Fc-Fusion Proteins: A Growing Class of Therapeutics - Austropa ...

Design of CD4IgG• Assembles into ahomodimer• Retains fidelity ofcleavage site by papainin hinge• Binds C1q• Binds Protein A• Is transported acrossplacenta Chamow et al., Biochemistry 29, 9885-9891 (1990)6

Structural variety of Fc-fusion proteins2008--At least 40 Fc-fusion cytokines describedJazayeri and Carroll, Biodrugs 22, 11-26 (2008)7

34 Therapeutic monoclonal antibody/Fc-fusion protein approvals—USATechnologyYearApproval2011 Benlysta Yervoy2010 Prolia/XgevaActemra2009 Arzerra StelaraIlaris Simponi2008 Nplate Arcalyst2007 Soliris2006 Vectibix Lucentis*2005 Orencia2004 ErbituxAvastinTysabri2003XolairBexxar**RaptivaAmeviveFc- Fusion Protein2002Zevalin**HumiraHuman20012000CampathMylotarg***Humanized1998Simulect Synagis Remicade HerceptinEnbrelChimeric1997RituxanZenapaxMouse19941984ReoPro*Orthoclone OKT38*Fab or (Fab) 2 antibody fragment**Immunoconjugate

USA-approved Fc-fusion proteinsBrandnameGenericnameSponsorYearapprovedConstructTargetbindingdomain IgdomainMW (kDa)Expression systemTargetClinicalindicationEnbrel etanercept Immunex/Amgen1998 TNFR2 ϒ1 Fc 150 CHO TNF Rheumatoidarthritis;juvenileidiopathicarthritis;psoriasisAmevive alefacept Biogen-Idec2003 LFA3 ϒ1 Fc 92 CHO CD2 PsoriasisOrencia abatacept BMS 2005 CTLA4 ϒ1 Fc 92 CHO CD28(indirect)Arcalyst rilonacept Regeneron 2008 IL1-RIIL1RAcPNplate romiplostim Amgen 2008 Peptidemimetic ofTPO9Rheumatoidarthritis;juvenileidiopathicarthritisϒ1 Fc 251 CHO IL1 Cryopyrinassociatedperiodicsyndromesϒ1Fc,fusionat Cterm59 E. coli TPOR Chronicidiopathicthrombocytopenic purpura

Potential production issues with Fc-fusion proteins • Upstream– Folding and secretion– Disulfide bond formation– Glycosylation• Non-Ig domains bring addition glycosylation• Downstream– Acid lability• Use of Protein A---High pH for elution– Rea et al., BioPharm Int’l, Mar supplement (2008)• Virus inactivation– Solvent/detergent– Reduced Protein A chromatographic capacity• Ghose et al., Biotechnol. Bioeng. 96, 768-779 (2006)– Increased proteolysis– Aggregation• Hydrophobic interaction chromatography– Glycoform heterogeneity• Highly sialylated forms often desirable10

Fc-fusion proteins in development• Receptor Fc-fusion– VEGF Trap• VEGFR:Fc, binds to VEGF-A, VEGF-B and PIGF,aflibercept, Ph 3, Regeneron/Sanofi-Aventis• Economides et al., Nat. Med. 9, 47-52 (2003)– Atacicept• Receptor for BLyS and APRIL, Merck-Serono– Belatacept• CTLA4 binds to B7, Bristol Myers Squibb• Differs from abatacept by 2 amino acids• Peptide Fc-fusion (peptibody)– AMG 386• peptide inhibitor of TIE2/ANG2:Fc, I-SPY 2TRIAL adaptive design in breast cancer, Amgen• Enzyme Fc-fusion– Factor VIII-Fc• Biogen-Idec– Factor IX-Fc• Biogen-IdecSee JM Reichert, Mabs 3, 76-99 (2011)11

Presentations• Engineering a CHO cell line for enhanced production of Fc fusionproteins and blood clotting factors– Pierre-Alain Girod, CSO, Selexis SA, Geneva, Switzerland• Assessment of Regeneron’s cytokine trap technology– Kevin Bailey, VP, Regeneron, Tarrytown, NY, USA• Product quality challenges during process improvements for anFc fusion protein– Barbara Woppmann, Sr. Engineer, Biogen-Idec, Cambridge, MA, USA• Challenges in upstream and downstream processing for Fcfusion proteins– Michiel Ultee, CSO, Laureate Bioservices, Princeton, NJ, USA12

