Solvias Prospects 01/2012

Leveraging the power of cocrystals

Characterization of Biologics –the deamidation of asparagine

New Partnership for integrated chemical development and manufacturing services

1 2

012

2 SoLviaS ProSPeCtS — 1/2012

3

Preface 4

New partnership for integrated chemical development and manufacturing services 9

Leveraging the power of cocrystals 12

Characterization of biologics – the deamidation of asparagine

20

accelerating chemical (process) development with HtS technology 24

News 26

events

Content

3SoLviaS ProSPeCtS — 1/2012

Dr. Hansjörg WaltHer — Ceo

Dear reader and valued customer

Welcome to the first issue of our customer magazine in 2012. i would like to take this opportunity to thank you for your trust and partnership in the past year and wish you and your loved ones the very best for the New Year.

With this issue we are pleased to announce the finalization of our partnership agree-ment with rohnerChem. although the two companies have a long history of close collaboration, with the formalization of our cooperation we can now provide you with a smooth solution from route development to commercial production. one overall project manager as a single point of contact ensures that the project meets deadlines and budget and customers are well informed at any point of time along the process.

over and above that, i’m glad that we can retain the tradition of our scientific experts not only providing their dedicated services to you as customers but also actively con-tributing to this magazine.

timo rager, project leader for solid-state development, shares his solution for an im-proved cocrystal screening based on thermodynamic considerations.

Deamidation represents a major degradation process in proteins. an article written by Frank Moffatt suggests the use of two characterization tools that are readily incorpo-rated into standard workflows, alongside commonly used techniques. the procedures developed by our scientists alena Böhmová and Maria Schwarz promise to produce complementary data during characterization, comparability and stability assessments.

in the field of high-throughput catalysis screening, Felix Spindler outlines how an efficient screening workflow can boost the development of industrial catalytic processes.

We wish you an inspiring read. Yours sincerely

Dr. Hansjörg Walther


Solvias is well established as an expert for chemical, analytical and solid-state de-velopment. in its own right, rohnerChem is recognized for its experience in scale-up and commercial scale custom synthesis. the signed partnership agreement merely formalized the existing close collaboration between the two companies and now enables customers to benefit even more from a seamless experience and a powerful combination of resources, expertise and technologies. through the commitment of an integrated service offering, clients will have only one overall project manager to supervise the entire project and ensure a smooth process from a to Z.

New partnership for integrated chemical development and manufacturing servicesSolvias and rohnerChem have entered into a strategic partnership that will close the chain of services from route scouting through to commercial manufacturing to deliver enhanced integrated chemical development and manufacturing services to customers. this partnership capitalizes on the complementary characteristics of Solvias’ expertise in chemical, analytical and solid-state development and rohnerChem’s experience in scale-up and commercial scale custom synthesis of complex, small molecule fine chemicals and aPis.


accelerating cHemical Developmentalready at the earliest stages of development upscaling requirements will be con-sidered for optimization allowing customers to benefit from faster commercialization and overall cost. With a strong focus on the development of lean and cost-effective processes for chiral molecules, Solvias relies on the extremely efficient high-throughput screening (HtS) which has been used successfully in asymmetric homogeneous catalysis, C-X-couplings, racemate resolutions, carbonylations, hydroformylations, bio-catalysis development, and various other transformations. to ensure the controlled production of a desired polymorphic form it is essential to primarily understand the product and be intimately familiar with the process. Solvias’ solid-state development group has the expertise and is fully equipped to assist in developing crystallization processes, perform polymorphism studies and provide guidance in the development of the best solid form for any aPi. after process optimization and the delivery of kg-quantities which can be performed at either partner’s Kilolab, commercial quanti-ties can be manufactured at rohnerChem’s FDa inspected facilities.

Both companies will continue to capitalize on their strong expertise in enantio-selective catalysis for the efficient production of building blocks and aPis and from the ideal fit of key technologies in catalytic, hazardous chemistry, high-pressure, organo-metallic, cryogenic and various other technology areas.

tHomas rosatzin — Ceo of rohnerChem, commented:

“Our customers will benefit substantially from this cooperation as we will be able to reduce timelines for customers and create a service platform with an extremely broad technology and asset toolbox from the first milligrams to commercial quantities.”


OCF3

CF3

OHCF3

CF3

20 bar H25°C

PN

O

Ru

PPh3

Cl Cl

Fe

Asymmetrichydrogenationunderhighpressure

a case stuDy – fast Development anD syntHesis of a cHiral proDuctGoal: Production of 400 kg of a chiral alcohol with an enantiomeric excess of 98 %. Solvias performed catalysis screening, optimized the reaction conditions and manu-factured and supplied the catalyst precursor. rohnerChem was responsible for pro-cess development, safety investigation and scale-up. Development and production of the 400 kg were successfully completed within two months. the reaction was carried out at 25°C and at 20 bar. the quality of the starting material and the handling of the homogeneous catalyst were identified as the most critical factors.

Hansjörg WaltHer — Ceo of Solvias, stated:

“As a service provider, our efforts are constantly focused on adding value to our customers’ businesses. The strategic partnership ideally supplements our integrated offer in chemical and analytical development and provides customers with a smooth solution from route development to commercialization.”


