development and large scale manufacturing of exosome-based
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Engineering Conferences InternationalECI Digital Archives
Integrated Continuous Biomanufacturing III Proceedings
9-20-2017
Development and large scale manufacturing ofexosome-based therapeuticsKonstantin KonstantinovCodiak Biosciences, USA
Follow this and additional works at: http://dc.engconfintl.org/biomanufact_iii
Part of the Engineering Commons
This 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 [email protected].
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/55
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Development and Large Scale Manufacturing of Exosome-Based Therapeutics
Konstantin Konstantinov, Ph.D.
SVP, Manufacturing & Process Sciences
Integrated Continuous Biomanufacturing III ConferenceHotel Cascais Miragem, Cascais, Portugal, Sep 17-21, 2017
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Cascais Rio
Beautiful Cascais
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2002: 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 dev
Brief history of my involvement with continuous
bioprocessing
2012: B&B – Integrated continuous
biomanufacturing (Genzyme)
2014: Progress with continuous Mab
production (Genzyme)
2015: Capacity of a fully integrated
continuous system (Genzyme)
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W. Ross Ashby (1956): An Introduction to Cybernetics
The principle of requisite variety
If 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 potential
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Biotherapeutic Modalities and Technologies for Their Manufacturing: Importance of Multi-Modality Production Platforms
5
Modality
#1
Modality
#2
Modality
#M
Technology
#1
Technology
#2
Technology
#T
T = M
. . . Modality
#1
Modality
#2
Modality
#M
Technology
#T
T = 1
. . .
Modality
#1
Modality
#2
Modality
#M
Technology
#1
Technology
#T
T << M
. . .
Preferred approach:Multi-modality tech platforms
Continuous bioprocessing
belongs here
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Exosome Science and Bioprocess Engineering
have evolved independently
Bioprocess Engineering
• Rich experience with MAbs & rProteins• Upstream/Downstream/Fill-finish• Cell Culture Engineering• Protein Recovery• Analytics & Characterization• Mature, yet fast advancing• Industry dominated
Exosome 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 potential
Codiak
• Bridge the two fields• Integrate the best science &
process technology • Hire people from both fields
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Exosomes: A New Biotherapeutic Modality
Introduction to Exosome Biology
Approach to Exosome Manufacturing
Recent Results and Path Forward
Biological
Research
CMC
Knowledge
7
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8Nikki Ross, Codiak BioSciences exosome isolation data
Exosomes: Next Generation Biologicals
EM image: Purified exosomes from human cells
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Exosome Biology
Subclass of Extracellular Vesicles
Produced by all living cells
Native information carrier between tissues
Protected from the immune system
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Mechanisms for Exosome Uptake by Cells
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Exosome 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 Vesicles
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Explosion in Exosome Knowledge
0
200
400
600
800
1,000
1,200
1,400
1,600
1,800
2,000
Peer Reviewed Exosome Publications 1983 – 2016
Since 1983,
~70%
Published
in Last
5 Years
12
2007 Citations: >4500
Professor Jan Lotvall,Chief Scientist, Codiak
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Rapidly Growing Number of Exosome Companies
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Exosomes: A New Biotherapeutic Modality
Introduction to Exosome Biology
Approach to Exosome Manufacturing
Biological
Research
CMC
Knowledge
14
Recent Results and Path Forward
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Challenges 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 process
