Download Identifying Unexpected Impurities in Drug Products

Identifying Unexpected Impurities in Drug Products
A Challenging Task
INTRODUCTION
Unexpected impurities, suddenly arising in e.g. a QC analysis, may create a lot of stress and anxiety
within different departments of a pharmaceutical company.
In a lot of cases, these unexpected impurities are – at first – not identified, as they are often initially
detected with non-specific detectors in generic analytical methods, such as HPLC-UV Chromatography.
Figure 1: Chromatogram with unknown peak
These impurities may be observed throughout the complete life cycle development of the drug product,
as can be observed from the Figure below. The parts indicated in italic and bold represent steps
whereas unexpected impurities require identification.
Figure 2: Life cycle development of a drug product with indication of applicability of impurity identification
Page 1 of 14
Toxikon Europe NV
Romeinsestraat 12
3001 Heverlee, Belgium
e-mail: [email protected]
Tel.: ++32 (0)16 400.484
WHAT IS IT?
As the regulatory department, as well as the quality department get involved in the root cause analysis
in order to evaluate the impact of this impurity on the drug quality and safety, the first question that
pops up is: “What is it?”. At first this appears to be an easy and straightforward question but it may turn
into a nightmare for the analytical chemistry department. For them, it means:
That the in-house validated process is not under control.
A complete shift in priorities because of the high urgency of the problem.
The necessity of having extensive expertise and access to the high-end analytical techniques
which allow structure elucidation, such as accurate mass measurements, such as LC-ToF or GCToF or other structural elucidation techniques like NMR.
A need to the access of analytical standards, even if they are not commercially available. This in
order to ultimately confirm the impurities identity in the routine QC analyses (such as
confirmation of retention time with HPLC-UV).
WHERE IS IT COMING FROM?
Apart from the “What is it?”-question, another question will arise: “Where is the unexpected impurity
coming from?”, a critical question if the presence of this unexpected impurity needs to be avoided. The
origin of the compound may also determine which Guidelines (and associated control limits) to follow in
the final evaluation of the drug impurity. Relevant guidelines include ICH Q3A (Impurities in New Drug
Substances), ICH Q3B (Impurities in New Drug Products), ICH Q3C (Impurities: Guideline for Residual
Solvents), ICH M7 (Assessment and control of DNA reactive impurities in pharmaceuticals to limit
potential carcinogenic risk), EMA, PQRI-PODP and PQRI-OINDP.
Impurities may be introduced into the drug product through different potential cause pathways, as
described in the root cause approach using the fishbone of Ishikawa.
Figure 3: Fishbone of Ishikawa
Impurities can originate from:
Unexpected degradation of the Active Ingredient (AI degradation compounds)
Page 2 of 14
Toxikon Europe NV
Romeinsestraat 12
3001 Heverlee, Belgium
e-mail: [email protected]
Tel.: ++32 (0)16 400.484
(genotoxic) impurities, present as a result of the synthesis of the active ingredient. (solvent
residues, catalysts, reaction products in synthesis, intermediate synthesis compounds…)
Chemical Compounds, introduced into the drug product as a result of an interaction between
the primary packaging and the drug product (Leachables).
Chemical Compounds, introduced in the drug product as a result of the contact between
processing materials and the product stream (storage bags, filters, tubing materials...)
Secondary Leachables, being formed as a result of a chemical reaction between a leachable and
drug product components (Active Ingredient, Excipients, Adjuvants, Preservatives…).
Leachables, coming from the secondary packaging (label, ink, adhesive, overwrap, cardboard
boxes…)
Impurities, introduced into the API during intermediate storage. (Leachables from drug
substance containers)
Drug product ingredient impurities (Active Ingredient, Excipients, Adjuvants, Preservatives).
These could be – but are not limited to – processing impurities, catalyst or solvent residues,
degradation compound of the ingredient and leachables of the containers used for storage of
the ingredients.
Based on a single absorbance peak in an HPLC-UV Chromatogram, it is not only almost impossible to
identify the compound, but it also becomes even more challenging to identify the source of this
contamination at first.
IS IT TOXIC?