Engineering CHO Cell LineFor Enhanced Productionof mAbs and Fc:FusionProteinsPierre-Alain Girod22nd ESACT Meeting 2011 in ViennaCONFIDENTIAL

Cell Line Development Process Desirables• Productivity• Stability• Speed• RobustnessCONFIDENTIAL

Cell Line Development Process Components• Expression vector• Transfection method• Host cell line• Selection method• Media• Culture conditionsCONFIDENTIAL

How Components Impact on Yield• Expression vector è expression level• Transfection method è copy number• Host cell line è secretion, p-t modifications• Selection method è strength of expression• Media è yield, structure• Culture conditions è yield, structureGENETICSPROCESSCONFIDENTIAL

Difficult to Express Protein ClassesGPCRskinasesion channelsblood plasma proteinsvaccinesAntibodies…CONFIDENTIAL

CONFIDENTIAL10VHc250 AntibodyCombinationsPool ScreeningTiterVH01VH02VH03VH04VH05VH06VH07VH08VH09VH10VL01VL02VL03VL04VL05VL06VL07VL08VL09VL10VL11VL12VL13VL14VL15VL16VL17VL18VL19VL20VL21VL22VL23VL24VL2525VLcXSLXV01G1SSLXV02G1SSLXV03G1SSLXV04G1SSLXV05G1SSLXV06G1SSLXV07G1SSLXV08G1SSLXV09G1SSLXV10G1SSLXV11G1SSLXV12G1SSLXV13G1SSLXV14G1SSLXV15G1SSLXV16G1SSLXV17G1SSLXV18G1SSLXV19G1SSLXV20G1SSLXV21G1SSLXV22G1SSLXV23G1SSLXV24 1SSLXV25G1SSLXV01G1SSLXV02G1SSLXV03G1SSLXV04G1SSLXV05G1SSLXV06G1SSLXV07G1SSLXV08G1SSLXV09G1SSLXV10G1SSLXV11G1SSLXV12G1SSLXV13G1SSLXV14G1SSLXV15G1SSLXV16G1SSLXV17G1SSLXV18G1SSLXV19G1SSLXV20G1SSLXV21G1SSLXV22G1SSLXV23G1SSLXV24 1SSLXV25G1SSLXV01G1SSLXV02G1SSLXV03G1SSLXV04G1SSLXV05G1SSLXV06G1SSLXV07G1SSLXV08G1SSLXV09G1SSLXV10G1SSLXV11G1SSLXV12G1SSLXV13G1SSLXV14G1SSLXV15G1SSLXV16G1SSLXV17G1SSLXV18G1SSLXV19G1SSLXV20G1SSLXV21G1SSLXV22G1SSLXV23G1SSLXV24 1SSLXV25G1SSLXV01G1SSLXV02G1SSLXV03G1SSLXV04G1SSLXV05G1SSLXV06G1SSLXV07G1SSLXV08G1SSLXV09G1SSLXV10G1SSLXV11G1SSLXV12G1SSLXV13G1SSLXV14G1SSLXV15G1SSLXV16G1SSLXV17G1SSLXV18G1SSLXV19G1SSLXV20G1SSLXV21G1SSLXV22G1SSLXV23G1SSLXV24 1SSLXV25G1SSLXV01G1SSLXV02G1SSLXV03G1SSLXV04G1SSLXV05G1SSLXV06G1SSLXV07G1SSLXV08G1SSLXV09G1SSLXV10G1SSLXV11G1SSLXV12G1SSLXV13G1SSLXV14G1SSLXV15G1SSLXV16G1SSLXV17G1SSLXV18G1SSLXV19G1SSLXV20G1SSLXV21G1SSLXV22G1SSLXV23G1SSLXV24 1SSLXV25G1SSLXV01G1SSLXV02G1SSLXV03G1SSLXV04G1SSLXV05G1SSLXV06G1SSLXV07G1SSLXV08G1SSLXV09G1SSLXV10G1SSLXV11G1SSLXV12G1SSLXV13G1SSLXV14G1SSLXV15G1SSLXV16G1SSLXV17G1SSLXV18G1SSLXV19G1SSLXV20G1SSLXV21G1SSLXV22G1SSLXV23G1SSLXV24 1SSLXV25G1SSLXV01G1SSLXV02G1SSLXV03G1SSLXV04G1SSLXV05G1SSLXV06G1SSLXV07G1SSLXV08G1SSLXV09G1SSLXV10G1SSLXV11G1SSLXV12G1SSLXV13G1SSLXV14G1SSLXV15G1SSLXV16G1SSLXV17G1SSLXV18G1SSLXV19G1SSLXV20G1SSLXV21G1SSLXV22G1SSLXV23G1SSLXV24 1SSLXV25G1SSLXV01G1SSLXV02G1SSLXV03G1SSLXV04G1SSLXV05G1SSLXV06G1SSLXV07G1SSLXV08G1SSLXV09G1SSLXV10G1SSLXV11G1SSLXV12G1SSLXV13G1SSLXV14G1SSLXV15G1SSLXV16G1SSLXV17G1SSLXV18G1SSLXV19G1SSLXV20G1SSLXV21G1SSLXV22G1SSLXV23G1SSLXV24 1SSLXV25G1SSLXV01G1SSLXV02G1SSLXV03G1SSLXV04G1SSLXV05G1SSLXV06G1SSLXV07G1SSLXV08G1SSLXV09G1SSLXV10G1SSLXV11G1SSLXV12G1SSLXV13G1SSLXV14G1SSLXV15G1SSLXV16G1SSLXV17G1SSLXV18G1SSLXV19G1SSLXV20G1SSLXV21G1SSLXV22G1SSLXV23G1SSLXV24 1SSLXV25G1SSLXV01G1SSLXV02G1SSLXV03G1SSLXV04G1SSLXV05G1SSLXV06G1SSLXV07G1SSLXV08G1SSLXV09G1SSLXV10G1SSLXV11G1SSLXV12G1SSLXV13G1SSLXV14G1SSLXV15G1SSLXV16G1SSLXV17G1SSLXV18G1SSLXV19G1SSLXV20G1SSLXV21G1SSLXV22G1SSLXV23G1SSLXV24 1SSLXV25G1SMab VariantsmAbs Expression