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Nanoparticle Tracking Analysis (NTA) is used routinely for exosome titer & size characterization
However, NTA does not differentiate exosomes from non-exosome particles
F1:Mean 162.5nm
Mode 112.1nm
2.6x1011 P/ml
F2:Mean 149.1nm
Mode 122.2nm
3.0x1011 P/ml
F3:Mean 173.3nm
Mode 109.4nm
3.0x1011 P/ml
F4:Mean 159.1nm
Mode 154.9nm
1.2x1011 P/ml
F1
F2
F3
F4
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A Typical Current (Academic) Process
Grow adherent cells (typically primary MSCs) in serum
Wash and remove old media, grow cells without serum
2000 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 cushion
110,000 g spin, 120 minutes, 4C (discard supernatant)
Resuspend pellet and wash in PBS
110,000 g spin, 70 minutes, 4C (discard supernatant)
Perform particle count, freeze in PBS or alternate solutionResuspend pellet and wash in PBS
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Nucleic Acids – DNA, RNA
Small Molecules
Antibody
CRISPR
Lipids
The Codiak Exosome Platform
SURFACE ENGINEERING LOADING SPECIFIC PAYLOADS
LOCAL OR SYSTEMIC DELIVERY COMBINATORIAL POTENTIAL
Agonist
Uptake
Immune Tolerance
Antagonist
Tropism
CD47mAb
receptorligand
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Plug-and-Play Manufacturing Technology:Integration of Codiak’s Biological and Production Platforms
Static Component (Production Process)
Bioreactor Purification Loading Formulation
Three Variable Components (Cell Lines, Payloads, Delivery Routes)
Advanced Upstream, Downstream, Analytics | Scalable | Low COGS
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Engineered
Cell Lines/ExosomesPayloads
Delivery
routes
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Exosomes: A New Biotherapeutic Modality
Introduction to Exosome Biology
Approach to Exosome Manufacturing
Biological
Research
CMC
Knowledge
20
Recent Results and Path Forward
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Targeting KRAS with siRNA-Loaded Exosomes
PBS Control Exo siKras G12D iExo
0 25 50 75 100 125 150 175 200
1X107
8X106
6X106
4X106
2X106
Tum
or
Ra
dia
nc
e (
p/s
ec
/cm
2/s
r)
Start
Treatment
0
21
Sacrifice
200 DaysLOADED
EXOSOMES
PBS
UNLOADED
EXOSOMES
Exosomes 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 Kalluri
Nature (2017) doi:10.1038/nature22341, Received 20 September 2016 Accepted 03 April 2017 Published online 07 June 2017
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Intra-venous Intra-peritoneal
Distribution of Exosomes in Healthy Tissues
40
30
20
10
0
g/m
L
Liver 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 g
0
1 0
2 0
3 0
4 0
g/m
L
Mouse NHP Mouse NHP
30
20
10
0
g/m
L
TestesLymphaticsPancreas 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 y
0
1 0
2 0
3 0
4 0
g/m
L
40Pancreas, 24 h
22
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time
Valu
e / I
nte
ns
ity /
Im
pact
+
- Ea
rly
en
thu
sia
sm
Re
ali
ty
hit
s Building the
fundamentalsSteady growth
Introduction of Advanced New TechnologiesInternet, monoclonal antibody, electric cars, gene therapy, etc.
Where is exosome technology and Codiak?
Internet
MoAb
Electric cars
Gene therapy
Exosomes
24
ICB
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Moving forward …
Continuous biomanufacturing
Exosomes+
Stay tuned!
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To the awesome ICB community -
academia & industry: Thank you !
ICB 3 organizers• Chetan Goudar• Suzy Farid• Paula Alves• Veena Warikoo
ECI & ICB colleagues & friends• Manuel Carrondo• Wei-Show Hu• Jamie Piret• Mike Betenbaugh• Barry Buckland• Barbara Hickernell• Tressa D’Ottavio
My Bayer days• Dr. Hans Henzler• Chetan Goudar• Jim Michaels• Chun Zhang• John Thrift• John Murphy• David Naveh
My Genzyme days• Veena Warikoo• Rahul Godawat• Chris Hwang• Claudia Buser• Weichang Zhou• Jason Walther• Jin Yin• Marcella Yu• Jean McLarty
Codiak• Scott Estes• Kathryn Golden• Agata Villager• Damian Houde• Mike Mercaldi
and many, many others …Academia: Prof. Cooney (MIT), Prof. Yoshida (Osaka U)