Once the compound is identified, the next question that will pop up is: “Is it Toxic?”. Associated
questions with this issue are:
Is it alarming to have this compound in the drug product as an impurity?
Is the safety of the patient at risk?
Is the quality or the performance of the drug product compromised by the presence of this
compound?
These are all questions which can be answered when performing a proper risk/toxicological assessment.
In a first step, typically, the scientific literature is reviewed for this compound to verify if any relevant
toxicological testing has been performed.
Toxicological information may range from the results of an AMES test, a Acute Toxicity (LD50)
information, Short- and Long-Term Toxicity information, Reproductive Toxicity testing, Immunogenicity,
Carcinogenicity, etc. If sufficient toxicological information is publicly available, a paper assessment of the
obtained information may be sufficient to address the presence of the compound in the drug product,
from a toxicological point of view.
However, in a lot of cases, no toxicological information on the substance will be available. In this case,
one could consider performing a Structure Activity Relationship (SAR) assessment on the compound via
e.g. a DEREK NEXUS assessment. This kind of Structure Activity Relationship Assessment could already
address the main concerns with regard to the toxicity of the compound
In some cases, (e.g. large volume parenterals), it is possible that authorities may request more
toxicological information on the detected compound in order to address the concern of the presence of
this compound in the drug product in a more detailed way. The outcome of a toxicological test like a 90day repeat dose study may address these regulatory concerns in a more appropriate way than a paper
risk/toxicological assessment.
Page 3 of 14
Toxikon Europe NV
Romeinsestraat 12
3001 Heverlee, Belgium
e-mail: [email protected]
Tel.: ++32 (0)16 400.484
CAN/SHOULD WE DO SOMETHING ABOUT IT?
The question “Can – or should – we do something about it” goes hand in hand with the answer to all
previous questions, summed up above. If the presence of a compound – at the detected concentrations
– would pose an unacceptable risk to the patient, or if it would seriously compromise the quality or
efficacy of the drug product, it is clear that necessary steps should be taken to – at least – reduce, or
eliminate the presence of this unexpected and undesired impurity. In order to allow performing a “rootcause analysis” on the presence of this particular impurity, it is of unambiguous importance to identify
the source of the compound in order to take preventive measures.
In a lot of cases, however, the presence of the compound may not pose any safety, quality or efficacy
issues for the drug product. In that case, a proper documentation of the impurity (its identity,
concentration, toxicological evaluation and quality impact) may be sufficient.
Page 4 of 14
Toxikon Europe NV
Romeinsestraat 12
3001 Heverlee, Belgium
e-mail: [email protected]
Tel.: ++32 (0)16 400.484
IDENTIFICATION OF IMPURITIES - THE TOXIKON SOLUTION
In order to make the identification process a little bit smoother, The “Structure Elucidation Team” of
Toxikon Europe has developed a 6 step approach to enhance the efficiency of the overall process:
STEP 1: UNDERSTANDING WHAT IS KNOWN ALREADY
STEP 2: IMPURITIES PROFILING OF THE DRUG PRODUCT USING THE TOX-RAY DATABASE
STEP 3: LINKING RESULTS OF THE IMPURITIES PROFILING STUDY TO CUSTOMERS CHROMATOGRAPHY
STEP 4: LINKING THE IMPURITIES TO THEIR SOURCE
STEP 5: CONFIRMATION OF THE IDENTITY OF THE IMPURITY AND ITS QUANTIFICATION
VIA GENERIC HPLC-UV METHOD – SYNTHESIS OF COMPOUNDS
STEP 6: PERFORMING A TOXICOLOGICAL ASSESSMENT
Each of the steps will briefly be explained in detail in the document. It should be noted that some steps
may not always need to be performed in a sequential way, some steps can run simultaneously in the lab,
which allows shortening the turnaround times for this type of studies.
All results from impurity elucidation studies can be presented as a formal report suitable for regulatory
submission.
STEP 1: UNDERSTANDING WHAT IS KNOWN ALREADY
Although an isolated view of an HPLC-UV Chromatogram, where the unexpected peak is showing up,
may not allow giving any further specifics about the compound and its identity, the context where this
impurity was detected may already include some clear indications in which direction to look and how to
optimize the identification strategy.
