.3 Therefore more and more attention is being paid to the development of an approach that would enable unnecessary analytical testing to be avoided but still provide a satisfactory evidence for the absence of MIs above determined limits. In June 2014 the ICH M7 guideline: Assessment and control of DNA reactive (mutagenic) impurities in pharmaceuticals to limit potential carcinogenic risk4 reached Step 4 of the ICH process and so became recommended for adoption to the three regulatory parties to ICH. The purpose of this guideline is to provide a practical framework that is applicable to the identification, categorization, qualification, and control of MIs. This guideline describes four potential approaches to the development of a control strategy for a drug substance. While options 1, 2 and 3 require inclusion of a test for the impurity in either the drug substance specification or in the specification for a raw material, starting material or intermediate, option 4 offers the possibility of not including an impurity in any of the specifications. This last control strategy relies on understanding the process parameters and their impact on residual impurity levels


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(including fate and purge knowledge) with sufficient confidence that the level of the impurity in the drug substance will be below the acceptable limit, such that no analytical testing is required. According to the guideline, a scientific risk assessment based on physicochemical properties and on process factors that influence the fate and purge of an impurity - including chemical reactivity, solubility, volatility, ionisability and any physical process step designed to remove impurities - can be used to justify an option 4 approach. The result of such a risk assessment could be expressed as an estimated purge factor for clearance of the impurity by the process.4 This concept was presented in detail by Teasdale et al.5 in 2010. In order to assess the carryover of potential MIs into API, AstraZeneca developed a tool based on the assessment of key physicochemical properties of the substance in question, relating them to the downstream processing conditions. A score is assigned for each of the physicochemical parameters (reactivity, solubility, volatility, ionisability and any physical process designed to remove impurities) (Table 1). These are then multiplied together to give a purge factor for each stage of the process. Multiplying the purge factors for individual stages yields an overall purge factor. In 2013 Teasdale et al.6 published further and more extended information on purge factor calculation, presented together with various risk assessment case studies. Following these publications, Elder et al.7 used the same approach to assess the ability to purge impurities in the synthesis of pazopanib hydrochloride. According to their results the tool very accurately predicted the purging capacity for the most reactive MIs. For the other, less reactive, MIs, measured and predicted values agreed reasonably well. Evidently from published data5-7, originator drug companies have made a very large contribution to the development of alternative approaches to the assessment of potential MIs. Since the generic industry is also facing difficulties when proving the efficient control of MIs, a tool that could reduce the need for


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analytical testing would be very welcome. Although the use of generic drugs is increasing, the recipe for success in generic industry is far from straightforward. Strong competition and low prices demand constant process optimization and improvement. The use of starting materials, reagents or intermediates with a mutagenic potential is often difficult to avoid since most syntheses involve use of electrophilic (alkylating) agents that are likely to react with DNA.5 When addressing the issue of potential MIs in the generic industry, guidelines on genotoxic impurities (GTIs) have to be followed. All actual and potential impurities have to be assessed and evaluated for mutagenic potential by using (Quantitative) Structure-Activity Relationships ((Q)SAR) methodologies. After implementation of the ICH M7 guideline4 the use of two complementary (Q)SAR methodologies (one expert rule-based and the other one statisticalbased) will be required. For any impurity with mutagenic potential control action would have to be taken, as proposed in the ICH M74. Scientific justifications implying the absence of MIs in the final drug substance have already been used for regulatory submissions, but the regulatory acceptance of the justifications was not always positive and the analytical data had to be presented instead. However, scientific arguments could be more acceptable to regulators if the quantitative or semi-quantitative element were included in the assessment.6 Promising results and potential benefit to the industry encouraged us to apply the semi-quantitative risk assessment tool presented by Teasdale et al.5 to the assessment of principal potential MIs in the synthesis of a developmental vortioxetine drug substance. Vortioxetine is a drug substance used for treating major depressive disorder and generalized anxiety disorder. It was developed by Lundbeck A/S and received its first global approval for the treatment of major depressive disorder in the USA in September 2013.8