Bottlenecks of rProtein ProductionGene copy Nb*(Hc + Lc)mRNA/gene*(Hc + Lc)IgG amount(batch culture; d3)mAb1 1.525 166 0.1mAb2 1.472 5707 22.7mAb3 7.740 35740 85.4Other1 0.493 6344 20Other2 4.21 29 7* Data normalized to GAPDH used as endogenous controlLimited integration è sequence-dependent ?Limited transcription è mRNA instability ?Limited translation è saturation of secretion pathway?CONFIDENTIAL

Approaches to Debottleneck Protein ProductionLimited integration è expression vector designLimited transcription è codon optimization, toxic motifsLimited translation è culture conditions, mediaoptimization, cell engineeringCONFIDENTIAL

Effect of Media and Culture Conditions on BCFBlood clotting factorTiter (mg/L)350300250200150100500BatchFed-BatchFed-Batch switch to 32°C at day 50 1 2 3 4 5 6 7 8 9 10 11 12Run time [days]Clone-dependent è more effort incell line selectionCONFIDENTIAL

Inefficient Processing and Secretion of mAbHigh producerLow producerChase (hrs) : 0 1 2 4 6 0 1 2 4 6IgG(HC) 2HC-LCTX-100solubleFree-HC(LC) 2Free-LCTX-100insolubleAggregated -LCCodon optimization è no increase in productivitySignal peptide è +10% using IL-SP with Low-ProducerEngineer cells to increase folding and processingCONFIDENTIAL

ER-protein folding machineries…Source: Khan, S. U. and M. Schroder (2008), Cytotechnology 57(3): 207-31.CONFIDENTIAL

Chaperones operating in the ER…Source: Khan, S. U. and M. Schroder (2008), Cytotechnology 57(3): 207-31.CONFIDENTIAL

Will use of HT chaperone systems allowus to enhance the secretion process ?Activity of Factors Tested:Vesicle traffickingRetro-translocationER calcium homeostasisGeneral metabolismPost-translational modificationsCONFIDENTIAL

Metabolically Engineered CellsLow-PHigh-P3025qP (pcd)20151050A B G H Control A G H ControlLakkaraju, A. K., C. Mary, et al. (2008). Cell 133(3): 440-51SRP14 slows elongation of the nascent chain tomaximize the efficiency of protein translocation into theER through the transloconCONFIDENTIAL

