Solvias Prospects 01 l 2011

Extractables and leachables in pharma –A serious issue

Solvias’ one-stop shop concept pays dividends

Genotoxic impurities – A long standing debate seeks resolution

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New FAcility Solvias in Kaiseraugst

2 SolviaS ProSPEctS — 1/2011

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Preface 4

News 7

Events 8

Genotoxic impurities – a long standing debate seeks resolution

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Extractables and leachables in pharma – a serious issue 29

rapid results in transmission Electron Microscopy (tEM) 32

Solvias’ one-stop shop concept pays dividends

content

3SolviaS ProSPEctS — 1/2011

Dr. HANsjörg wAltHer — cEo

Dear reader and valued customer

Welcome to our new issue of the Prospects magazine! Spring is not only in the air, it is also in our step as we delight over the warm weather and the excitement to be serv-ing customers out of our new headquarters. We made it! all analytical services are now located at our new labs in Kaiseraugst. this move and the transition was a major step in enhancing our service offer, and we are happy to announce that it all went smoothly with a minimum effect on our daily business. the successful FDa audit in april was the “icing on the cake.”

today you hold the first issue of the Solvias magazine created at our new location in your hands. it therefore comes as no surprise that it is packed with exciting news and developments devoted to analytical chemistry, a service area which is now central to our offer out of the new location.

But, it is not exclusively about analytical chemistry – although there’s nothing in here without. We have included a real life case study which describes a customer’s expe-rience using our “bundled-services” offer for chemical and analytical development and how they benefited from these. last but not least we are thrilled with the results of our customer satisfaction survey. Not only was the participation high, the results underline the fact that the strategy we have been following is highly approved by our clients. Many sincere thanks for this vote of confidence! Yours sincerely

Dr. Hansjörg Walther


New X-rAy PowDer DiFFrActometerSolvias installs a new fully GMP-qualified XrPD instrument with unprecedented high resolution and suitability for highly potent substances.

through this investment, Solvias boosts its highly recognized capabilities in solid-state development, particularly in X-ray powder analysis. the new X-ray powder diffractometer is a Stoe StaDi P instrument. a main feature of the new diffractometer is the cutting-edge detector (Mythen 1K), which allows high resolution and fast data collection. as opposed to most oth-er instruments which measure in reflection mode, the new in-strument measures in transmission mode, significantly reduc-ing the effects of preferred orientation. Preferred orientation results from the slight squeezing of the sample during prepa-ration in order to obtain a flat sample surface. it can lead to incorrect peak heights and in unfavorable cases even to ooE/ooS results.

the copper X-ray source is equipped with a germanium mono-chromator yielding a monochromatic radiation (cu-Kα1). this results in narrow peak widths and much improved resolution of overlapping peaks. Well resolved peaks are essential to ob-tain low detection limits of unwanted polymorphic forms and for successful indexing of a powder diffraction pattern. if in-dexing is successful and all experimentally found peaks can be explained with calculated peaks from the indexed unit cell, this

News

solviAs customer sAtisFActioN survey successFully coNcluDeDthanks to the very useful input and feed-back we received from our customers, we are really pleased to announce the re-sults of the customer satisfaction survey we recently conducted.

a thorough review and analysis of the results show that over 95 % of the respondents were happy to very happy with our services. a flattering result!

the qualitative data generated revealed that scientific and technical know-how, the quality of the work, on time delivery, as well as the offer of a ‘one-stop services provider’ are impor-tant criteria for customers’ decisions to work with Solvias. this result confirms the strategy we have been following. this survey has been extremely valuable as it drives us even more to foster a close collaboration with our customers. Fur-ther enhancing our communication and customer care con-tinue to enjoy the number one position on our list of priorities.

Please remember, your input is essential for us to structure our internal processes such that we can serve your needs more specifically and flexibly. thank you once again for your es-teemed feedback, don’t hesitate to keep it coming!

X-raypowderdiffractometer


News

is strong evidence that the analyzed sample contains only a single crystalline form. High resolution also has the potential to lower loDs of polymorphic impurities.

a further highlight of the new Stoe instrument is the possibility of measuring samples in spinning glass capillaries. Using this technique, powder diffraction data of high enough quality to be used in structure determination can be obtained. in favor-able cases this can replace single crystal X-ray analysis.

in collaboration with the instrument vendor, Solvias developed a new safety cell for the instrument with disposable components. this unique feature allows the investigation of highly potent and highly toxic substances while still maintaining the same high qual-ity of data. the instrument is fully qualified under GMP and can be used for development work and routine quality control alike.

imProveD moNocloNAl ANtiboDy (mAb) cHArActerizAtioN by cAPil-lAry isoelectric FocusiNg (cieF) FollowiNg PAPAiN DigestioN

at the yearly conference on well characterized biopharmaceuti-cal products, WcBP, in Washington D.c. earlier this year, Solvias presented a novel approach to characterizing monoclonal antibodies.Monoclonal antibodies typically undergo a number of posttranslational modifications/degradation during produc-tion and storage. Many of these modifications result in forma-tion of charge variants, i.e. species with a change of the net

Figure1:cIEFofCetuximabbeforeandafterdigestionwithpapain

Figure2:Schematicrepresentationofcleavageofantibodiesbypapain

charge of the mab. relevant examples include removal for c-terminal lysine, acetylation of the N terminus, sialylation/de-sialylation, deamidation. Each of these pathways can potentially lead to one or more charge variants. thus, all pathways combined can result in a highly complex mixture of different species.

to improve resolution of the charged variants, digestion of the mab using various enzymes has been previously used, includ-ing digestion with pepsin, trypsin, lys-c, and papain. Papain digestion results in the generation of Fab and Fc fragments of the mab see figure1. analysis of the fragments by iEX and oth-er analytical tools has been performed extensively in the past. However, only very limited data is available on ciEF of Fab and Fc fragments.

at the WcBP conference, Solvias presented data on ciEF af-ter papain digestion of various commercial and development mabs. two well separated peak families due to Fab and Fc are readily observed seefigure2. in all investigated mabs, additional variants were visible that were masked using conventional ciEF with no digestion. the results show the potential of ciEF with papain digestion as a general tool for quality control and char-acterization of mabs.

copies of the poster can be requested at [email protected].


New corPorAte locAtioN reADy to serveMore space and “all under one roof” leads to an improved service offer for customers

With the move to our new location in Kaiseraugst, we have enlarged our laboratories to provide our clients with a much wider spectrum of services which include analysis of large and small molecules, solid-state development, and process analyti-cal technology. Many employees, who in the past worked at various locations, are now shaping Solvias’ future united under one roof. the new building with 6,400 square meters of labora-tory space (14,000 square meters of gross floor space) accom-modates the entire analytical service area of Solvias. Most of the services have already been relocated and were successfully inspected by Swissmedic and the FDa.

Within the scope of the relocation we have also made signifi-cant investments in devices and infrastructure, with new walk-in chambers for stability studies being only one such example. these chambers complement the existing climate cabinets, massively increasing the storage capacity for these studies.

NewlocationatKaiseraugst

News

over and above that, to satisfy market demands for early stage projects requiring limited sample quantities, Solvias has intro-duced salt programs with significantly reduced sample quan-tities. to achieve this, we apply the latest technology in high-throughput screening.

Finally, a corporate center of excellence for chemical develop-ment catalysis completes the integrated services. this center provides special services such as the production of aPis for clinical phase ii a, which for now remains at the Basel location.

in a nutshell, our new corporate location is equipped to better meet the needs of our customers and the ever-changing mar-ket requirements faster and more flexibly.