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METHODOLOGY: The same approach as that described by Teasdale et al.5 has been used for assessing the presence of four potential MIs in the vortioxetine synthetic process, with the exception of one minor modification regarding the physical process parameter. For each MI we determined a score for following physicochemical-properties at each step of the synthesis: reactivity, solubility, volatility, ionizability, together with recrystallization as an additional physical process designed to eliminate impurities. The solubility term in our assessment relates to the solubility of the impurity of concern during the isolation process whereby, if impurity is freely soluble in the process solvent, it will remain within the solvent when the product is filtered. In addition to the isolation processes, recrystallization is performed in the stage 3a of the synthesis. According to Teasdale et al.5 recrystallization would be described within the solubility parameter. However, we designed and introduced a step of recrystallization into the synthesis of the vortioxetine drug substance in order to eliminate any residual process impurity, so we have included it within the physical process parameter (Table 1). The theoretical purge factors for the individual stages were multiplied together to obtain overall purge factors for the four MIs. The theoretical purge factors were then compared with measured values and with the results of depletion studies of MIs during the various stages of the synthetic process. The results are presented in Tables 3-5 and 7.


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Table 1. Physicochemical parameters and associated purge factors (adapted from Teasdale et al.5). Copyright 2010 American Chemical Society. Physicochemical parameter

Purge factors

reactivity

highly reactive = 100 moderately reactive = 10 low reactivity/unreactive = 1

solubilitya

freely soluble = 10 moderately soluble = 3 sparingly soluble = 1

volatility

boiling point > 20 °C below that of the reaction/process solvent = 10 boiling point ± 10 °C that of the reaction/process solvent = 3 boiling point > 20 °C above that of the reaction/ process solvent = 1

ionisability

ionisation potential of MI significantly different from that of the desired productb

physical processes - present = 3 recrystallization absent = 1 a

This relates to solubility within the context of an isolation process whereby the impurity in question, if highly soluble, will remain within mother liquors and hence be purged from the desired product. b This relates to a deliberate attempt to partition the desired product/GI between an aqueous and organic layer, typically achieved by manipulation of the pH to change the ionized/unionized state of one of the components.

DISCUSSION: Vortioxetine drug substance is prepared by a 3-step synthetic route as presented in Scheme 1.


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Scheme 1. Synthesis of vortioxetine drug substance

In the first step of synthesis 2-chloronitrobenzene (I) is used as the starting material and in the third step of synthesis reagent bis (2-chloroethyl)amine hydrochloride (III) is introduced to form the piperazine ring in the 2-((2,4-dimethylphenyl)thio)aniline (IV) intermediate. All three substances, I, III and IV, are potential MIs, but with different points of formation/ introduction to the synthetic process. In addition to the mutagenic potential of III itself, other potential MIs can be formed due to its presence in the reaction. Since III and IV are formed/ introduced at the final coupling stage of synthesis, they theoretically pose higher risk of being carried over into the final drug substance. 3-Chloronitrobenzene (II) and 4-chloronitrobenzene are also potential MIs that


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can be present as impurities in the starting material 2-chloronitrobenzene (I). Because of the different positions of the substituent (ortho-, meta- and para- position), the three aromatic compounds do not have the same reactivity properties. While 2- and 4-chloronitrobenzene react by the same mechanism, significantly lower reactivity during the same reaction conditions is observed in the case of 3-chloronitrobenzene. Because of the similar properties of 2- and 4chloronitrobenzene it is to be expected that the presence of both can be predicted by estimating the purge factor of 2-chloronitrobenzene (I) alone. For this reason, only 2-chloronitrobenzene (I) and 3-chloronitrobenzene (II) have been included in the assessment of purge factors. As already noted, other potential MIs can be formed during the synthesis of vortioxetine drug substance; however, only the four principal ones (I, II, III and IV), that are introduced directly into the synthesis or formed as reaction intermediates, are discussed here. The four potential MIs are presented in Table 2, with their chemical names, structures, CAS numbers and mutagenicity data. Although the Ames test has been reported to be negative for II, we have included it in the assessment on the basis of its structural alert that is also present in I. A theoretical purge factor for II has been calculated to show the impact of reactivity parameter on the purge factor determination; however, experimental data is not presented. A theoretical overall purge factor of 8.1 x 106 has been determined for I that is introduced into the first stage of synthesis (Table 3). The purge factor is dominated mainly by the high reactivity of I under given process conditions. Empirically it was shown that the reaction between I and 2 occurs immediately and completely, thus a value of 100 was used to describe the reactivity of I in stage 1. Furthermore, any residual I could be reduced in stage 2 by the reducing agent Fe. Based on the reported negative bacterial mutagenicity assay9 for 2-chloroaniline, an amino analogue of I formed at this stage, we have classified it as a Class 5 impurity (non-mutagenic) (according to the classification proposed by


