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CHAPTER-5

INVESTIGATIONS ON GENOTOXIC IMPURITIES IN

ACTIVE PHARMACEUTICAL INGREDIENTS

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5.1 INTRODUCTION

Indeed, when safety of pharmaceutical products is of utmost

importance, it is enforced to pay particular attention to the quality of

drug substances and other raw materials used in the formulations. API

synthesis encompasses multiple reaction steps for conversion of basic

starting materials to the products. Each reaction involves reactive

intermediates, reagents, catalysts, byproducts, solvents, etc. It is

imperative to mention that no reaction under this universe is 100%

selective and specific and eventually, results in minute levels of

unintended products, which carry from one stage to the other. Some of

these may be present in low levels in the final Active pharmaceutical

ingredient (API) and drug product as impurities. A subset of these

impurities may have Genotoxic potential and are called Genotoxic

impurities.

Genotoxic impurities [178] (GTIs) are the chemical compounds that

may be mutagenic and could potentially damage DNA with an

accompanying risk of cancer [179]. In order to be biologically effective,

API should elicit physiologic response and APIs are toxic in general. In

21st Century, Regulatory authorities generally expect sponsors of clinical

trials and commercial marketing authorizations to demonstrate the

removal of potentially genotoxic impurities (PGIs) or control them to

minute levels in the ppm range.

228

There had been two incidents, necessitates change in regulatory

authorities stand point and realized the significance of GTIs. First one is

the well-publicized case of Roche‟s Antiviral molecule, Nelfinavir,

marketed under the brand name of Viracept [180a]. In another case,

wherein the drug substance was recrystallised from acetone, and the

applicant had failed to consider potential contamination of mesityl oxide

arising from this. The application was rejected by the European

Medicines Agency‟s (EMEA) during 2007 [180b].

Batches of the Nelfinavir manufactured at Roche‟s plant in

Switzerland were apparently contaminated with traces of ethyl

methanesulfonate (EMS) arising from reactor cleaning procedures,

wherein trace levels of methanol is reacted with Methane sulphonic acid

to yield EMS [181]. In terms of assessing the risk to patients, Roche has

investigated based on the toxicology of EMS and patient exposure limits

and finally, Roche had recalled all nelfinavir (Viracept) manufactured at

its Swiss plant with immediate effect in June 2007 [180]. EMS is a well

established genotoxic agent that has been used extensively as model

compound in experimental work to establish the responsiveness of the

test system under investigation and found that EMS induces DNA

damage by a direct mechanism, acting as a mono functional ethylating

agent [180].

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5.1.1 Regulatory concern

The European Medicines Agency‟s (EMEA) was the pioneering

regulatory body to impose detailed guidelines to handle GTIs which came

into operation at the beginning of 2007 [182]. The USFDA subsequently

released a draft guideline in Dec, 2008 [183]. Essentially both of these

guidelines mention the recommended approaches to deal with GTIs,

especially its control limits in the form of Threshold of Toxicological

concern (TTC), [179a] wherein 1.5 microgram per day daily intake of

impurity is considered as virtually safe dosage, while low and high limits

are case specific based on the toxic potential of a given compound.

Therefore, GTIs have to be controlled below the TTC limit. Observers

[184-186] has critically reviewed the history of the evolving guidance on

genotoxic impurities.

Process chemistry is the interplay of various challenges like Safety

(Process and substances harmful to health), Environment (substances

harmful), and Intellectual property (Patent infringement aspects),

Regulatory (Compliance with various geographies regulations),

Economics (Lowest cost), Quality (Highest quality) Robustness and

Throughput. From the decades, a Process chemist is well versed with

these challenges adequately. At this juncture, “Genotoxic impurities”

have become new ripple creators in a steady flow of Process chemistry

arena and it requires altogether a different skill set to deal with it.

230

In absence of a defined guideline for handling of Genotoxic impurities

during synthesis of APIs, a chemist should demonstrate an unique skill

set by harmonizing the chemistry, toxicology, regulatory and analytical

aspects related to minute level impurities. Hence, this part of thesis is

devoted towards our efforts to review and devise a pragmatic framework

to address PGIs during synthesis of APIs with case studies.

