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sø- og handelsretten

sø- og handelsretten kendelse afsagt den 8. marts 2019 sag bs-34191/2018-shr sag bs-1208/2019-shr roche a/s f. hoffmann-la roche ag og genentech, inc (advokat sture rygaard for alle) mod samsung bioepis nl b.v. biogen (denmark) manufacturing aps (advokat klaus ewald madsen og advokat johnny petersen for begge) og msd danmark aps (advokat morten achilles bruus) 2 denne afgørelse er truffet af vicepræsident mads bundgaard larsen og de sagkyndige medlemmer henrik peder bendiksen og john meidahl petersen. sagens baggrund og parternes påstande denne sag om midlertidigt forbud og påbud i henhold til retsplejelovens kapitel 40, der blev anlagt den 13. september 2018, vedrører, hvorvidt betingelserne for at nedlægge forbud og påbud mod samsung bioepis nl b.v., biogen (denmark) manufacturing aps og msd danmark aps, for så vidt angår fremstilling, salg og markedsføring af det bio- similære lægemiddel ontruzant, er opfyldt, herunder navnlig om stridspatentet dk/ep 1 308 455 t3 om en bestemt stofsammensætning af antistoffet hummab4d5-8 (trastuzu- mab) er gyldigt og krænket. stridspatentet er udstedt den 22. marts 2006 med prioritet den 6. maj 1998 og udløber den 3. maj 2019. roche a/s, f. hoffmann-la roche ag og genentech, inc har nedlagt følgende påstan- de: 1. det forbydes biogen (denmark) manufacturing aps at fremstille, udbyde, brin- ge i omsætning eller anvende lægemidlet ontruzant®, omfattet af markedsfø- ringstilladelse nr. eu/1/17/1241/001, jf. bilag plesner5, og/eller trastuzumab til brug herfor, eller importere eller besidde sådant ontruzant® og/eller trastuzu- mab til brug herfor med sådant formål så længe patent nr. dk/ep 1308455 er i kraft. 2. det påbydes biogen (denmark) manufacturing aps at tilbagekalde alle allerede skete leverancer af ontruzant®, jf. markedsføringstilladelse nr. eu/1/17/1241/001, jf. bilag plesner5, fra samtlige modtagere og at anmode samtlige modtagere om at tilbagekalde de skete leverancer fra deres aftagere. 3. det forbydes samsung bioepis uk limited, samsung bioepis nl b.v. og msd danmark aps i danmark at udbyde, bringe i omsætning eller anvende lægemid- let ontruzant®, omfattet af markedsføringstilladelse nr. eu/1/17/1241/001, jf. bi- lag plesner5 og bilag plesner35, eller importere eller besidde det med et så- dant formål så længe patent nr. dk/ep 1308455 er i kraft. 3 4. det påbydes msd danmark aps at tilbagekalde allerede skete leverancer af on- truzant®, jf. markedsføringstilladelse nr. eu/1/17/1241/001, jf. bilag plesner5, fra samtlige modtagere, herunder grossister, hospitaler, apoteker og de relevan- te afdelinger på hospitalerne, hvortil levering er foretaget, eller som, msd dan- mark aps er bekendt med, anvender ontruzant®. 5. det påbydes msd danmark aps at anmode sine aftagere i danmark af on- truzant®, jf. markedsføringstilladelse nr. eu/1/17/1241/001, jf. bilag plesner5, om at tilbagekalde og returnere til msd danmark aps de pågældendes leveran- cer af det nævnte lægemiddel fra samtlige modtagere, herunder grossister, ho- spitaler, apoteker og de relevante afdelinger på hospitalerne, eller som, msd danmark aps er bekendt med, anvender ontruzant®. 6. det påbydes msd danmark aps omgående med øjeblikkelig virkning at afmel- de prisregistrering af lægemidlet ontruzant®, jf. markedsføringstilladelse nr. eu/1/17/1241/001, jf. bilag plesner5, hos lægemiddelstyrelsen. de midlertidige forbud og påbud påstås principalt nedlagt uden sikkerhedsstillelse; subsidiært mod en af retten fastsat sikkerhed. roche gør gældende, at roche skal tillægges sagsomkostninger i henhold til retspleje- lovens § 421, og der skal således tages stilling til sagsomkostninger i forbindelse med forbudssagen. biogen (denmark) manufacturing aps har over for påstand 1 og 2 principalt nedlagt på- stand om, at forbud og påbud nægtes fremme, subsidiært at begæringen om forbud og påbud fremmes mod tilvejebringelse af en af sø- og handelsretten fastsat sikkerhed. samsung bioepis nl b.v., har over for påstand 3 principalt nedlagt påstand om, at for- bud og påbud nægtes fremme, subsidiært at begæringen om forbud og påbud fremmes mod tilvejebringelse af en af sø- og handelsretten fastsat sikkerhed. 4 msd danmark aps har over for påstand 3 – 6 principalt nedlagt påstand om, at forbud og påbud nægtes fremme, subsidiært at begæringen om forbud og påbud fremmes mod tilvejebringelse af en af sø- og handelsretten fastsat sikkerhed. oplysningerne i sagen sagens parter og de omhandlede lægemidler roche a/s og f. hoffmann-la roche ag er en del af roche-koncernen. f. hoffmann-la ro- che blev oprindeligt etableret i 1896 af fritz hoffmann-la roche. roche-koncernen ope- rerer globalt under to divisioner, en lægemiddeldivision og en diagnostikdivision, og beskæftiger mere end 90.000 ansatte i mere end 100 lande. lægemiddeldivisionen be- skæftiger sig med forskning og udvikling af originale lægemidler, og over halvdelen af roches produktudvikling vedrører biologiske lægemidler. roche har på nuværende tidspunkt mere end 15 biologiske lægemidler på markedet i eu. f. hoffmann-la roche har en eksklusiv licens til at markedsføre herceptin i europa. i danmark sælges lægemidlet herceptin af roche a/s. genentech inc. er et datterselskab i roche-koncernen. genentech udviklede lægemidlet herceptin og sælger dette i usa. genentech er ejer af stridspatentet. roche a/s og f. hoffmann-la roche ag og genentech inc. vil i det følgende samlet blive benævnt ”roche”. biogen (denmark) manufacturing aps (herefter biogen) er en del af biogen-koncernen, som er en global producent af biologiske lægemidler med hovedsæde i massachusetts, usa, og med kontorer i 30 lande og samarbejdspartnere i 70 lande. biogen-koncernen blev etableret i 1978 og er verdens ældste uafhængige bioteknologiske selskab. biogen- koncernen beskæftiger ca. 7.500 medarbejdere på verdensplan. samsung bioepis nl b.v (herefter bioepis) er en del af den koreanske lægemiddelsvirk- somhed samsung bioepis co. ltd. 5 samsung bioepis co. ltd er et joint venture mellem det koreanske selskab samsung bi- ologics co. ltd. og det amerikanske selskab biogen idec therapeutics inc. samsung bio- epis er en del af samsung koncernen. samsung bioepis co ltd. har ikke noget datterselskab i danmark. samsung bioepis co. ltd. har udviklet en række biosimilære lægemiddelkandidater med fokus på immunologi, onkologi og stofskiftesygdomme. fire af sådanne produkter er hidtil blevet lanceret i europa. de lancerede produkter omfatter en biosimilær version af herceptin, der markedsføres under varemærket ontruzant. det europæiske læge- middelagentur udstedte den 15. november 2017 markedsføringstilladelse for ontruzant. markedsføringstilladelsen indehaves af samsung bioepis nl b.v. det er oplyst, at den første europæiske lancering af ontruzant var i storbritannien den 8. marts 2018. msd danmark aps (herefter msd) er en del af merck & co. koncernen, som er en af verdens største lægemiddelselskaber. merck & co. koncernen har hovedsæde i new jersey, usa, og er repræsenteret i mere end 140 lande, herunder danmark. samlet set har merck & co. koncernen ca. 69.000 ansatte og havde i 2017 en samlet om- sætning på 40,1 mia. usd. msd har rettighederne til at sælge ontruzant på det danske marked. herceptin er et biologisk lægemiddel, der indeholder aktivstoffet trastuzumab. et biosi- milært lægemiddel er et biologisk lægemiddel, der svarer til et allerede godkendt biolo- gisk lægemiddel - referencelægemidlet. de aktive stoffer i biologiske lægemidler er ofte større og mere komplekse, end det er tilfældet for kemiske lægemidler, og det er derfor ikke muligt på nuværende tidspunkt at lave identiske kopier. trastuzumab, der er et monoklonalt antistof, blev første gang godkendt til medicinsk anvendelse i usa i 1998. et monoklonalt antistof er et antistof, der er udviklet til at gen- kende og binde sig til en specifik struktur, som findes i bestemte celler i kroppen. 6 trastuzumab er designet til at binde selektivt til her2-receptoren, som er overudtrykt i ca. en fjerdedel af brystkræfttilfældene. ved at binde til her2 aktiverer trastuzumab im- munsystemets celler, som derefter dræber tumorcellerne. trastuzumab forhindrer også her2 i at udsende signaler, som får tumorcellerne til at vokse. stridspatentet roche fik den 22. marts 2006 udstedt patent dk/ep 1 308 455 t3 (herefter stridspatentet eller 455-patentet) på en ”sammensætning omfattende anti-her2-antistoffer”. patentet blev udstedt på grundlag af europæisk patentansøgning nr. 02029008.6, der blev indle- veret den 3. maj 1999 og havde prioritet fra 6. maj 1998 (us 84459 p/488-patentet). strids- patentet udløber den 3. maj 2019. patentets krav 1 har i den engelske version følgende ordlyd: “1. a composition comprising a mixture of anti-her2 antibody and one or more acidic variants thereof, wherein the amount of the acidic variant(

  1. s)is less than about 25%, 25 and wherein the acidic variant(
  2. s)are predominantly deamidated variants wherein one or more asparagine residues of the anti-her2 antibody have been deamidated, and wherein the anti-her2 antibody is hummab4d5-8, and wherein the deamidated variants have asn30 in cdr1 of either or both vl regions of hummab4d5-8 converted to aspartate.” den danske oversættelse af stridspatentet blev bekendtgjort den 31. juli 2006. 455-paten- tet har følgende patentkrav af betydning for sagen: ” 1. sammensaetning omfattende en blanding af anti-her2 antistof og en eller flere sure varianter deraf, hvor masngden af sur variant er mindre end 5 ca. 25%, og hvor de sure varianter hovedsageligt er deamiderede varianter, i hvil- ke en eller flere asparaginrester i anti-her2 antistoffet er blevet deamideret, og hvor anti-her2 antistoffet er hummab4d5-8, og hvor de deamiderede varianter har asn30 i cdr1 i en af eller begge 10 vl- regioner i hummab4d5-8 omdannet til aspartat.” af stridspatentet fremgår blandt andet: ” beskrivelse opfindelsens område denne opfindelse angår generelt proteinoprensning. beskrivelse af beslægtet teknik storskala, økonomisk oprensning af proteiner er et stigende vigtigt problem for bioteknologiindustrien. generelt produceres proteiner af cellekulturer ved brug af enten mammale eller bakterielle cellelinier modificeret til at producere proteinet af interesse ved insertion af et rekombinant plasmid indeholdende genet for dette 7 protein. eftersom cellelinierne, der anvendes, er levende organismer, skal de fødes med et komplekst vækstmedium indeholdende sukre, aminosyrer og vækstfaktorer, sædvanligvis tilført fra præparater af animalsk serum. separation af det ønskede protein fra blandingen af forbindelser, der er givet til cellerne og biprodukterne fra cellerne selv til en renhed, der er tilstrækkelig til anvendelse som et humant terape- utika giver en ekstrem udfordring. procedurer til oprensning fra celledebris afhænger først og fremmest af pro- teinets ekspressionssite. nogle proteiner kan secerneres direkte fra cellen til det om- givende vækstmedium, andre fremstilles intracellulært. for sidstnævnte proteiner er første trin i oprensningsprocessen lyse af cellen, hvilket kan udføres ved en ræk- ke fremgangsmåder inklusiv mekanisk forskydning, osmotisk chok eller enzymati- ske behandlinger. sådanne brud frigiver hele cellens indhold i homogenatet og pro- ducerer yderligere subcellulære fragmenter, som er svære at fjerne pa grund af de- res lille størrelse. disse fjernes generelt ved differentialcentrifugering eller ved fil- trering. det samme problem opstår, skønt i en mindre skala, med direkte secernere- de proteiner pa grund af cellers naturlige død og frigivelse af intracellulære værts- celleproteiner i forløbet for proteinproduktionskørsel. nar en klaret opløsning indeholdende proteinet af interesse er blevet opnå- et, separeres det normalt fra andre proteiner produceret af cellen ved brug af en kombination af forskellige kromatografiske teknikker. disse teknikker separerer blandinger af proteiner pa basis af deres ladning, hydrofobicitet eller størrelse. ad- skillige forskellige kromatografiresiner er tilgængelige for hver af disse teknikker, hvilket muliggør præcis skræddersyning og oprensningsforløb til det særlige prote- in, der er involveret. essensen i hver af disse separationsmetoder er, at proteiner kan føres til enten at bevæge sig ved forskellige hastigheder ned gennem en kolonne og således opnå en fysisk separation, der øges, som de passerer yderligere ned gennem kolonnen eller til at adhaerere selektivt til separationsmediet, hvorved de elueres forskelligt af forskellige solventer. i nogle tilfælde separeres det ønskede protein for urenheder, når urenhederne specifikt adhærerer til kolonnen og proteinet af inter- esse ikke gør, det vil sige proteinet af interesse er til stede i "gennemløbet". ionbytningskromatografi er en kromatografisk teknik, som almindeligvis anvendes til oprensning af proteiner. i ionbytningskromatografi tiltrækkes ladede afsnit pa overfladen af soluten af modsatte ladninger h¨æftet til en kromatografisk matrix, forudsat at den ioniske styrke i den puffer er lav. elueringen opnås generelt ved at øge ionstyrken (dvs. konduktiviteten) i pufferen for at konkurrere med solu- ten for de ladede sider i ionbyttermatrixen. ændring af ph og derved ændring af so- luttens ladning er en anden made at opnå eluering af solutten. ændringen i konduk- tiviteten eller ph kan være gradvis (gradienteluering) eller trinvis (trinvis eluering). tidligere har disse ændringer været progressive, dvs. ph eller konduktiviteten øges eller reduceres i en enkelt retning. … sammendrag af opfindelsen den foreliggende opfindelse tilvejebringer en ionbytterkromatografisk fremgangsmåde, hvor et polypeptid af interesse bindes til ionbyttermaterialet med en initialkonduktivitet eller ph, og derpa vaskes ionbyttermaterialet med en inter- mediaerpuffer med en forskellig konduktivitet eller ph eller begge. ved et specifikt punkt efter disse mellemvaske, og i modsætning til standard ionbytterkromatogra- fipraksis, vaskes ionbyttermaterialet med en vaskepuffer, hvori aendringen i kon- duktivitet eller ph eller begge fra den intermediaere puffer til vaskepufferen er en modsat retning af aendringen i konduktivitet eller ph eller begge opnaet i de tidli- gere trin. kun efter vask med vaskepufferen er ionbyttermaterialet klar til at poly- peptidmolekylet af interesse kan elueres ved påførsel af elueringspufferen med en konduktivitet eller ph, eller begge, som er forskellig fra konduktiviteten eller ph el- ler begge af pufferne, der er anvendt i de tidligere trin. denne nye tilgang til ionbytterkromatografier er saerlig anvendelig i situa- tioner, hvor et produktmolekyle skal separeres fra et meget taet relateret kontami- 8 nerende molekyle ved fuldproduktionsskala, hvor både renhed og højt udbytte af polypeptidproduktet er ønsket. opfindelsen tilvejebringer således en sammensætning omfattende en blan- ding af anti-her2-antistof som defineret i krav 1. dette kan tilvejebringes ved en fremgangsmade til at oprense et polypeptid, som er et anti-her2-antistof, fra en sammensætning omfattende polypeptidet og en kontaminant, hvilken fremgangs- måde omfatter følgende trin udført efter hinanden: (