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Müller et al.10 and the ICH M7 guideline4). Reaction monitoring showed that reduction of I goes to completion, however, the reduction reaction is only moderately fast and takes 16 hours to complete. For this reason we used a value of 10 to describe the reactivity of I in stage 2. Residual I potentially present in stage 3 could react with IV in the aromatic nucleophilic substitution reaction of chloronitrobenzenes with amines; however, it would not react with III. The reactivity of I with IV was examined experimentally. Reaction was carried out under the same conditions as reaction between III and IV. After 1 hour 37 % of product was detected in the reaction mixture. Subsequent monitoring has shown that the reaction did not go further, i.e. equilibrium was achieved at 37 % of product formed. Since the reaction does not go to completion, a reactivity value of 1 was used for I in stage 3a. Additional purging of impurities is expected due to the isolation of intermediates in all three stages of the synthesis. When determining theoretical purge factors, we included isolation steps within the solubility parameter, as proposed by Teasdale et al.6. Although I is freely soluble in the process solvent DMSO, under the reaction conditions, its solubility decreases during the isolation step during which the product is filtered after the addition of water to the reaction mixture. A more conservative factor of 3 was therefore used to describe the solubility of I in stage 1 (isolation). In stage 3a, the crude 6 is recrystallized in order to remove any residual impurity not eliminated in earlier steps. Teasdale et al.6 described the recrystallization process within the solubility parameter. However, we decided to describe the recrystallization process by physical process parameter since it is introduced in addition to already present isolation steps (precipitation followed by filtration) and has been designed for purification purposes. When assigning the value for the recrystallization process, a value of 3 was used if recrystallization is present and 1 if not. In this case a scale from 1-10 could be used, as in the case of solubility, but


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we decided to use more conservative values in order to avoid overestimation. The recrystallization process introduced in stage 3a is the principal point of impurities purification in presented synthetic process. For this reason potential impurities would be controlled in the recrystallized vortioxetine hydrochloride (compound 6). In the subsequent stage (stage 3b) of synthesis vortioxetine hydrochloride is further transformed to the final vortioxetine drug substance. However, this stage is of minor significance with regard to the impurities purification and the purge factor calculation for this stage was included here only for representative purpose. The limit for any GTI present in vortioxetine drug substance is 75 ppm (based on the TTC limit of 1.5 µg/day and considering a daily dose of 20 mg), which means that a purge factor of 100,000 would indicate the ability of the process to reduce the level of GTI to 75 ppm.


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Table 2. Chemical names and structures, CAS numbers and mutagenicity information for potential MIs in the synthesis of vortioxetine drug substance Impurity of Chemical name and structure concern

CAS number

Mutagenicity data

I

88-73-3

DEREK Nexus 4.1.0 and SARAH Nexus 1.2.0 (Lhasa Ltd.) prediction: Derek: Aromatic nitro compound alert Sarah: Positive prediction (100% - supported by exact match from training set) Other genotoxicity data (literature data): Positive Ames test in Salmonella typhimurium strain TA98, Ta100 and E.coli WP2UVRA with metabolic activation Positive carcinogenicity studies11

II

121-73-3

DEREK Nexus 4.1.0 and SARAH Nexus 1.2.0 (Lhasa Ltd.) prediction: Derek: Aromatic nitro compound alert Sarah: Negative prediction (100% - supported by exact match from training set) Other genotoxicity data (literature data): Negative Ames test12, 13


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III

821-48-7

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DEREK Nexus 4.1.0 and SARAH Nexus 1.2.0 (Lhasa Ltd.) prediction: Derek: Alkylating agent and Nitrogen or sulfur mustard alerts Sarah: Positive prediction (100% - supported by exact match from training set for Bis(2-chloroethyl)amine) Other genotoxicity data (literature data): Positive Ames test14

IV

1019453-85-0

DEREK Nexus 4.1.0 and SARAH Nexus 1.2.0 (Lhasa Ltd.) prediction: Derek: No alerts Sarah: Positive prediction Other genotoxicity data (literature data): No literature data.


