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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, 1049-001 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
stage1a,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 cells2. 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 backbone1a,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 nucleophilic4. Chemical
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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.
1.1. 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 registration1a. 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 GTIs5.
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 changes5b,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 it6. 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 cancer5b,5c; this risk is justified by the
anticipated health benefits for the patient in
taking the medicine5c,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
classes5a-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
patients5b.
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In 2013 the ICH M7 guideline was
published; it provides guidance on analysis of
structure activity relationships (SAR) for
genotoxicity.
1.2. 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.
1.3. 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 matrix13.
1.4. 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 GTI14. 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.
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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 developed15.
2. 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.
2.2. Recrystallization
The recrystallization process was based on
the WO9800437 patent.
50 mL of a solution having 10000 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.
2.3. 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
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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
MPTS pKa value is -2,58.
2.4. MIPs synthesis
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
used as cross linking agent and
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.
EGDMA and AIBN were added to the
polymerization solution, which was purged with
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 38-63 µm was used to evaluate the
binding properties of the polymers. The
polymers were dried in the oven overnight at
40ºC. The same fraction was used for the
characterization of the scavengers. The non-
imprinted polymer was prepared in the same
way as described above, but without the
template molecule. To study the binding
properties of the different scavengers prepared,
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
solutions, and the DMAP concentration in
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
MIP1 4 4 40 2% 65ºC for 24 hours
MIP2 4 4 40 2% 40ºC for 12 hours, then the
temperature was increased
(5ºC/30 minutes) to 65ºC for
an additional 3 hours
MIP3 1 4 10 1%
MIP4 1 4 20 1%
NIP4 0 4 20 1%
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
250 ppm. To confirm that Meta was not
adsorbed by the polymers, 75 mg of MIP was
added to 1,5 mL of a solution of 1000 ppm
DMAP and 10000 ppm Meta in DCM. After the
solution was stirred at 60 rpm for 24 hours, it
was assayed by HPLC-UV.
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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 post-
stream reaction containing 10.000 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.
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)
0
100
200
300
400
500
600
AP
I (m
g)
Step
Recovered API
API loss in washing solutions
Another API losses
0
100
200
300
400
500
600
AP
I (m
g)
Step
Recovered API
API lost in washing solutions
Another API losses
0
10
20
30
40
50
GTI
(m
g)
Step
Another GTI losses
GTI removed in washing solutions
Recovered GTI
0
10
20
30
40
50
60
GTI
(m
g)
Step
Another GTI losses
GTI removed in washing solutions
Recovered GTI
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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.
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.
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 10000 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.
3.3. 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.
3.3.1. 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
0
100
MIP4 MIP3 MIP2 MIP1
% b
ind
ing
100 ppm 50 mg 100 ppm 25 mg1000 ppm 50 mg 1000 ppm 25 mg
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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.
3.3.2. 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.
3.3.3. 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.
3.4. 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%).
Figure 4 – GTI removal and API losses using OSN.
0,00
20,00
40,00
60,00
80,00
100,00
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14% o
f G
TI r
emo
val a
nd
AP
I lo
sses
Dilution ratio
Remoção de GTI Perdas de API
20% API losses
95% GTI removal
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.
3.5. MPTS mitigation
3.5.1. 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
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.
5. Acknowledgements
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
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