  3. a)binde polypeptidet til et ionbyttermateriale ved brug af en loading- puffer, hvor loadingpufferen er ved en forste konduktivitet og ph; (
  4. b)vaske ionbyttermaterialet med en intermediærpuffer med en anden konduktivitet og/eller ph, saledes at kontaminanten elueres fra ionbyttermaterialet; (
  5. c)vaske ionbyttermaterialet med en vaskepuffer, som er ved en tredje konduktivitet og/eller ph, hvor ændringen i konduktivitet og/eller ph fra intermediærpufferen til vaskepufferen er i en modsat retning af ændringen i konduktivitet og/eller ph fra loadingpufferen til inter- mediærpufferen; og (
  6. d)vaske ionbyttermaterialet med en elueringspuffer med en fjerde kon- duktivitet og/eller ph, saledes at polypeptidet elueres fra ionbytter- materialet. den første konduktivitet og/eller ph kan være den samme som den tredje konduktivitet og/eller ph. … beskrevet heri er ligeledes en fremgangsmåde til oprensning af et antistof fra en sammensætning omfattende antistoffet og en kontaminant, hvilken frem- gangsmåde omfatter loading af sammensætningen pa en kationbytterresin, hvor mængden af antistof, der loades pa kationbytterresinen er fra ca. 20 mg til ca. 35 mg antistof pr. ml kationbytterresin, og eventuelt yderligere omfattendemeluering af antistoffet fra kationbytterresinen. fremgangsmaden omfatter fortrinsvis yderligere et intermediaert vasketrin til eluering af en eller flere kontaminanter fra ionbytterre- sinen. dette intermediære vasketrin kommer normalt fbr trinnet, hvor antistoffet elueres. i sammensætningerne i opfindelsen som defineret i krav 1 er mængden af sur(
  7. e)variant(
  8. er)i sammensætningen mindre end ca. 25% og fortrinsvis mindre end 20%, for eksempel intervallet fra ca. 1% til ca. 18%. eventuelt omfatter sammen- saetningen yderligere en farmaceutisk acceptabel bærer. … figur 5 viser kvaliteten af rhumab her2 i hver kationbytterkromatografi poolprøve elueret ved carboxysulfonkationbytterresin højtryksvæskekromatografi (csx hpiex). toppe a, b og 1 er deamiderede former af rhumab her2. top 3 er nondeamideret rhumab her2. top 4 er en kombination af c-terminal lysin-holdi- ge og iso-aspartat-varianter af rhumab her2. … detaljeret beskrivelse af de foretrukne udførelsesformer definitioner "sammensætningen", der skal oprenses heri, omfatter polypeptidet af inter- esse og en eller flere kontaminanter. sammensætningen kan være "delvist oprenset" (dvs. være underkastet et eller flere oprensningstrin, såsom protein a kromatografi, som i eksempel 1 nedenfor) eller kan opnås direkte fra en værtscelle eller organis- me, der producerer polypeptidet (f.eks. kan sammensætningen omfatte høstet celle- kulturvæske). … en "variant" eller "aminosyresekvensvariant" af et startpolypeptid er et po- lypeptid, som omfatter en aminosyresekvens, der er forskellig fra den for startpoly- peptidet. … 9 en "sur variant" er en variant af et polypeptid af interesse, som er mere sur (f.eks. bestemt ved kationbytterkromatografi) end polypeptidet af interesse. et ek- sempel pa en sur variant er en deamideret variant. en "deamideret" variant af et polypeptidmolekyle er et polypeptid, hvor en eller flere asparagin rester i det originate polypeptid er blevet omdannet til aspartat, dvs. den neutrale amidsidekaede er blevet omdannet til en rest med en generelt sur karakter. deamideret hummab4d5 antistof fra eksemplet nedenfor har asn30 i cdr1 i enten den ene eller begge vl-regioneme deraf omdannet til aspartat. ud- trykket "deamideret human dnase" som anvendt heri betyder human dnase, der er deamideret ved asparaginresten, der forekommer i position 74 i aminosyrese- kvensen for den native modne humane dnase (us patent 5.279.823; udtrykkeligt in- korporeret heri ved reference). udtrykket "blanding", som anvendt heri med henvisning til en sammensæt- ning omfattende et anti-her2 antistof, betyder tilstedeværelsen af både det ønske- de anti-her2 antistof og en eller flere sure varianter deraf. sure varianter kan ho- vedsagiigt omfatte deamideret anti-her2 antistof med mindre mængder andre su- re varianter. det er for eksempel fundet, at i præparater af anti-her2 antistof op- nået fra rekombinant ekspression er så meget som ca. 25% af anti-her2 antistoffet deamideret. … udtrykket "antistof" anvendes i den bredeste forstand og dækker specifikt monoklonale antistoffer (inklusiv fuld-længde monoklonale antistoffer), polyklona- le antistoffer, multispecifikke antistoffer (f.eks. bispecifikke antistoffer) og antistof- fragmenter, sa længe de udviser den ønskede biologiske aktivitet. antistoffet heri er rettet mod et "antigen" af interesse. fortrinsvis er antige- net et biologisk vigtigt polypeptid og administrering af antistoffet til et pattedyr, der lider af en sygdom eller lidelse, kan resultere i en terapeutisk forbedring i det patte- dyr. … udtrykket "hypervariabel region" anvendt heri refererer til aminosyrere- sterne i et antistof, som er ansvarlige for antigen-binding. den hypervariable region omfatter aminosyrerester fra en "komplementærbestemmende region" eller "cdr" (dvs. resterne 24-34 (li), 50-56 (l2) og 89-97 (l3) i det variable domæne i den lette kæde og 31-35 (hi), 50-65 (h2) og 95-102 (h3) i det variable domæne i den tunge kæ- de; … "skelet" eller "fr"-rester er de raster i det variable domæne, som er forskel- lige fra resterne i den hypervariable region, som defineret heri. cdr og fr-resterne i rhumab her2 antistoffet ifølge eksemplet nedenfor (humab4d5-8) er identifice- ret i carter et al., proc. natl. acad. sci. usa, 89:4285

(1992). … generelt er cdr-resterne direkte og væsentligst involveret i pavirkning af antigen- binding. … udtrykket "ionbyttermateriale" refererer til en fast fase, som er negativt la- det (dvs. en kationbytterresin) eller positivt ladet (dvs. en anionbytterresin). lad- ningen kan påføres ved at hæfte en eller flere ladede ligander til den faste fase, for eksempel ved kovalent kobling. alternativt eller yderligere kan ladningen være en iboende egenskab ved den faste fase (f.eks. i tilfældet af kisel, som har en total ne- gativ ladning). … en "kationbytterresin" refererer til en fast fase som er negativt ladet og som således har frie kationer til bytning med kationer i en vandig opløsning, der passeres over eller gennem den faste fase. en negativt ladet ligand koblet til den fa- ste fase til dannelse af kationbytterresinen kan for eksempel vaere et carboxylat el- ler sulfonat. kommercielt tilgaengelige kationsbytterresiner inkluderer carboxy- 10 methylcellulose, bakerbond abx™, sulfopropyl(sp) immobiliseret på agarose (for eksempl sp-sepharose fast flow™ eller sp-sepharose high per- formance™ fra pharmacia) og sulfonyl immobiliseret på agarose (f.eks. s-sep- harose fast flow™ fra pharmacia). udtrykket "anionbytterresin" anvendes heri som reference til en fast fase, der har positive ladninger, dvs. med en eller flere positivt ladede ligander, såsom quaternære aminogrupper, hæftet dertil. … med mindre andet er indikeret, refererer udtrykket "her2", når det anven- des heri, til human her2 protein og "her2" refererer til human her2-gen. det hu- mane her2-gen og her2-protein er for eksempel beskrevet i semba et al., pnas (usa), 82:6497-6501
(1985)og yamamoto et al,. nature 319:230-234
(1986)(genebank accessions nummer x03363). udtrykket "hummab4d5-8", når det anvendes heri, refererer til et humani- seret anti-her2-antistof omfattende aminosyresekvensen for den lette kæde ifølge seq id no 1, og aminosyresekvensen for den tunge kæde ifølge seq id no 2 eller aminosyresekvensvarianter deraf, som bibeholder evnen til at binde her2 og inhi- bere vækst af tumorceller som overudtrykker her2 (se us patent nr. 5.677.171). … at "eluere" et molekyle (f.eks. polypeptid eller kontaminant) fra et ionbyt- termateriale betyder fjernelse af molekylet derfra ved at aendre ionstyrken i puffe- ren, der omgiver ionbyttermaterialet, saledes at pufferen konkurrerer med mole- kylet for de ladede sites pa ionbyttermaterialet. "behandling" refererer til bade terapeutisk behandling og profylaktiske el- ler præventive tiltag. de, der har behov for behandling, inkluderer de, der allerede har en lidelse, såvel som de, hvor lidelsen skal forebygges. en "lidelse" er en hvilken som helst tilstand, som kan gavnes ved behandling med polypeptidet, der er opren- set som beskrevet heri. denne inkluderer kroniske og akutte lidelser eller sygdom- me inklusiv de patologiske tilstande, som prædisponerer pattedyret for den pagæl- dende lidelse. … måder til udførelse af opfindelsen … til rekombinant produktion af polypeptidet isoleres og indsættes nuclein- syren, der koder derfor, i en replikerbar vektor for yderligere kloning (opformering af dna) eller til ekspression. dna kodende for polypeptidet isoleres let og sekven- teres ved brug af konventionelle procedurer (f.eks. hvor polypeptidet er et antistof ved brug af oligonucleotidprober, som er i stand til at binde specifikt til generne ko- dende for den tunge og lette kæde i antistoffet). mange vektorer er tilgængelige. vektorkomponenterne inkluderer generelt, men er ikke begrænset til, en eller flere af de følgende: en signalsekvens, et replikationsstartområde, en eller flere markør- gener, et enhancerelement, en promotor og en transkriptionstermineringssekvens (f.eks. som beskrevet i us patent 5.534.615, der specifik er inkorporeret heri ved re- ference). egnede værtsceller til kloning eller ekspression af dna i vektorerne heri er de prokaryote, gær- eller høje eukaryote celler beskrevet ovenfor. … interessen har imidlertid været størst for vertebrate celler, og propagering af vertebrate celler i kultur (vævskultur) er blevet en rutineprocedure. eksempler på anvendelige mammale værtscellelinier er abenyre cv1-linie transformeret med sv40 (cos-7, atcc crl 1651); human embryonisk nyrelinie (293 eller 293 celler subklonet til vækst i suspensionskultur, graham et al., j. gen. virol., 36:59
(1977)); babyhamster nyreceller (bhk, atcc ccl 10); kinesiske hamsteræggestokkeceller/- dhfr (cho, urlaub et al., proc. natl. acad. sci. usa, 77:4216
(1980)); muse-sertoli- teller (tm4, mather, biol. reprod., 23:243-251
(1980)); abenyreceller (cv1 atcc ccl 70); nyreceller fra grøn marekat (vero-76, atcc crl-1587); humane cervicale 11 carcinomaceller (hela, atcc ccl2), hundenyreceller (mdck, atcc ccl 34); bøffelrotteleverceller (brl 3a, atcc crl 1442); humane lungeceller (w138, atcc ccl 75); humane leverceller (hep g2, hb 8065); brysttumor fra mus (mmt 060562, atcc ccl 51); tri-celler (mather et al., annals n.y. acad. sci., 383:44-68