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Table 3. Theoretical purge factor calculation for potential MIs in the synthesis of vortioxetine drug substance MI of concern

Synthetic stage

Reactivity (R)

I

Stage 1

100

Stage 2

II

III

IV

Solubility (S)

Volatility (V)

Ionisability (I)

3

1

1

1

300

10

3

1

1

1

30

Stage 3a

1

3

1

1

3

9

Stage 3b

10

10

1

1

1

100

Overall purge factor

8.1 x 106

(Isolation)

Physical processes (Pp) (Recrystallization)

Theoretical purge factor

Stage 1

1

3

1

1

1

3

Stage 2

10

3

1

1

1

30

Stage 3a

1

3

1

1

3

9

Stage 3b

1

10

1

1

1

10


8100

Stage 3a

10

1

1

1

3

30

Stage 3b

10

1

1

1

1

10


300

Stage 3a

10

10

1

1

3

300

Stage 3b

1

10

1

1

1

10


3000


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The actual level of I was measured during kilo-lab experiments in intermediate 3 and isolated intermediate IV. The level of I depleted from 1.6 % present in 3 to < QL (5 ppm) in isolated IV, which means that the level of I is already significantly reduced in stage 2. During the development of the synthetic process depletion studies were additionally conducted to show elimination of impurities during the isolation and recrystallization steps introduced into the synthesis. A certain amount of I was spiked in stage 3a and stage 3b and its level was measured after the isolation/recrystallization. The results are presented in Table 4. Table 4. Depletion of substance I during the stages of synthesis Stage

Concentration of I before isolation/ recrystallization [%]

Concentration of I after isolation/ recrystallization [ppm] a

Depletion [%]

Purge factor

Stage 2

1.6a

< QLc

˃ 99

3200

Compound 3

Isolated compound IV

2.0b

< QLc

˃ 99

4000

Crude compound 6

Recrystallized compound 6

1.9b

< QLc

˃ 99

3800

Compound 6

Isolated drug substance

Stage3a

Stage 3b

Overall purge factor 4.9 x 1010 a

Determined by the analytical methods presented in Supporting information.

b

Based on the amount of the impurity spiked in each synthetic stage separately.

c

QL is 5 ppm

Results show more than 99 % depletion of impurity I during the isolation (Stage 2 and Stage 3b) and recrystallization processes (Stage 3a). The overall purge factor calculated from the results of depletion studies is 4.9 x 1010. If comparing the theoretical value and the value based on


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depletion studies (8.1 x 106 versus 4.9 x 1010), we can see that the calculated purge factor underpredicts the purge capacity of the process by a factor of 6000. Under-prediction is especially significant in the case of recrystallization step (theoretical value of 9 versus 4000). Even a conservatively calculated purge factor predicts the ability of the process to significantly reduce the level of I, therefore it is proposed that the risk assessment tool based on purge factor calculation could be used to evaluate the risk of substance I being carried over into the final drug substance. The theoretical purge factor for substance II (8100) is clearly lower than that for I. This difference was expected because of the significantly lower reactivity of II under the given process conditions. The reactivity of II in stage 1 was examined empirically. Under the same reaction conditions no reaction products were observed. Moreover, the reaction did not occur even upon heating and after carrying out overnight. For this reason a value of 1 was used for reactivity of II in stage 1. Low reactivity is also related to low mutagenic potential which is reflected in a negative Ames test result.12, 13 According to the ICH M74 and EMEA’s document Questions and answers on the 'Guideline on the limits of genotoxic impurities'15, the negative Ames test overrules the structural alert and no further studies are required, providing the level remains below ICH Q3A/B limits. Thus, actual values and depletion of II was not examined as in case of I, III and IV. For III, a theoretical purge factor of 300 was calculated. III is introduced in the last stage of synthesis and has a moderate reactivity under the given process conditions. Even though the isolation process is present, the impurity cannot be purged from the desired product since its solubility in the process solvent is very low. However, it can be eliminated through the recrystallization process. The level of III in crude 6 has been measured by the analytical method presented in Supporting information and the results show that it is present at a level of 1036 ppm. Depletion studies have also been conducted by spiking III in stage 3a and stage 3b and