7SolviaS ProSPectS — 1/2011

events

BoS cmc

June 16, 2011 Copenhagen, Denmark

FeSt der moleküle

June 17/18, 2011 Basel, Switzerland

BoS non-clinical

September 20, 2011 London, UK

chemoutSourcing conFerence

September 12–15, 2011Ocean Place Resort – Long Branch, NJ, US

Find out more about • chemical and analytical development• gmP manufacturing• catalysis

aaPS national conFerence

October 23–27, 2011 Washington DC, US

cPhi WorldWide

October 25–27, 2011 Frankfurt, GermanyVisit us at Booth 42C14

Find out more about • Polymorphism, Salt, and crystallization• chemical and analytical development• gmP manufacturing• catalysis

SolviaS Science day

November 3, 2011Basel, Switzerland

TeNTaTiVe PROGRam – SPeaKeRS CONFiRmeD

General Lecture• dr. Wolfgang albrecht (head of chemical research,

ratiopharm gmbh, germany)

Session: Chemical Development and Catalysis• Prof. dr. matthias Beller (leibniz institute for catalysis,

university of rostock, germany)• Prof. dr. karl anker Jørgensen (center for catalysis,

university of aarhus, denmark)• Prof. dr. andreas kirschning (institute of organic chemistry,

leibniz university of hanover, germany)• Prof. dr. ian Paterson (department of chemistry,

university of cambridge, uk)• ligand contest Winner 2011 Session: Solid-State Development• Prof. dr. Josef Breu (university of Bayreuth, germany)• Prof. dr. michal Borkovec (department of inorganic, analytical, and

applied chemistry, university of geneva)• dr. heinz-Josef deussen (leo Pharma, Ballerup, denmark)• Prof. dr. gérard coquerel (university of rouen, France)• dr. andreas Schreiner (novartis, Switzerland) Session: Pharmaceutical and Biopharmaceutical analysis• Prof. dr. gabrielle dallmann (dallmann consultancy,

ceo Pharmatching gmbh, Freiburg, germany)• Prof. dr. michael Przybylski (department of chemistry,

university of konstanz, germany)• Prof. dr. Peter ruppersberg (ceo valtronic, Switzerland)• dr. kai Sheffler (thermo Fischer Scientific, germany)• Prof. dr. tudor arvinte (therapeomic inc, Bio center,

university of Basel, Switzerland)


Dr. AlDo HörmANN — Product innovation Manager

ADDressiNg tHe issueFour out of 13 major objections to drug substances in marketing authorizations re-sulted from issues on genotoxic impurities not properly addressed in the application as was recently published in a review on quality concerns raised during European centralized marketing authorizations1. the same publication lists 56 other concerns related to impurities in drug substance, again genotoxic impurities being one major cause for concern. the European Directorate for the Quality of Medicines & Healthcare (EDQM) has published a list of the top 10 deficiencies in the certification of suitabil-ity to monographs of the European Pharmacopoeia for the last quarter of 20092, and genotoxic impurities ranked fourth on this list. clearly, genotoxic impurities are a hot topic owing to the potential for substantial issues to arise during the approval pro-cess of new drugs.

Genotoxic impurities – A long standing debate seeks resolution Genotoxic impurities in drugs is a serious issue for the pharmaceutical industry. low acceptable limits lead to technical challenges in both analytical and process development. the lack of fully harmonized guide-lines adds regulatory uncertainty. this article addresses the major issues surrounding this hot topic and provides an overview of the status quo along with potential solutions.Author: Dr. Aldo Hörmann

DEFiNitioN oF a GENotoXic coMPoUND a compound that interferes with the genetic material at any level (DNa, chromosomes) is considered genotoxic. it has the potential to cause cancer.

Mutagenic compounds: interact with DNa inducing a change in the nucleic acid sequence (gene mutation).

clastogenic compounds: interact with chromosomes inducing breakages or rearrangements of chromosomes (chromosomal aberration).


rEGUlatorY DocUMENtS WitH rElEvaNcE to GENotoXic iMPUritiES impurities in general• icH Q3a (r2): impurities in New Drug Substances, october 25, 2006• icH Q3B (r2): impurities in New Drug Products, June 2, 2006• icH Q3c (r3): impurities: Guideline for residual Solvents, November 2005

control of potential genotoxic impurities• EMEa/cHMP/QWP/251344/2006, Guideline on the limits of genotoxic

impurities, adopted June 28, 2006• EMa/cHMP/SWP/431994/2007 rev. 3, Questions and answers on the Guide-

line on the limits of genotoxic impurities, adopted September 23, 2010• FDa Draft Guidance for industry, Genotoxic and carcinogenic impurities in

Drug Substances and Products: recommended approaches, December 2008• Pharmeuropa, vol 20, No. 3, July 2008, Potential Genotoxic impurities

and European Pharmacopoeia monographs on Substances for Human Use

test methods for potential genotoxicity• icH S2a: Specific aspects of regulatory Genotoxicity tests for

Pharmaceuticals, april 1996• icH S2B: a Standard Battery for Genotoxicity testing of Pharmaceuticals,

July 1997• icH S2(r1): DraFt consensus Guideline (will eventually replace icH S2a

and S2B): Guidance on Genotoxicity testing and Data interpretation for Pharmaceuticals intended for Human Use, March 6, 2008


• FDa includes carcinogenic impurities (many carcinogens are non-genotoxic, Sar for carcinogenicity not reliable).

• FDa includes additional safety margins for pediatric populations.• FDa allows use of safety data from “surrogates” (i.e. structurally similar

compounds) instead of default ttc.• FDa requires safety testing for any impurity at a level above the icH

qualification limit, even if Sar does not create a structural alert.• FDa questions the applicability of the 1.5 µg/day ttc approach for

some routes of administration (dermal, ophthalmic).• FDa specifically includes products under development and defines ttc

for clinical trials while the EMa guideline is less clear although some aspects are addressed in the Q&a document.

A History oF regulAtioNsthe discussion of how to tackle the issues surrounding genotoxic impurities in drugs has been going on for numerous years. the icH Q3 guidelines outline a framework of how to deal with impurities in general but are not specific about how to deal with genotoxic impurities. a milestone was reached when Pharmaceutical research and Manufacturers of america (PhrMa) published a highly recognized paper on the sub-ject in 20063. the same year, the European Medicines agency (EMa) finalized the “Guideline on the limits of Genotoxic impurities” which came into force on Janu- ary 1, 2007. a question and answer document was soon published by the EMa and has been revised several times since. the document answers various questions from the industry that remained unclear in the EMa guideline. clearly, the EMa guideline together with the Q &a document set a landmark for what regulators expect from the pharmaceutical industry and largely define today‘s approach to genotoxic impurities.

The discussion of how to tackle the issues surrounding genotoxic impurities in drugs has been going on for numerous years.

the FDa published a guidance for industry on genotoxic and carcinogenic impuri-ties which has still not progressed beyond a draft status. this guidance is very similar to the EMa guideline and the Q&a documents but a few important differences exist seeboxbelow.

Box:majordifferencesbetweentheEmaandFDa(draft)guidance


Figure1:DecisionTreeforassessmentofacceptabilityofgenotoxicImpuritiesaccordingtotheEmaguidelineongenotoxicimpurities

1. Impuritieswithstructuralrelationshiptohigh-potencycarcinogensaretobeexcludedfromtheTTCapproach

2. Ifcarcinogenicitydataavailable:Doesintakeexceedcalculated10–5cancerlifetimerisk?3. Case-by-caseassessmentshouldincludedurationoftreatment,indication,patientpopulationetc.* abbreviations:

NOEL/UF–NoObservedEffectLevel/UncertaintyFactorPDE–PermittedDailyExposureTTC–ThresholdofToxicologicalConcern

Genotoxic impurity

test data indicate concern, e.g.ames test positive/DNa reactive

calculate PDE* (NoEl /UF* analysis):Safe exposure?