More efficient targeting to the ER by SRPHigh-P Low-PHigh-P Low-PChaperone -- A G H -- A G HTX-100solubleTX-100insolubleIgG(HC) 2HC-LCFree-HCFree-LCSpecific productivity (pcd)50403020100-- G -- GCo-expression of SRP14:+ 400% on « Difficult-to-express mAb »+ 40% on « Easy-to-express mAb »CONFIDENTIAL

Volumetric Productivity in Fed Batch CultureHigh-PLow-P3.02.52.52.0IgG titer (g/L)2.01.51.0IgG titer (g/L)1.51.00.50.500 5 10 15Run time (days)00 5 10 15Run time (days)1.2E+071.2E+07Cell density (c/ml)1.0E+078.0E+066.0E+064.0E+062.0E+06Cell density (c/ml)1.0E+078.0E+066.0E+064.0E+062.0E+060.0E+000 5 10 15Run time (days)0.0E+000 5 10 15Run time (days)CONFIDENTIAL

Factors Impacting on Production of TNFR:FcVolume adjusted titer (g/L)2.01.81.61.41.21.00.80.60.40.205 3 2 1+30% Qpin total cellpool0 5.00E+07 1.00E+08 1.50E+08IVCFive chaperones show an increase in productivity(1 in general metabolism, 2 in trafficking, 2 in p-t modifications)ABCDEFGHIJKLMNOCtrlCONFIDENTIAL

ConclusionsBottlenecks are specific to each proteinAppropriate chaperones can augment the secretionof recombinant proteinDevelopment of a SuperCHO host cell line by coexpressionof chaperones has limitationCHO genome sequence will enable geneticengineering deactivation/activation of chaperonesystemsCONFIDENTIAL

Thankyou!pierre-alain.girod@selexis .comCONFIDENTIAL

Bioprocessing Challenges ofFusion ProteinsWorkshop on Fusion ProteinsESACT Conference, 15 May 2011, ViennaMichiel E. Ultee, Ph.D., CSOLaureate Biopharmaceutical Services

Fusion Proteins are Created byMolecular Biologists, Not Nature• Not selected by eonsof evolution fordesirable propertiessuch as– Expression rate– Stable structure– Different portions ofthe molecule notinterfering with othersCoagulation Factor VIIConfidentialSlide 2

First Example: Fc-Fusion + mFactor VIIfrom Iconic Therapeutics• Mutated Factor VII attached tohuman IgG 1 Fc• Designed such that the FactorVII would bind tissue factor(TF) and the Fc would provideeffector function• Made in BHK cell line forefficient post-translationalmodification (γ-CarboxyglutamicAcid)mFVIIhingeIgG 1 FcConfidentialSlide 3

Second Example: 3-Part Fusion Proteinfrom Enobia Pharma• A proprietary, bioengineeredcombination of human IgG 1 Fc,human Alkaline Phosphatase,and a bone-targeting acidicpeptide (D 10 or Asp 10 )• Native structure is a dimer of anIgG-like structure.• Alkaline Phosphatase inactivatesbelow pH 5• The D10 peptide produces astrongly negatively chargedregionRef: DW Rea, ME Ultee, SX Chen, TL Loisel,Biopharm Intl, Mar 2008 Supp, p20-25Confidential Slide 4

Temper Expectations on Expression Rates• Examples of Fc Fusion Proteins vs. IgG atLaureate:Fusion Partner Cell Line Titers in mg/LMembrane Protein CHO 200-550Enzyme 1 + Asp10 CHO 250-300Enzyme 2 + Asp10 CHO 125-150Mutated Factor VII BHK 6-8N/A : IgG Mab CHO 1000-3000ConfidentialSlide 5

Increasing Titers of Fusion Proteins• Same as for OtherRecombinant Proteins• Basal media and feedingstrategies• Use of growth factorssuch as LR3-IGF-1 &Hydrolysates• Bioreactor parameters –CO 2 levels, pH, agitatorspeeds, sparging and airoverlayrates, temperature• Seeding DensityConfidential Slide 6Titer (mg/L)6005004003002001000Slide 6Evaluation of Initial Seeding Density- Titer518488450477SF1 SF9 SF10 SF11