ROhneRChem ‒ SWiSS reLiaBiLitY iN DeveLoPMeNt aND MaNuFaCturiNG

rohnerChem is located in Pratteln close to Basel, Switzerland. the company has been established in 1906. We develop and manufacture a wide range of com-plex organic chemicals, advanced intermediates and aPis. the plant consists of one r&D center, a pilot plant and two production buildings with a total reactor vol-ume of more than 150 m3. our pilot and production plants operate at a tempera-ture range of –90 to 200°C and a pressure range of 0.02 to 64 bar. available reactors (50 L–10,000 L) are stainless steel, hastelloy, inconel 686 and glass-lined. rohnerChem is focused on rapid development and flawless upscaling of fine chemicals and aPis in close cooperation with its customers. over the last decade, more than CHF 100 million have been invested into the company, enabling us to offer truly state-of-the art equipment, capabilities and services, from the initial development quantities up to hundreds of mtons.

our core tecHnologies anD services• organometallic and cryogenic

chemistry• aPi manufacturing (FDa, Swissmedic)• transition-metal catalysis• rapid process development, flawless

upscaling, and economy-of-scale production

• High-pressure hydrogenations and carbonylations

• Handling of solids• Hydrazine chemistry and heterocycles• Functional polymers• Hazardous and malodorous reagents

and reactions

RohnerChemsmanufacturingfacilities


SOlviAS ‒ iNteGrateD CHeMiCaL aND aNaLYtiCaL DeveLoPMeNtSolvias is an entirely independent and privately owned company located in Basel, Switzerland. the teamwork and cooperation of more than 280 highly qualified employees lead to successful chemical and analytical development projects.

state-of-tHe-art manufacturing infrastructure• Multi-purpose reactors from 30–100 L scale (–70°C up to 350°C), non-GMP and cGMP • Multiple autoclaves up to 300 bar (0.05 to 50 L)• reactor materials ranging from glass, glass-lined steel, stainless steel, PtFe,

tantalum and monel to hastelloy B and C• Chromatography for up to 20 kg of separation material

integrateD cHemical anD analytical Development• Process research and development• Salt and cocrystal screening• Process optimization• reference substance supply• Polymorph screening• Manufacturing of

- tox material (non-GMP/cGMP) - cGMP starting materials - aPis for clinical phase

• Method development and validation (iCH)

• analytical characterization• reference standard qualification• extractables/leachables• Quality control analysis• Stability studies

Qualitythe company’s laboratories have been successfully inspected by more than 300 customers, the Swiss authorities (Swissmedic) and the FDa.

available tecHnology• asymmetric homogeneous catalysis• C-X-coupling• Chiral ligands (more than 600 chiral

ligands and catalysts)

• Biocatalysis• Fluorination chemistry (HF, SF4, F2)• Hazardous and high-pressure chemistry

rohnerChemGempenstrasse 6, 4133 Pratteln, [email protected], www.rohnerchem.com, tel. +41 61 825 11 11

Solvias aGrömerpark 2, 4303 Kaiseraugst, [email protected], www.solvias.com, tel. +41 61 845 60 00


Cocrystal formation can help optimize the physicochemical properties of an active pharmaceutical ingredient. this fact was underscored in December 2011 with the FDa publication of a Draft Guidance for industry about the regulatory classification of pharmaceutical cocrystals.

a primary motivation to prepare cocrystals is the search for drug substances with im-proved solubility in the biological environment. Solubility properties are, however, not only relevant for the application of cocrystals but also for their preparation. a cocrystal is preferably crystallized from a solution, and the composition of the reaction mixture has a critical impact on the success of the crystallization process. as can be seen from Figure1, the pure cocrystal is formed only in a limited range of compositions. in other domains of the phase diagram, the cocrystal is obtained with admixtures of the aPi or the cocrystal former. Still other combinations of aPi, cocrystal former and solvent provide the pure aPi or the pure cocrystal former as the only solid phase. Knowing the phase diagram is therefore essential for the successful preparation of a cocrystal with optimal yield and purity.

Furthermore, the solubilities of the individual components (aPi and CCF) should also be taken into account in cocrystal screening experiments. otherwise, the cocrystal may easily be missed. a method that is considered particularly reliable consists in suspension equilibration experiments with saturated solutions. But the effect of a solvent on cocrystal screening can be even more complex: Some solvents may form a solvate with one of the components or with the cocrystal. this solvate formation will compete with cocrystal formation and may prevent the dis covery of a cocrystal Figure2.

Leveraging the power of cocrystalsCocrystals open a myriad of possibilities to optimize the physico-chemical properties of solid dosage forms. However, discovering cocrystals is a prerequisite to its development. Solvias has the solution with an improved screening method based on thermodynamic considerations for finding cocrystals.

Dr. timo rager — Project LeaderTimoRagerreceivedhisPhDwithatopicinpolymerchemistryin1997.Followingseveralpostdoctoralstays,hejoinedSolviasin2005asaprojectleaderforsolid-statedevelopment.

Dilution of the solvent will lower the risk of solvate formation or – at least – decrease the stability of a potential solvate.


Figure2:TheformationofasolvateoftheAPIcanleadtoasignificantdecreaseinsizeofthecocrystaldomaininthephasediagram.Thiscocrystalphasemaythereforeeasilybemissed.

Figure1:Exampleofaphasediagramencompassinganactivepharmaceuticalingredient(API),acocrystalformer(CCF),andasolvent.

Solvate

How then to deal with this risk in screening experiments in a most efficient way, i.e., without performing an extensive investigation of solvate formation of a new aPi or performing many parallel experiments in a number of solvents?Solvates are only stable in the presence of a high concentration of the corresponding solvent. therefore, dilution of the solvent will lower the risk of solvate formation or – at least – decrease the stability of a potential solvate. Considering that it is not known in advance which solvent is critical to solvate formation, the right way to dilute is through the combination of a large number of solvents. the concentration of each solvent in the mixture will then be low.