Is the compound already present in previous QC-chromatograms of the drug product, but has
now increased in concentration?
Page 5 of 14
Toxikon Europe NV
Romeinsestraat 12
3001 Heverlee, Belgium
e-mail: [email protected]
Tel.: ++32 (0)16 400.484
Did the peak show up after a change in the process, e.g. equipment, ingredient, packaging?
Figure 4. HPLC-UV chromatograms and spectra of an API with a spectrally related (1) and non-related (2 )impurity
originating from the manufacturing process.
Is there any indication that the compound could be related to the API (e.g. degradation
compound)? An increase in concentration of the impurity, together with a decreasing
concentration of the API, could be an indication that the impurity is API related. Also, similarities
in the UV-Spectrum between the impurity and the API, may be an indication that it is API
related.
Could the compound be related to the packaging (i.e. can it be a leachable); was the compound
also observed in the blank solution which was not in contact with the container / closure
system?
Is there a clear relationship observed between the ageing period and the concentration of the
compound? If there is no relationship between the ageing and the impurity concentration, it
may be an indication that the impurity was already present before the final fill and finishing. On
the contrary, if the concentration increases over time, this may be an indication that it is an API
degradation product or a leachable.
Based upon the specifics of the HPLC-UV method, is it possible to draw any conclusion on the
polarity, volatility or expected molecular weight range of the compound?
What is the estimated concentration of the compound, or what is the signal-to-noise level,
observed in Sponsors chromatography.
Is the HPLC-UV method, in which the impurity was detected, transferable to an LC/MS system?
The answers to these questions – extracting some information from the customer – will give the Toxikon
Identification Lab some guidance how to optimize the testing strategy for the identification of the
unexpected impurity. The full list of questions, assisting in optimizing an impurities identification
protocol, is available upon request (if interested, please send an e-mail to [email protected] and request
for the impurities identification questionnaire).
Page 6 of 14
Toxikon Europe NV
Romeinsestraat 12
3001 Heverlee, Belgium
e-mail: [email protected]
Tel.: ++32 (0)16 400.484
STEP 2: IMPURITIES PROFILING OF THE DRUG PRODUCT USING THE TOX-RAYTM DATABASE
It is clear that an HPLC-UV analysis, although very valuable in a Quality Control environment, may not
tell the whole story regarding the composition of the drug product, especially at low concentrations – or
at trace levels. Indeed, compounds will only be detected in HPLC-UV when they contain chromophores
in their molecular structure which will absorb (UV-)light at the selected wavelength. If this is the case,
and the concentration of the compound is sufficiently high, the peak will be detected in the HPLC-UV
chromatogram.
However, in a lot of cases, impurities will either be not detected via HPLC-UV, or their response to the
UV-detector – or the selected wavelength – will be quite low. In addition, some impurities will not be
compatible with the chromatography of the Quality Control HPLC-UV method and will hence not be
detected. The latter situation, however, is beyond the scope of an identification project of an
unexpected impurity that was previously detected in HPLC-UV.
An Impurities Profiling Study, as described by the ICH Q3A and Q3B Guidelines, tries to map all drug
product impurities in a very broad way, this even at trace levels. Understanding the impurities profile of
a drug is a crucial first step in the identification study of an unknown compound, for two different
reasons:
An impurities profiling study will give a detailed overview of what is present as an impurity. It is
very likely that the unexpected impurity, detected in the HPLC-UV chromatogram, will have
been detected and reported.
Other impurities, detected in the drug product, may assist in finding the source of the
unexpected impurity, when the compound is not readily identified.
In the impurities profiling study, the impurities, present in the drug product, will be determined at trace
analysis level, using generic screening methods.
For organic compounds, typically an orthogonal analytical approach will be used combining three
different complementary techniques:
1. Headspace GC/MS allows determination and identification of volatile, low molecular weight
organic compounds (VOC) such as monomers, residual solvents, volatile degradation products
and other volatile compounds.