(1982)); mrc 5-celler; fs4-celler og en human hepatomalinie (hep g2). … dyrkningsbetingelserne, såsom temperatur, ph og lignende, er de, der tidligere er anvendt med værtscellerne til ekspression, og de vil være klare for fagmanden. … efter vask med intermediærpufferen vaskes kationbytterresinen eller den- ne reækvilibreres med vaskepufferen, som har en konduktivitet eller ph-værdi, el- ler begge, som er mindre end den for intermediærpufferen (dvs. konduktivitet eller ph-værdi, eller begge, er ændret i en modsat, dvs. omvendt, retning i forhold til det foregående trin, ulig ionbytterkromatografi-trin i litteraturen). i eksemplet nedenfor havde vaskepufferen cirka samme konduktivitet som loadingpufferen (dvs. inter- vallet fra ca. 5,2 til ca. 6,6 mmhos), og dens konduktivitet var derfor lavere end den i intermediærpufferen. i en anden udførelsesform kan man reducere konduktivite- ten i vaskepufferen til en konduktivitet, som er lavere end eller højere end den for loadingpufferen, forudsat at konduktiviteten i vaskepufferen er lavere end den i in- termediærpufferen. i en anden udforelsesform kan ph i vaskepufferen være lavere end ph-værdien i intermediærpufferen (f.eks. kan ph i vaskepufferen være ca. 5,0). … en terapeutisk formulering omfattende polypeptidet, eventuelt konjugeret med et heterologt molekyle, kan fremstiiles ved at blande polypeptidet med den on- skede renhedsgrad med valgfri farmaceutisk acceptable bærere, excipienser eller stabilisatorer […] … polypeptidet oprenset som beskrevet heri eller sammensætningen omfat- tende polypeptidet og en farmaceutisk acceptabel bærer anvendes derpå i forskelli- ge diagnostiske, terapeutiske eller andre anvendelser, der er kendte til sådanne po- lypeptider og sammensætninger. for eksempel kan polypeptidet anvendes til at be- handle en lidelse i et pattedyr ved at administrere en terapeutisk effektiv mængde af polypeptidet til pattedyret. de følgende eksempler er givet som illustreringer og ikke begrænsninger. beskrivelserne af alle citeringer i specifikationen er inkorporeret heri. eksempel 1 fuldlængde human igg rhumab her2 (humab4d4-8 i carter et al., proc. natl, acad. sci., 89:4285-4289
(1992)omfattende aminosyresekvensen for den lette kæde ifølge seq id nr. 1 og aminosyresekvensen for den tunge kæde ifølge seq id nr. 2) blev produceret rekombinant i cho-celler. efter proteinproduktion og se- cernering til cellekulturmediet blev cho-cellerne separeret fra cellekulturmediet ved tangentialstrømningsfiltrering (prostack™). protein a-kromatografi blev derpa udført ved at påføre den høstede cellekulturvæske (hccf) fra cho-cellerne direkte pa en ækvilibreret prosep a™ søjle (bioprocessing, ltd.). efter protein a-kromatografi udførtes kationbytterkromatografi ved brug af en sulfopropyl (sp)sepharose fast flow™ (spsff) kolonne (pharmacia) for yderligere at separere det ønskede anti-her2-antistofmolekyle. kromatografiope- rationen blev udført ved en bindings- og elueringsmetode. spsff-kolonnen blev fremstillet til loading ved efterfølgende vask med re- genereringspuffer (0,025 m mes/ 1,0 m naci, ph 5,6), efterfulgt af ækvilibrerings- puffer (0,025 m mes/ 50 mm naci, ph 5,6). kolonnen blev derefter loaded med pro- tein a-pool, indstillet til en ph-værdi pa 5,60 ± 0,05 og en konduktivitet pa 5,8 ± 0,2 mmhos. inden eluering blev kolonnen vasket i tre trin
(1)loadingpuffer (0,025 m mes/ 50 mm naci, ph 5,6) i minimum 1 kolonnevolumen;
(2)intermediærpuffer (0,025 m mes/ 70 mm naci, ph 5,6) indtil toppen af en 280 nm top blev nået og
(3)12 vaskepuffer (0,025 m mes/ 50 mm naci, ph 5,6) i minimum 1,2 kolonnevolumen. rhumab her2 blev derpå elueret fra kolonnen med elueringspuffer (0,025 m mes/ 95 mm naci, ph 5,6). 280 nm elueringsprofilen har en skulder i den forreste ende (figur 3). ved vendepunktet i denne skulder starter opsamling og fortsætter i yder- ligere 5 kolonnevolumener. kolonnen blev derpa regenereret med regenererings- puffer (0,025 m mes/ 1,0 m naci, ph 5,6). … materialer og metoder kolonne- og load-fremstiliing: en reduceret skala spsff-kolonne blev pakket. dimensionerne var: 27,0 ml volumen, 1,0 cm i diameter og 34,5 cm bedhøjde. ph- værdi i en alikvot af protein a-pool blev titreret til 5,6 med 1,5 m tris base. konduk- tiviteten blev reduceret ved tilsætning af et lige volumen sterilt vand til injektion (swfi). … totalt seks kromatografikdrsler blev udfprt med load-densiteter pa 15, 20, 25, 30, 35 og 40 mg rhu-mab her2 pr ml spsff-resin. … bestemmefse af rhumab her2 antistofvarianter (csx hpiex): rhumab her2 spsff kromatografikolonnen resolverer antistofvarianter. fraktioner for hver af un- dersøgelseskromatografierne blev testet for den relative mængde variant antistof ved csx hpiex kromatografi. en bakerbond wide-pore™ csx hpiex kolon- ne (4,6 x 250
  1. mm)blev kprt ved 1 ml/min ved 55°. den mo bile fase blev dannet af en tertiær gradient (label 2). 13 kolonnen køres ved 1 ml/min ved 55°c. a-pufferen var 0,025 m mbs, ph 5,9; b-pufferen var 1 m ammoniumacetat, ph 7,0 og c-opløsnlngen var sterilt vand til injektion. kolonnen blev ækvilibreret med gradientens initialbetingelser (49% a; 1% b og 50% c), og 200 µl prøve fortyn- det med swfi og indeholdende <300 µg protein, blev injiceret. hvert resulterende kromatogram blev integreret for at bestemme det procentvise areal af hver top for hver fraktion (tabel 3 og figur 5). … resultat og diskussion deamideret og andre sure varianter af rhumab her2 blev produceret, når antistoffet blev fremstillet ved rekombinant dna-teknologi (se for eksempel csx top a, b og 1 i figur 5). de deamiderede og andre sure varianter udgjorde ca. 25% (beregnet som arealet under den integrerede kurve eller profil opnået ved csx-kro- matografi) af sammensætningen opnået fra det initiale protein a kromatografitrin. det blev opdaget, at ionbytterfremgangsmåden beskrevet heri, kunne anvendes til at reducere mængden af deamideret og andre sure varianter i anti-her2-sammen- sætningen, dvs. til ca. 13% eller mindre (dvs. mængden af sure varianter i fremstil- lingen underkastet kationbytterkromatografi, som beskrevet heri, blev reduceret med ca. 50% eller mere). et absorbansspor fra en kationbytterkolonne kørsel, udført som beskrevet ovenfor, er vist i figur 3. denne fremgangsmåde resolverede en deamideret variant af anti-her2-antistof, som kun adskiller sig en lille smule fra non-deamideret anti- her2-antistof. stigningen i konduktivitet fra de initielle betingelser til intermediær- 14 vasken startede elueringen af det deamiderede anti-her2-antistof. fortsat vask ved denne konduktivitet blev imidlertid fundet at eluere non-deamideret anti-her2-an- tistof, hvilket resulterede i tab af produkt. ved at fortsætte direkte fra intermediær- pufferen til elueringspufferen blev observeret enten en uacceptabel lav fjernelse af deamideret anti-her2-antistofproduktet, hvis fraktionssamling begyndte tidligere eller uacceptable lave udbytter af anti-her2-antistofprodukt, hvis fraktionssamling blev forsinket, til deamideret anti-her2-antistof var reduceret. det blev opdaget, at ved at gå tilbage til lavere konduktivitet som anvendt initialt, fortsatte eluering af deamideret anti-her2-antistof uden signifikant anti-her2-antistof produktelue- ring. … kvaliteten af rhumab her2 i alle elueringspools er ækvivalent bestemt ved csx hpiex analyse (figur 5). sammenlignet med loadmaterialet er der en berigelse af non-deamideret antistof (top 3), ingen ændring i mængden af iso-asp102 eller lys450 antistof (top 4) og en reduktion i mængden af asp30 deamideret antistof (top- pene a, b, 1 og andre). 15 …” antistoffet trastuzumab var beskyttet som en ny kemisk forbindelse ved patent dk/ep 0590058 (carter) (internationalt publikationsnummer wo1992022653) med titlen ”hu- maniseret heregulin-antistof”. patentet udløb den 15. juni 2012. det supplerende beskyt- telsescertifikat nr. cr 2004 00004 udløb den 29. juli 2014. den europæiske patentansøgning og behandlingen heraf ved epo den 23. december 2002 sendte roche en patentansøgning til epo vedørende pa- tentansøgningsnummer 02029008.6. patentansøgningens krav 1 angik en sammen- sætning omfattende en blanding af anti-her2 antistof og en eller flere sure vari- anter deraf, hvor mængden af sur variant(
  2. er)er mindre end 25 %. det fremgik af krav 9, at mængden af sur variant(
  3. er)skulle bedømmes ved brug af en c sx hpiex-kolonne (carboxy sulfion cation exchange high pressure liqued chromato- graphy). den 12. marts 2004 sendte epo en vurdering til roche, hvori der om krav 9 blev anført: ”in claim 9, the applicant attempts to characterise the composition by the technique used (c sx hpiex) to quantify the acidic cariant(
  4. s)present in the composition. however, claim 9 contains no information as to the ingredients of the compositions. consequently, claim 9 does not meet the requirements of art. 84 epc. it is reminded that the technique used to characterise the acidic variant(
  5. s)and define their amounts in the composition is actually irrelevant to the composi- tion itself. the applicant is invited to delete claim 9 or to redraft it as process claim.” 16 det er oplyst, at roche herefter udtog krav 9 og sammenskrev kravene 1, 5, 6 og 7 til det nuværende krav 1, hvorefter patentet blev offentliggjort med patentnum- mer dk/ep 1 308 455 (455-patentet). den 20. december 2006 indleverede synthion b.v en indsigelse mod 455-patentet, idet de anfægtede at opfindelse havde nyhed og opfindelseshøjde. den 5. oktober 2010 traf epo’s indsigelsesafdeling en afgørelse, hvor de fandt stridspatentet ugyl- digt på grund af manglende nyhed. roche appellerede afgørelsen fra epo’s indsigelsesafdeling til epo’s technical bo- ard of appeal, der traf afgørelse i sagen den 16. april 2015. det fremgår af afgørel- sen, at board of appeal ved vurderingen inddrog følgende dokumenter:  d4: wo92/22653 (carter)  d6: wo97/04801 (andya)  d7: harris waterside, siderne 1-7  d20: harris
(1995) d22: harris
(2001) erklæring fra d. wang. epo’s technical board of appeal fandt, at opfindelsen havde nyhed og opfindel- seshøjde, og appelkammeret omgjorde derfor indsigelsesafdelingens afgørelse og opretholdt strispatentets gyldighed. af den nærmere begrundelse herfor, fremgår bl.a.: “[…] novelty (article 54 epc) 2. the subject-matter of claim 1 relates to a composition comprising a mixture of the anti-her2 antibody hummab4d5-8 and one or more acidic variants thereof, wherein the amount of the acidic variant(
  1. s)is less than about 25%, and wherein the acidic variant(
  2. s)are predominantly deamidated variants, and wherein the deamidated variants have asn30 in cdr1 of either or both vl regions of hummab4d5-8 converted to aspartate. 3. novelty of the claimed subject-matter was challenged by the respondent on the basis of the reconstituted, previously lyophilised, rhumab her2 formu- lations disclosed in example 1 of document d6. 4. for an invention to lack novelty, all the claim's features must be disclosed in the prior art. if the prior art consists of a written description, as in the present case, what is made available to the public is the information content of the written description (see decision g 2/88, reasons, point 10). it is a generally ac- cepted principle that for lack of novelty, there must be a direct and unambigu- ous disclosure, either explicit or implicit, in the state of the art which would 17 inevitably lead the skilled person to subject-matter falling within the scope of what is claimed. in this context "implicit disclosure" means disclosure which any person skilled in the art would objectively consider as necessarily implied in the explicit content (see e.g. decision t 1523/07, reasons, point 2.4). 5. example 1 of document d6 (see pages 18 to 27) reports the development of a lyophilized formulation comprising full length humanised antibody humab4d5-8, which is referred to as rhumab her2 (see page 18, line 35 to page 19, line 2). it was undisputed that document d6 does not explicitly dis- close the feature of claim 1 that the acidic variants are predominantly deami- dated variants, wherein the deamidated variants have asn30 in cdr1 of ei- ther or both vl regions of hummab4d5-8 converted to aspartate. 6. the question to be answered is thus whether or not this feature is implicitly disclosed in the sense that it can be directly and unambiguously derived from what is explicitly disclosed in document d6. 7. from page 19 to page 24, document d6 describes the various lyophilised for- mulations of the rhumab her2 antibody. on page 19 document d6 discloses (see lines 13 to 15) that: "[i]n early screening studies, the stability of several lyophilized recom- binant humanized anti-her2 antibody (rhumab eer2) formulations was investigated after incubation at 5°c (proposed storage condition) and 40°c (accelerated stability condition). in the liquid state, rhumab her2 was observed to degrade by deamidation (30asn of light chain) and isoaspartate formation via a cyclic imide intermediate, succinimide (102asp of heavy chain)." 8. from page 24, line 11 onwards document d6 describes experiments which ad- dress the stability of the antibody after reconstitution and during storage. for this purpose, four different types of reconstituted lyophilised formulations of rhumab her2 were prepared and the stability of these reconstituted formu- lations in terms of deamidation or succinimide formation was determined. in this context document d6 discloses (see page 26, lines 14 to 18) that: '’[a]s mentioned previously, the major degradation route for rhumab her2 in aqueous solutions is deamidation or succinimide formation. the loss of native protein due to deamidation or succinimide formation was assessed for the four reconstituted rhumab her2 formulations. analysis of rhumab her2 deamidation and succinimide formation was performed using cation exchange chromatography." 9. figures 5 to 8 depict the results of the cation exchange chromatography in terms of % native protein against time (days) for the different formulations at different temperatures. the % native protein is defined as the peak area of the native (not degraded) protein relative to the total area as measured by cation exchange chromatography (see legends of figures 5 to 8 on page 4, lines 20 to 38). pursuant to figure 5 the amount of the native protein is around 82% at the beginning of the experiment (time zero). 10. the respondent argued that the value of 82% native protein reported in fig- ure 5 implied the presence of a maximum of 18% variants, i.e. less than about 25% as stipulated by claim 1. these variants could be either acidic or basic. as the only acidic variant disclosed in document d6 was the variant resulting from deamidation at asn30 in the light chain of rhumab her2, document d6 anticipated the claimed subject-matter. 11. in relation to this line of argument the board observes that the experiments described at page 26 of document d6 are concerned with comparing the prop- erties of reconstituted, previously lyophilised, formulations over time. docu- ment d6 is silent about the nature of the degraded antibody present in these reconstituted formulations of rhumab her2. 12. document d6 mentions on page 19 that in the liquid state rhumab her2 was observed to degrade by deamidation at position asn30 of the light chain and 18 isoaspartate formation at position 102asp of the heavy chain (see point 7 above). in the context of the studies performed on the reconstituted formula- tions document d6 refers back to this statement (see point 8 above). however, the statement on page 19 is made in the context of "early screening studies" and the skilled person has no reason to conclude that the same degradation takes necessarily place in the reconstituted formulations. from the statement on page 19 it is also not derivable that (