We have tested this approach with the well-known aPi carbamazepine, which forms a number of solvates and cocrystals. Saturated solutions of 19 cocrystal formers were prepared in a mixture of nine solvents that are known to form solvates with carbama-zepine (acetic acid, acetone, dioxane, DMF, DMSo, ethylene glycol, formic acid, sulfo-lane, and water). Solid carbamazepine was suspended in these saturated solutions, and the precipitate was analyzed by X-ray powder diffraction after a couple of days. the corresponding cocrystal was recovered in 16 experiments whereas the other three experiments generated a mixture of cocrystal and acetic acid solvate. the hit rate in this set of experiments was therefore greater than 80 %.

Another issue that can make cocrystal screenings tedious arises from large differences in solubility. Solvent mixtures

can also provide a solution to this problem.


another issue that can make cocrystal screenings tedious arises from large differences in solubility. a suitable solvent has to be determined for each cocrystal former, and the amount of suspended solid has to be adapted for each experiment. Solvent mixtures can also provide a solution to this problem: as can be seen from Figure3, the solubility differences between different cocrystal formers in a solvent mixture are comparatively small. thus, the same solvent – and therefore the same amount of aPi – may be used for a number of different cocrystallization experiments.

Based on all these aspects, we consider suspension equilibration experiments in complex solvent mixtures to be a reliable and rapid method for the first step of cocrystal development, i.e., the discovery of new cocrystals. We offer this method as a complementary approach in our most recent screening projects. •

Figure3:Solubilitiesofcarbamazepineand19cocrystalformersinwater ,isopropanolandthesolventmixture

Literature: T.Rager,R.Hilfiker,Cryst.GrowthDes.10(2010)3237-3241

o-acetylsalicylic acidadipic acid

Benzoic acid(+)-Camphoric acid

CarbamazepineFumaric acidGlutaric acidGlycolic acid

1-Hydroxy-2-naphthoic acidMaleic acidL-Malic acid

Malonic acidNicotinamide

oxalic acid2-oxoglutaric acid

SaccharinSalicylic acidSuccinic acid

DL-tartaric acidL-tartaric acid

Solubility [mol/mol]

10–5 10–4 10–3 10–2 0.1 1.0


introDuctionProteins are complex, heterogeneous and unstable and yet they are part of the most intimate molecular architecture and functions within our bodies and represent the most significant growth sector of the innovative pharmaceutical market, driven largely by recombinant monoclonal antibodies (Mabs).

Besides oxidation, aggregation, and fragmentation, deamidation is another major degradation process in proteins. the amides asn and Glu are prone to deami dation but the rate of deamidation is generally much faster for asparagine (asn) than for glutamic acid (Glu). “Simple” deamidation (amide carboxylic acid) changes the charge of a protein, lowers the isoelectric point (pi) and increases the mass by 1 Da. the surface and structure of the protein may also be altered. in the case of biologic medicines this may lead to aggregation which in turn could risk an immune response or a reduction in biological activity.

the degradation pathway of deamidation plays a crucial role in autoimmune diseases, cancer, neuro-degeneration and aging. on average proteins contain 4.4 % for asn1 so the potential for deamidation associated with asn is almost universal. Both natural antibodies and recombinant Mabs alike show significant deamidation at asn 384 in the conserved Fc region2. Deamidation has been described as a “biological” or

“molecular clock”3 but as we will see every “clock” runs at a different speed for example, from the age of 25, asn in human α a-crystallin has t½ of 77 years4.

Characterization of biologics – the deamidation of asparagineDeamidation represents a major degradation pathway of therapeutic proteins, and plays a crucial role in autoimmune diseases, cancer, neuro-degeneration and aging. this article suggests the use of two characterization tools that are readily incorporated into standard workflows, alongside commonly used techniques. in our experience the procedures are likely to produce complementary data during characterization, comparability and stability assessments.Author: Dr.FrankMoffatt

Dr. frank moffatt — Business Development ManagerSinceJuly2010BusinessDevelopmentManageratSolviasAG.FromJuly2007toJuly2010ProductManagerforBiopharmaceuticalsatSolvias.


Currently regulators demand that biosimilar applications include comprehensive comparability to an innovator product. a scientifically sound and well presented physico-chemical characterization package may minimize the demands for addition-al non-clinical and clinical data. We suggest the use of two less commonly used char-acterization tools that in our experience are likely to produce additional useful data concerning deamidation. the methods have been validated for quality control pur-poses. the first is a whole protein or top down method, the other a bottom-up meth-od based upon peptide mapping.

in practice, deamidation is not as “simple” as the hydrolysis of asn to aspartic acid (asp) Scheme1. often isoasp is the major product and sometimes the reaction stops at the intermediate succinimide (asu) stage.

A scientifically sound and well presented physico-chemical characterization package may minimize

the demands for additional non-clinical and clinical data.

ThemethodsweredevelopedinthelaboratoriesofSolviasbyDipl.Ing.AlenaBöhmováandPDDr.MariaA.Schwarz


tHe cHemical reactivity of asparagine in proteinsSoLutioN CoNDitioNSinsulin is composed of two disulphide linked chains, is one of the lowest molecular mass protein therapeutics and has been studied in great depth10. the a-chain has 21 amino acids and the B-chain 30 amino acids. However, the deamidation behavior is even more complicated than shown in Scheme 1. a covalent dimer is formed by a chemical reaction between the C-terminal asn via the asu intermediate and the N-terminal Phenylalanile (Phe) of a second molecule. at pH 2–3 deamidation occurs on the a chain at position 21 rendering the insulin inactive so that within six months at 25°C deamidation is 90 % complete. at neutral pH deamidation occurs on the B chain at position 3 but all three forms asn, asp, and isoasp are equally active. Note that at low pH only asp is formed and not isoasp.

in general it is observed that deamidation increases as the ionic strength or temperature increases (at pH 7.4), is slowest at pH 4–6 and is buffer dependent11. therefore forced degradation to generate deamidation products is generally accom-plished at basic pH.