2. GC/MS (after appropriate sample preparation) allows the determination of the semi-volatile
organic compounds (S-VOC), such as plasticizers, low MW polymer oligomers, small polymer
additive degradation compounds, lubricants, photo-initiators, adhesive compounds, inks, …
3. UPLC - High Resolution Accurate Mass (HRAM) Spectrometry (after appropriate sample
preparation, if necessary) enables the determination of non-volatile organic compounds (NVOC), such as API-degradation compounds, interaction compounds of API/excipients, with
leachables, high MW polymer oligomers, additives, anti-oxidants, slip agents, acid scavengers,
cross-linking agents, UV-stabilizers, pigments, etc…
In some cases – i.e. when there is an indication that the impurity may have polar functional groups, such
as acids and carbonyls, derivatization GC/MS may be considered as a fourth analytical method to render
the impurities profiling more complete.
Page 7 of 14
Toxikon Europe NV
Romeinsestraat 12
3001 Heverlee, Belgium
e-mail: [email protected]
Tel.: ++32 (0)16 400.484
For inorganic compounds, typically ICP-MS, GF-AAS and ICP-OES (after appropriate sample preparation,
if applicable) will be used for the determination of elemental metal impurities.
Toxikon Europe has developed a “Screener Database” (TOX-RAYTM) containing over 3000 Volatile, SemiVolatile and Non-Volatile Organic Compounds which allows performing a high level of first pass
identification in Impurities Profiling Studies. Although the TOX-RAY database finds its origin in
Extractables & Leachables testing, the database has been expanded to other classes of impurities, such
as Genotoxic Impurities, API’s and their degradation Compounds, reaction products between leachables
anddrug product ingredients, etc…. The compound identification in the TOX-RAYTM Database is based on
both the confirmation of the retention time of the compound and on its mass spectrum (or accurate
mass for UPLC-HRAM MS), using hyphenated chromatographic techniques (Headspace-GC/MS, GC/MS
and UPLC-MS(HRAM)). The TOX-RAYTM Database is not only built from commercially available standards,
it also contains synthesized compounds or compounds which were isolated from a material extracts. In
addition, the “TOX-RAYTM Database” identifies Most Probable Compounds (M.P.C.) and Tentatively
Identified Compounds (T.I.C.). These M.P.C. and T.I.C. are compounds – often encountered in Leachable
and Impurities Profiling Studies – where no analytical standards are available but where additional
structural information can be given.
Polars
Derivatisation
GC/MS
Semi volatiles
GC/MS
Volatiles
HS-GC/MS
TOX-RAYTM
Screening Database
>3000 Impurities
Inorganics
ICP/OES
ICP/MS
GF/AAS
Non Volatiles
UPLC/HRAMS
Figure 5. Overview of the TOX-RAY
Page 8 of 14
TM
screening database
Toxikon Europe NV
Romeinsestraat 12
3001 Heverlee, Belgium
e-mail: [email protected]
Tel.: ++32 (0)16 400.484
The TOX-RAYTM database is a very powerful discriminatory tool to rapidly – and very cost effectively –
screen a drug product for potential impurities in a very broad way. Using the TOX-RAYTM database, an
impurities profiling study can be completed in less than a week, if necessary. However, it should be
noted that, although the TOX-RAY database is a very useful instrument in identifying impurities very
rapidly, there is no absolute guarantee that the unexpected compound of interest will be fully identified
during this initial stage of the study. However, when this would be the case, the identification problem
will have been solved in a very cost effective way, since only routine analyses were involved. In this way,
the high-end analytical techniques which are normally necessary to identify the impurities, (such as
NMR) may not have been necessary.
A detailed protocol for an Impurities Profiling study is available on request. (if interested, please send an
e-mail to [email protected] and request for the protocol of an impurities profiling study).
STEP 3: CHALLENGING THE RESULTS OF THE IMPURITIES PROFILING STUDY AGAINST CUSTOMER
CHROMATOGRAPHY
When performed, the mapping of the impurities in the drug product against the TOX-RAYTM Database
can significantly narrow down the number of candidates for the unexpected compound of interest.
These results, however, must be challenged against the customer’s chromatography to discriminate the
targeted impurities from the other possibilities.
