gmp reagents for regenerative medicine: process research ... · 7/15/2008 · • optimized mix of...

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Mohan C VemuriThermo Fisher ScientificDirector, R&DStem Cell Biology

7/9/2015

GMP Reagents for Regenerative Medicine: Process Research and Development

2

Agenda

• MSCs for clinical cell therapy• Global trends toward serum free manufacturing• Increasing yield through scale up technologies• CTS platform & advantages• Case studies

3

Isolate

Cells for Therapy

Expand

Differentiate

Deplete

SafetyQualify

Potency

Key themes• Expertise• Compliance• Productivity

iPSC

NSC

Keratinocytes

Hepatocytes

Neuronal

ESC

MSC

HSC

Fibroblasts

Endothelial

4

Human Mesenchymal Stem Cells

5

Therapeutic applications of MSCs

Human MSCs have become of interest for clinical application due to:• Immunosuppressive attributes• Capacity for homing and engraftment• Wide-range mesoderm differentiation potential

Potential MSC Therapies:• Graft versus Host Disease• Crohn’s Disease• Bone Defects/ Genetic Disease• HSC Transplantation• Cardiac repair

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Cell Therapy Clinical Trials - 2014

31%

47%

13%

4%

2%3%

Major Cell Types

MSC total (90)Immune cell total (140)BM MNC (38)Adipose SVF (12)Skin cells (7)Cord Blood MNC (9)

Bersenev Alexey. Cell therapy clinical trials – 201 report. CellTrialsblog. February 1, 2014. Available: http://celltrials.info/2014/02/01/2013-report/

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Global trends toward serum free manufacturing

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Trends towards serum free culture

• Supply of FBS is predicted to be unable to support the cell therapy industry if more than one blockbuster therapy hits the market

• The use of serum carries with it the risk of transmission of adventitious agents

• Lot to lot variability presents a challenge for manufacturing reproducibility and adds cost from a Quality and lot selection perspective

• Serum can potentially cause xenogeneic immune response in patients

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Classical Human MSC MediaBasal media with 10-20% FBS

Reduced Serum Complete MediaMesenPRO RS™ (reduced-serum + GFs)

Alternative Human MSC MediaBasal media with 10% Human Serum or

Human Platelet-Rich Plasma Lysate

(1) Serum-Free or (2) Serum-Free and Xeno-Free

The Evolution of Human MSC Medium

• Eliminate need for pre-qualification of reagents

• Decrease lot-to-lot variability and increase performance

• Eliminate the need for xenogeneic serum

• Eliminate need for pre-qualification of reagents

• Decrease lot-to-lot variability and Increase performance

• Eliminate the need for serum

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Serum-supplemented Media

• Pros:• General growth promoting properties for many cell types

• FBS: historically used with many cell types• Pooled human serum: used with primary cells (T Cells, etc.)

• Commonly used, most historical functional data generated with serum-containing formulations

• Cons:• Undefined (complex mixtures of components)• Potential for lot-to-lot variability• Risk of contamination by adventitious agents• Lot size limitation

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Why Serum-free?

• Advantages:• Defined components and reduced variability• Reduce exposure to adventitious agents

• Serum-free → xeno-free → animal origin-free (AOF)• Advantage

• Path to 510K• GMP

• Large scale lot production

• Challenges:• Cost-of-goods (COGs)• Recombinant proteins/sourcing/function• Impact on cell phenotype and function

12

Serum-free Alternatives

• Serum-free:• may contain native proteins from animal sources

• Xeno-free:• may contain native proteins from human sources only• Platelet lysate from pooled donors, functional, but undefined

• Animal origin-free (AOF):• No native proteins from animal sources (including human)• May contain recombinant proteins/growth factors/etc.

• Chemically-defined:• No proteins or undefined components (proteins, hydrolysates)

13Ng et al., Blood, 15 July 2008, Vol. 112, No. 2, pp. 295-307.

• Analysis of active pathways in human MSCs suggests that PDGF, TGFβ and FGF are important signaling pathways for hMSC proliferation and differentiation

Chondrogenic

Basal Chondro

Adipogenic

Basal Adipo

Osteogenic

Basal Osteo

Gene Expression Analysis to Facilitate Media Design

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Advanced Human MSC Media Systems

• MesenPRO RS - Reduced (2%) Serum-Containing Medium

• StemPro™ MSC SFM - Serum-Free MSC Medium

• StemPro MSC SFM XenoFree - Serum-Free/Xeno-Free Medium

A complete line of cGMP-manufactured advanced MSC media

For research use only. Not intended for human or animal therapeutic or diagnostic use.