No further action

reduce tosafe level

Sufficient evidence for threshold- related mechanism of genotoxicity

Use alternativew/o genotoxic

impurity

restrict orreject

applied use

level as low asreasonably practicable?

Presence of genotoxic impurityunavoidable?

Does estimated intakeexceed ttc* of

1.5 μg/day2?

intake level >1.5 μg/day acceptable3?

Negligible/acceptable

risk

Negligible risk1

reduce to aslow as reason-

able level

=pharmaceuticalassessment

=toxicologicalassessment

emA’s DecisioN tree For AssessmeNt oF AccePtAbility oF geNotoXic imPurities

Yes

Yes

Yes No

No

No

No

No

No

Yes

Yes


>Figure2:Decisiontree

forhandlinggenotoxic

orcarcinogenicimpuri-

tiesaccordingtothe

FDadraftguidance

FDA’s DecisioN tree

1. Safetythresholdapproachforgenotoxicandcarcinogenicimpuritiesisnotapplicabletocompoundswithadequatedatatoderivecompound-specificriskassessmentorforthosewithSaRstohigh-potencycarcinogens.Inaddition,theapproachmaynotbeappropriateforsomeroutesofadministration(e.g.,dermal,ophthalmic)becauseofthelackofarelevantdatabasefromwhichathresholdlimitcanbederived.

identify impurity

observed level exceeds relevant icH qualification threshold or is less

than icH qualification threshold but displays a structural alert?

No further action

consider alter-nate synthetic

pathway

able to prevent formation of impurity?

conduct appropriate genotoxicity assays

impurity considered genotoxic based on assay

results/weight of evidence?

adequate evidence for threshold mechanism?

consider restricting or rejecting proposed use

based on risk-benefit ratio

Set specification based on calculated permitted daily exposure

reduce the level of impurity to that associated with a daily exposure

≤ qualification threshold for geno-toxic and carcinogenic impurity1

(1.5 μg per day)

No

No No

Yes

Yes

Yes

No

Yes


to resolve differences between regulators worldwide and to further clarify uncertain-ties about dealing with genotoxic impurities, the icH has initiated a project to come up with the new guideline, icH M7 6. a document for public consultation is expected in 2012, and the finalized document is currently expected for November 2013.

geNotoXic imPurities – issues AND solutioNsBoth the EMa and the FDa guidelines provide a decision tree for assessing the ac-ceptability of genotoxic impurities Figures1and2. For the purposes of this overview we will focus on the tree from the FDa guideline shown in Figure2.

the first step is to identify the impurity. the chemical structure is then checked by an appropriate structure-activity relationship (Sar) software (e.g. Derek) for any structur-al alerts, i.e. for features in the chemical structure that indicate genotoxic potential. if a structural alert is raised, the substance qualifies as a potential genotoxic impurity at this stage. only if the substance raises no structural alert and its level is below the icH qualification threshold seeglossary are no further actions needed.

at this stage it makes sense to perform a genotoxicity assay (e.g. ames test) to ex-perimentally confirm potential genotoxicity (even though this step actually occurs later in the FDa decision tree). No further action is required if genotoxicity is not con-firmed. if, however, genotoxicity is confirmed, an evaluation should be made as to whether another synthetic pathway might be used that would avoid the formation of genotoxic impurities. in practice this will often be impossible or perhaps not com-mercially viable in which case a reduction of the genotoxic impurities to acceptable levels will be required. this step is critical and demands the combined efforts and know-how from both process development and the analytical department.

Both the FDa and the EMa guidelines distinguish between substances with enough evidence for a threshold-related mechanism of genotoxicity see glossary and those without sufficient evidence. only in very few cases is there enough data available to support a threshold-related mechanism. in those cases, acceptable levels can be derived based on the principles outlined in icH Q2c for class 2 solvents.


in the vast majority of cases not enough evidence for a threshold-related mechanism is available. then the concept of the threshold of toxicological concern (ttc) will be applied. the basis for this concept is that a dose of 1.5 µg per day for any substance is expected to cause not more than 1 case of cancer in 105 persons over their entire lifetime which is considered an acceptable risk. For clinical development and/or for short-term use of the drug, it is often acceptable to increase the ttcseetable. it should be noted here that the ttc of 1.5 µg per day does not apply to some high-potency genotoxic carcinogens, including flatoxin-like-, N-nitroso-, and azoxy-compounds for which no ttc is defined.

in some cases it might be hard to achieve acceptable levels of genotoxic impurities based on the ttc concept. then a case-by-case evaluation will be performed weigh-ing the potential risks of genotoxic impurities against the benefits of the drug. in par-ticular, for life-saving drugs, e.g. in cancer treatment, the risk of genotoxic impurities will usually be negligible compared to the benefit.

uNcertAiNty AND coNcerNs oF tHe iNDustry although the EMa and FDa guidances helped a lot in clarifying many questions about genotoxic impurities, there still remain uncertainties in the pharmaceutical community concerning:

• Which systems should be used for Sar (for identification of structural alerts)?• Different Sar software used across the industry and by regulators is leading

to unpredictable situations.• What are the relevant toxicological end points in Sar? current thinking favors

mutagenicity as other end points (e.g. cancerogenicity) are considered unreliable.

DUratioN oF EXPoSUrE

Single dose < 1 month < 3 months < 6 months < 12 months

allowable daily intake

120 μg 60 μg 20 μg 10 μg 5 μg

From the EMa Q&a document. a very similar table is in the FDa guideline applying to clinical trials. instead of “Single dose” an exposure time < 14 days is defined for ttc 120 µg.

Table:Thresholdoftoxicologicalconcern(TTC)asafunctionofoverallexposuretime


• can surrogate data be used to predict the genotoxic potential and to set a specification? the FDa guideline in principle allows this but the EMa’s position is not perfectly clear and cases have been reported where surrogate data was not accepted4.

• inclusion of carcinogenic impurities in the FDa draft guidance leads to confusion as many carcinogens are non-genotoxic and should therefore be regulated elsewhere.

• How many steps back in the synthesis should one go to evaluate potential occurrence of genotoxic impurities?

• lack of full harmonization between regulators worldwide.• Some guidance on genotoxic metabolites is lacking.

Furthermore, doubts have been raised whether today’s approach by regulators might be overly conservative5. this includes even general doubts about the biological rel-evance of non-threshold related mechanisms and thus the ttc concept. also, for structurally related substances the ttc applies to the sum of all substances, not to the individual substance. this is generally regarded as too conservative 4, 5.

clearly there are high expectations for the icH M7 process to resolve most of above issues and to find a generally well accepted agreement between regulators and in-dustry6. Undoubtedly, experience in how regulators deal with submissions in practice is a distinct advantage.

For the reader interested in the details of genotoxic impurities, the recent book edited by a. teasdale is highly recommended7.