SeQueNCeasnGly is particularly sensitive, 70–80 % deamidation occurred with t½ of 8 hours during tryptic digestion at 37°C12 whereas asnval & asnile have a t½ of >200 days13.

Mabs have numerous asn residues. Most resist deamidation in the conserved regions (Fc) of an igG14 :• Deamidation observed SNG, eNN, LNG, & LNN• Deamidation not observed GNt, tNY, YNP, WNS, SNF, CNv, SNt, WNS, FNW, HNa, FNS,

SNK, GNv, HNH, SNY, LNW, SNL, NNF, DNa, GNS, & FNr

Scheme1:Deamidationofasparagine

O

O

O

OHO

NH

NH

HN

CH2

O

O

O

NH2

O

NH

NH

HN

CH2

O

OOO

HN

OH

HNNH

CH2

Isoaspartic acid

CH2NO

O

OO

NH

NH

Succinimide intermediate

Aspartic acid

Asparagine


Figure1:Forceddeami-dationinaMAbshownbycIEF

HiGHer orDer StruCtureat 37°C and pH 8 the rate of deamidation of asn-67 in native ribonuclease a is more than 30-fold slower than that of the reduced, unfolded protein15 demon strating that the location within the native protein at the β-turn (residues 66–68) suppresses deamidation.

an antibody fragment (50 kDa Fc protein) was compared to a 22 amino acid tryptic peptide16. the relevant sequence is ---SN382 GQPe N387 N388 YK. the rate of deami-dation at position 382 was 30 times greater in the peptide than in the protein. However deamidation at position 387 was only observed in the protein. Deami dation at 388 was not seen as expected since the next amino acid is tyr.

a metHoD for cHarge variation in mabs – papain DigestionCapillary isoelectric focusing (cieF) is often the method of choice for revealing charge variation. an electropherogram of a Mab before and after heat stressing reveals an increase in species of lower pi caused by deamidation Figure1. Digestion of Mabs with papain cleaves apart the Fab and Fc domains.

0.08

0.06

0.04

stressed/forced deamidation

unstressed

0.02

0.00

–0.02

abso

rban

ce

pl

–0.04

–0.06

–0.08

–0.10

–0.125.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0

–iN

t+

–iN

t+

–7.4

4 –7.5

8–7

.76 –7

.94

–8.10

–8.2

4

Deamidation


We have studied numerous Mabs and in all instances found that an acidic Fc cluster is resolved from a more basic Fab cluster Figure2, confirming earlier reports17. Whereas proteins sometimes precipitate near their pi which interferes with ieF, we have not observed this complication with papain digests. additionally the compli cation of ag-gregates often seen in a whole antibody ieF is absent in the fragmented antibody method. Finally in many cases more peaks are observed in the digest samples when compared with whole Mabs meaning that more information about deamidation is obtained. in a whole antibody one cannot distinguish between a single deamidation in the Fc region and in the Fab region. if both occur this is clearly shown in the papain digests.

When comparing two samples whether from different batches, different processes, stability samples or biosimilars the location of any differences to the Fab or Fc part is biologically relevant. therefore the effect of deamidation upon potency and can be studied rationally. the Fc region is responsible for effector functions like antibody dependent cytotoxicity (aDC) and antibody-dependent cell-mediated cytotoxicity (aDCC) therefore differences in this region could be investigated further by cell based bioassays whereas differences in the variable specific antigen recognition region (Fab) could be followed-up with a simple binding assay such as an eLiSa. therefore, based upon scientifically defensible information expensive studies of irrelevant bio-logical pathways can be avoided.

Figure2:AnalysisofapapaindigestofaMAbbycIEF

acidic variants (av)

aggregates

basic variants (bv)

bv of Fab

av of Fab

bv of Fcav of Fc

pH

intact

digested

fcfab

abso

rban

ce

Pixel

–0.30

–0.20

–0.10

0.00

0.10

0.20

0.30

0.40

0.50

2000 400 600 800 1000 1200 1400 1600 1800 2000


Figure3:DeamidationproductsobservablebyAsp-Npeptidemapping

cHarge (DeamiDation) localization by peptiDe mapping using asp-nusually deamidation sites are located by peptide mapping. the most commonly used trypsin digests contain peptides which are analyzed by HPLC-MS. trypsin cleaves at the C-terminus of Lys and arg, the natural abundance of which are 8 % and 5 % respectively. typically tryptic peptide maps generate a “needle in a haystack” scenario in which deamidations may be concealed. on the other hand, asp-N cleaves at the N-terminus of asp, the natural abundance of which is 5 %1. in addition 6–8 pH is the optimum for asp-N activity whereas the optimum for trypsin is pH 8–9 18. there-fore asp-N peptide maps are simpler than tryptic maps with fewer deamidation arti-facts. asp-N does not cleave after isoasp but isoasp peptides show up with a mass increase of 1 Da. this is illustrated for the “P4 peptide” of ribonuclease a Figure3.

Capillary electrophoresis is particularly informative about asp-N digests because isoasp containing peptides always have a slower electrophoretic mobility in a cathodic electric field compared to the original asn peptide. Changes in the profile of an asp-N peptide map after forced deamidation of a Mab can be seen Figure4. Ce-MS is particularly useful to identify all peptides including deamidation products.

DVQAVCSQKNVACKNGQTNCYQSYSTMSIT (53–82)

1 Site deamidationTheor. max. 3 new peptides1 new isoAsp peptide2 new fragment peptides

Asn IsoAsp IsoAsp

[ ]

Asn AsnIsoAsp

Asn Asn Asn

2 Site deamidationTheor. max. 8 new peptides≤ 1 new isoAspx2 peptides≤ 2 new isoAsp peptides≤ 4 new single cleavage peptides≤ 1 new double cleavage peptides


Figure4:Anelectro-pherogramofanAsp-NdigestofaMAbbeforeandafterforceddeami-dation(increasesshowninred)

summary

• We have outlined two useful methods for the deamidation of asn in proteins.