  3. i)deamidation in the reconstituted for- mulations of example 1 involves, position asn30 or (
  4. ii)that the result of the deamidation, if it is at position asn30, is necessarily a conversion of asn30 to asp30. no conclusion can thus be drawn about the nature of any particular variant which might be present in the reconstituted formulation at time zero, or at any other point in time, from the written description of example 1 of doc- ument d6. 13. the board concludes from the above that the feature that the acidic variants are predominantly deamidated variants, wherein the deamidated variants have asn30 in cdr1 of either or both vl regions of hummab4d5-8 converted to aspartate, is not directly and unambiguously disclosed in document d6. 14. in a second line of argument the respondent submitted that when the starting materials were clearly defined and the method by which these starting mate- rials were reacted, processed or separated was clearly defined, then the result of that process was directly and unambiguously disclosed, although inher- ently. 15. as discussed above (see points 11 and 12), the starting material of the experiments conducted in example 1, i.e. the composition of the reconsti- tuted formulations of rhumab her2 at time zero of the study, and in partic- ular the nature of the variants of the antibody present in the reconstituted for- mulation, is not clearly defined in document d6. accordingly, the respon- dent's second argument fails for this reason alone. 15. it follows from the above, that document d6 does not directly and unambigu- ously disclose all the features of claim 1. therefore, the subject-matter of this claim, and of dependent claims 2 to 6, is not anticipated by document d6. the main request fulfills the requirements of article 54 epc. […] inventive step closest prior art 18. the closest prior art for assessing inventive step is normally a prior art docu- ment disclosing subject-matter conceived for the same purpose or aiming at the same objective as the claimed invention and having the most relevant tech- nical features in common, i.e. requiring the minimum of structural modifica- tions (see case law of the boards of appeal of the epo, 7th edition 2013, sec- tion i.d.3.1). 19. the purpose of the claimed invention is the reduction of the amount of acidic variants occurring in anti-her2 antibody preparations. 20. the respondent proposed that either document d6 or document d7 repre- sented the closest prior art, while the appellant considered that document d7 represented the closest prior art. 21. document d6 is concerned with providing stable lyophilized antibody for- mulations that contain specific excipients which protect against further physi- cal and chemical degradation of the antibodies contained in the lyophilised reconstituted formulations (see page 1, lines 4 to 6; example 1). although doc- ument d6 discloses (see page 19, lines 13 to 15) that rhumab her2 may de- grade in the liquid state by deamidation at asn30 of the light chain and suc- cinimide formation at aspl02 of the heavy chain, it is silent about removal of the resulting variants. 22. document d7 is a collection of slides of a presentation given at a conference. the waterside monoclonal conference, relating to chromatographic tech- 19 niques for the characterisation of rhumab her2. document d7 discloses that a composition comprising rhumab her2 and charge variants thereof can be resolved by monos cation exchange chromatography (see lower slide on page 4). an analysis of the individual peaks with respect to the presence of asn30 and/or asp30 is shown in the lower slide on page 6. in the upper slide on page 7, document d7 reports that peak 1, which comprises the acidic variant, has 82% specific activity in a pl85her2 binding assay while peak 3, which com- prises the native rhumab her2, shows 100% specific activity in the binding assay. in the same slide it is reported that 25% of the pool has deamidated asn30 and that it was decided not to remove the deamidated material. docu- ment d7 also discloses that deamidation increases when harvested cell cul- ture fluid (hccf) is held and that therefore harvests are taken straight through to purification (see page 7, upper slide). 23. the board concludes that document d6 does not have the same purpose as the claimed invention as it is concerned with the stabilisation of reconstituted lyophilised antibody preparations and does not aim at reducing the amount of undesired variants, such as acidic variants. document d7 on the other hand recognises the issue of the occurrence of deamidated/acidic variants and thus relates to same purpose as the invention. accordingly document d7 repre- sents the closest prior art. technical problem to be solved 24. the technical problem to be solved in view of document d7 is the provision of rhumab her2 compositions with improved properties. the solution con- sists in the provision of a composition as defined in claim 1 in which the level of a particular type of acidic charge variant is reduced to below 25%. obviousness 25. when considering whether or not the subject-matter constitutes an obvious solution to the technical problem, the question to be answered is whether or not the skilled person, in the expectation of solving the technical problem de- fined in point 24 above, would have modified the teaching in the closest prior art document d7 so as to arrive at the invention in an obvious manner. 26. in that context, the appellant argued that document d7 would not have pro- vided any motivation to the skilled person to remove the deamidated mate- rial. 27. however, document d7 discloses that deamidation of asn30 decreases the binding activity of rhumab her2 in a p185her2 binding assay and that har- vests are taken straight through to purification to avoid an increase in deami- dation (see point 22 above). thus, in the board's view, document d7 teaches that deamidation at position asn30 has a negative impact on the activity of the rhumab her2 antibody preparation and also that the occurrence of deamidation should be minimised. the skilled person would thus have been motivated to remove the deamidated material, i.e. the acidic variants, from the antibody preparation disclosed in document d7 in order to solve the prob- lem formulated above. the question which remains to be answered is whether or not he would also have had a reasonable expectation of success. 28. the respondent submitted that the skilled person would have easily suc- ceeded in the task by performing a monos cation exchange chromatography as disclosed in document d7 to resolve the acidic variants from the native an- tibody. by collecting the native rhum&b her2 only after elution of the acidic variants, the acidic variants would have been removed from the antibody preparation. absent any disclosure of the conditions of the monos cation ex- change chromatography in document d7, the skilled person would have used routine conditions which formed part of his common general knowledge. 20 29. in this context the board notes that it is undisputed that the skilled person, when trying to implement the teaching of document d7, would also have been aware of document d20. this document discloses the same profile for the cation exchange chromatography of rhumab her2 (see figure 2) as docu- ment d7. document d20 moreover provides detailed instructions about the conditions for the monos cation exchange chromatography (see section 2.2. on page 130). 30. however, the respondent itself has provided evidence that it was unable to obtain the cation exchange profile as given in figure 2 of document d20 by following these instructions: see point 5 of declaration d26. according to points 6 and 7 of declaration d26 a different column material, namely baker- board csx, was required to actually obtain the cation exchange profile de- picted in figure 2 of document d20, which is the same as the one depicted in document d7. thus, as shown by declaration d26, the skilled person aiming at solving the problem defined in point 24 above by following the teaching of document d7 and aware of routine conditions of monos cation exchange chromatography of rhumab her2 as disclosed in document d2q would not have succeeded in separating the acidic variants from the native antibody molecule. 31. the respondent further submitted that if the skilled person had found that he was not able to separate the acidic variants from the native antibody by monos cation exchange chromatography he would have found a suitable method in document d13. the respondent relied in particular on figure 7, which depicts the results of an anion exchange chromatographic separation of recombinant human growth hormone (hgh) and its deamidated isoforms. 32. the board accepts that the skilled person looking for techniques to separate an antibody from its deamidated isoforms would have been aware of docu- ment d13. the board observes however that document d13 addresses the is- sues arising in the context of the separation of small amounts of proteins (see page 57, paragraph bridging the columns). it reports on separation studies performed with novel stationary phases, termed mci gel protex, with inter alia standard protein mixtures and hgh and concludes that protex stationary phases are suitable for the separation of proteins with subtle differences, such as variants and isoforms (see page 63, left hand column, last paragraph). 33. the board considers it at least questionable whether the skilled person, start- ing from document d7, which deals with the large scale production of rhumab her2 (see page 4, upper slide), and faced with the problem set out above (see point 24), would have taken account of the separation techniques disclosed in document d13 for the separation of small amounts of proteins. 34. assuming, in the respondent's favour, that the skilled person would have taken into account the techniques of document d13 he would, in the board's view, have had no reason to infer that a separation technique that worked for hgh would also work for the rhumab her2 antibody preparation. the mole- cules separated in document d13, hgh and its deamidation isoforms, are con- siderably smaller than the rhumab her2 antibody and its acidic variants. in the case of rhumab her2 only one or two charges are changed in the acidic variant relative to the large net charge of the native antibody. the relative dif- ference in charge between the native antibody and its variant is thus small and the skilled person would have had no reason to assume that the condi- tions disclosed in d13 for the separation of hgh and its isoforms would re- solve rhumab her2 and its, isoforms. indeed, pursuant to the patent, a wash step with an intermediate buffer of lower conductivity is necessary to sepa- rate the acidic variant from the native antibody in an cation exchange chro- matography (see paragraph [0114]). this step is not disclosed in document d13. 21 35. in summary, the board is not persuaded that either of the two methods sug- gested by the respondent would have led the skilled person to the successful separation of the acidic variants from the native rhumab her2 antibody. the skilled person would thus have had no reasonable expectation of success. 36. for these reasons, the board concludes that the subject-matter of claim 1 is not obvious. accordingly, the subject-matter of claim 1, and by the same token that of dependent claims 2 to 6, involves an inventive step within the mean- ing of article 52.