Figure 6: Q-Exactive from ThermoFisher Scientific
To unequivocally identify impurities during this phase, Toxikon currently chooses to transfer and
hyphenate the customer’s chromatography to (U)HPLC and hybrid high resolution accurate mass
spectrometry using the Q-Exactive Technology of ThermoFisher Scientific.
Coupling the customer chromatography to this high-end instrumentation capacitates the acquisition of
useful data on the impurities, even if they slipped through the net during the profiling study.
The combination of fast scanning speeds (up to 12 Hz) with a large mass and dynamic
range(above 5000), high sensitivity and very high mass resolution (up to 140 000), robust mass accuracy
Page 9 of 14
Toxikon Europe NV
Romeinsestraat 12
3001 Heverlee, Belgium
e-mail: [email protected]
Tel.: ++32 (0)16 400.484
(down to below 1 ppm), and the multiplexing ability to obtain both targeted and untargeted
fragmentation data, assures that even minor components can be detected with simultaneous
acquisition of valuable structural information.
Transferring the customer’s analytical HPLC QC-method to its mass spectrometry compatible congener,
however, remains critical in this process. Based on the reflection in STEP 1 and possibly results from
STEP 2, however, our team of chromatography and mass spectrometry specialists can develop an
efficient strategy to overcome this hurdle. In a nutshell, key method transfer steps involve:
Selection of column hardware and stationary phase with appropriate selectivity.
Selection of the appropriate volatile mobile phase(s) to achieve an optimal ionization of the
target compounds while keeping conformity with the chromatographic selectivity of the original
method. This can include, e.g. choice of pH, solvents, volatile buffer composition and if
necessary volatile ion pairing reagents.
Selection of the appropriate ion source / ionization technique and related parameters to
interface the (U)HPLC to the mass spectrometer.
Selection of the appropriate sample preparation or analytical techniques to tackle where
necessary ion suppression and interferences caused by matrix components such as salts and
detergents.
Once the above considerations have been verified – or chromatographic changes have been
implemented – the LC-UV chromatogram can be reproduced using an LC-HRAM Q-Exactive Orbitrap
system, initially using a Diode Array Detector (DAD). If the method transfer was successful,
consequently, the unexpected impurity in the chromatogram should be observed in the LC-DAD mode.
At that point, one can start to focus on obtaining more data on the compound itself. This can be done,
using the following sequence of steps:
Checking if the UV-DAD spectrum, obtained for the unexpected impurity, can be linked to the
API, other drug product ingredients or to the impurities of the impurities profiling study,
detected and identified with UPLC-HRAM chromatography (using the TOX-RAYTM database).
Optimizing the ionization mode of the instrument (APCI+, APCI-, ESI+, ESI-) for the compound of
concern. Although it is not a guarantee that all compounds will ionize in one of the four
potential ionization modes – especially at very low concentration levels – in most of the
identification projects Toxikon has undertaken so far, it was possible to select and optimize an
ionization mode that achieved adequate ion formation and sensitivity. With both APCI and ESI at
our disposal, we can cover the ionization of a broad range of compounds going from nonpolar to
very polar analytes and within a large mass range . In the event that no ionization mode would
be successful for the target compound, other routes of identification (such as isolation via
fraction collection with subsequent, GC/MS, GC/ToF, NMR analyses) will need to be considered.
Once High Resolution Accurate Mass and MS-MS based fragmentation data have been obtained
for the compound, the identification may begin.
o Does the high resolution accurate mass spectrum of the compound allow determining
the elemental formula as a starting point?
o Do the observed fragmentation mass spectra yield structural information regarding the
compound through fragmentation mechanisms?
o Can the elemental formula of the compound – or one of more of its fragments – be
linked to one of the compounds, detected and identified in the impurities profiling
study?
Page 10 of 14
Toxikon Europe NV
Romeinsestraat 12
3001 Heverlee, Belgium
e-mail: [email protected]
Tel.: ++32 (0)16 400.484
o
o
o
Is there a correlation between the similarity in UV-data and MS-data obtained for the
compound in the impurities profiling study?