5.2 REVIEW OF LITERATURE

Even though vast amount of literature [178-180, 187] on “Genotoxic

impurities” and “Potential Genotoxic impurities” is available,

Pharmaceutical industry is still in evolution stage of understanding to

take up corresponding studies. Currently available ICH guidance

[188,189] that address issues related to impurities and residual solvents

include ICH Q3A(R2), ICH Q3B(R2), and ICH Q3C(R3) inadequately

covers the details on PGIs. In addition, the European Medicines Agency‟s

(EMEA) Committee for Medicinal Products for Human Use (CHMP)

published a guideline regarding limits of genotoxic impurities [190,191].

It describes an approach for assessing genotoxic impurities of unknown

carcinogenic potential or potency based on the TTC concept, also

attempts to provide guidance to industry on how to address

specifications for impurities possessing genotoxic potential, as functional

groups that render starting materials and synthetic intermediates useful

as reactive building blocks may also be responsible for the genotoxicity.

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Hence, it is important for any API to ensure and maintain differentiated

quality requirements of different regulatory bodies worldwide, and to

cater best quality product to the patients [192].

There are four important phases in dealing with PGIs, which are

Identification, classification, qualification and control methodology. The

following are some of the guidelines and tools that would be helpful to handle

these phases. (a) Muller‟s classification for identification of structural alerts of

genotoxicity [180a]. (b) EMEA/CHMP/QWP/ 251344/2006 Guidelines [190a],

(c) Software assessment for genotoxicity evaluation (Toxtree- which is based on

Crammer & Benigni/Bossa Rules and OSIRIS Property Explorer (Organic

Chemistry Portal) [193], and (d) Toxicology information tools [194] (Merck

Index, MSDS, Pharmaceutical Substances Volumes, Toxicology databases) were

used for estimation of limits of control for structural alerts. These programs

have been demonstrated to be highly predictive for genotoxicity [195,196].

Afore mentioned phases of Identification, classification, qualification

and control strategies are described herein succinctly.

5.2.1 Identification and classification of genotoxic impurities

Chemistry skills should be appended with Toxicology, Regulatory and

Analytical in each of these phases and more specifically in identification

and classification. For a given API, thorough review of synthesis details,

set of all possible impurities due to starting materials, key intermediates,

in-situ reaction products, reagents, catalysts, byproducts, and metals is

essential so as to arrive at PGIs identification. All these chemicals should

be subjected to a rigorous screening against precedent structural alerts

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[180a] and known toxic compounds [183]. Once the PGIs are identified,

the next step is to classify them across five classes as outlined below.

Impurities are classified into one of the five classes using data (either

published in the literature or from genotoxicity testing) and comparative

structural analysis to identify chemical functional moieties correlated with

mutagenicity. The five classes are as suggested by PhRMA group [197] are

mentioned in Table-5.1

Table 5.1: Classification of genotoxic impurities

Identification of class-2 and 3 PGIs can be done through thorough

screening of the process, reactants and reagents used in the synthesis of API

with the help of knowledge-based expert systems for structure–activity

relationships if the alerting functional moiety is not present in the structure of

the parent API. Further, genotoxicity also depends on the structural

constraints, chemical environment and or experimental data in the

assessment. Regulatory action should not be persuaded, only based on the

presence of alerting structure. It is important to deal the approach, case by

case and precedence data (Ames test results, closely related structures,

proprietary) should be considered to arrive at conclusion.

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Some widely recognized alerts for DNA reactivity, i.e., mutagenic

activity are depicted in Figure-5.1 (this list is not exhaustive).

DESCRIPTION OF GROUPS

Legends : A= Alkyl, Aryl, or H ; Halogen = F, Cl, Br, I ; EWG = Electron withdrawing

group(CN,CO, ester, etc.,)

1. Aromatic Groups : N-Hydroxyaryls

[Purines or Pyrimidines, Intercalculators. PNAs or PBAHs]

2. Alkyl and Aryl Groups:

3. Heteroatomic Groups:

Figure 5.1: Basic functional groups for identification of structural alerts

5.2.2 Categorization, qualification and risk assessment of impurities

After completion of identification and classification the next phases

are qualification across the classes and finally to carve out a control

strategy. A representative decision tree suggested by PhRMA‟s is

mentioned in Figure-5.2 [197].