• the papain method for Mabs takes a top-down protein level view, has some practical advantages in comparison to the intact protein, and gives information that is bio-logically relevant.

• the asp-N method is more generally applicable to any protein and derivatives thereof, including antibody drug conjugates, PeGylated or HeSylated proteins and so on, facilitating the identification of the precise location of any unstable asn, and providing data that is complementary to tryptic digestion. Furthermore, the same principle of asp-N digestion can also be used for finding Gln deamidation by analogous digestion with Glu-C.

• For both methods Ce has some advantages compared with chromatographic methods.

• Ce-MS enables deamidation products to be identified and any sites susceptible to deamidation to be determined.

• in both cases the enzyme step is the key that unlocks the information. thereafter alternative chromatographic modes of separation such as HPLC or uPLC or ieC may sometimes be preferred over the electrophoretic methods presented herein.

the final critical point is, that with very limited additional effort, the methods fit within standard workflows and are likely to produce useful information. •

Minutes4 5 6 7 8 9 10 11 12 13 14 15 16 17

AU

-0.0025

-0.0020

-0.0015

-0.0010

-0.0005

0.0000

0.0005

0.0010

0.0015

0.0020

AU

-0.0025

-0.0020

-0.0015

-0.0010

-0.0005

0.0000

0.0005

0.0010

0.0015

0.0020UV - 214nmDS_unstressed_asp_P1_redP1_06_red.dat

UV - 214nmDS_stressed_asp_P2_redP2_03_red.dat

D:\32Karat\Projects\mycograb\Data\090423\P1_06_red.dat, UV - 214nm

D:\32Karat\Projects\mycograb\Data\090423\P2_03_red.dat, UV - 214nm

p1

p2

p3

p4

p5p6 p7

p8

deamidationrelated

D:\32Karat\Projects\mycograb\Data\090423\P1_06_red.dat, uv - 214nmD:\32Karat\Projects\mycograb\Data\090423\P2_03_red.dat, uv - 214nm


References

1. Klapper,M.H.,“Theindependentdistributionofaminoacidnearneighborpairsintopolypeptides”,BiochemicalandBiophysicalResearchCommunications,1977,78,(3),1018–1024

2. LiuY.D.,vanEnkJ.Z.,FlynnG.C.,“HumanantibodyFcdeamidationinvivo”,Biologicals,2009,37,(5),313–3223. RobinsonN.E.,RobinsonA.B.,“MolecularClocks:DeamidationofAsparaginylandGlutaminylResiduesinPeptides

andProteins”,AlthousePress,CaveJunction,OR,US,2004,www.deamidation.org4. TakemotoL.,BoyleD.,“Deamidationofspecificglutamineresiduesfromalpha-Acrystallinduringagingofthe

humanlens”,Biochem.1998,37,(39),13861–138655. ClarkeS.,“Propensityforspontaneoussuccinimideformationfromaspartylandasparaginylresiduesincellular

proteins”,Int.J.,PeptideProteinRes.,1987,30,808–8216. StephensonR.C.,ClarkeS.,“SuccinimideFormationfromAspartylandAsparaginylPeptidesasaModelforthe

SpontaneousDegradationofProteins”,J.Biol.Chem.,1989,264,6164–61707. GeigerT.,ClarkS.,“Deamidation,Isomerization,andRacemizationatAsparaginylandAspartylResiduesinPeptides”,

1987,262,(2),785–7948. TeshimaG.,StultsJ.T.,LingV.,Canova-DavisE.,“IsolationandCharacterizationofaSuccinimideVariantofMethionyl

humangrowthhormone“,1991,266,(21),13544–135479. ViolandB.N.,SchlittlerM.R.,KolodziejE.W.,TorenP.C.,CabonceA.,SiegelN.R.,DuffinK.L.,ZobelJ.F.,SmithC.E.,

TouJ.S.,“Isolationandcharacterizationofporcinesomatotropincontainingasuccinimideresidueinplaceofaspartate129”,ProteinSci.,1992,(12),1634–1641

10. FoyeW.O.,LemkeT.L.,WilliamsD.A.,“Foye’sPrinciplesofMedicinalChemistry”,LippincottWilliamsandWilkins,WoltersKluwerHealth,2007

11. RobinsonN.E.,RobinsonA.B.,“Predictionofproteindeamidationratesfromprimaryandthree-dimensionalstructure”,Proc.Natl.Acad.Sci.,2001,98,4367–4372

12. KrokhinO.V.,AntonoviciM.,EnsW.,WilkinsJ.A.,StandingK.G.,“Deamidationof-Asn-Gly-sequencesduringsamplepreparationforproteomics:consequencesforMALDIandHPLC-MALDIanalysis”,Anal.Chem.,2006,78,(18),6645–6650

13. RobinsonA.B.,RobinsonL.R.,“Distributionofglutamineandasparagineresiduesandtheirnearneighborsinpeptidesandproteins”,Proc.Natl.Acad.Sci.,2001,98,703–711

14. CheliusD.,RehderD.S.,BondarenkoP.V.,“Identificationandcharacterizationofdeamidationsitesintheconservedregionsofhumanimmunoglobulingammaantibodies”,Anal.Chem.,2005,77,(18),6004–6011

15. WearneS.J.,CreightonT.E.,“Effectofproteinconformationonrateofdeamidation:RibonucleaseA”,Proteins:Structure,Function,andBioinformatics,1989,5,(1),8–12