In particular, for life-saving drugs, e.g. in cancer treatment, the risk of genotoxic impurities will

usually be negligible compared to the benefit.


glossAry/AbbreviAtioNs

alarP as low as reasonably Possible. a concept pushed mainly by EMa to reduce the levels of genotoxic impurities to a level as low as can reasonably be done, e.g. by changes to the synthesis, optimization of impurity removal etc. it is however recognized that a substance at levels below the ttc does not need to be reduced further.

ames test an in-vitro test for genotoxicity developed by Bruce ames. it is also called “bacterial reverse mutation test” (e.g. icH S2 guidelines). the test is based on certain mutated strains of Salmonella bacteria that cannot synthesize histidine and thus can grow only in the presence of histidine. Upon addition of a genotoxic compound, the genetic material is mutated such that the bacterium can produce histidine itself. consequently, a genotoxic substance will allow growth of the bacterium even in the absence of histidine.

icH identification threshold

the limit above which an impurity should be identified according to icH guide-lines Q3a and Q3B. the threshold depends on the daily dose of the drug. For drug substances with doses ≤ 2 g/day, the identification threshold is 0.10% of the drug substance or 1.0 mg/day whichever is lower. a different threshold applies for degradation products in drug products (see icH Q3B).

icH qualification threshold

the limit defined in icH guidelines Q3a and Q3B above which an impurity should be evaluated for biological safety. the threshold depends on the daily dose of the drug. For drug substances with doses ≤ 2 g/day, the qualification threshold is 0.15% of the drug substance or 1.0 mg/day whichever is lower. a different threshold applies for degradation products in drug products (see icH Q3B).

Sar Structure activity relationship. the connection between a chemical structure and its biological activity. it tries to relate biological activity with the presence, absence and combination of structural groups. Sar is often used to predict toxicity based on structure. various software is available to perform Sar, including Derek, MDl-QSar, McaSE/Mc4Pc, toPcat, aDMEWorks.

Structural alert a group or feature in the chemical structure that triggers a toxicity concern in Sar.

threshold-related mechanism

in general, regulators assume that genotoxic substances can potentially damage DNa at any exposure level and that there is no threshold at which the substance carries potential risk. However, it is widely recognized that there are defense mechanisms against genotoxic events in living organisms, this mechanism be-coming effective at very low levels of genotoxic impurities up to a certain threshold. in this case extrapolation from high-dose studies to come up with safe levels of genotoxic substances would lead to unrealistically low levels far below the threshold. one would rather use a no-observed-effect approach to define the limit. in many cases it is however very difficult to prove that a threshold exists and consequently in most cases a non-threshold-related mechanism must be assumed leading to the ttc approach.

ttc threshold of toxicological concern. a level at which any chemical will not result in a risk of significant carcinogenicity or other toxicological effects. For genotoxic substances, the generally accepted ttc is 1.5 µg/day which gives rise to a risk of cancer of not more than 1:105 over a human’s entire lifetime. this risk is consid-ered acceptable due to the generally beneficial nature of pharmaceuticals.the ttc does not apply to some structural classes of highly carcinogenic risk including aflatoxin-like-, N-nitroso-, and azoxy-compounds.


There are high expectations for the ICH M7 process to find a generally well accepted

agreement between regulators and industry.

coNclusioNDespite significant progress on the regulatory side, the debate about genotoxic im-purities continues. to come up with solutions for genotoxics in drug development programs that are commercially viable but still address the concerns for safety in a manner that will be accepted by the regulators, experience and know-how in various areas are required. the most relevant are:

• advanced and extensive analytical development resources for unambiguous identification and characterization of materials

• Highly sensitive quantitative techniques for trace analysis• Expert consideration of the chemistry of process development in particular

reagents, possible side-reactions and potential impurities – especially reactive substances

• Expert assessment of the toxicological aspects• Quality of data generated within a robust quality system that will deliver

information in a form likely to be accepted by regulatory bodies • a quality system that will stand up to close scrutiny of regulators and

as a service provider, or sponsors/clients• regulatory support that presents a compelling argument based upon

sound data generated within a clear regulatory strategy

as a specialist provider of chemical and analytical development services for the pharma-ceutical industry, Solvias can help in these fields and will be happy to discuss any projects with you. •

References 1. J.J.Borg,J.-L.Robert,g.Wade,g.aislaitner,m.Pirožynski,E.abadie,T.Salmonson,P.V.Bonannoa,“WhereisIndustry

gettingItWrong?aReviewofQualityConcernsRaisedatDay120bytheCommitteeformedicinalProductsforHumanUseduringEuropeanCentralisedmarketingauthorisationSubmissionsforChemicalEntitymedicinalProducts,”J.Pharm.PharmaceutSci(www.cspsCanada.org)12(2):181–198,2009.

2. EuropeanDirectoratefortheQualityofmedicines&HealthCare,“Toptendeficiencies,NewapplicationsforCertificatesofSuitability(End2009),”documentPa/PH/CEP(10)65,Strasbourg,June2010.

3. müllerL.,mautheR.J.,RileyC.m.,andinom.m.,DeantonisD.,BeelsC.,DegeorgeJ.,DeKnaepa.g.m.,EllisonD.,FagerlandJ.a.,FrankR.,FritschelB.,gallowayS.,HarpurE.,HumfreyC.D.N.,Jacksa.S.J.,JagotaN.,mackinnonJ.,mohang.,NessD.K.,O’Donovanm.R.,Smithm.D.,Vudathalag.,YottiL.,“arationalefordetermining,testing,andcontrollingspecificimpuritiesinpharmaceuticalsthatpossesspotentialforgenotoxicity,”RegToxPharm44,198–211,2006.

4. a.Teasdale,Presentationatthe2010aaPS-PSWCmeeting,NewOrleans,November14,2010,seewww.usp.org/pdf/EN/meetings/workshops/aapsWorkshopPresentationsDay2.pdf.Thislinkalsocontainsothercurrentrelevantcontributionstothesubjectofgenotoxicimpurities.

5. D.Snodin,g.K.Vudathal,“genotoxicImpurities:aCaseforaRegulatoryRethink,”aaPSNewsmagazine,February2009.

6. ICHFinalConceptPaperonm7,datedNovember27,2009andICHFinalBusinessPlanonm7,datedmay6,2010.BothdocumentswereendorsedbytheICHSteeringCommitteeonJune9,2010.

7. a.Teasdale(editor),“genotoxicImpurities:StrategiesforIdentificationandControl,”JohnWiley&Sons,February2011.


the challenges of leachables have been well known in the food industry for a long time but despite strict regulations, problematic cases gain public attention. Potential exposure to bisphenol a, for example, led the canadian authorities to ban polycar-bonate infant bottles. the EFSa discovered that Germans were exposed to 4-methyl-benzophenone in their chocolate muesli. the source was printing ink on the outside of the cardboard box. recently, Scott Mabury (University of toronto) found that pop-corn bags leach polyfluoroalkyl phosphoric acids, which are absorbed by humans, accumulate in the body and can be metabolized to perfluorinated carboxylic acids which are potentially carcinogenic and hormone disrupting.

Extractables and leachables in pharma – A serious issueleachables are trace amounts of chemicals originating from containers, medical devices or process equipment that end up as contaminants in medicinal products resulting in exposure to patients. We will explain why this is of so much concern and how testing for leachables as well as extractables should be conducted.Author: Dr.Frankmoffatt

Dr. FrANk moFFAtt — Business Development Manager

SinceJuly2010BusinessDevelopmentmanageratSolviasag.FromJuly2007toJuly2010ProductmanagerforBiopharmaceuticalsatSolvias.DEFiNitioN oF EXtractaBlES aND lEacHaBlES

• Extractables are chemical entities, both organic and inorganic, that will extract from components of a container closure system or device into solvents under controlled conditions. they are used to identify and quantify potential leachables.

• leachables are chemical entities, both organic and inorganic, that migrate from components of a container closure system or device into a drug product over the course of its shelf-life.