Is there any correlation between the Mass Spectral data of the unexpected impurity and
the different ingredients of the drug product (API, excipients, adjuvants, buffers, protein
stabilizers, preservatives…)
Is there any correlation between the mass spectrum or fragmentation pattern of the
unexpected impurity and any of the 3.000 mass spectra, present in the TOX-RAYTM
database or other libraries?
It may be obvious that there are different potential outcomes of this phase of the study.
The best result would be, of course, when a direct correlation can be made between the accurate mass
spectral data, obtained for the unexpected impurity, and on one of the compounds, reported and
identified in the impurities profiling study. In this case, one could immediately proceed with STEP 5,
which is the confirmation of the impurity in the generic HPLC-UV method (see below). The success rate
of this approach is largely dependent upon the quality of the compound database and the number of
relevant compounds it contains. The TOX-RAYTM database allows Toxikon to offer a high success rate in
identifying the compound quite rapidly since TOX-RAYTM contains over 3.000 very relevant compounds,
and it is still expanding permanently through our R&D activities with compounds that are relevant for
impurities research. This is especially the case if the impurity is related to any material that was in
contact with the drug product, either at the level of production or in the final storage of the drug
product.
However, it cannot be guaranteed for 100% that every drug product impurity is present in the database.
In this case, a tentative identification can be performed based upon the interpretation of mass spectral
data and the correlation one can perform with other mass spectral information, such as mass spectra of
the API, API degradation compounds, drug product ingredients, common impurities for the API, polymer
additives, etc…. It is evident that, if the identification of the compound is tentative, other analytical
techniques (such as fraction collection, NMR, GC-ToF, FTIR) will need to be involved to further elucidate
the structure.
Figure 7. Approach for unknown impurity identification flow chart.
Page 11 of 14
Toxikon Europe NV
Romeinsestraat 12
3001 Heverlee, Belgium
e-mail: [email protected]
Tel.: ++32 (0)16 400.484
STEP 4: LINKING THE IMPURITIES TO THEIR SOURCE
Once the unexpected impurity has been identified, the next question becomes: “what is the source?”.
When the impurity is a degradation product of the active ingredient, the situation is quite clear.
Impurities can then be linked to degradation (the decomposition of the material), synthesis (raw
materials, solvent, intermediate and byproducts), formulation (interaction with excipients), dosage form
(pH, leaching), method (pH, temperature, e.g. autoclave method), ageing (interaction amongst
ingredients, hydrolysis, oxidation, photolysis, decarboxylation, pH) and environment (temperature, light,
relative humidity).
All these processes are integrated in in-house software and database, containing over more than 3000
potential impurities and able to generate potential pathways for both API and excipients. The obtained
theoretical models are subsequently linked to the practical observed structures.
When the unexpected impurity is not API-related, there is a relatively high probability that the impurity
has been introduced into the drug product as a result of a contact between a material and the drug
product either during the final storage or during (bio)pharmaceutical production. These compounds can
be:
Chemical Compounds, introduced into the drug product as a result of an interaction between
the primary packaging and the drug product (Leachables).
Chemical Compounds, introduced in the drug product as a result of the contact between
processing materials and the product stream (storage bags, filters, tubing materials...)
Secondary leachables, being formed as a result of a chemical reaction between the leachable
and drug product components (Active Ingredient, Excipients, Adjuvants, Preservatives…).