(1)and 56 epc. […]” kendt teknik (før stridspatentets prioritetsdag) biogen, bioepis og msd har overordnet gjort gældende, at stridspatentet er ugyldigt på grund af manglende nyhed og opfindelseshøjde i forhold til kendt teknik på prioritets- tidspunktet. de for sagen væsentlige dele af modholdene er medtaget og gennemgået i nedenstående eksperterklæringer af professor paul dalby og nigel k. h. slater, samt i parternes sammenfattende processkrifter. modholdene udgøres af følgende kendte tek- nik: patent dk/ep 0590058/wo1992022653 (carter) antistoffet trastuzumab blev beskyttet i ovenstående patent med prioritetsdag
  1. juni 1991 under titlen ”humaniseret heregulin-antistof”. patentet udløb den
  2. juni
  3. det supplerende beskyttelsescertifikat nr. cr 2004 00004 udløb den
  4. juli
  5. paten- tet beskriver flere udgaver af humaniseret antistofpolypeptider, herunder hum- mab4d5-8, samt en metode til at frembringe de humaniserede antistoffer. patentet in- deholdt endvidere en vurdering af hummab4d5-8 som den mest potente humanisere- de variant af det murine antistof mumab4d5, samt en angivelse af konsensussekvens for immunglobulinet, samt en struktur model herfor. patent wo 97/04801 (andya) patentet blev udstedt den
  6. februar 1997 på baggrund af en patentansøgning indleve- ret den
  7. juli
  8. patentet krævede prioritet fra den
  9. juli 1995 og
  10. marts
  11. patentets titel er ”stabile isotonic lyophilized protein formulation”, og patentet indehol- der en beskrivelse af, hvorledes proteinopløsninger kan frysetørres og derefter senere rekonstitueres som opløsning. patentet anfører to eksempler på antistofsammensætnin- ger, der er frysetørret og dernest rekonstrueret og analyseret, hvoraf det ene forsøg ”an- ti-her2 formulation” vedrører trastuzumab (hummab4d5-8). harris
(1995)22 artiklen "processing of c-terminal lysine and arginine residues of proteins isolated from mam- malian cell culture" af reed j. harris, der blev publiceret i journal of chromatography a i 1995, omhandler blandt andet frembringelse af antistoffet rhumab her2 ved brug af cho-celler. artiklen indeholder endvidere en gengivelse af tre kromatogrammer udført ved kationsbytningkromatografi på tre prøver af rhumab her2, samt en analyse af re- sultaterne heraf. harris waterside og komplet harris waterside harris waterside er en kopi af en powerpoint præsentation (siderne 1-7) vedrørende et foredrag afholdt af reed j. harris på the waterside monoclonal conference, der blev af- holdt den 22-25 april
  1. præsentationen vedrørte kromatografi teknikker til karakte- riseringen af det humaniserede monoklonale antistof rhumab her
  2. komplet harris waterside er den fulde udgave af ovenstående porwerpoint præsenta- tionen, dvs. siderne 1-
  3. den komplette udgave indeholder dermed yderligere 15 sider. der er mellem parterne enighed om, at komplet harris waterside ikke blev fremlagt un- der indsigelses- eller appelsagen ved epo, der alene var i besiddelse af harris watersi- de, siderne 1-
  4. powell “a compendium and hydropathy/flexibility analysis of common reactive sites in proteins: reactivity at asn, asp, gln, and met motifs in neutral ph solution”, er et kapitel skrevet af michael f. powell m.fl., der blev publiceret i
  5. kapitlet omhandler fastlæggelse af grænserne for reaktionerne for asn, asp, gln og met med henblik på at forudsige sand- synligheden for en bestemt deamidering i de såkaldte ”hot spots”. til brug for diskussionerne om den kendte teknik, fagmandens almindelige viden på prioritetstidspunktet mv. er endvidere fremlagt følgende publiceret eller hidrørende fra før prioritetsdagen: schmidt, carina
(1989)“the purification of large amounts of monoclonal antibodies”, journal of biotechnology 11, s. 235-252. 23 harris, e l v og angal. s
(1990), “protein purification applications: a practical approach”, oxford university press, s. 151-156. carter, paul m.fl.
(1992)“humanization of an anti-p185her2 antibody for human cancer ther- apy”, proc. natl. acad. sel. usa, vol. 89 s. 4285-4289. rodrigues, maria l.
(1995)“development of a humanized disulfide-stabilized anti-p185her2 fv-β-lactamase fusion protein for activation of a cephalosporin doxorubicin prodrug”, can- cer research 55, s. 63-70. harris, reed j. m.fl.
(1996)“identifying unexpected protein modifications”, mass spectrom- etry in the biological sciences. carter, paul
(1996)“targeting the product of the her2/neu protooncogene for therapy”, breast cancer advances in biology and therapeutics – 21st meeting of the international asso- ciation for breast cancer reseach. fra tiden før prioritetsdagen er envidere fremlagt det ikke offentligt tilgængelige doku- ment u.s. biological license application (bla) for herceptin (item 4: vol 1, s. 48-50, 67 og 83), dateret den 24. april 1998, vedrørende kationbytterkromatografier udført på her- ceptin. dokumenter om den tekniske lære mv. publiceret eller hidrørende fra efter prioritetsda- gen: weitzhandler, michael m.fl.
(1998)“protein variant separations by cation-exchange chro- matography on tentacle-type polymeric stationary phases”, journal of chromatography a, 828, s. 365-372. dionex
(1999), “monitoring protein deamidation by cation-exchange chromatography”, ap- plication note 125. 24 weitzhandler, michael m.fl.
(2001), “protein variant separations using cation exchange chro- matography on grafted, polymeric stationary phases”, proteomics 1, s. 179-
  1. supplement til u.s. biological license application (bla) for herceptin, dateret
  2. au- gust 2011, vedrørende ændring af testemetoden fra ion-exchange chromatography (iec) til high-performance ion-exchange chromatography (hpiec) samt kromato- grammer og sammenligning af de to testmetoders resultater. (ikke offentligt tilgæn- geligt). harris, reed j. m.fl.
(2001)“identification of multiple sources of charge heterogeneity in a re- combinant antibody”, journal of chromatography b, s. 233-245. harris, reed j.
(2002), “the ideal chromatographic antibody characterization method”, pow- erpoint præsentation på ibc’s ottende internationale konference om antibody produc- tion and downstream processing: creating succesful antibody-based biopharmaceuti- cal products. supplement til u.s. biological license application (bla) for herceptin, dateret 31. marts 2004, vedrørende udført kationonbytningkromatografi og resultaterne heraf. (ikke of- fentligt tilgængeligt). vlasak, j. og ionescu, r.
(2008)”heterogeneity of monoclonal antibodies revealed by charge- sensitive methods”, current pharmaceutical biotechnology 9, s. 468-481. wang, dongyuan
(2011), erklæring vedrørende eftergørelse af kationbytningkromato- grammerne i harris
(1995). seokkyun, kim m. fl.
(2017)”drifts in adcc-related quality attributes of herceptin®: im- pact on development of a trastuzumab biosimilar”, mabs vol. 9, no. 4, s. 704-714. buick, richard
(2017), erklæring udarbejdet til brug for inter partes review i usa ved- rørende u.s. patent no. 6,339,142. 25 lee, jihun m.fl.
(2018)”evaluation of analytical similarity between trastuzumab biosimilar ct- p6 and reference product using statistical analyses”, mabs vol. 0, nr. 0, s. 1-25. ontruzant som tidligere anført er ontruzant en biosimilær version af roches lægemiddel hercep- tin, der er et biologisk lægemiddel indeholdende aktivstoffet trastuzumab. der er fremlagt tre analyser af ontruzant med henblik på at fastlægge den kromatogra- fiske profil af produktet, herunder fastlæggelse af renheden og mængden af sure vari- anter. i rapport dateret 9. august 2018 testede solvias ontruzant (150
  1. mg)på en kationbytter- kromatograf af mærket dionex propac wcx-10 med henblik på blandt andet at fastlæg- ge den kromatografiske profil af produktet. herceptin blev anvendt som referencestan- dard. følgende fremgår blandt andet af rapportens testresultater: 26 27 i rapport dateret 16. november 2018 testede solvias ontruzant (150
  2. mg)blandt andet med henblik på at karakterisere og fastlægge mængden af sure varianter i produktet. herceptin blev analyseret sideløbende med ontruzant. der blev udført ”tryptic peptide mapping” og trypsinspaltning (”tryptic digest”). solivas anvendte cex hplc samt en propac wcx-10-kolonne. af rapportens testsresultater fremgår bl.a. følgende: 28 29 i rapport af 18. december 2018, udarbejdet af eag laboratories, blev der foretaget ana- lytiske tests på produkterne herceptin og ontruzant på en bakerbond wide-pore car- boxy sulfon hpiex-kolonne ved 55 °c, hvorefter testresultaterne blev sammenlignet. af rapportens resultater fremgår blandt andet: 30 eksperterklæringer fremlagt af parterne der er til brug for denne sag og tidligere sager afgivet en række eksperterklæringer. professor paul dalby har til brug for en tidligere sag ved commercial court of brussels afgivet eksperterklæring af 29. december 2017. af denne fremgår: ”[…] b. documents considered in this declaration 5. i was provided with the following documents:
  3. a)ep-b9 1 308 455 (the "patent");
  4. b)pfizer's submission dated 20 october 2017 (english translation);
  5. c)the expert declaration of dr buick (dated 29 august 2017);
  6. d)genentech's submission dated 1 august 2017 (english translation);
  7. e)wo 97/04801 ("wo 801");
  8. f)wo 92/22653 ("wo 653"); and
  9. g)r.j. harris, presentation at the waterside monoclonal conference april 22-25, 1996 ("waterside"); 6. through genentech's lawyers, i have been provided with all the documents i need to express the opinions i set out below regarding the validity of the patent in the light of the issues raised in pfizer's submission. c. the technical field to which the patent relates 7. the patent relates to compositions of the antibody hummab4d5-8 (inn designa- tion trastuzumab; commercialized under the name "herceptin ®") which are char- acterized in that the amount of acidic variants of the antibody in the composition is less than about 25% and that the acidic variants are predominantly variants in which asparagine at position 30 (asn30) in cdr1 of either or both vl regions is deamidated and converted into aspartate. 8. the patent describes a purification method based on cation exchange chromatogra- phy which allows the efficient separation of the native antibody from such acidic variants and thereby provides antibody compositions in which the amount of the acidic variants is reduced. the antibody compositions are intended for pharmaceu- tical use. 9. more specifically, the antibody hummab4d5-8 is an antibody which is directed against the antigen her2 (see paragraph [0063] of the patent). her2 is a receptor which belongs to the family of epidermal growth factors. it is overexpressed in some types of cancer, for example in certain types of breast cancer, which are referred to as "her2-positive". hummab4d5-8 (trastuzumab) has the capacity of inhibiting the growth of tumor cells which overexpress her2 (see paragraph [0063] of the patent) and is thus in particular used to treat her2positive breast cancer patients. […] e. deamidation of asn30 in hummab4d5-8 and the patent 12. it was known in the art that after protein synthesis degradation of a protein can occur, including in the form of deamidation of certain amino acids, in par- ticular by removal of an amino group (-nh2) by hydrolysis from the side chain of asparagine (asn) or glutamine (gln) residues. in the case of as- paragine (asn), such a deamidation results either in a succinimide, an aspar- 31 tate or an isoaspartate residue (l return to this specific point below). the loss of the nh2 group to form aspartate or isoaspartate results in a protein mole- cule which is more acidic than the native amidated form and which has a dif- ferent net charge and, consequently a different pl value. there is also a very small (1da) increase in mass. 13. the patent is concerned with variants of the anti-her2 antibody hummab4d5-8 which are deamidated to form aspartate at residue asn30 in cdr1 of the vl regions of the antibody and which are, thus, more acidic than the native nondeamidated form of the antibody 14. depending on whether asn30 is deamidated in only one or in both vl regions of hummab4d5-8, the resulting acidic variants differ from the native anti- body by one or two charges. 15. it is described in the patent that, apart from the acidic variants which are deamidated at position asn30 and in which the asparagine residue is con- verted into aspartate, other variants of hummab4d5-8 occur during the re- combinant production in cho cells, e.g. basic variants in which aspartate at position 102 of the heavy chain is converted into succinimide or isoaspartate or variants in which an additional lysine residue occurs at position 450 (see patent in table 3). f. deamidation of asn30 in hummab4d5-8 and wo 801 16. i have been asked to comment on pfizer's position on knowledge of deamida- tion, particularly at asn30 in hummab4d5-8, prior to the patent. by way of example pfizer states at paragraph 113 of the october submission: "trastuzumab was known at the priority date of ep'455 to degrade mainly by deami- dation of the amino acid asparagine at asn30 and isomerization of the amino acid as- partate at asp102" (see also paragraph 124 and footnote 125). 17. i have reviewed wo 801 and i disagree that the skilled person would under- stand the disclosure of wo 801 as pfizer states. 18. first, it is important to focus on the factors that affect deamidation. this chem- ical reaction occurs naturally at a slow rate at asparagine and glutamine residues. asparagine deamidates, in general, faster than glutamine. 19. in hummab4d5-8 there are many potential sites of deamidation. in the light chain alone there are 42 potential sites of deamidation (being the number of asparagine and glutamine residues in the two light chains). the rate of deami- dation at each site in hummab4d5-8 is determined by the following factors: i. primary sequence: in a peptide lacking a higher order structure, the major determinant of the rate of deamidation is the amino acid residue immediately adjacent to asparagine on the c-terminal side. deamidation proceeds more quickly if asparagine is followed by a small, flexible amino acid. as such, the asn-gly (asparagine-glycine) tandem is most prone to deamidation. in addition, specific side chains, such as of serine and histidine may promote deamidation. ii. tertiary (three dimensional) structure: the rate of deamidation is determined by the conformational restriction imparted by the higher- order structure and solvent accessibility to the residue (which deter- mines accessibility to the acid or base catalyst, see next point). iii. ph: the chemical mechanism of deamidation is catalysed by either an acid or a base, therefore, an inversed bell-shape ph-rate profile is typically observed, i.e. the reaction is faster at higher ph and also at lower ph, with a minimum around ph 4 to 6. iv. other conditions: deamidation is faster at higher temperature. ionic strength and buffer components also affect the rates of deamidation. the overall effects are likely to be due to the combined impact on the reaction itself and also on the protein structures. 32 20. the effect of all these factors is impossible to predict for a complex protein such as hummab4d5-8. without clear analytical information it is not possi- ble to conclude that hummab4d5-8 does mainly degrade by a particular pathway. degradation of hummab4d5-8 may proceed at another potential deamidation site (rather than asn30 of the light chain) or at other potential sites of (nondeamidation) degradation e.g. aspartate isomerization to isoas- partate. 21. wo 801 does not provide the necessary information. 22. for example, wo 801 does not disclose on page 19 (or elsewhere) what the mentioned asn30 deamidation degrades to. in general deamidation can pro- duce three products:
(1)succinimide;
(2)isoaspartate; or
(3)aspartate. any combination of one or more of the three products may be found if an as- paragine is susceptible to deamidation
  1. based on wo 801 the skilled person would not know what specific degrada- tion products would form when degrading at asn
  2. g. buick
  3. i have been provided with the declaration of dr buick ("buick"). i have been asked to comment on whether the described experiments show what a skilled person would have undertaken given wo 801 or the waterside slides.