Fusion Proteins Tend to AggregateCompared to IgG Antibodies• Sometimes a fusionprotein is aggregated 10-30% in harvest media• Standard low-pHconditions used to elutean antibody off of aProtein A capture caninduce majoraggregation of a fusionproteinSEC-HPLC from Protein A Elutionof mFVII-Fc ProteinConfidentialSlide 7

Reducing Aggregation from Protein-AChromatography• Mid-pH Wash: pH 4.7-5.5– Helps reduce Host-CellProteins that may coaggregatewith Fc-Protein• Immediate neutralization ofeluate:– Necessitates alternative viralinactivation step such assolvent-detergent• Elution: Use highest pHthat will still effect elutionUse of pH 4.9 Wash to Reduce HCP on mFVII-FcIC1 013108 Column5 Continuous001:10_UV1_280nm IC1 013108 Column5 Continuous001:10_Cond IC1 013108 Column5 Continuous001:10_pHIC1 013108 Column5 Continuous001:10_Fractions IC1 013108 Column5 Continuous001:10_InjectIC1 013108 Column5 Continuous001:10_LogbookmAU15010050InjectionpH 4.9 WashpH 3.7 ElutionWa Elutishin nggwithwith 1.01M MArgi Arginine nine,,Stri0.2 0.2ppinMMgCitr Citrwithate, ate,0.1pH pHM4.9 4.0Citrate,WapHshin2.5gwithPBS0X1 X2 X3 X4 A1 A2 A4 A6 A8 A11 B11 B9 B7 B5 B3 B1 C20 50 100 150 200 250 mlConfidentialSlide 8

Other Strategies for Protein A Elution• Different acidic buffers:Acetate, Glycine,Citrate, Succinate• Anti-aggregatingExcipients– Polysorbate-80 (Tween-80) at 0.02-0.20%)– Arginine (0.1-1.0M)• Use Alkaline pHpH 11 Elution Conditions Found Effective forAP-Fc-D10 Fusion ProteinRef: DW Rea, ME Ultee, SX Chen, TL Loisel,Biopharm Intl, Mar 2008 Supp, p20-25ConfidentialSlide 9

Removal of Aggregates• Ceramic Hydroxylapaptite (CHT) often effective.CHT of Protein A Eluate MP-FcSEC-HPLC of Monomeric FractionMonomerAggregateDimer10MNaPhos+0.25MNaCl pH7.2ElutionMainPeakTail 1Tail 350mMNaPhosphateStrip0.1MNaOHF4 F64000 5000 6000Confidential Slide 10

Removal of Aggregates -- SEC• When bind-elute techniques are not effective, Size-Exclusion Chromatography can bemAUIC1SPDXPRODUCT001:10_UV1_280nm IC1SPDXPRODUCT002:10_UV1_280nm IC1SPDXPRODUCT003:10_UV1_280nmPURIC1 080508 Superdex200 Product001:10_UV1_280nm IC1 200L Superdex PRODUCT001:10_UV1_280nm400Black 200L Pilot #1Orange 200L Pilot #2Blue 200L cGMP #1Red 200L cGMP #2Green 200L cGMP #3Superdex 200pgChromatography of FVII-Fc300DimerMonomer200Aggregate1000Confidential0 50 100 150 minSlide 11

Typical IgG Separation Techniques MaybePrevented by the Fc Fusion Partner• Ex: Anion-Exchange– Used in F/T Mode for IgG’s to remove HCP & DNA– AP-Fc-D 10 Fusion protein bound irreversibly to AEXdue to D 10 region• Ex: Hydroxylapatite (CHT)– Used to remove aggregates and HCP– AP-Fc-D 10 Fusion protein bound to CHT but recoverywas only 40%ConfidentialSlide 12

Even Nanofiltration Can be Problematic• Standard process formost IgG’s and otherrecombinant proteinsto instill a robust viralremoval step• mFVII-Fc fusionprotein showed poorrecovery or slow flowrate on some filters%ConfidentialSlide 13

Conclusions• Fc-Fusion proteins are hybrid, multi-functional,“designer proteins” intended to combine thefeatures or two or more proteins• In spite of having an IgG-Fc portion, they aregenerally not like IgG’s, from as seen by either theproduction cells or the biopharmaceuticalscientist!• Fc-Fusion proteins can be “tamed” by acombination of persistence, innovation, and goodluck.ConfidentialSlide 14