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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