  4. the experiments described in buick involve the expression, purification and analysis of a complex protein. it is therefore a combination of many complex experiments. each of them has the ability to affect the end result.
  5. in my view the report fails to show that a skilled person would have produced the same protein as dr buick or analyzed it as he describes.
  6. despite the experiments undertaken in buick being complex and multistage there is very little information provided and thus i am unable to fully analyze the differences between buick and what the skilled person would have done. there may be additional points over those discussed here that are not evident from the materials and methods disclosed in buick.
  7. buick states that commercially available herceptin was used to "calibrate" the experiments. buick fails to explain what changes were made to the protocols to "calibrate" them or provide any data for experiments performed without such calibration. such a reference compound is generally useful as a guide when developing methods such as analytical chromatography.
  8. buick describes the use of chinese hamster ovary ("cho") cells to produce the hummab4d5-8 antibody. cho cells are now a very popular cell line for the production of antibodies but there are, and were in 1998, many other suit- able cell lines frequently used for antibody production. for example, zena- pax/daclizumab (fda approved in 1997) was produced using a murine nso myeloma cell line and reopro/abciximab (fda approved 1994) was produced using a murine myeloma sp2/0 cell line.
  9. in the light of this i do not see a reason for the skilled person to ignore the in- struction in wo 653 to use human embryonic kidney cells. hek293 cells (the specific human cell line mentioned in wo 653) are significantly different from cho cells. further, they were a well-accepted cell line, particularly as they are of human origin.
  10. the choice in buick of cho cells will affect the profile of hummab4d5-8 vari- ants produced during the cell culture and purification of the protein, includ- ing the acidic variants and deamidated variants. this is because different cell lines require different conditions and those conditions (such as ph, ionic strength and other conditions) affect the rate of deamidation as i have ex- plained above.
  11. even when using an identical cell line the profile of hummab4d5-8 variants will be affected by changes to the rate and level of expression of the recombi- 33 nant protein. factors that affect the rate and level of expression, and which have been chosen in buick not based on information in wo 801, are: i the expression vector pcdna3.1, which contains a particular tran- scription promotor driving expression of the recombinant protein; ii the sequence of the transcribed dna which has been "optimized" from the required amino acid sequence (different sequences will tran- scribe better depending on the host cell, but buick does not describe the approach taken); and iii the choice of transient transfection over creating a stable cell line and the particular transient transfection method used.
  12. in buick mono s column chromatography is used to analyze the protein pro- duced by the cho cells. as a basis for the mono s conditions buick reports that harris 1995 was used to provide the protocol (because waterside only re- ports that mono s was used without any conditions). however, buick reports performing the mono s chromatography at 22° c but in harris 40° c was used. this is a significant difference and could impact the resolution of the chro- matography as is demonstrated in a pair of 2015 papers by fekete et al. h. separating acidic variants
  13. i have also been asked to comment upon the difficulties that the skilled per- son would encounter if they tried to separate highly similar variants such as the asn30 deamidated variants of hummab4d5-
  14. such separation was not a matter of routine for the skilled person.
  15. the acidic variants of hummab4d5-8 which are deamidated at asn30 only differ from the native antibody in one or two charges depending on whether asn30 is deamidated in only one or in both vl regions of the antibody. sepa- ration of such highly similar proteins was not regarded as a matter of routine at the priority date of the patent. further, the theoretical knowledge of chro- matography available to the skilled person would not have helped them to find the correct chromatography conditions which would have in fact pro- vided separation of the deamidated acidic variants from the native antibody.
  16. it was not usual, at the priority date, to see reports in the literature of separa- tions where the proteins being separated differed by a single charge. i do not recall seeing anything that is similar to the separation of antibody deamidated acidic variants. there were reports in the art about the separation of single charge variants of very small proteins. however, an extrapolation of the methodology used for such very small proteins to a large protein having the size of an antibody is anything but straightforward. this is because the num- ber of charged groups on a protein surface scales with the size (molecular weight) of the protein. therefore discriminating 1 or 2 net charge changes in a very small protein is proportionally easier than for a very large protein like an antibody, because the % change in net charge, and subsequent impact on pi, is greater for the smaller protein.
  17. thus, in summary, it is my view that it would not have been a matter of rou- tine for the skilled person to obtain a separation of the native antibody from the acidic variants which are deamidated at position asn
  18. rather, the skilled person would have had to find the very specific chromatography conditions (including the appropriate chromatography material and conditions for the chromatography run, like flow-rate, buffers for elution and washing etc.) which would have provided separation. the information available in the prior art in this respect in connection with very small proteins would have been no help to the skilled person when facing the problem of having to separate asn30 deamjdated variants of the antibody hummab4d5-8 from the native antibody for the reasons set forth above. […]” 34 professor paul dalby har i erklæring (scientific statement) af den
  19. maj 2018 udtalt sig om visse aspekter af analytisk kromatografi, hvoraf fremgår: ”[…] opinion
  20. in ep 455 chromatography is used for at least two different purposes. first, chromatography is used preparatively, during the purification process of the claimed antibody composition. furthermore chromatography – more pre- cisely cation exchange chromatography, or "carboxy-sulfon high performance ion exchange chromatography" (in ep 455 abbreviated as "csx hpiex") – is used analytically, to determine the quantity of acidic variants. the two chro- matography methods should be distinguished from each other.
  21. the output of chromatography is referred to as a "chromatogram". it is a plot that consists of several different peaks that may be of various shapes. plotted on the x-axis is the retention time and plotted on the y-axis is a signal being proportional to the concentration of the specific analyte separated. for a per- son skilled in the art it is clear when analysing a typical chromatogram result- ing from the csx chromatography described in ep 455 that the acidic variants, namely variants that are more acidic than the native antibody according to csx chromatography, elute before the native antibody and therefore are to be found left of the main peak containing the native antibody.
  22. the person skilled in the art is aware that the specific amount of variants re- sulting from chromatographic analysis may deviate depending on the (chro- matography) column used. he is thus aware that the use of different columns for analysis of the same antibody composition can yield differing results. reading a specified amount of acidic variants in the claim as in ep 455 he would therefore turn to the description for information about which column was used to determine that specific amount of acidic variants set out in the claim.
  23. the person skilled in the art reading ep 455 would also be aware that anti- body compositions produced by means of biological processes in cells (can) contain different variants, which may not be distinguishable from other vari- ants (or the native antibody) with the methods available at the respective date. in other words, the person skilled in the art knows that due to future techni- cal progress in chromatography there may be columns in the future which, when analysing the identical antibody composition, might resolve additional antibody variants that could not be resolved with previously used methods.” den
  24. juni 2018 afgav professor paul dalby en erklæring til brug for düsseldorf land- gericht, hvoraf følger: ”… specifically, i have been asked whether a biological product (herzuma) that pro- duces a chromatogram with the same elution times of peaks as herceptin must also have the same type of deamidations as herceptin, namely has asn30 in cdr1 of ei- ther or both vl regions of hummab4d5-8 converted to aspartate in corresponding chromatographic peaks.
  25. firstly, the chromatographic peaks are dependent on the overall charge and hence pl(isoelectric point) of the protein, which is sensitive to deamidation of asn to asp/isoasp.
  26. differences between asp and isoasp at any position would lead to chromato- graphic elution at different times, as ion exchange chromatography is able to re- 35 solve between asp and isoasp, as is observed in table 4-1 in hl11, for peaks 1 vs 2, and for 3 vs 4 which differ in both cases only in asp102 vs isoasp
  28. if two different antibody preparations have the same amino acid sequence, and they produce chromatographic elution profiles with a set of peaks having the same elution positions (for example as in figure 6 of hl21), then the most rea- sonable explanation is that each corresponding peak with the same peak position (eg time in fig 6 of hl21) contains the same variant, from, for example, deami- dation at asn30 to asp.
  29. if two different antibody preparations have the same amino acid sequence, it is highly improbable that they would have the same chromatographic profile while also having differences in their deamidation profiles. only modifications that cannot be resolved by the chromatographic method would not change the chro- matographic profile. while there may be certain types of degradation that are not detectable through this type of chromatography this does not include deamida- tion (for example, asn to asp vs asn to isoasp), which can be resolved by the particular chromatography used in test procedure q12412, described in genen- tech's submission to the fda (hl11) from page 27 onwards.