Leachables, coming from the secondary packaging (label, ink, adhesive, overwrap, cardboard
boxes…)
Impurities, introduced into the API during intermediate storage. (Leachables from drug
substance containers)
As Toxikon is a leading lab in Extractables and Leachables testing, more than 6.000 E&L-Projects have
been performed in the last 10 years. In order to structure the information and knowledge on these
materials and their composition, Toxikon has developed an internal database, called TOXI-PEDIA. The
TOXI-PEDIA database links a.o. extractables, leachables and related impurities to materials of construct,
used in the manufacture of primary & secondary packaging and of components using in
(bio)pharmaceutical manufacturing. The link between the identity of a specific material and its
extractables composition is considered as being strictly confidential. However, more generic information
which can be provided (e.g. the impurity is a rubber component, or coming from the secondary
packaging (such as an overwrap)…) may give some guidance in these impurity identification projects as
of where in the biopharmaceutical manufacturing process, or in final storage, the impurity may have
been introduced, this without breaching confidentiality. Knowing the source of the impurity may be a
first crucial step in solving the issue…
Page 12 of 14
Toxikon Europe NV
Romeinsestraat 12
3001 Heverlee, Belgium
e-mail: [email protected]
Tel.: ++32 (0)16 400.484
STEP 5: CONFIRMATION OF THE IDENTITY OF THE IMPURITY AND ITS QUANTIFICATION VIA GENERIC
HPLC-UV METHOD – SYNTHESIS OF COMPOUNDS
The ultimate confirmation of the identity and the determination of the concentration of the unexpected
impurity should be considered at 3 levels:
1. By injection of an analytical standard of the impurity into the (UP)LC-HRAM method, developed
on the Q-Exactive Orbitrap LC/MS of ThermoFisher, it should be confirmed that both the
retention time and the mass spectrum (i.e. accurate mass of the molecular ion and/or fragments
thereof) is identical for the impurity detected in the drug product, and the analytical standard,
ideally spiked to a drug product that does not contain the impurity. When both retention time
and accurate mass details of the parent ion and its fragments have been confirmed, the
compound can be considered as being identified.
2. A final analysis should be performed by the pharmaceutical company, to confirm the retention
time match between the drug product impurity and the analytical standard, using the generic
HPLC-UV Chromatography.
3. The generic HPLC-UV method should be validated for the impurity (ICH Q2R1), which would
allow a subsequent determination of concentration in the drug product, and would include
validation parameters as linearity, accuracy, range, precision and LOD/LOQ. It is evident that the
a good understanding of the accurate concentration of the impurity in the drug product is
primordial in order to perform a solid toxicological assessment (STEP 6)
A crucial point in the final confirmation of identity of the impurity is the availability of the analytical
standard. In case the compound is commercially available, the product can simply be purchased and a
final confirmation of identity and concentration can be initiated.
The situation becomes more challenging when the identified compound is not commercially available as
an analytical standard. In this case, the only way to obtain the chemical compound is either through
chemical synthesis or via isolation. It is needless to say that these routes are always very cumbersome
for a pharmaceutical company, as they often do not have the resources to perform the chemical
synthesis or isolation in-house.
Toxikon has developed its own tailored chemical synthesis services, this to provide an integrated
identification service to our customers. The TOXYNTHTM Synthesis Services are offered through
NEOSOME LifeSciences, a subsidiary of – and fully owned by – Toxikon Corporation. The synthesized
impurities can be released at Toxikon with a GMP certificate of compliance.
STEP 6: PERFORMING A TOXICOLOGICAL ASSESSMENT
The Toxikon Group can assist its customers in the toxicological assessment of the identified impurity at
different levels:
1. At Toxikon Europe, there is a direct link to the DEREK NEXUS software, where – once the
compound has been identified and a chemical structure is available – a Structure Activity
Relationship (SAR) assessment report can be generated.
Page 13 of 14
Toxikon Europe NV
Romeinsestraat 12
3001 Heverlee, Belgium
e-mail: [email protected]
Tel.: ++32 (0)16 400.484
2. The result of a DEREK NEXUS SAR-assessment can then be taken up in a full toxicological
evaluation report, in which the broader toxicological literature of the compound (or related
compounds) is reviewed. Subsequently, a risk assessment is performed, based upon the “worst
case” administration regimen of the drug product (the total volume of the container, the
volume per administration, the number of administrations per day and the duration of the
treatment). This should allow verifying the amounts, found for the impurity in the drug product,
against the (expected) Permitted Daily Exposure for the compound.
3. In case no toxicological data are available for the identified impurity, and there is a regulatory
necessity to provide more experimental toxicological data, Toxikon can fully assist you with a
broad array of in-vitro (e.g. AMES) and in-vivo (e.g. Sub-Acute or Chronic Toxicity, as a 90 day
repeat dose study) toxicological testing.
Page 14 of 14
Toxikon Europe NV
Romeinsestraat 12
3001 Heverlee, Belgium
e-mail: [email protected]
Tel.: ++32 (0)16 400.484