Development and large scale manufacturing of exosome-based

Engineering Conferences InternationalECI Digital Archives

Integrated Continuous Biomanufacturing III Proceedings

9-20-2017

Development and large scale manufacturing ofexosome-based therapeuticsKonstantin KonstantinovCodiak Biosciences, USA

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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

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

2

Cascais Rio

Beautiful Cascais

3

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)

4

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

5

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

6

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

7

Exosomes: A New Biotherapeutic Modality

Introduction to Exosome Biology

Approach to Exosome Manufacturing

Recent Results and Path Forward

Biological

Research

CMC

Knowledge

7

8Nikki Ross, Codiak BioSciences exosome isolation data

Exosomes: Next Generation Biologicals

EM image: Purified exosomes from human cells

9

Exosome Biology

Subclass of Extracellular Vesicles

Produced by all living cells

Native information carrier between tissues

Protected from the immune system

9

10

Mechanisms for Exosome Uptake by Cells

1111

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

12

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

13

Rapidly Growing Number of Exosome Companies

Exosomes: A New Biotherapeutic Modality

Introduction to Exosome Biology

Approach to Exosome Manufacturing

Biological

Research

CMC

Knowledge

14

Recent Results and Path Forward

15

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

16

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

17

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

18

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

18

19

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

19

Engineered

Cell Lines/ExosomesPayloads

Delivery

routes

Exosomes: A New Biotherapeutic Modality

Introduction to Exosome Biology

Approach to Exosome Manufacturing

Biological

Research

CMC

Knowledge

20

Recent Results and Path Forward

21

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

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

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

Moving forward …

Continuous biomanufacturing

Exosomes+

Stay tuned!

25

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)