  30. thus, when presented with a trastuzumab composition that has a highly similar chromatographic profile to herceptin (hl 11, fig 4-2, p. 78), i think there is no reasonable explanation other than that the deamidation product profiles would be the same as set out in hl11 table 4-1.” professor nigel k. h. slater har til brug for denne sag afgivet eksperterklæring af
  31. ok- tober
  32. af denne fremgår: ”… instructions and documents considered
  33. for the purpose of preparing this report, i have been provided with and have read copies of the following documents: (a) a scientific review article entitled “processing of c-terminal lysine and arginine residues of proteins isolated from mammalian cell culture”, by reed j. harris, published in the journal of chromatography a, 705
(1995), pages 129-134 (“harris 1995”); (
  1. b)a presentation entitled “chromatographic techniques for the characteriza- tion of human monoclonal antibodies: rhumab her2”, presented by reed j. harris at the waterside monoclonal conference in april 1986 (“wa- terside”); (
  2. c)patent application wo 97/04801 (“wo ‘801”); (
  3. d)a book chapter written by michael f powell of genentech entitled “a compendium and hydropathy/flexibility analysis of common reactive sites in proteins: reactivity at asn, asp, gln and met motifs in neutral ph solu- tion” (“powell”); and (
  4. e)a declaration by dr richard buick. 3. i have been asked to comment on what the skilled person, as at 06 may 1998, would have understood from these documents, and what steps it would have been obvious for him or her to take in light of the document and his or her ‘common general knowledge’ (“cgk”). i discuss the skilled person and the cgk below. 4. i have also been provided with a copy of ep 1 308 455 b9 (the “patent”) and asked to consider whether the alleged invention(
  5. s)of any of the claims of the patent are novel and/or obvious to the skilled person in light of the documents identified above. 36 the skilled person 5. i have been told that the skilled person is someone with practical experience and an interest in the subject matter of the patent. i understand that the skilled person is a legal construct (he/she may in fact be a team) who is familiar with the cgk of the relevant scientific discipline. i understand that he/she reads documents with care but has no capacity for inventive thought. 6. since the patent concerns compositions containing an anti-her 2 antibody following protein purification, the skilled person will be familiar with the molecular biological synthesis of proteins, including antibodies, and the com- mon practice of purifying proteins, including to separate out native and de- graded protein variants. such methods of protein purification include ion ex- change chromatography. 7. i believe that, except where i have indicated otherwise, the views i express in this report would be representative of those of the skilled person. common general knowledge 8. i have been asked to summarise the cgk of the skilled person as at may 1998. it has been explained to me that cgk is that information which is generally known by the skilled person and is regarded as a good basis for further ac- tion. i understand that certain information may form part of the cgk if it is information that the skilled person would refer to as a matter of course, even if he/she cannot remember it, but which they understand is generally re- garded as a reliable basis for further work by the majority of those who are engaged in the art at the relevant time. 9. except where i have stated otherwise, i believe that the information in the re- mainder of this section would have been well known to, and accepted by, the skilled person in may 1998. it would therefore have formed part of his/her cgk at that time. when i refer to ‘1998’ below (unless i indicate otherwise), that is shorthand for ‘may 1998’. preparation of therapeutic proteins and antibodies 10. in may 1998, the skilled person would have been aware of a variety of meth- ods to purify medicinal products intended for clinical administration. the skilled person would have been aware that purification is necessary in order to ensure safety, efficacy and consistency in clinical use and to meet quality control specifications required by regulatory authorities. the skilled person would also have known that purification steps inevitably lead to a reduction in yield of the target protein; accordingly, there is always a balance to be struck between achieving the desired levels of antibody purity and yield. 11. biologicals, including antibodies, have particularly onerous regulatory re- quirements in comparison with small molecule chemical compounds due to the inherent variability that can be introduced as a result of recombinant ex- pression in living cellular expression systems. it was well known in 1998 that biological products are by definition heterogeneous, and so their purification and composition have to be carefully monitored. this applies equally to the manufacturing process for the ‘reference’ biological (e.g. herceptin® (trastuzumab)) and for any biosimilar thereof. 12. it was well known in 1998 that trace contaminants, including viruses and cel- lular debris, are often present in the cell culture media following harvesting of the protein of interest from the host cell. it was also well known that recom- binant expression of therapeutic proteins and antibodies often results in a composition containing variants of the native protein (including antibodies) of interest. such variants may be caused by degradation of the native protein, and/or by post-translation modification. post-translational modifications such as glycosylation are modifications made to a protein after the polypeptide 37 chain has been synthesised; these modifications are not encoded by the dna encoding the protein. 13. by may 1998, several forms of post-translational modification and protein degradation mechanisms were well known, including the process by which certain amino acids are deamidated following protein synthesis. post-transla- tional modification and protein degradation events were known to affect both the structural and biological properties of proteins and antibodies. it was also known that such modifications and events could have an effect on the speci- ficity and strength of binding of an antibody to its respective epitope, espe- cially if the modification was in the parts of the antibody responsible for anti- gen binding, i.e. the complementarity determining regions (“cdrs”). 14. therefore, the skilled person in may 1998 would be mindful of the require- ment to purify proteins, including therapeutic antibodies, in order to remove as many as possible of the variant species and degraded particles from the na- tive form. the degree of purity of the therapeutic antibody, including as re- gards variant species, and the consistency between different batches of prod- uct would have been amongst the quality control checks that the regulatory bodies would have scrutinised carefully (to ensure that the production process is sufficiently controlled in order to achieve consistently safe and effi- cacious product). deamidated variants 15. by the priority date of the patent, in may 1998, it was well known that native proteins could be degraded to form acidic variants, including deamidated variants formed by the degradation of certain amino acids. it was known that this was a potential issue for antibodies (and other proteins) produced by re- combinant expression for therapeutic use. deamidated, acidic forms were one of a number of protein variants that the skilled person would have known about in the late 1990s and for which the process and quality control depart- ments of biopharmaceutical companies would have been vigilant. 16. deamidation is a chemical reaction in which an amino functional group (- nh2) in the side chain of the amino acids asparagine or glutamine is removed or converted to another functional group. asparagine is converted to aspartic acid (also referred to as aspartate) or isoaspartic acid and glutamine is con- verted to glutamic acid (also referred to as glutamate). the resulting deami- dated form is more acidic than the native amidated (amine-containing) form and is therefore known as an ‘acidic variant’. 17. these processes are spontaneous (non-enzymatic) and are influenced by the protein or peptide’s tertiary structure (the way the protein folds determine which asparagine and glutamine residues are surface exposed), sample ph, buffer composition and storage conditions. by 1998, the skilled person would have understood that deamidation of asparagine residues is one of the most common post-translational modifications occurring in therapeutic proteins produced by recombinant dna technology. 18. the chemical process by which deamidation occurs was well known in 1998 and is shown in figures 1 and 2, below. 38 figure 1: asparagine (asn) deamidation, aspartate (asp) isomerisation and succinimide formation. taken from harris et al., identifying unexpected pro- tein modifications. figure 2: deamidation of asn by direct hydrolysis of the amide side chain to asp (bottom route) and by succinimide (cyclic imide) formation to produce either asp or iso-asp (top route). taken from powell “a compendium and hy- dropathy/flexibility analysis of common reactive sites in proteins: reactivity at asn, asp, gln and met motifs in neutral ph solution”. 19. as indicated above, it was known in may 1998 that the presence of deami- dated acidic variants might influence the stability and biological activity of therapeutic antibodies. this would be of particular concern if asparagine residues are present in the cdr of an antibody, as the binding properties of the antibody to its respective epitope (e.g. its specificity or avidity) may be af- fected. that said, a change of an amino acid in the constant region of an anti- body (e.g. the fc ‘tail’) may affect the antibody’s interaction with other com- ponents of the immune system and thus the immune response. this is because the constant region is responsible for interacting with and activating other components of the immune system through its capability to bind fc-receptors. such a modification could thereby affect the activity and/or stability of the therapeutic protein. accordingly, if the native antibody possesses asparagine or glutamine residues and therefore has the potential to deamidate, particu- larly in the cdr, it would be essential to carry out an analysis of the impact of the presence of this variant and to ensure that the process, including the amount of deamidated variants, is appropriately controlled. as noted this would also have been important for medicines regulators, who would have wanted to know about batch-to-batch consistency and degradation of thera- peutic proteins. deamidation of a protein that is prone to it will start in cell culture and will continue for as long as the protein remains in an aqueous en- vironment (although this could be slowed down e.g. by storing the finished product in a freezer prior to thawing and use). there was therefore a desire to get therapeutic proteins as pure as possible during purification, in order to 39 maximise the amount of un-deamidated native protein and thus improve the shelf-life of the protein dispatched for clinical use. 20. in summary, in may 1998 there was (for good reason) a general interest and desire to avoid deamidation or to reduce the amount of deamidated variants in compositions known to be susceptible to deamidation (e.g. proteins con- taining asparagine residues). 21. if i consider the amino acid sequence of the anti-her2 antibody, hummab4d5 (also referred to as humab4d5-8), given in figures 7a and 7b of the patent, i can confirm that the skilled person would have considered that this antibody is susceptible to deamidation due to the presence of asparagine (
  6. n)residues in its amino acid sequence. the sequence of humab4d5-8 was published by carter et al. in 1992 (“carter 1992”). carter 1992 was a seminal paper in the development of the antibody that became known as herceptin® and identified the cdrs. the skilled person would have noted in particular the presence of an asparagine in cdr1 of the light chain (vl-cdr1), at posi- tion 30 (asn30). protein purification and chromatography 22. recombinant expression systems and the separation and purification of anti- bodies and other proteins produced by them were well established by 1998. 23. in summary, recombinant protein expression involves introducing an expres- sion vector containing the dna coding sequence for an antibody or other pro- tein of interest into a host cell by stable or transient transfection. numerous cell expression systems were routinely available in 1998, including a number of immortalised mammalian cell lines which could be propagated in cell cul- ture. well known and widely used cell lines included, for example, the hu- man embryonic kidney-293 (“hek”), hela human cervical carcinoma, and chinese hamster ovary (“cho”) cell lines. 24. typically, the protein of interest would be secreted from the host cell into the cell culture medium from which it would be harvested. centrifugation or fil- tration would typically be used as a first step to remove cells and debris from the culture medium. next, protein a affinity chromatography was typically used to purify monoclonal antibodies synthesised by recombinant expression. 25. affinity chromatography works on the basis of highly specific interactions be- tween two recognition sites. typically, one of the sites is immobilized onto a solid matrix to form the stationary phase. the target molecule, together with any contaminants, is passed over the stationary phase. as a result of the in- teractions between the immobilized site and the target molecule, the target molecule is retained on the solid phase whilst contaminants flow through the column. the target molecule is subsequently ‘eluted’ from the stationary phase by disrupting the specific interaction, for example by dropping the ph of the buffer. 26. protein a chromatography is a specific form of affinity chromatography used to purify antibodies from other proteins. it relies on the ability of protein a, a protein derived from the cell wall of the bacterium staphylococcus aureus to bind to a sequence found in the constant region of igg antibodies. by may 1998, protein a chromatography was a well-established, standard purification process for separating monoclonal antibodies from other proteins present in the supernatant harvested from cell culture systems. protein a chromatogra- phy typically leads to a high purity of antibody in the eluate, with a yield of around 90-95%. nevertheless, further purification steps are likely to be re- quired for a commercial process for manufacturing a therapeutic antibody product. ion-exchange chromatography 27. ion-exchange chromatography is used to separate charged proteins by ex- ploiting differences in their overall net surface charge. proteins have an over- 40 all charge dependent on how many basic or acidic amino acid residues are present on their surface and how these residues are arranged in the tertiary structure. ion-exchange chromatography enables the separation of similar molecules that would be otherwise difficult to separate, as the charge of the molecule of interest can be readily manipulated, for example by changing the buffer ph. the principles have long been well established; nevertheless ion- exchange chromatography has been continuously improved since it was de- veloped in the 1950s. 28. the ph at which a molecule has no net charge is called its isoelectric point, or pi. in a buffer with a ph greater than the pi of the protein of interest, the pro- tein will carry a net negative charge; therefore, a positively charged anion ex- change resin would be chosen to capture this protein. conversely, in a buffer with a ph lower than the pi of the protein of interest, the protein will carry a positive net charge; thus, a negatively-charged cation exchange resin would be used. when an ion exchange chromatography column is loaded with a sample at a particular ph, all proteins that are appropriately charged will bind to the resin. for example, if an anion exchange resin is chosen, all proteins that are negatively charged at the loading buffer ph will bind to the positively charged column resin. 29. a typical ion-exchange chromatography process can be summarised as fol- lows: after loading a protein or antibody composition onto an ion exchange chromatography column, the column is washed to remove unbound proteins. the protein of interest (having bound to the column) is then eluted using ei- ther a salt gradient or by changing the ph. the protein of interest (in purified form) is then collected as it elutes from the column. the degree of purity of the eluate (the protein fraction that emerges from the column) can then be de- termined by analytical techniques, including additional chromatographic steps, as discussed further below. 30. in may 1998, the skilled person would have regarded ion-exchange chro- matography as a routine and reliable technique for the purification of anti- bodies. the skilled person would have known how to determine the optimum conditions to achieve the desired purity for each antibody of interest by rou- tine buffer screening – i.e. by manipulating the ph and conductivity of the different buffers used during the chromatography in order to maximise the purity of the target protein (whilst still delivering a good yield). 31. i consider that it would have been entirely possible for the skilled person in 1998, carrying out routine trial and error, to establish suitable chromatogra- phy conditions to have separated an antibody from variants having only a slight difference in charge. i note that in neither waterside nor harris (dis- cussed below) is there any suggestion that the cation-exchange chromatogra- phy used to separate variants with only a small charge difference was unusu- ally difficult; on the contrary, the focus is entirely on the results, with the method used being treated in a matter of fact way. the patent 32. i have read the patent, which is entitled “a composition comprising anti-her2 antibodies”. i note that it is mostly concerned with a method to purify proteins and antibodies. indeed, paragraph [0001] of the patent states that “this inven- tion relates generally to protein purification” and paragraph [0012] states that the purification method is useful “in situations where a product molecule must be sep- arated from a very closely related contaminant molecule…”. 33. i have been told that the patent is a divisional of an earlier patent application (ep 1 075 488), which also relates to the specific method explained in the de- scription of the patent. 34. the patent was filed on 06 may 1999 and has priority date of 06 may 1998. 41 the claims of the patent 35. although the description given in the patent largely concerns processes for purifying proteins, the claims are directed only to certain specific composi- tions of anti-her2 antibody and its deamidated variants, and not to the method or process by which such compositions are produced. 36. claim 1 of the patent reads as follows: “a composition comprising a mixture of anti-her2 antibody and one or more acidic variants thereof, wherein the amount of the acidic variant(