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CLASS 5 : No alert

Not tested

EstablishedNot established

Not possible

CLASS 1 : Genotoxic

carcinogens

Eliminate impurity

Risk assessm

(Staged) TTC

CLASS 2 : Genotoxic

but unknown

Threshold mechanism

PDE(e.g. ICH Q3 appendix 2)

CLASS 3 : Alert unrelated

to parent

Impurity genotoxic ?

1

Controle as an ordinary impurity

CLASS 4 : Alert related

to parent

API genotoxic 2

Y

N

N

Limited data

Figure 5.2: Decision tree for qualification of genotoxic structural

alerts

As a result of qualification of PGIs across five classes, the control limit

for these impurities shall be between TTC and ICH limit (known impurity

at less than 0.15% and unknown impurity at below 0.10% in general) as

ordinary impurities. In most of the cases, PGIs should be controlled at

TTC level, which is dosage specific. For instance, when the maximum

daily dosage (MDD) is 100 mg per day then the calculated TTC value is

150 ppm, whereas for maximum daily dosage of 2gms per day, the

calculated TTC is 0.75 ppm. Therefore, higher the MDD, lesser the

control limits. So as to control the impurities at minute levels, some of

the key challenges encountered by a process chemist during synthesis

and Analytical chemist during estimation are illustrated below.

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The challenges encountered by a process chemist comprises; (1)

Avoiding PGIs as reagents, starting materials, synthetic intermediates

and byproducts in chemical process though an important consideration,

it is not always feasible, or desirable. (2) Some of the functional groups

that render starting materials and synthetic intermediates useful as

reactive building blocks are also responsible for their genotoxicity due to

their ready reactivity. More specifically, while designing a non-infringing

route of synthesis to overcome the patent restrictions in generic

competition, PGIs shall become road blocks (3) prevention of mesylate or

tosylate salt formation during process development would restrict the

process chemist to have leverage upon salt isolation. (4) Often it is

impossible to eliminate PGIs completely. (5) Culminates to increase in

cost and delay in development schedules.

The challenges encountered by the Analytical chemist comprises; (1)

Detection in minute (ppm/ppb) levels demands exploration of various

new and sensitive techniques. (2) Selection of Appropriate Techniques

(HPLC, LC-MS, LC-MS-MS, GC-MS, NMR, etc) which should be employed

for regular testing [198]. (3) The need for analytical measurements with

adequate selectivity and sensitivity. It shall be more difficult, in case of

non-availability of chromophores, wherein non-conventional Analytical

methods must be the choice (4) Analytical quantification studies would

be restricted because of the reactivity and instability of GTIs.(5) Each

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new sample matrix, (e.g. different intermediate or drug substance)

presents new challenges requiring additional development.

5.2.3 Genotoxic impurity control strategy

Toxicology assessment identifies genotoxic compounds in a route that

need to be addressed. A proposed control strategy for Genotoxic

impurities as a function of where they enter the synthetic scheme is

illustrated in the form of a “Decision Tree” elsewhere as shown in Figure-

5.3 [199]

Conf irmed GTI* enter Decision tree

Is GTI introduced

in f inal

step

Is GTI introduced

in penultimate

step

Is GTI introduced >4 steps

Test API and impose limit based on toxicology assessment

Found in penultimate at a level

Demonstrate absence and/or removal ef f iciency or establish specif ication for

registration

Test to demonstrate absence or rejection ef f iciency (or provide chemical rationale for

removal if possible)

Provide chemical rationale for removal

(or test, if necessary)

*Where GTI is an intermediate,reagent,observed by-product or Likely by-product

NY Y

Y

Y

NN

N

Figure 5.3: Control strategy for genotoxic impurities

Based on the structural alerts suggested by Muller et al, most of the

key reactive intermediates which are usually employed to facilitate

smooth chemical transformation are found to be PGIs and hence, it is a

bitter pill to the synthetic chemist to avoid them during synthesis.

237

Therefore, it is imperative to ensure its complete conversion during

reaction sequence or reject them using proper work up method.