16. SinhaS.,ZhangL.,DuanS.,WilliamsT.,VlasakJ.,IonescuR.,ToppE.,“EffectofsecondarystructureondeamidationintheFcfragmentofanIgG1monoclonalantibody”,ProteinScience,2009,18,(8),1573–1584

17. SantoraL.C.,KrullI.S.,GrantK.,“Characterizationofrecombinanthumanmonoclonaltissuenecrosisfactor-alphaantibodyusingcation-exchangeHPLCandcapillaryisoelectricfocusing”,Anal,Biochem.,1999,275,(1),98–108

18.SimpsonR.J.,“ProteinsandProteomics:ALaboratoryManual”,ColdSpringHarborLaboratoryPress,200319.DataincollaborationwithChristianNeusuess,AalenUniversity

acknowledgements: the methods were developed in the laboratories of Solvias by Dipl. ing. alena Böhmová and PD Dr. Maria a. Schwarz


accelerating chemical (process) development with Hts technologythe integration of high-throughput screening (HtS) experiments in chemical research and development enables rapid evaluation of numerous potential catalysts.

tecHnical note

a reliable prediction for the best catalyst/ligand system is only possible when close analogies exist. as a consequence, the task of identifying the best catalysts typically means a lot of experimental work. the integration of high-throughput experimenta-tion, which now offers the possibility of empirically evaluating very large numbers of catalysts within a very short time, has boosted the development of industrial catalytic processes in recent years.

Solvias has been conducting high-throughput screening programs for many years and has performed a plethora of asymmetric hydrogenation projects. the combina-tion of profound expertise in this field and the availability of a very efficient screening workflow is the key for the very high success rate we have achieved (based on Solvias’ technical assessments). Project dedicated designs were successfully performed and resulted in the identification of one or more hits for further development typically within five to eight working days. Solvias approved applications for HtS experiments include but are not limited to:• Homogeneous asymmetric hydrogenation• C-X-coupling reactions (C-C, C-N, C-o-coupling)• Carbonylation/hydroformylation reactions• Heterogeneous hydrogenation• enzymatic catalysis (currently limited to the screening of wild type keto reductases

and hydrolases)• organocatalysis• Crystallization (Screenings for resolution of racemates and purification)• Polymorph and salt screening studies• racemic resolution (classical resolution via diastereomeric crystallization)

the range of the screening workflow can readily be extended to any metal catalyzed transformation such as hydrosilylation, boration reactions.these modules are provided to customers as stand-alone screening services, but are also used as an integrated part for development projects, whereby Solvias provides support for early drug development including process r&D as well as the supply of final aPis manufactured under cGMP.

Dr. felix spinDler — technical expert CatalysisFelixSpindlerstudiedattheETHZurichandreceivedhisPh.D.degreein1982withathesisinthefieldofhomogeneouscatalysis.In1983hejoinedtheCatalysisDepartmentofCiba-Geigy’sCentralResearchLaboratoriesinBasel,whereheworkedonthedevelopmentofthehomogeneouscatalysistechnology,particularlyasymmetrichydrogenation,andligandsynthesis.Sincethespin-offofSolviasin1999,hehasbeencollaboratingwithnumerouscustomersonindustrialapplicationsofthecatalysistechnology.


aPPLiCatioN oF HtS For aSYMMetriC HYDroGeNatioNas a leading provider of chiral ligands, Solvias has built up a library of approximately 700 ligands for asymmetric hydrogenation (65 % proprietary and iP-protected ligands, 35 % third-party iP-protected and off-patent ligands). of these, 150 state-of-the-art ligands (and their enantiomers), which are available on multi-kg scale, are primarily used in screenings. the remaining ligands are considered ‘research ligands’, most of them being synthesized for the first time only on gram scale.to efficiently identify the most promising catalyst(s) and ligand(s), Solvias has de veloped a screening concept using Key Ligands and High Priority Ligands. Key Ligands are so-called ‘privileged’ ligands, which show excellent performance in a wide range of hydrogenation reactions. Key Ligands and High Priority Ligands are typically evaluated with the initial HtS plate, and High Priority Ligands and the rest of the ligand library with the second HtS plate, partly aiming at the optimization of the steric and electronic properties of the catalyst ‘lead’. the rationale of this strategy is to identify the catalyst candidate(s) for further development with one or two HtS plates (96 or 192 experiments).For each screening project with preferred hydrogenation substrates such as enamides, acrylic acids, non-functionalized ketones, and many more, Solvias uses pre-designed dedicated experimental setups which guarantee high levels of success. For unprecede-nted substrates, dedicated expert designs are foreseen. this dual strategy has proved to be very efficient and successful. For example, the three hydrogenation substrates depicted above were evaluated three times using al-most the identical experimental setup, and despite their different structural proper-ties, high levels of enantiomeric excess (≥ 92 % ee) were obtained for each substrate after performing just one HtS plate.

Scheme1:Substrates

N

NR1

O

R1

R1R

O O OR'R1

94% ee 92% ee (ester)/>97% ee (acid) 93% ee


aPPLiCatioN oF HtS For HeteroGeNeouS HYDroGeNatioN the identification of a technically and economically feasible catalyst for the chemose-lective hydrogenation of a functionalized chloro-substituted benzaldimine was the main project target of a customer in the fine chemical industry.