  7. s)is less than about 25%, and wherein the acidic variant(
  8. s)are predominantly deamidated variants wherein one or more asparagine residues of the anti-her2 antibody have been deamidated, and wherein the anti-her2 antibody is hummab4d5-8, and wherein the deamidated variants have asn30 in cdr1 of either or both vl re- gions of hummab4d5-8 converted to aspartate.” 37. claims 2-4 specify compositions where the proportion of acidic variants is fur- ther reduced. claim 5 specifies a composition according to claim 1 by refer- ence to specified light- and heavy-chain amino acid sequences. claim 6 sim- ply requires that the compositions further comprise a pharmaceutically ac- ceptable carrier. 38. i note that the term ‘acidic variant(s)’ referred to in the claims is defined in paragraph [0024] of the patent as “a variant of a polypeptide of interest which is more acidic (e.g. as determined by cation exchange chromatography) than the polypep- tide of interest. an example of an acidic variant is a deamidated variant.” at para- graph [0025], a deamidated variant is described as “a polypeptide wherein one or more asparagine residue(
  9. s)of the original polypeptide have been converted to aspar- tate, i.e. the neutral amide side chain has been converted to a residue with an overall acidic character.” 39. none of the claims of the patent define how the composition of anti-her2 an- tibody is to be made, or how the percentage of acidic variants in the composi- tion is to be measured by analytical means. nor do the claims of the patent set any requirement for the claimed antiher2 antibody to have any particu- lar activity or satisfy any functional parameter. indeed, the patent is silent as to what advantages an antibody composition according to the claims would have over any other anti-her2 antibody compositions (or, more specifically, why the ‘permitted’ level of acidic variants (“less than about 25%”) has been set as it has). 40. even considering the patent as a whole, i believe it would have been unclear to the skilled person why claim 1 specifies “less than about 25%” (or why claims 2 to 4 specify the levels of acidic variants they do). although the skilled per- son would have been aware of the potential problems of acidic variants of a native antibody generally, there is no information given in the patent as to why it may be advantageous for the limitation to be set at these (or any other) specific levels. 41. likewise, it is unclear to me why claim 1 of the patent specifies that acidic variants are predominantly deamidated variants wherein one or more as- paragine residues of the antiher2 antibody have been deamidated at asn-30 in cdr1 of either or both vl to aspartate. the description provided by the patent 42. the background and description given in the patent is largely confined to a discussion of well known, routine methods for carrying out the purification of proteins and antibodies prepared by recombinant expression, and of anti- her2 antibody in particular. most of the discussion, and the only example provided, describes using ion exchange chromatography. for example, para- graph [0011] states that disclosed “herein is an ion exchange chromatographie [sic.] method wherein a polypeptide of interest is bound to the ion exchange material 42 at an initial conductivity or ph and then the ion exchange material is washed with an intermediate buffer at a different conductivity or ph, or both.” 43. paragraph [0012] states that the purification method is useful in “situations where a product molecule must be separated from a very closely related contaminant molecules at full manufacturing scale, where both purity and high recovery of polypeptide product are desired”. 44. in summary, the purification method explained in description of the patent involves the following steps: (
  10. a)the polypeptide of interest is bound to an ion exchange material us- ing a loading buffer (step a); (
  11. b)the column is then washed with an intermediate buffer to elute the contaminants (step b); (
  12. c)the ion-exchange material is further washed with a wash buffer (step c); (
  13. d)finally, the ion exchange material is washed with an elution buffer to elute the polypeptide of interest (step d). 45. at paragraph [0013](c), it is stated that “the change in conductivity and/or ph from the intermediate buffer to the wash buffer is in an opposite direction to the change in conductivity and/or ph from the loading buffer to the intermediate buffer”. para- graph [0014] explains that where cation exchange is used “the conductivity and/or ph of the intermediate buffer is/are preferably greater than the conductivity and/or ph of the loading buffer; the conductivity and/or ph of the wash buffer is/are preferably less than the conductivity and/or ph of the intermediate buffer; and the conductivity and/or ph of the elution buffer is/are preferably greater than the conduc- tivity and/or ph of the intermediate buffer”. it suggests that “preferably, the con- ductivity and/or ph of the wash buffer is/are about the same as the conductivity and/or ph of the loading buffer”. paragraph [0015] indicates that, preferably, elution of the contaminant and of the polypeptide is achieved by modifying the conduc- tivity of the intermediate and elution buffers, while keeping the ph approxi- mately the same. 46. the above steps are depicted in the flow diagram of figure 1 example in the patent 47. a worked example of this purification process is provided in example 1 of the patent (paragraphs [0102] to [0119]). although described as ‘example 1’, it is the only example provided. 48. paragraph [0102] indicates that the experiments in example 1 were carried out using a full length recombinant humanized anti-her2 monoclonal antibody (“rhumab her2”), humab4d5-8 as disclosed in carter 1992. i understand that humab4d5-8 is trastuzumab, the active ingredient of herceptin. para- graph [103] explains that humab4d5-8 was expressed in cho cells, which, as i mentioned at paragraph 23, were (and remain) a commonly used cell line for recombinant protein expression (including in commercial manufacture). initial purification steps were then carried out. tangential-flow filtration (a process in which the material to be filtered is passed tangentially over a filter membrane, through which the filtrate passes) was used to separate the cells from the culture medium. the harvested cell culture fluid (i.e. the filtrate from the tangential-flow filtration) was then purified by protein a affinity chro- matography. 49. paragraph [0104] states that, following these initial steps, cation exchange chromatography was performed using a sulphopropyl (sp)-sepharose fast flow™ (“spsff”) column. the materials and methods section states (para- graph [0106]) that a reduced-scale spsff column (27.0 ml volume, 1.0 cm di- ameter and 34.5 cm bed height) was used. at [0105] it is explained that, after equilibrating the column, the protein a pool (adjusted to ph 5.60±0.05 and a 43 conductivity of 5.8±0.2 mmhos) was loaded. the column was then washed us- ing the following buffers (each at ph 5.6 and containing 0.025 m mes):
(1)loading buffer (50 mm nacl) for a minimum of 1 column volume;
(2)intermediate buffer (70 mm nacl) until an apex of a 280 nm peak was reached; and
(3)wash buffer (50 mm nacl) for a minimum of 1.2 column volumes. 50. after washing, rhumab her2 was eluted with elution buffer containing 95 mm nacl. 51. the patent notes that the elution profile (at an absorbance of 280
  1. nm)in fig- ure 3 has a shoulder on the leading edge (i.e. the small ridge to the left of the main peak), and that pooling started at the inflection point of this shoulder and continued for five column volumes. the column was then regenerated with regeneration buffer. paragraph [0106] explains that the equilibration, load, and initial wash steps were performed at a linear flow rate of 200 cm/h, while chromatography steps were performed at 100 cm/h. 52. i set out figure 3 below. this is described at paragraph [0019] as an absorbance trace from a cation exchange chromatography run of example 1 “at full man- ufacturing scale”. it is unclear what is meant by this as the materials and meth- ods section describes example 1 as having been carried out using a labora- tory-scale 27 ml column. this could certainly not be used at ‘full manufactur- ing scale’. accordingly, this statement appears to me to be an error, and i be- lieve the skilled person would have treated it as such. 53. as can be seen from the trace above (on which time runs downwards), after loading the material in a 50 mm buffer, the 70 mm nacl intermediate buffer is applied. this starts to cause protein elution and, at the apex of the 280 nm peak, a 50 mm wash buffer is applied. after a time, the elution falls back to base line, until the application of the 95 mm elution buffer which causes the 44 elution of the main peak. the trace then drops again as all the protein that elutes at this concentration comes off. finally, a high concentration (1.0
  2. m)‘re- generation buffer’ is applied to wash out any remaining proteins. 54. paragraph [0114] states that “the increase in conductivity from the initial condi- tions to the intermediate wash began to elute the deamidated anti-her2 antibody. however, continued washing at this conductivity was found to elute nondeamidated anti-her2 antibody, resulting in a loss of product”. it continues that “by going back to lower conductivity as used initially, the elution of deamidated anti-her2 antibody continued, without significant antiher2 antibody product elution”. the skilled person would have considered it possible that the 50mm wash might flush out deamidated variants loosened by the 70mm buffer, but would have also expected some of these variants to rebind to the column during this step, given that it has the same conductivity as the buffer used for column loading. in any event, it would have been routine for the skilled person to have ad- justed the ph and conductivity of the buffers to seek to elute the deamidated forms preferentially. for example, he or she could have used a conductivity gradient to determine which conductivity of buffer to use in each of the wash steps. 55. the skilled person looking at figure 3 would have been somewhat concerned about the shape of the largest peak, as it is clear that it contains at least two species. there is a small shoulder on the leading (top) edge, which would have been understood as containing acid variants. there is also an inflection two- thirds up the trailing (lower) edge, suggesting a further species is present. 56. paragraphs [0109] to [0112] of the patent discuss an analysis of fractions of pu- rified hummab4d5-8 composition that were collected after the ion-exchange chromatography process has been completed. it is explained that the fractions were analysed using carboxy sulfon cation exchange high pressure liquid chromatography (“csx hipex”). the skilled person would have understood that it was used to determine the proportion of acidic variants in the antibody composition, with the ultimate purpose being to assess how successful the ap- plied method has been in separating the acidic variants from the native anti- body. 57. table 3 and figure 5 give the results obtained by csx hpiex chromatographic analysis of the rhumab her2 composition. i set these out below. as regards table 3, the skilled person would understand that the column on the left, “csx peak”, refers to the individual peaks of the chromatogram obtained by csx hpiex analysis, while the column on the right refers to the characterisation of the substances in these individual peaks. the species referred to as “rhumab her2 variant” represent variant species of the polypeptide of interest. the patent does not say how the individual variants referred to in table 3 have been determined. 58. from the last sentence in paragraph [0112], the skilled person skilled would understand that the area under the peaks that are visible in the chromatogram 45 obtained by csx hpiex chromatography (which is not itself shown in the patent), were integrated to determine the percentage area of each peak for each fraction. in this context, it is important to note that the patent does not disclose the integration method used (whether visual integration, software- based auto-integration or adjusted software-based integration). the areas from under the individual peaks are, however, plotted in figure 5 (shown be- low). 59. from the legend given on figure 5, the skilled person would understand that the black dots (‘load’) represent the areas under the peaks for the antibody composition (obtained by expression, filtration and protein a chromatography as de- scribed above)– i.e. the ‘raw material’. he or she would understand that the bars represent the areas under the peaks for the ‘pool’ fraction that had been subjected to the ion-exchange chromatography process described in the patent – i.e. the ‘product’. the differently shaded bars represent the analyses of the pool from each of the six different loading densities (ranging from 15 to 40 mg antibody per ml of column resin, as described at paragraph [0107]) ap- plied to the spsff column. the skilled person would notice that the different loading densities do not appear significantly to affect the content of the pool material. 60. comparing the heights of the black dots and of the columns, the skilled per- son would have noticed that the pool fraction had been enriched in native an- tibody relative to the load material: the area under csx peak 3 (i.e. the fully processed antibody) is increased from about 68% to 82% of the total. this is primarily at the expense of the peaks representing acidic variants (peaks a, b and 1) and ‘others’. whilst this is clearly an improvement, i do not believe that the skilled person would have considered it to be particularly impressive. 61. the person skilled in the art could readily have read off from the y-axis of the graph in figure 6 the area under each csx peak as a percentage of the total protein content (whether with a ruler or callipers, or, as would have been a simple exercise by 1998, by scanning the figure and measuring the digitized version), for both the ‘load’ and ‘pool’ material. he or she would have re- ferred to the description of the peaks in table 3 to understand what each con- tained. i have measured the following values for the ‘load’ material using digital callipers, giving the description from table 3 alongside for reference: 46 62. the skilled person would understand from this that: (
  3. a)peak 3 represents fully processed (i.e. native) hummab4d5-8, which amounted to about 68% of the total protein in the load mate- rial. (
  4. b)peak 4 represents basic variants, with an isoaspartate at position 102 and/or and additional c-terminal lysine (both in the heavy chain). the area under this basic peak is 7.22% of the load material. (
  5. c)peaks a, b and 1 contain acidic variants, with the deamidation of asn30 in the light chain. these three acidic peaks amount to 16.71% of the load material. (
  6. d)‘others’ amount to 8.31% of the total. the definition from table 3 suggests that ‘others’ represents a mixture of variants. these include variants with succinimide at asp102 (i.e. replacing a negatively charged, acidic side chain with a neutral side chain) and/or “multi- ple permutations found in peaks 1 and 4”. the latter suggests a mixture of acidic (from peak 1) and basic (from peak 4) variants. 63. this would suggest that, if all of the ‘others’ were assumed to be acidic vari- ants, the total proportion of acidic variants in the ‘load’ material would amount to 25.02%. that would be consistent with the statement at paragraph [0113] that “deamidated and other acidic variants constituted “about 25%” of the composition obtained from the initial protein a chromatography step. however, i consider that the skilled person would have considered it highly unlikely that all of the variants making up the ‘others’ were acidic, given the pointer in table 3 to it being a mixture of acidic and basic variants. i believe that the skilled person considering figure 5 would have concluded that the ‘load’ material contained (rounding to the nearest whole number) between about 17% and 25% of acidic variants. 64. claim 1 of the patent specifies a level of acidic variants of “less than about 25%” for the claimed composition, with the suggestion that this can be made by the ion-exchange method described in the patent. as i have mentioned above, it would have been unclear to the skilled person why the 25% level is specified. in any event, the skilled person reading the patent would under- stand that the starting material loaded onto the spsff column itself already contained at most around 25% of acidic variants (and possibly as little as 17%). he or she would therefore have questioned what contribution the patent made given that routine filtration and protein a chromatography steps used to prepare the starting material already result in a composition according to the claims (i.e. even before ion exchange chromatography is applied). the prior art harris 1995 65. harris 1995 is a review article from the journal of chromatography a entitled “processing of c-terminal lysine and arginine residues of proteins isolated from mam- 47 malian cell culture”. the author is reed j harris from the analytical chemistry department at genentech, inc. the introduction explains that variants of re- combinant proteins may result from in vivo posttranslational modification or spontaneous (non-enzymatic) protein degradation. examples of protein degradation are listed, including aspartate isomerization and deamidation of asparagine or succinimide formation. harris then discusses several examples of c-terminal lys/arg processing. 66. harris 1995 discusses various experiments, in particular in relation to rhumab-her2. section 2.3 (‘materials) states that rhumab her2 is “a recom- binant humanized antibody produced in transfected cho cells” and refers (refer- ence [8]) to carter 1992 published in may 1992. although carter 1992 dis- cusses several recombinant humanized anti-her2 antibodies, humab4d5-8 is very clearly singled out as the most potent. for example, the closing sen- tence of the discussion section concludes that the preferential cytotoxicity against tumor cells “augurs well for the ongoing treatment of human cancers over- expressing p185her2 by using humab4d5-8”. the skilled person would have un- derstood the rhumab her2 antibody in harris 1995 to refer to humab4d5- 8. 67. i note that carter 1992 used hek cells, rather than the cho cells referred to by harris 1995, as the antibody expression system. the skilled person reading harris 1995 would not have attributed any great significance to the change between these well-established cell lines. the antibodies would have identi- cal amino acidic sequences and hence fold to give the same three-dimensional architecture. the structure of the paired heavy and light chain variable do- mains that form the antigen-binding surface, and thus the antigen-binding ac- tivity, will be the same. the only differences will be in the fine details of n-

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