Based on the stage in which PGI is introduced in a given route of

synthesis, primarily dictates the control mechanism. While final (N-stage)

and penultimate stages (N-1) introduction must be controlled in API spec

and N-2 to N-3 stages introduction should be empirically justified based

on chemical rationale supported with cutoff studies report. Based on PGI

identification, process chemist may attempt to change the route of

synthesis during scheme design which is noblest, develop a robust

downstream process to limit the impurity below the concern level, and in

case of PGI is not controlled; Ames test can be performed to confirm the

toxicology data of API. In case the toxicology testing suggests that, it is

mutagenic then control must be established and monitored by any of the

options stated above.

5.3 OBJECTIVE OF THE PRESENT WORK

Impurity profiling of APIs that include genotoxic impurities is critical

activity and these changes are making the product development

(including NCEs and Generic drugs) as well as regulatory approval of

drugs more complex. This dragged our attention towards working on

potential genotoxic impurity monitoring in few active pharmaceutical

ingredients and present as a chapter in this dissertation to help the

chemists working in the field.

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In our studies, we focused on devising a systematic frame work for

identification, classification, qualification and control strategy on PGIs

which involves identification of structural alerts, qualification of

structural alerts as genotoxic impurities and finally demonstrated their

control in the drug substance. For this, we selected the same APIs

(Lacidipine, Valacyclovir, and Ganciclovir) on which, we have already

reported our synthetic studies and process development approaches in

the previous chapters.

5.4 RESULTS AND DISCUSSION

Our attempts on control of genotoxic impurities in Lacidipine (1),

Valacyclovir (49) and Ganciclovir (73) are summarized in different

subsections as follows.

5.4.1 PGIs assessment and their control in Lacidipine (1)

During process development of lacidipine, 1 twelve process related

impurities were effectively controlled at isolation processes or purification

procedures through better understanding of their solubility variation from the

desired intermediates or drug product.

Our approach on handling PGIs in 1 started with evaluation of total

synthesis including raw materials, intermediates, catalysts, reagents,

solvents, metals, by-products, degradants etc. The screening was done

carefully by evaluating the presence of structural alerts published in

scientific literature [187a] along with data on the chemical class for

239

closely related structures, also by employing some generic rule-based

software [187b-f]. The study revealed that seven structural segments are

having potential genotoxicity as shown in Figure-5.4.

Figure 5.4: Potential genotoxic impurities during lacidipine

synthesis

5.4.1.1 Origin of genotoxic structural alerts

Of the seven identified structural alerts for genotoxicity, tert-butyl

bromoacetate (21) and o-phthalaldehyde (3) are key starting materials for

the synthesis of 1. Bromoaceticacid (138) is a starting material for the

synthesis of tert-butylbromoacetate (21) as depicted in Scheme-5.1.

Scheme 5.1: Synthesis of tert-butyl bromoacetate (21)

Tetrachloroorthoxylene (36) is an intermediate during the

manufacture of o-phthalaldehyde (3) that is generated by the

chlorination of o-xylene (35). 2-Carboxy benzaldehyde (44) is an impurity

aroused due to over chlorination followed by hydrolysis during the

synthesis of 3 (Scheme-5.2).

240

Scheme 5.2: Synthesis of o-phthalaldehyde (3).

2-Vinyl benzaldehyde 28 is an undesired product during Wittig

reaction when the decarboxy impurity, 27 reacts with 3 (Scheme-5.3).

Hence, 28 is an in situ impurity during the synthesis of lacidipine (1).

Scheme 5.3: Plausible formation of 2-vinylbenzaldehyde

Total synthesis of lacidipine (1) with an emphasis on PGIs

introduction into the synthesis is depicted in Scheme-5.4.

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Scheme 5.4: Origin of PGIs during synthesis of lacidipine (1)

5.4.1.2 Control of PGIs

As per the guidlelines, potential Genotoxic impurities should be

controlled below TTC (Threshold of Toxicology Concerned). TTC limit is

calculated by a simple methodology, which consists of dividing 1.5 (a

default safe dosage as per regulatory guidelines) with maximum daily

dosage of given drug with (Eq-1).

GTI Limit= 1.5/ Max daily dose in grams……………………….Eq-1

GTI limit for Lacidipine = 1.5/ Max Daily dose in grams.

= 1.5/0.006

= 250 ppm

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The limit as per TTC calculation for Lacidipine is 250ppm, meaning that

each structurally dissimilar PGI should not cross this limit in the API.