• leachables are usually present in drug product matrices as complex mixtures at trace levels relative to the active pharmaceutical ingredient (aPi).


in the case of plastic containers, typical extractables and leachables are additives and processing aids such as antioxidants and other stabilizers, plasticizers, emulsifiers, colorants but also monomers and oligomers of the plastic polymer and all kinds of reaction products. Useful information can often be obtained from vendors regard-ing the formulation of the polymer packaging, but the issue remains complex as the full manufacturing chain from raw materials all the way to a plastic container involves different specialized manufacturers at many steps and full traceability is hard to obtain. Quite generally, containers meant to protect a drug from environmental contamination are actually themselves a source of contamination.

in pharma, not only containers are a source of leachables. combination products such as inhalers and pens or even more sophisticated medical device equipment like insulin pumps and implants may all leach unwanted chemicals. also with the advent of disposable equipment, mainly in the manufacturing of biopharmaceuticals, an-other source of leachables has entered the arena.

Containers meant to protect a drug from environmental contamination are actually themselves

a source of contamination.

at what stage in the development of a drug E/l becomes an issue, depends on the development program. certainly, when the final container closure system and/or de-vice is to be selected, E/l programs become mandatory.

smAll molecule Drugs AND biologics trigger DiFFereNt coNcerNsUnlike small molecules, biologics can be immunogenic. leachables might interact with a protein in such a way as to trigger an immune response as observed in the case of Eprex1. in rare cases patients generated an immune response to an essential natural human protein EPo. the effect had nothing to do with the toxicological prop-erties of the leachables. the consequence was a 100 or so patients requiring blood transfusions for the rest of their lives.

another recent example is the reaction of the rubber processing chemical thiuram disul-fide via disulphide exchange with captopril, a thiol containing angiotensin-converting enzyme inhibitor 2. rubber is an exceptionally rich source of diverse classes of process-ing additives and in both of the above examples rubber was the source of leachables.


leAcHiNg is Not limiteD to PrimAry PAckAgiNg or to liquiDsleaching is facilitated by the exposure of surfaces to liquid. this is well reflected by the generally accepted risk matrix in Figure1. However mass transfer via the solid state is well known. Similar to the chocolate muesli case, benzophenone and other photo-initiators 1-benzoylcyclohexanol and 2-hydroxy-2-methylpropiophenone from inks used on the labels of HDPE bottles were found to migrate into a solid product3 mak-ing regulatory agencies sensitized to this possibility.

oFFiciAl guiDeliNes AND iNterest grouPsBoth the USP and the EP contain chapters that deal with the testing of plastic mate-rials, including extraction tests. However only very limited guidelines on E/l for final dosage forms are available from regulators. Probably the most relevant documents are the EMa guideline on plastic immediate packaging materials4 which provides a decision tree for when in the EU E/l studies are required and the FDa guideline on container closure systems5. additionally there are guidelines on genotoxic and carci-nogenic impurities in drug products in general6, 7. None of these guidelines provide any details on how to perform E/l studies.

to fill the gap, various industrial/regulatory groups offer consensus views that help construct convincing arguments that the drug product is safe. Probably the leading group of opinion formers is the influential PQri working group on leachables and extractables. this group published a recommendation for E/l for orally inhaled nasal drug products (oiNDP)8, 9. only recently, PQri also came up with tentative recom-

Degree of concern associated with the route of administration

likelihood of packaging component-dosage form interaction

HigH meDium low

HigHest

inhalation aerosols and Solutions; injections and injectable Suspensions

Sterile Powders and Powders for injections, inhalation Powders

HigH

ophthalmic Solutions and Suspensions, transdermal ointments and Patches, Nasal aerosols and Sprays

low

topical Solutions and Suspensions, topical and lingual aerosols, oral Solutions, and Suspensions

topical Powders, oral Powders

oral tablets and oral (hard and soft Gelatin) capsules

Figure1:RiskassessmentTableforvariousdosageforms(adoptedfromref.19)


mendations of parenteral and ophthalmic drug products (PoDP)10. the BioProcess Systems alliance (BPSa) covers the aspects of E/l of disposable bioreactors11. last but not least the ElSiE group should be mentioned which is generating databases of extractables and toxicological profiles for the group members12.

AssessmeNt oF eXtrActAbles Forms tHe bAsis oF A successFul leAcHAbles stuDyExtractable studies are conducted to understand what substances are contained in a material that have the potential to leach into the drug product. the start is a “worst- case scenario” as shown by a “controlled Extraction Study” under conditions that de-pend upon the context. the chemical composition of surfaces that come in contact with the drug product, the solubilizing power of the drug product, the duration and conditions of contact are all relevant considerations. this testing is performed under exaggerated conditions of time and temperature in the laboratory using common, neat solvents that bracket the solvating power of the drug. then, the therapeutic in-dication dosage form, route of entry into the body and frequency as well as the life

coMPoNENtS oF aN EXtractaBlES StUDY 1) Extraction

• suitable solvents – optimized for the polymer/material• vigorous extraction technique(s)• to asymptotic levels

2) Multiple analytical techniques, Gc, lc with different detectors3) identify principally by MS4) Quantify extractables greater than or equal to the aEt (based on the Sct)

expectancy of the patient help establish an acceptable level of risk to be argued on a scientific basis. reliable identification and sensitive analytical techniques are essen-tial. Expert experimental design avoids unnecessary work and the generation of arti-facts through chemical reactions that may trigger false concerns.

To fill the gap, various industrial/regulatory groups offer consensus views that help construct convincing arguments

that the drug product is safe.


tecHNiques, DAtA geNerAtioN AND iNterPretAtioNthe first major analytical challenge is to identify the substances in the extracts. Efficient identification is critically dependent upon having automated work-flows. the major analytical tools are Gc and lc coupled to MS (MS/MS).

Headspace Gc-MS highly volatilesGc-MS semi-volatileslc-MS non- volatilesic anions and cations (inorganic or organic)icP-MS elements including heavy metals

various modes of separation are required owing to the diversity of physical chemical properties encountered. For identification purposes, mass spectrometry is clearly the main work-horse. High mass accuracy is often helpful to narrow down the number of possible chemical structures. an accurate mass combined with a database of typi-cal plastic additives (e.g. NiSt for Gc-MS, proprietary databases) provides a powerful basis for assignment of chemical structures to chromatographic peaks. the quality of any proprietary database is also a decisive factor in the success of the identifica-tion exercise. Following an identification based upon MS, a comparison against an authentic reference standard might be required in order to finally confirm an identity.

The first major analytical challenge is to identify the substances in the extracts.

accurate quantitation requires comparison against a reference standard but in the absence of such a material relative or semi-quantitation is often an acceptable means to estimate the levels of specific extractables.

toXicology AND risk AssessmeNtin order to assess the risk to human health of a substance found in an extractable study, the toxicology of this “potential leachable” must be considered. a toxicological database of extractables is being compiled by ElSiE starting with 15 priority com-pounds: antioxidants, anti-slip agents, plasticizers, cross-linking agents, lubricants, monomer, starting material, surfactants, and acid scavengers (calcium stearate).

toxicological information is not often readily available for a number of substances. However, for additives that are considered by the US Environmental Protection agency (EPa) to be high production volume compounds, there is a lot of public toxicological information available. compounds that are regulated for food contact applications


also often have publically available tox data. the PQri best practices have established some toxicological thresholds for extractables and leachables for orally inhaled and nasal drug products (oiNDP)13, 14:

1) the Qualification threshold (Qt) is the level (5 µg/day) below which a given leachable is not considered for safety qualifications unless it presents a structure-activity (Sar) concern.