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measuring its level after the isolation/recrystallization. The results (Table 5) show significant depletion of III during both stages of synthesis.

Table 5. Depletion of substance III during the stages of synthesis Stage

Concentration of III before isolation/ recrystallization [%] a

Concentration of III after isolation/ recrystallization [ppm] b

Depletion [%]

Purge factor

Stage3a

0.5

< QLc

˃ 99

714

Crude compound 6


1.0

24

˃ 99

417

Compound 6

Isolated drug substance

Stage 3b

Overall purge factor 297738 a

Based on the amount of the impurity spiked in each synthetic stage separately.

b

Determined by the anayltical method presented in Supporting information.

c

QL is 7 ppm.

According to the results more than 99 % depletion of III is observed during stage 3a and stage 3b of the synthetic process. The overall purge factor calculated from the results of depletion studies is approximately 1000-times higher than the theoretical purge factor (300). By assigning a value of 3 for the recrystallization process we clearly under-predicted the ability of the process to eliminate impurities. Under-prediction of the purge capacity of a process was discussed by Teasdale et al.5, who recorded a particularly significant difference between predicted and measured factor in the case of the isolation stage. However, authors suggested retaining a more conservative scale in order to compensate for any variance in processes. Even though the


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calculated purge factor is very conservative in predicting substance III carry over, the presence of the impurity in the drug substance cannot be excluded based on the calculated factor of 300. Thus, a control strategy in line with ICH M74 should be considered for III. Based on its positive bacterial mutagenicity assay, III is a known mutagen. However, the carcinogenic potency or TD50 of this substance is not reported and it is therefore classified as a Class 2 substance, i.e. known mutagen with unknown carcinogenic potential. According to the ICH M7 guideline4, the TTC approach would usually be used for mutagenic impurities where no carcinogenicity data are available. However, III is an demethylated analogue of nitrogen mustard (CAS number 51-75-2) which is a known mutagenic carcinogen with TD50 in rats of 0.0114 mg/kg/day.16 Nitrogen mustard and its derivatives (chemical group of bis(ß- haloalkyl)amines) are bifunctional alkylating agents which, although widely used in chemotherapy of cancer, are themselves highly carcinogenic.17 Their reactivity is based on the formation of the highly electrophilic quaternary aziridinium ion intermediate.18 Some of the nitrogen mustard analogues with known carcinogenicity data are presented in Table 6.


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Table 6: Carcinogenicity data for some of the nitrogen mustard analogues (reproduced from Carcinogenic Potency Database16) Chemical name and structure

Chlorambucil

Cyclophosphamide

Nitrogen mustard

Prednimustine

Melphalan

CAS number

305-03-3

50-18-0

51-75-2

29069-24-7

148-82-3

Ames test

positive

positive

positive

-

positive

0.896

2.21

0.0114

19.2

0.0938

0.133

5.96

-

-

0.15

0.133

2.21

0.0114

19.2

0.0938

Harmonic Rat mean of TD50 Mouse (mg/kg/day) Most sensitive species (µg/day)