After deriving a limit of control, key challenge is to develop sensitive

analytical methods to monitor these impurities. The identified PGIs

(Table-5.1) are sourced from different reactants of and intermediate

stages of the synthetic process of 1 as captured in Scheme-5.4. Control

of such impurities in a multi pot synthesis is relatively an easy task due

to the advantage of having multiple isolation procedures for the

intermediates. Our approach, being a one pot synthesis, control of PGIs

is a strapping challenge. Mole ratios of each Raw material, intermediates

(1.2 moles of 21 to TPP (24) ensured maximum conversion of 21; 1.5

equivalent of 3 to 25 ensured utmost reaction completion intern

conversion of 4; 3 moles of 5 guaranteed the conversion of 4 to 1) and

isolation solvent selection (21 & 138 are primarily washed out during

isolation of 25 in Toluene; residual carryover of these impurities gets

eliminated during the final crystallization of 1 using IPA), reaction

conditions to ensure maximum conversion of input materials, work up

procedures (during witting reaction in biphasic alkaline medium,

substantial amounts of 5, 28, 44 remained back in alkaline medium

because of its high solubility in alkaline medium during product

extraction in DCM) coupled with final crystallization process using IPA

have ensured that seven PGIs are well within the TTC limit of 250ppm.

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Detailed process optimization efforts have been illustrated elsewhere in

the thesis (refer chapter 2).

The PGIs investigated in lacidipine, 1 are as follows. Table 5.2: Control of Genotoxic impurities in Lacidipine.

S. No. Impurity Chemical

name

Structure LOD

(ppm)

LOQ

(ppm)

Result

(ppm)

1♀ 2-Formyl benzoic

acid*

23 70 Below LOD

2♀ o-Phthalaldehyde*

5 15 Below LOD

3♀

(E)-3-(2-formyl

phenyl)-2-propenoic

acid-1,1-dimethyl

ethyl ester*

22 65 Below LOD

4♀ 2-Bromo acetic acid BrCH2COOH 64.4 222.2 Below LOD

5♀ 2-Vinyl Benzaldehyde*

39.9 137.7 Below LOD

6♀ Tetra chloro ortho

xylene*

48 144 Below LOD

7♂ Tertiary butyl bromo

acetate*

28 100 Below LOD

♀ 1-6: Liquid Chromatography with UV Detector.

♂ 7: Gas Chromatography with Flame Ionization Detector

* Comes under Class-3 classification

As evident by the batches trend data, PGIs were well controlled below

250 ppm to meet the regulatory requirement. Hence, the effectiveness of

the process for synthesis of 1 as mentioned in preceding sections to

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control PGIs made the toxicity confirmation testing for identified PGIs

insignificant. Hence, the option of toxicity testing for PGIs was not

preferred.

Out of 7 potential Genotoxic impurities, 3 and 44 were analyzed

using a single and all the other impurities are estimated separately.

The brief summary of supplementary case studies related to the new

and improved synthesis of valacyclovir, 49 and new synthesis of

ganciclovir, 73 is mentioned in here.

5.4.2 PGIs assessment and their control in Valacyclovir HCl (49)

The new synthesis of 49 involves reaction of imidazole (64) with

sodium azide and sulfuryl chloride to furnish 65, which is then treated

with 57 to give α-azido acid derivative of L-valine (66) as shown in

Scheme-5.5.

Scheme 5.5: Synthesis of Valacyclovir HCl (49)

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The obtained 66 is then condensed with 45 in presence of DCC and

triethylamine to obtain a novel intermediate, 67 which is subjected to

reduction with raney nickel to yield the valacyclovir freebase. The

freebase is then treated with aqueous hydrochloric acid to form

Valacyclovir hydrochloride (49) as depicted in Scheme-5.5.

This synthetic scheme was subjected to the systematic frame work

for potential genotoxic impurities and devised control strategy is

summarized herein. The calculated TTC limit is 0.75ppm (MDD=2gm)

Table 5.3: Control of PGIs in Valacyclovir HCl (49)

S. No. Chemical name Structure LOD

(ppm)

LOQ

(ppm)

Result

(ppm)

1♀

(S)-2-azido-3-

methylbutanoic

acid*

0.20 0.60 Below

LOD

2♀

(S)-2-((2-amino-6-

oxo-1H-purin-9(6H)-

yl)methoxy)ethyl2-

azido-3-

methylbutanoate*

0.70 2.10 Below

LOD

3♂ Sodium Azide*** 0.50 1.50 Below

LOD

4♀ 1H-imidazole-1-sulfonyl azide*

0.50 1.50 Below LOD

5♂ Sulfuryl chloride* SO2Cl2 0.50 1.50 Below

LOD

* Comes under Class-3 *** Comes under Class-1 ♀ 1, 2, 4: Liquid Chromatography with UV Detector.