2) the Safety concern threshold (Sct) is the dose (0.15 µg/day) below which a leachable would present negligible concern for adverse carcinogenic and noncarcinogenic effects.

3) Known highly toxic substances such as PaHs, nitrosamines and 2-mercaptoben-zothiazole) are considered to be “special case compounds” for orally inhaled and nasal drug applications and should be considered on a case-by-case basis.

4) By using one of these toxicological thresholds as well as dosing information, the analytical Evaluation threshold (aEt) can be calculated. the aEt is the level at or above which an oiNDP pharmaceutical development team should identify and quantify a particular extractable and/or leachable and report it for potential toxi-cological assessment.

acceleratedsolventextraction


above limits apply irrespective of the duration of the exposure. a short-term expo-sure (e.g. vaccination) would however be clearly less risky than a life-long exposure (e.g. a drug against hypertension). For drugs that are life-saving in an acute situation or in cases where life expectancy is limited much higher limits have in the past been accepted. risk assessment is a multi-variate task and sound scientific reasoning must be used to demonstrate that any risks are acceptable.

leAcHAblesthe leachable study is conducted in order to monitor any substances that leach out of a material into the final drug product in its commercial packaging (primary and secondary) and are thus exposed to patients. the smart way to conduct a regulatory leachables study is as part of a regulatory stability study. the substances of concern and to be controlled will have been identified via toxicological consideration of the ex-tractables data. a simple example of how analytical evaluation threshold is set follows:

if we take the Sct proposed by PQri of 0.15 μg per day for an individual leachable in an oiNDP, an MDi with 0.5 ml of drug product in a canister that has 200 actuations

AbbreviAtioN Notes

analytical Evaluation threshold

aEt Sensitivity of analytical method for leachables studies (see PQri)

Drug Product DP Formulated active ingredient

orally inhaled and Nasal Drug Product

oiNDP common dosage forms

Metered Dose inhaler MDi Device delivering a specific amount of medication to the lungs

Safety concern threshold Sct acceptable daily intake for a carcinogen (see PQri)

total Daily intake tDi Mass ingested per day per person

threshold of toxicological concern

ttc Same as tDi

coMMoN aBBrEviatioNS USED iN EXtractaBlES aND lEacHaBlES


with a recommended daily dose of 10 actuations would have an estimated aEt of 6 μg/ml and a final aEt of 3 μg/ml.

the impact of drug formulation upon leachables was shown in a study15 using rubber exposed to 11 common formulations. Key findings were drug product additives that alter the polarity of the formulation could impact the leachables profile, for example; tween 80 significantly increased the leachables 2-methylpentane, 3-methylpentane, hexane, methylcyclopentane, and cyclohexane but had no impact on BHt. the bulk-ing agents sucrose, mannitol, and trehalose also led to different levels of leachables but trehalose gave the minimum levels.

in the following sections a few special topics are mentioned that deserve special attention.

PreFilleD syriNgesthese devices are increasingly popular. Potential contaminants are inorganic oxides and ions from borosilicate glass, organics from the rubber plunger, Fe, cr, Mn, Ni and Mo from the stainless steel needle; adhesives (organic oligomers and polymers such as acrylates that are processed via curing process); silicone oil (polydimethyl silox-ance) to lubricate the barrel, syringe tools including tungsten pin and nylon pins. Metals and ions such as Fe and Mn may catalyze oxidation of proteins. Where poly-mers are used rather than glass the usual scenario of investigation organic molecule E/l will apply. interactions with ions or other molecules might lead to aggregation in the case of biologics.

coNtrol or quAliFicAtioN oF mAteriAlsthe Polymerforum Group engages the polymer supplier chain in understanding of the needs of pharma. a minor change in a polymer manufacturing chain might trig-ger product recalls and lengthy investigations. control of materials via extractables testing should use validated analytical methods (in accordance to icH guidelines) that are also capable of detecting new substances.

Many materials are used such as glass, natural or synthetic rubbers, polyethylene, polyethylene terephthalate, and polypropylene (see US Pharmacopeia <661>), poly-vinylchloride, polymethylacrylates, polyolefins, co-polymers such as acrylonitrile-bu-tadienestyrene. these materials are themselves complex formulations containing numerous impurities and additives that serve a variety of purposes therefore repre-senting thousands of possible chemical structures. regulators also have compliance requirements for container closure systems that are also medical devices. various sections of iSo10993 apply to such combination products. companies must pro-vide quantitative data on the chemical composition of the materials going into their devices. in all cases, E/l are not limited to raw materials but also include any compo-sitional changes that are likely to occur in use.


AbbreviAtioN Notes

american association for Pharmaceutical Science

aaPS international forum – exchange of knowledge among scientists – discovery, development and manufacture of pharmaceutical productswww.aaps.org

Bio-Process Systems alliance BPSa inter-company group supporting the use of disposable reactor technologywww.bpsalliance.org

European Food Safety authority

EFSa an EU official bodywww.efsa.europa.eu

Extractables and leachables Safety information Exchange

ElSiE inter-company group producing databases of 1) extractables from selected plastics 2) toxicology for selected substanceswww.elsiedata.org

international Pharmaceutical aerosol consortium on regulation and Science

iPac-rS international association of innovator and generic companies that develop, manufacture or market orally inhaled and nasal drug productswww.ipacrs.comwww.ipacrs.com/PDFs/Device%20Paper.pdf

aaPS inhalation technology Focus Group

itFG www.aaps.org/inside/focus_groups/IT/index.asp

Polymerforum annual meetings and continuous dialogue in be-tween polymer suppliers and the pharma industrywww.ipacrs.com/PDFs/materials/8-Polymer%20Forum%20-%20Tjader.pdf

National institute of Standards and technology

NiSt US agency responsible for advancing measure-ment science, standards, and technologywww.nist.gov

Product Quality research institute

PQri collaboration between FDa’s center for Drug Evaluation and research, industry, and academiawww.pqri.org

orGaNizatioNS iNvolvED WitH EXtractaBlES aND lEacHaBlES


DisPosAble bioreActorsare we only concerned with the final container/delivery system? Unfortunately not! With the increasing popularity of disposable bioreactors for the production of biolog-ics the various plastics – bags, tubing, filters are sources of potential contamination. it may be possible to adopt a process validation approach to show that contaminants are removed by down-stream processing. interestingly, the BPSa11, 16 advocates the use of a less extreme range of extraction conditions than recommended for drug products.

An essential principle is “fit for purpose“ solutions meeting regulatory aims without “over-engineering.”

tHe solviAs APProAcHSolvias has worked on E/l over the years and offers expert service incorporating smart practices for sample preparation such as accelerated solvent extraction and a full range of analytical technologies. Beyond the NiSt database, Solvias has a large in-house proprietary mass spectral database with over 2000 entries for using standardized lcMS and GcMS methods for the automated reliable identification of polymer additives and their degradation products.

an essential principle is “fit for purpose” solutions meeting regulatory aims without “over-engineering.”

eXPertise AND tecHNology Are tHe oNly sHortcutsSerious contamination of food and medicine may rapidly become global knowledge via today’s information superhighways. the challenges should not be underestimated and must be addressed scientifically and methodically via the decision-trees such as are described by PQri.

the key overarching E/l concepts are risk assessments and control systems. aEts are based upon Scts. Studies are performed in a tiered multidisciplinary approach that aims to satisfy the regulators that the risks have been identified and controlled.


resources can be minimized and risks managed through adopting state-of-the-art methodologies within fully engineered work-flows designed by experts and sup-ported by automation. thereafter effective advocacy and consultation with the regu-lators maximizes the probability of winning market authorization.

