Konstantinov, Konstantin

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Engineering Conferences InternationalECI Digital ArchivesIntegrated Continuous Biomanufacturing III Proceedings9-20-2017Development and large scale manufacturing ofexosome-based therapeuticsKonstantin KonstantinovCodiak Biosciences, USAFollow this and additional works at: http://dc.engconfintl.org/biomanufact_iiiPart of the Engineering CommonsThis Abstract and Presentation is brought to you for free and open access by the Proceedings at ECI Digital Archives. It has been accepted for inclusionin Integrated Continuous Biomanufacturing III by an authorized administrator of ECI Digital Archives. For more information, please contactfranco@bepress.com.Recommended CitationKonstantin Konstantinov, "Development and large scale manufacturing of exosome-based therapeutics" in "Integrated ContinuousBiomanufacturing III", Suzanne Farid, University College London, United Kingdom Chetan Goudar, Amgen, USA Paula Alves, IBET,Portugal Veena Warikoo, Axcella Health, Inc., USA Eds, ECI Symposium Series, (2017). http://dc.engconfintl.org/biomanufact_iii/55Development and Large Scale Manufacturing of Exosome-Based TherapeuticsKonstantin Konstantinov, Ph.D.SVP, Manufacturing & Process SciencesIntegrated Continuous Biomanufacturing III ConferenceHotel Cascais Miragem, Cascais, Portugal, Sep 17-21, 2017 2Cascais RioBeautiful Cascais32002:  B&B – USP/DSP integration using annular chrom (Bayer)1996:  B&B – large scale continuous NS0 process for Mab (Bayer)1989:  B&B - SCP continuous process optimization & control (Osaka U)1970s:  SCP continuous process using methanol (ICI)2004:  ACS industrial award to Bayer for perfusion technology devBrief history of my involvement with continuous bioprocessing2012: B&B – Integrated continuous biomanufacturing (Genzyme)2014: Progress with continuous Mab production (Genzyme)2015: Capacity of a fully integrated continuous system (Genzyme)4W. Ross Ashby (1956): An Introduction to CyberneticsThe principle of requisite varietyIf a system is to be stable, the number of states of its control mechanism must be greater than or equal to the number of states in the system being controlled.“ …. only variety can destroy variety "An Expert Approach for Control of Fermentation Processes as Variable Structure Plants.  J. Ferment. Bioeng., 70, 48-57 (1990)Exosomes:  A new modality with a tremendous therapeutic potential5Biotherapeutic Modalities and Technologies for Their Manufacturing:  Importance of Multi-Modality Production PlatformsModality #1Modality #2Modality #MTechnology#1Technology #2Technology #TT = M. . . Modality #1Modality #2Modality #MTechnology #TT = 1. . .Modality #1Modality #2Modality #MTechnology#1Technology #TT << M. . .Preferred approach:Multi-modality tech platformsContinuous bioprocessing belongs here 6Exosome Science and Bioprocess Engineering have evolved independentlyBioprocess Engineering• Rich experience with MAbs & rProteins• Upstream/Downstream/Fill-finish• Cell Culture Engineering• Protein Recovery• Analytics & Characterization• Mature, yet fast advancing• Industry dominatedExosome Science• New field• Rapidly advancing, but not yet mature• Driven by academia (2016 ISEV:  95% academic participants)• Few companies, mostly startups• No large scale applications• Tremendous therapeutic potentialCodiak• Bridge the two fields• Integrate the best science & process technology  • Hire people from both fields7Exosomes: A New Biotherapeutic ModalityIntroduction to Exosome BiologyApproach to Exosome ManufacturingRecent Results and Path ForwardBiologicalResearchCMCKnowledge8Nikki Ross, Codiak BioSciences exosome isolation dataExosomes:  Next Generation BiologicalsEM image: Purified exosomes from human cells9Exosome BiologySubclass of Extracellular VesiclesProduced by all living cellsNative information carrier between tissuesProtected from the immune system10Mechanisms for Exosome Uptake by Cells11Exosome Terminology Can Be Confusing• Many “-somes” in the literature- Exosomes, endosomes, ectosomes, oncosomes, dectosomes, connectosomes, gohstosomes, vexosomes, FedExosomes, …• No standard definition translatable into characterization methods• Typical exosome attributes- Size (40~200 nm)- Canonical surface proteins (CD9, CD47, CD63, CD81, etc.)- Relevant functional assays (uptake, biodistribution, etc.)