Lower value


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As presented in Table 6, TD50 values of nitrogen mustard analogues range from 0.0114 to 19.2 mg/kg/day. All substances are tertiary amines, having in common the bis (2-chloroethyl) amine fragment, but differ in tertiary N-substituent. According to the ICH M7 guideline4, a compound specific calculation of acceptable intakes may be applied for mutagenic impurities with no carcinogenicity data which are structurally similar to a known carcinogen compound class (classspecific acceptable intakes) provided that a rationale for chemical similarity and supporting data can be demonstrated. For this reason we have examined the similarity of III with nitrogen mustard and its analogues and the suitability of the TTC based control approach. Substance III, a demethylated analogue of nitrogen mustard, is a secondary amine that rather forms tertiary aziridine species than a reactive quaternary aziridinium ion as it is the case with nitrogen mustard. The tertiary aziridine formed from III lacks a permanent positive charge and is significantly less reactive than the quaternary counterpart. As a consequence, its ability to alkylate DNA is significantly lower.18 Based on the difference in the reactivity and consequently in the toxicity between the methylated and demethylated form, the control approach based on chemical similarity to a known carcinogen compound class of nitrogen mustards is not fully justified. As long as carcinogenic data for III is not available, a TTC approach seems to be more appropriate for the control of substance III. For compound IV a theoretical purge factor of 3000 was determined. Depletion of IV was studied during the recrystallization of crude 6 in stage 3a. The results (Table 7) show more than 99 % depletion and purge factor of 2000. A comparison of the theoretical value of 300 calculated for stage 3a with the results from depletion study shows that the calculated purge factor underpredicts the purge capacity of the recrystallization process by a factor of ~10.


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Table 7. Depletion of substance IV during the stages of synthesis Stage

Concentration of IV before isolation/ recrystallization [%] a

Concentration of IV after isolation/ recrystallization [%] b

Depletion [%]

Purge factor

Stage3a

1%

< QLc

˃ 99

2000

Crude compound 6


Overall purge factor 2000 a

Based on the amount of the impurity spiked in the synthetic stage.

b

Determined by the analytical method presented in Supporting information.

c

QL is 5 ppm.

CONCLUSION: The presence or possible formation of potential MIs has to be considered during drug substance synthesis development, which applies not only to the originator but also to the generic pharmaceutical industry. Of the different possible strategies, the most appropriate one has to be chosen in order to demonstrate control of impurities of concern. Based on the case study presented in this paper, the theoretical purge factor calculation approach could have an important role in identifying impurities that are more likely to be carried over into the final drug substance. The pharmaceutical companies could then focus on the impurities of significant concern and perform analytical testing only where necessary. For the potential MIs with low carryover risk, determination of a theoretical purge factor could constitute an essential part of the common scientific justifications that are based on knowledge of the process. Based on the synthetic process presented here, it is proposed to the authors of this novel approach to consider introducing recrystallization process as individual physical process designed to remove impurities. For assigning a value for recrystallization process the solubility of impurity in question should be followed as before; however, higher values or


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different scale could be considered, since the purification capability of the recrystallization process is often very high. Based on the personal communication with Teasdale19, the under-prediction of the purge factor tool in case of recrystallization process as pointed out in this paper is addressed in the latest version of the in silico purge factor tool. The tool implements recrystallization as an individual physical process designed to remove impurities. A scale from 1 to 100 can be used when assigning the purge factor for the recrystallization process.


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ABBREVIATIONS API, active pharmaceutical ingredient; GTI, genotoxic impurity; DMSO, dimethyl sulphoxide; DNA, deoxyribonucleic acid; MI, mutagenic impurity; QL, quantitation limit; (Q)SAR , (Quantitative) Structure-Activity Relationships; TTC, Threshold of Toxicological Concern.

ASSOCIATED CONTENT Supporting Information. Following contents of material is supplied as Supporting Information: Depletion of Substance I, Depletion of Substance III, Depletion of Substance IV, Analytical methods for determination of substance I, Analytical method for determination of substance III, Analytical method for determination of substance IV. This material is available free of charge via the Internet at http://pubs.acs.org.” AUTHOR INFORMATION Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡These authors contributed equally. Nevenka Lapanja,

‡

Borut Zupančič, ‡ Renata Toplak Časar, ‡ Damir Orkič, Matjaž Uštar, Astrid Satler, Sabina Jurca and Bojan Doljak


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ACKNOWLEDGMENTS We would like to thank Andreja Veskovič for providing mutagenicity data and Andrew Teasdale for the discussion on in silico purge factor tool development. We acknowledge funding of this project by Lek Pharmaceuticals d.d. which, in the collaboration with Faculty of Pharmacy, enabled the realization of this research.

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