taking into account all of the above considerations, it can be challenging to effective-ly design a program that will adequately address and evaluate the E/l issues associ-ated with the container closure system, delivery devices, and processing equipment. it is very important to partner with a cro/cto that has the knowledge and experi-ence to bring your product to market in an efficient manner. •

References1. NicoleCasadevall,1,“Epoetin-inducedautoimmunepureredcellaplasia,”TheHematologyJournal,2004,5,104–1092. CorredorC,TomasellaFP,YoungJ.“Druginteractionswithpotentialrubberclosureextractables.Identification

ofthiol-disulfideexchangereactionproductsofcaptoprilandthiurams,”JChromatogra.,2009,1216,(1),43–483. XueguangFang,NickCherico,DamonBarbacci,angelam.Harmon,mattPiserchioandHollyPerpall“Leachable

StudyonSolidDosageForm,”americanPharmaceuticalReviewNov/Dec20064. EmEa,CPmP/QWP/4359/03“guidelineonplasticimmediatepackagingmaterials,”London,may19,20055. FDaguidanceforIndustry“ContainerClosureSystemsforPackagingHumanDrugsandBiologics,”may19996. FDaDRaFTguidanceforIndustry:genotoxicandCarcinogenicImpuritiesinDrugSubstancesandProducts:

Recommendedapproaches,Dec3,20087. EmaguidelineEmEa/CHmP/QWP/251344/2006,gUIDELINEONTHELImITSOFgENOTOXICImPURITIES,London,

June28,2006,www.emea.europa.eu8. D.L.Norwood,“Understandingthechallengesofextractablesandleachablesforthepharmaceuticalindustry–

safetyandregulatoryenvironmentforpharmaceuticals,”am.Pharm.Rev.,2007,10,(2),32–399. DanielL.Norwood,etalPharmaceuticalResearch,“BestPracticesforExtractablesandLeachablesinOrallyInhaled

andNasalDrugProducts:anOverviewofthePQRIRecommendations”Vol.25,No.4,april200810. PQRIPODPE&LWorkshop,September22–23,2011,Washington,DCUSaagendacanbefoundatwww.pqri.org11. “Recommendationsfortestingandevaluationofextractablesfromsingle-useprocessequipment,”2010

seewww.bpsalliance.org12. ELSIEwww.elsiedata.org/13. PQRILeachablesandExtractablesWorkinggroup,“SafetyThresholdsandBestPracticesforExtractablesand

LeachablesinOrallyInhaledandNasalDrugProducts,”Sept8,200614. D.Ball,J.Blanchard,D.Jacobson-Kram,R.mcClellan,T.mcgovern,D.L.Norwood,

m.Vogel,R.Wolff,andL.Nagao,“Developmentofsafetyqualificationthresholdsandtheiruseindrugproductevaluation,”Toxicol.2007,Sci.97(2):226–236

15. XiaochunYu,PhD,DonDeCou,PhD,DerekWood,StevenZdravkovic,HeatherSchmidt,LaurieStockmeier,RobertPiccoli,PhD,DanielRude,XiaoyaDing,PhD“aStudyofLeachablesforBiopharmaceuticalFormulationsStoredinRubber-StopperedglassVials,”BioPharmInternational,Volume23,Issue4,apr1,2010

16. ToddKapp,JohnBoehm,JeffChase,JeffCraig,KenDavis,Vikasgupta,JohnStover,andSteveWilkowski,S.annemontgomeryandKevinOtt,“RoadmaptoImplementationofSingle-UseSystemsaBPSaWhitePaperformanufacturingDecision-makerswithinEnd-UserOrganizations,”BioProcessInternational,apr2010


rapid results in transmission electron microscopy (tem)Morphological aspects of aPi and biopharmaceuticals at the ultrastructural level deliver important information for troubleshooting and quality control. Well-characterized products provide deeper insight into the behavior of the product.Author: Dr. Nadine Mattes

iNtroDuctioNQuality control processes are of utmost concern in pharma-ceutical production. Deviations in product characteristics may have a direct impact on the health of consumers. the quality management process requires the acquisition of a lot of data including quantification, purity, biological activity, product-related and physico-chemical properties. But what does the product look like in its ultrastructure? this important character-istic is often neglected. visualizing the aPi in its micro- or nano-structure is an important benefit for characterization and may provide essential information about the behavior of the prod-uct regarding its bioavailability, pharmacokinetic and pharma-codynamic. the morphological information given by micro-graphs often speaks louder than words.

Solvias has been a specialist in visualizing aPi at the light as well as at the electron microscopic level for several years. reso-lution at the nanometer scale is a big advantage in using trans-mission electron microscopy (tEM). although specimen prep-aration for tEM is often very complex and time consuming, there are methods that are capable of delivering results within one or two days as the following examples show.

Dr. NADiNe mAttes — Solvias lab Manager Microscopy

NadinematthesdidherPhDinthefieldofhealtheffectsofnanoparticlesattheUniversityofBernfollowedbymanagingthelightandelectronmicroscopylabatthedepartmentofVeterinaryanatomyattheUniversityofBern.Since2006shehasbeenworkingingLPenviron-mentdevelopingandvalidatingligandbindingassaysforsamplesderivedfromclinicalandpre-clinicalstudies.In2010shejoinedSolviasagwhereshetookovertheresponsibilityfortheelectronandlightmicroscopylab.


tem APPlicAtioNs leADiNg to FAst resultsSizE DEtErMiNatioN oF NaNoParticlESFigure1 shows a sample with intrinsic contrast effects as it con-tains complex Fe particles adsorbed on a tEM holder. Nanopar-ticles with diameters of less than 2 nm can be visualized at a magnification of 100,000x or higher.

Using software-based image processing the size of the nanoparticles may be determined and also the size distribu-tion can be evaluated.

Figure1:Fe-complexparticles,unstained

Figure2:Virus-likeparticles,negativestained

Figure3:Bacterium,negativestained

vacciNE ProDUctioN: UltraStrUctUral cHaractErizatioN oF SUrFacE StrUctUrESSurface proteins can be made visible using negative contrast-ing techniques in combination with tEM.

Figure2 shows virus-like particles. the lipid membrane as well as the particles’ envelope where the specific surface antigens can be displayed. the image shows that the particles have a spherical shape and intact lipid membranes. the typical enve-lope structure is the evidence that the virus-like particles show the morphology expected. the existence of surface structures such as the envelope is essential for the effect of the product.

Figure3 shows an inactivated bacterium. the thick cell wall and a long flagella for movement are displayed. Furthermore, short pili on the cell surface can be visualized. the existence of the pili is important for the efficient effect of the product.

0.2 μm

individual particles

lipid membranevirus envelope

Matrix

Bacterial wall

Pill on the cell surface

Flagella

0.5 μm

100 nm


cHaractErizatioN oF aGGrEGatES or aGGloMEratE ForMatioN it may also be a quality issue if particles in the product form ag-gregates or agglomerate spontaneously. Figure4 shows particles as individual particles as well as large aggregates. the ratio of aggregates/agglomerates to individual particles can be deter-mined. the morphology of the particles and aggregates/ag-glomerates can be visualized.