♂ 3, 5: Ion Chromatography

In another embodiment, the improved synthesis of 49 involving

condensation of acyclovir (45) and N-carbobenzyloxy-L-valine (52, Cbz-

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L-valine) followed by de-protection of resulted N-Cbz protected

valacyclovir (53) using palladium catalyst.

Scheme 5.6: Improved synthesis of Valacyclovir HCl (49)

The key starting material Cbz-L-Valine (52) synthesis involves

protection of amino group of L-Valine (57) with benzyloxy carbonyl

chloride as mentioned in Scheme-5.6.

This synthetic scheme was subjected to the systematic frame work

for potential genotoxic impurities and devised control strategy is

summarized herein.

Table 5.4: Control of PGIs in Valacyclovir HCl

S. No. Chemical name Structure LOD (ppm)

LOQ (ppm)

Result (ppm)

1♀ Cbz-L-Valine*

0.60 1.80 Below

LOD

2♀ Valacyclovir, N-

Cbz-L-valinate*

0.70 2.1 Below

LOD

*: Comes under Class-3 ♀ 1, 2: Liquid Chromatography with UV Detector.

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5.4.3 Genotoxic impurities and their control in Ganciclovir (73)

The newly developed synthesis of ganciclovir (73) involves protection

of aldehyde group in acrolein (125) using triethyl orthoformate and

ammonium nitrate to give acrolein diethyl acetal (130) which is

subjected to oxidation using potassium permanganate to give

glyceraldehyde ethyl acetal (131) and it is further treated with trityl

chloride and Chloromethyl acetate (124) for tritylation and

acetoxymethylation respectively to give desired glycerol derivative, 133.

The intermediate 135 is obtained by the condensation of 133 with

diacetyl guanine, 78b which is synthesized by acetylating commercially

available guanine (78a) using acetic anhydride and acetic acid. In the

next step, acetyl group of 135 is deprotected under basic conditions

using sodium hydroxide and methanol to afford the deacetylated

compound 136, which is further subjected to hydrolysis under acidic

conditions to remove acetal and trityl group using the trifluoroacetic

acid to furnish the aldehyde 137 in situ upon reduction with sodium

borohydride in a tandem manner in methanol yields the final compound

ganciclovir (73) as depicted in Scheme-5.7.

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Scheme 5.7: Synthesis of Ganciclovir (73)

This synthetic scheme was subjected to the systematic frame work

for potential genotoxic impurities and devised control strategy is

summarized herein. Calculated TTC is 2.1ppm (MDD=700mg).

Table 5.5: Control of PGIs in Ganciclovir (73)

S. No. Chemical name Structure LOD (ppm)

LOQ (ppm)

Result (ppm)

1♀ Acrolein**

1.5 4.5 Below

LOD

2♂ Chloromethylacetate*

2.0 6.0 Below

LOD

* Comes under Class-3 ** Comes under Class-2

♀ 1: Liquid Chromatography with UV Detector. ♂ 2: Gas Chromatography

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5.5 CONCLUSIONS

Addressing “Genotoxic impurities” in the active pharmaceutical

ingredients which are already marketed and intended to market is an

uphill task to a process chemist. In order to offer a product to the society

which is devoid of Genotoxic impurities, as acceptable to regulatory

agencies, avoiding the usage of raw materials, reagents, solvents having

potential genotoxic alerts is the most idealistic approach. But, in real life

scenario, it would be difficult and complex activity to a chemist.

Therefore, as demonstrated with the case studies in this chapter, it is

recommended to carry out initial assessment for potential genotoxic

impurities and avoid the source of PGIs during route selection to a

possible extent and design the process to eliminate or control them with

optimal reaction conditions as per the defined limits with the help of

sensitive analytical techniques.