NR

Cl

R1NHR

Cl

R1H2

[cat]

Scheme2:Imine

the purpose was to identify a catalyst for the efficient hydrogenation of the imine without affecting both the chloro-substituent and labile C-C bond present in sub-stituent r. in the technical assessment, Solvias recommended evaluating three different types of catalysts:• Heterogeneous catalysts such as commercially available Pd, Pt, raney-Nickel catalysts

(despite their known ability for dehalogenation and/or C-C bond cleavage reactions)• Homogeneous ir catalysts. Such ir catalysts are known to hydrogenate aldimines

with high levels of chemoselectivity in the presence of numerous functional groups.• Modified heterogeneous catalysts. the development of such catalysts using organic

as well as inorganic compounds is clearly an expert approach.

the key question was how to efficiently evaluate these innumerous catalyst candi-dates? Solvias decided to run an initial HtS plate with 16 homogeneous ir catalysts and 80 heterogeneous catalysts in combination with eight different modifiers among others such as sulfur and phosphorous containing compounds. the 96-well plate was split into two parts; one portion of the catalysts was tested at 1 bar/room temperature and the second portion at 10 bar/ 25°C.this screening revealed that a limited number of modified and non-modified hetero-geneous and homogeneous ir catalysts are the most promising candidates. at the client’s request Solvias continued to further investigate the heterogeneous catalysts. Because the ratio between catalyst and modifier is essential for an optimum chemoselectivity, Solvias performed a second HtS plate with heterogeneous catalysts and modifiers, whereby the concentrations of the modifiers also were varied. a Pt catalyst modified with a distinct heterocyclic compound proved to be the catalyst system of choice with respect to both activity and chemoselectivity. a series of optimization experiments in single reactors using this modified catalyst concluded this project. ultimately, Solvias could experimentally demonstrate that the targeted hydrogenation is feasible yielding the desired amine with high levels of purity (>95 % selectivity of crude product prior to workup).


CoNCLuSioNSthe combination of expertise and high-throughput experimentation technology can at the same time accelerate and increase the success rate of development projects. in addition, high-throughput experimentation offers even more benefits. Due to the downscaling, significantly less material per experiment is required, specifically lower amounts of expensive precious metal-based catalysts, ligands and starting materials. Furthermore, the reproducibility of the results is considerably improved, because each experiment is set-up under standardized conditions by the robot. overall, Solvias’ customers who use the high-throughput screening workflow in combination with chemo- and bio-catalysis or crystallization studies have a sustainable profit: compressed time and cost of early-stage development boost the development of the final aPis or intermediates. •

the automated workflow with a capacity of 192 experiments per day consists of the following parts:• a glove box for the experimental setup under inert conditions• a core module with a two-arm liquid handler, 9 compartments for well plates that can be used for

stirring, heating and cooling (range: –20 to 90°C) and an external compartment for shaking slurries at ambient temperature

• 96-well plate reactors, which can be operated up to 100 bar and 100°C using non- corrosive gases• a centrifuge for the efficient removal of solvents from a 96-well plate• a ligand hotel with pre-batched ligand and catalyst vials• a heated shaker for the 96-well plate reactors which can accommodate up to four different reactors

on individual levels of pressure and temperature• Devices for the rapid analyses of reaction mixtures (SFC, HPLC and GC)

a powerful software suite handles and monitors the complete experimental execution and the analyses starting from the experimental design to the dispensing routines of the robot and the analytical data, and finally, to the visual presentation of the results.

SolviasHTSServicesbasedonacustomizedworkflowwhichhasbeendesignedbySymyxTechnologies,Inc.(nowFreeslateInc.)onthebasisofSolviasrequirements.


News

anD tHe european outsourcing aWarD 2011 goes to… solviasthe award which was received in the category “Most improved Process/Plant Facility”, recognized Solvias’ work for vitae Pharmaceuticals, a clinical stage biopharmaceutical company. the award- winning project provided a fully integrated approach to pharmaceutical product development.

vitae selected Solvias to conduct initial process r&D studies, analytical and solid-state chemistry development and the aPi supply for preclinical studies. Several process improvements were successfully achieved during the development program, all of which significantly helped to reduce the overall cost, increase pu-rity and facilitate the synthesis of the aPi. the availability of auto-mated fast screening tools for catalysis and crystallization devel-opment, proven expertise in chemical and analytical de velopment as well as the ability to deliver the aPi for clinical phase i and ii studies, contributed to the success of the project. over and above modern, state-of-the-art technology and equipment, the dedica-tion and skills of the individual scientists involved were key drivers for the smooth project progression. Dr. Doris Pupowicz, Custom Project Manager at Solvias highlighted this aspect: “What made the real difference were the excellent working and personal relationships that were built up between vitae’s and Solvias’ staff allowing for an efficient exchange of information without having to overcome any barriers hence leading to rapid responsiveness from both ends.”

cphi 2011: solvias Wins sustainability aWarDSolvias was announced winner of the exhibitor Sustainability award at the CPhl Worldwide, iCSe, innoPack and P-MeC europe 2011 exhibitor Party and innovation awards Ceremony on october 25, 2011.

Nominations for the award were invited from all 1,600 exhibitors at the event. Companies needed to demonstrate innovative efforts to prevent and reduce the environmental impact of their exhibi-tion stand. entries were judged according to the following criteria: minimizing environmental impact, economic indicators, commit-ment to change, commitment to community and commitment to conservation. Solvias ticked all the boxes.Solvias’ incorporation of effective green solutions included the use of lightweight and modular stand material, the minimization of long-distance shipping and the use of recyclable material. this not only satisfied the “green” criteria but equally important, demon-strated that green solutions can be economically attractive.