- However, above attributes overlap with other Extracellular Vesicles12Explosion in Exosome Knowledge02004006008001,0001,2001,4001,6001,8002,000Peer Reviewed Exosome Publications 1983 – 2016Since 1983, ~70%Published in Last 5 Years 2007  Citations: >4500Professor Jan Lotvall,Chief Scientist, Codiak13Rapidly Growing Number of Exosome CompaniesExosomes: A New Biotherapeutic ModalityIntroduction to Exosome BiologyApproach to Exosome ManufacturingBiologicalResearchCMCKnowledge14Recent Results and Path Forward15Challenges in Exosome Manufacturing• New scientific field, so far driven mostly by academia.  Low level of maturity and standardization• Limited analytics & characterization• Insufficient sample purity complicating data interpretation• High complexity• Non-scalable exosome production process16Nanoparticle Tracking Analysis (NTA) is used routinely for exosome titer & size characterizationHowever, NTA does not differentiate exosomes from non-exosome particles F1:Mean 162.5nmMode 112.1nm2.6x1011 P/ml F2:Mean 149.1nmMode 122.2nm3.0x1011 P/ml F3:Mean 173.3nmMode 109.4nm3.0x1011 P/ml F4:Mean 159.1nmMode 154.9nm1.2x1011 P/ml F1F2F3F417A Typical Current (Academic) ProcessGrow adherent cells (typically primary MSCs) in serumWash and remove old media, grow cells without serum2000 g spin, 30 minutes, 4C to remove cells (discard pellet)12000 g spin, 45 minutes, 4C to remove cell debris (discard pellet)Dilute supernatant in sucrose cushion110,000 g spin, 120 minutes, 4C (discard supernatant)Resuspend pellet and wash in PBS110,000 g spin, 70 minutes, 4C (discard supernatant)Perform particle count, freeze in PBS or alternate solutionResuspend pellet and wash in PBS18Nucleic Acids – DNA, RNASmall MoleculesAntibodyCRISPRLipidsThe Codiak Exosome PlatformSURFACE ENGINEERING LOADING SPECIFIC PAYLOADSLOCAL OR SYSTEMIC DELIVERY COMBINATORIAL POTENTIALAgonistUptakeImmune ToleranceAntagonistTropismCD47mAbreceptorligand19Plug-and-Play Manufacturing Technology:Integration of Codiak’s Biological and Production PlatformsStatic Component (Production Process)Bioreactor Purification Loading FormulationThree Variable Components (Cell Lines, Payloads, Delivery Routes)Advanced Upstream, Downstream, Analytics | Scalable | Low COGSEngineered Cell Lines/ExosomesPayloadsDelivery routesExosomes: A New Biotherapeutic ModalityIntroduction to Exosome BiologyApproach to Exosome ManufacturingBiologicalResearchCMCKnowledge20Recent Results and Path Forward21Targeting KRAS with siRNA-Loaded ExosomesPBS Control Exo siKras G12D iExo0 25 50 75 100 125 150 175 2001X1078X1066X1064X1062X106Tumor Radiance (p/sec/cm2/sr)Start Treatment0Sacrifice200 DaysLOADED EXOSOMESPBSUNLOADED EXOSOMESExosomes facilitate therapeutic targeting of oncogenic KRAS in pancreatic cancerSushrut Kamerkar, Valerie S. LeBleu, Hikaru Sugimoto, Sujuan Yang, Carolina F. Ruivo, Sonia A., Melo, J. Jack Lee & Raghu KalluriNature (2017)    doi:10.1038/nature22341, Received 20 September 2016   Accepted 03 April 2017   Published online  07 June 2017Intra-venous Intra-peritonealDistribution of Exosomes in Healthy Tissues403020100g/mLLiver Spleen Pancreas Colon LungL iv e r S p le e n P a n c r e a s C o lo n L u n g01 02 03 04 0g/mLMouse NHP Mouse NHP3020100g/mLTestesLymphaticsPancreas Colon KidneyP a n c r e a s L y m p h a t i c s T e s t e s C o l o n K i d n e y3 04 0g/mLPancreas, 24 h22timeValue / Intensity / Impact+- Early enthusiasmReality hits Building the fundamentalsSteady growthIntroduction of Advanced New TechnologiesInternet, monoclonal antibody, electric cars, gene therapy, etc. Where is exosome technology and Codiak?InternetMoAbElectric carsGene therapyExosomes24ICBMoving forward …Continuous biomanufacturingExosomes+Stay tuned!25To the awesome ICB community -academia & industry:  Thank you !ICB 3 organizers• Chetan Goudar• Suzy Farid• Paula Alves• Veena WarikooECI & ICB colleagues & friends• Manuel Carrondo• Wei-Show Hu• Jamie Piret• Mike Betenbaugh• Barry Buckland• Barbara Hickernell• Tressa D’OttavioMy Bayer days• Dr. Hans Henzler• Chetan Goudar• Jim Michaels• Chun Zhang• John Thrift• John Murphy• David NavehMy Genzyme days• Veena Warikoo• Rahul Godawat• Chris Hwang• Claudia Buser• Weichang Zhou• Jason Walther• Jin Yin• Marcella Yu• Jean McLartyCodiak• Scott Estes• Kathryn Golden• Agata Villager• Damian Houde• Mike Mercaldiand many, many others …Academia:  Prof. Cooney (MIT),  Prof. Yoshida (Osaka U)

Development and large scale manufacturing of exosome-based therapeutics

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Development and large scale manufacturing of exosome-based therapeutics

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