Figure4:Particlesformingaggregates,negativestained

tem APPlicAtioNs For r&D there are several sophisticated methods for sample prepara-tion. Bulk material can be embedded in resin, and ultrathin sections of <100 nm thickness can be made. images of heavy metal stained ultrathin sections of e.g. tissue, plant material or polymers can be established. the method can also be com-bined with the analytical method of immunogold-labeling (for identification of specific proteins, gold particles with 3–15 nm diameter are coated with antibody against the protein of inter-est) but depends on the availability of gold-labeled antibodies.the sophisticated method of immunogold-labeling can also be used for the applications listed above for further analytical results and added value to the morphology information given by the images.

the method and applications listed can be offered to support characterization of the product. as the examples above show it might be important to involve morphology as one characteris-tic in early phases towards later registration and production. •

1 μm


solviAs’ oNe-stoP sHoP coNcePt PAys DiviDeNDs

vitae Pharmaceuticals took advantage of Solvias’ new and extended manufacturing capabilities and the deal illustrated how the service bundling concept serves to promote Solvias’ core expertise in process design.

March 21, 2011Volume 89, Number 12, pp. 20–21

Rethinking A Renin InhibitorCase study #3: Vitae turns to Solvias for a new synthetic route as well as Phase II clinical production.Rick Mullin

In 2009, as its lead drug candidate completed Phase I clinical trials as a kidney disease treatment, Vitae Pharmaceuticals de-cided to change its manufacturing game plan. The Fort Wash-ington, Pa.-based computational drug discovery firm had earlier enlisted Solvias to help design the drug’s final production steps. Hoping to reduce costs, it entrusted Solvias to overhaul how the starting materials were made as well.

Solvias, a Swiss contract services firm, was making changes of its own at the time. The company was in the midst of opening a kilogram-scale facility that produces pharmaceutical chemi-cals at current Good Manufacturing Practice standards. Its goal was to add early-stage manufacturing to the process design and analytical services it offered drug companies.

By adding cGMP manufacturing to its menu, Solvias was aiming to establish the proverbial one-stop shop for early drug devel-opment. Vitae executives liked what they saw and decided they would hire Solvias for yet a third task: production of the drug’s active pharmaceutical ingredient (API) for future clinical trials.With the expanded contract, Vitae became the second customer to take advantage of Solvias’ new manufacturing capabilities. The deal illustrated how the service-bundling concept serves to promote Solvias’ core expertise in process design.

Solvias began work on a new synthesis of the starting materi-als at the end of the Phase I clinical trials. At that time, the drug, called VTP-27999, was being manufactured by Carbogen Amcis, another Swiss firm, with starting materials from a Chinese sup-

plier. Vitae is now moving forward with Phase II production in-corporating new chemistry from Solvias that cuts the number of steps in its synthesis of the starting materials roughly in half.VTP-27999 is an orally available renin inhibitor with four chiral centers. The molecule is as complex as its target. Renin produc-tion is the first and rate-limiting step in the renin-angiotensin system, the primary biochemical pathway for regulating blood volume, arterial pressure, and vascular function. Renin affects critical organs – including the heart, kidney, and brain – at a tis-sue level. Tekturna, a Novartis drug for hypertension, is the only other renin inhibitor on the market.

According to David A. Claremon, vice president of chemistry at Vitae, controlling manufacturing costs is crucial, given the com-petitiveness of the therapeutic markets that a renin inhibitor might enter. For example, Vitae may also develop VTP-27999 as a hypertension drug, a market in which it would go up against low-cost alternatives. He says Solvias has pushed the compound in the right direction, arriving at elegant chemistry that signifi-cantly cuts the cost of production.

Solvias’ initial involvement was in optimizing the final synthetic steps, Claremon says. “We started with them in 2008 on scout-ing out catalyst routes and crystallization conditions for API production. That was starting from the back end of the synthe-sis, getting a suitable salt for the API, working on the three-step manufacturing process. Then we got them involved in identify-ing the routes to making the two starting materials.”

When Vitae learned of Solvias’ new kilo lab, it began calculat-ing the advantages of having the firm that designed the process also manufacture the clinical materials. “They would be able to provide a process well on its way to being a commercial process,” Claremon says. “And that’s what we are aiming for.”

Carbogen manufactured the API for initial clinical studies using starting materials synthesized via traditional medicinal chem-istry, Claremon recalls. “We also supplied these materials to Sol-vias, which began a second cGMP campaign,” he says. “But by this time, they were also developing methods to make the two components using asymmetric chiral catalysis.”

The molecules involved are a 3-substituted piperidine ring and a 3-substituted pyran ring. According to Claremon, the Solvias process induces chirality in racemic starting materials via asym-metric hydrogenation. The process moves forward with two mol-ecules, each with a pair of chiral centers. “The possible number of diastereoisomers is 24, if we proceed randomly,” he says. “But we control all the stereochemistry to give us a single enantiomer and diastereoisomer at the end of the process. No resolution is required in any of the work Solvias has identified for us. There is no waste associated with having materials to throw away from the resolution.”


Michael Quirmbach, vice president for U.S. business develop-ment at Solvias, says the firm is familiar with the complexities of renin inhibitor chemistry, having developed a scalable route for Tekturna, the Novartis drug. “The molecules are completely different,” he says. “But they are still targeting the enzyme renin. These are very complex molecules.”

Quirmbach describes the initial process for making the starting materials as “textbook chemistry.” He adds, “It was good, but it was long.” Solvias went to work on developing the chiral centers of the starting materials and the solid-state characterization of the API. Initial cGMP runs have been completed, he says.

Adding cGMP production to its process development services is a natural evolution for Solvias, Quirmbach says. “Prior to offer-ing cGMP kilo lab services, we had been focusing on early-stage process development and supplying non-cGMP toxicology ma-terial,” he explains. “Companies had been coming to us with challenging molecules for our process development and solid-state capabilities. We have 150 analytical chemists, with every-thing done in-house.” Solvias is counting on its strength in pro-cess chemistry to boost its entry into manufacturing, an area in which many of its competitors have long played.

At the same time, Solvias will continue to partner with other manufacturers to provide commercial-scale API production based on processes it develops. The new kilo lab has a capacity of about 20 kg. “We will be focusing on development, proof of concept, and first production for clinical trials,” Quirmbach says. “Once you have a good procedure available, a lot of other com-panies can do the manufacturing. The key is to have the devel-opment expertise and integrative project management skills.”

Vitae, as a venture-backed drug discovery firm that is dependent on contractors for a range of chemistry and manufacturing ser-vices, sees advantages to the one-stop shop offered by Solvias, Claremon explains. “Solvias has incredible in-house expertise in developing the means of doing chirally catalyzed chemistry,” he says. “They have automated equipment and the catalysts in hand. There aren’t many groups in the world who have all that, as well as analytical support and automated crystallization. “This was an important one,” Quirmbach acknowledges about the Vitae contract. “Vitae was only our second customer at the time for these bundled services. It was important to find custom-ers that believe we have the right qualifications and can deliver on single-source services for early drug development.”

Solvias currently has about 15 customers paying for bundled services, including manufacturing.

Chemical&Engineering NewsISSN 0009-2347Copyright ©2011 American Chemical Society

EN SUITE Solvias now packages cGMP production with its core process design services.


solviAs – your reliAble PArtNer iN tHe PHArmAceuticAl iNDustryif you are looking for increased capacity or profound know-how for your development or manufacturing activities, our experience and proven track record gives you confidence that your projects will be expertly performed and deliv-ered on time.

With our services, products and technologies in the field of analytical, chemical and biopharmaceutical development, we provide integrated solutions to enhance the value chain of our customers.

• analytical Services • Biopharmaceutical analysis• Polymorphism, Salts, and crystallization• chemical Development and GMP Manufacturing• catalysis• Process analytical technology (Pat)• Patent and Search Services

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4303

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solvias Ag

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Switzerland

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Solvias Prospects 01 l 2011