News

visible anD subvisible particles – particulate matter analysis at solviasHealthcare companies that develop and produce injectable products are faced with having to meet the increasingly strin-gent requirements of the united States Pharmacopeia (uSP) and the european Pharmacopoeia (Ph. eur.) regarding particulate and other foreign matter in the visible and subvisible size range. Solvias has the infrastructure, capacity and service portfolio to meet the growing demand for visible and subvisible particulate testing under iSo and cGMP quality standards.

visible particles Whereas the uSP simply states that parenteral formulations and ophthalmic solutions must be “essentially free of visible particles” emanating from the manufacturing or filling process (Chapter 1), the visible particles test is defined more thoroughly in Ph. eur. 2.9.20, with clear instructions for the assembly of a matt black and non-glare white panel. For the evaluation of visible particles the illumination is crucial. the light intensity is checked by a luxmeter to ensure that it is in the specified region for the test procedure. trained personnel and reproducible test conditions are a prerequisite for reproducible test results. the lower size limit of visible particles is generally in the range of 50 –100 μm, depend-ing on the observer.

subvisible particlesBeside visible particles, pharmaceutical products such as injec-tions (uSP <788> and <789>, Ph.eur. 2.9.19, Particulate Contami-nation: Sub-visible Particles, iCH Guidelines Q4B) or ophthalmic solutions (uSP <789>) need to be tested for particulate matter in the subvisible range. the uSP describes two methods: the light

Subvisibleparticledeterminationwithparticlecounter.

obscuration test and the microscopic particle test. the first test is typically carried out in a laminar flow cabinet with a particle coun-ter (Syringe by Klotz GmbH) under conditions minimizing contamination by any extraneous particulate matter.

the light obscuration test method, which is based on measur-ing the light-blockage (shadow) caused by the particles, allows the particles in the liquid media to be counted according to number and size. at the outset, glassware and containers are rinsed with a particle-free water supply that is integrated in the cabinet. the solution under investigation then flows through a thin capillary which is illuminated by a laser perpendicular to the capillary and a sensor at the other end of the capillary de-tects whether the light is blocked or not. the number of parti-cles is thus counted and their size is recorded at the same time.

in fact, the light obscuration test equipment is not exclusive to the uSP or Ph. eur. tests. Besides its use in manufacturing control it can also be used for development issues (non-GMP). in stability studies this method is capable of detecting protein agglomer-ates in the μm range in protein solutions. the rather large amount of substance is only required for sampling plans in batch control, the sample volume required for one measure-ment can be reduced to 1 mL. the measurement procedure is not limited to uSP/Ph. eur. parameters. Light obscuration can be freely adapted to the particle size of interest in the range of 150 μm. the method has a wide range of uses over and above the testing of medical or pharmaceutical products and can also be used to control the cleanliness of water, chemicals or beverages.

in its broadest application the light obscuration method is applicable for particle-size analysis and therefore perfectly complements the well-established particle analysis methods practiced at Solvias.


iNForMeX

February14–17,2012NewOrleans,USAVisitusatBooth808

find out more about • Polymorphism, salt, and crystallization• Chemical and analytical development• GMP manufacturing• Catalysis

aCHeMa

June18–22,2012Frankfurt,GermanyVisitusatBoothN7,hall4.1

find out more about • Process analytical technologies• Fiber-optic probes• Chemical and analytical development

CHeMoutSourCiNG CoNFereNCe

September10–13,2012LongBranch,NJ,USA

find out more about • Polymorphism, salt, and crystallization• Chemical and analytical development• GMP manufacturing• Catalysis

events

News

Dr. syuzanna r. Harutyunyan Wins solvias liganD contestthe jury of the annual Solvias Ligand Contest awarded the 2011 prize to Dr. Syuzanna r. Harutyunyan, assistant Professor in Synthetic organic Chemistry at the university of Groningen. the award ceremony and presentation of the prize-winning contribution took place during the 10th Science Day.

the jury honors Dr. Syuzanna r. Harutyunyan for her contributions in the area of asymmetric catalysis and the application of Solvias ligands with the following laudation: “in recognition of enlarging the scope of Cu-Josiphos and Cu-taniaphos catalyzed asymmetric addition reactions. Both the allylic substitutions with alkyl lithium reagents as well as the 1,2 addition of Grignard reagents to enones have an interesting synthetic potential.”

the Solvias Ligand Contest challenges researchers all over the world to submit new and improved applications of Solvias ligands and catalysts. to pick the winner, the jury assesses the novelty, scientific rigor and originality of the work submitted, as well as its practical applicability in organic synthesis.Please remember, your input is essential for us to structure our internal processes such that we can serve your needs more spec-i fically and flexibly. thank you once again for your esteemed feedback, don’t hesitate to keep it coming!

LigandContest2011,Dr.SyuzannaR.HarutyunyanwithDr.BenoitPuginattheawardpresentation.

publisher’s detailsSolvias aGrömerpark 24303 KaiseraugstSwitzerland

editor-in-chiefSilke oeschgerPhone +41 61 845 61 41, [email protected]

Design Furore visuelle Kommunikation GmbH, Basel

picture creditsPatrik Hänggi, Solvias archive, eva Schaub, rohnerChem, Juri Junkov

How to unsubscribeto unsubscribe from this magazine, please send an e-mail with your contact details to [email protected]

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

Solv

ias

aG

Mar

ketin

g an

d Sa

les

röm

erpa

rk 2

4303

Kai

sera

ugst

Switz

erla

nd

Phone +41 61 845 60 00Fax +41 61 845 69 [email protected]

pdf

solvias ag

römerpark 2

4303 Kaiseraugst

Switzerland

Phone +41 61 845 60 00

Fax +41 61 845 69 00

www.solvias.com

[email protected]

Company

Nam

e

Position

address

Postal code/city

Country

telephone

Fax

e-mail

PLeaSe SeN

D M

e iNFo

rMatio

N a

Bou

t:

a

nalytical Services

Biopharm

aceutical analysis

Polym

orphism, Salts, and Crystallization

Chem

ical Developm

ent and GM

P Manufacturing

i w

ould like to receive prospects as e-paper

printpdf

Catalysis

Process a

nalytical technology (Pat)

Patent and Search Services

Solvias in G

eneral

print

go

green

Sign up for our e-paper

Documents

Solvias Prospects 01/2012