Table 1. MSC culture product selection guide

Complete Media

Culture System

Basal Media

Supplements

Cell / Tissue Species

Cell / Tissue Source Substrates Passaging

Serum- Classical

Media

DMEM (low glucose) / alpha MEM

MSC-Qualif ied FBS

Human, mouse, sheep, pig,

canine, horse, rat

Bone Marrow Adipose

Cord Blood Umbilical Cord

Placenta

TryPLE, Trypsin,

StemPro® Accutase

Reduced-serum MesenPRO RS Human, mouse,

sheep, pig

Bone Marrow Adipose

Umbilical Cord

TryPLE, Trypsin,

StemPro® Accutase

Serum-free StemPro® MSC SFM Human Bone Marrow

Adipose Umbilical Cord

Hair Follicle

CELLstart, Fibronectin, Attachment

Factor TrypLE

Serum-free / Xeno-free StemPro® MSC SFM XenoFree Human

Bone Marrow Adipose

Umbilical Cord Cord Blood Pericytes

Fibroblasts

CELLstart, Coating Matrix,

Fibronectin TrypLE

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Adipocytes(Oil Red O)

MesenPRO RS Medium• Reduced (2%) serum with consistency and

variability• Eliminates need to pre-qualifying FBS • Utilizes product-specific human MSC quality

control assay• Maintains multipotent MSC phenotype• Gene expression profiles matches classical

medium

Osteoblasts(Alizarin Red S)

Chondrocytes(Alcian Blue)

Reduced Serum MSC Culture System


NOTE: Recommended seed density = 5x103 cells/cm2

16Chase et al., Stem Cell Research & Therapy 2010, 1:8.

A

B

a b

0

200

400

600

800

1000

1200

1400

1600

1800

SCM D+PBT D D+P D+B D+T D+PB D+PT D+BT

Growth Factor Supplementation

Rel

ativ

e Fl

oure

scen

ce U

nits

(RFU

)

C

0

5

10

15

20

25

Set-Up 6 7 8 9 10 11 12 13

Passage

Net P

opul

atio

n Do

ublin

gs

SFM SCM

P = PDGF-BB

B = bFGF

T = TGFβ1

• Optimized mix of recombinant growth factors provides an enhanced synergistic effect on human BM-MSC proliferation.

• Optimized formulation provides ability for continual propagation.

• More spindle-shaped morphology, supporting high density cell growth.

• System utilizes a humanized substrate (CELLstart™) and a recombinant trypsin alternative (TrypLE™)


Serum-Free MSC Culture: StemPro MSC SFM CTS Medium

NOTE: Recommended seed density = 1x104 cells/cm2

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StemPro MSC SFM XenoFree

Pass

age

1DMEM + 10%

MSC-Qualified FBSPa

ssag

e 9

Input cells = passage 5 human Bone Marrow MSCs (4-donor pool)

Recommended seed density = 5-7x103 cells/cm2

Adipocyte (Oil Red O)

Chondrocyte (Alcian Blue)

Osteoblast (Alkaline Phosphatase)

Passage 5 Multi-lineage Mesoderm Differentiation with StemPro Differentiation Regents

Expa

nsio

n an

d D

iffer

entia

tion

Xeno Free MSC Culture: StemPro MSC SFM XF Medium

StemProMSC SFM XenoFree: Doubling Time(Human BM-MSC)

0

20

40

60

80

100

120

1 2 3 4 5 6 7 8 9

Passage

Dou

blin

g Ti

me

(Hou

rs)

DMEM + 10% MSC-Qualified FBS StemPro MSC SFM XenoFree

StemPro MSC SFM XenoFree: Net Population Doublings(Human BM-MSC)

0

2

4

6

8

10

12

14

16

18

20

Set-up 1 2 3 4 5 6 7 8 9

Passage

Net

Pop

ulat

ion

Dou

blin

gs

DMEM + 10% MSC-Qualified FBS StemPro MSC SFM XenoFree

Multi-passage expansion of human bone marrow-derived MSCs


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Media System Catalog Number Additional Reagents

StemPro MSC SFM XF A10675 CELLstart CTS, GlutaMAX‐I CTS, TrypLE Select CTS, gentamicin (optional)

StemPro MSC SFM CTS A10332 CELLstart CTS, GlutaMAX‐I CTS, TrypLE Select CTS, gentamicin (optional)

MesenPRO RS 12746 GlutaMAX‐I CTS, TrypLE Select CTS, gentamicin (optional)

DMEM low glucose + 10% MSC‐Qualified FBS

1105412662

GlutaMAX‐I CTS, TrypLE Select CTS, gentamicin (optional)

StemPro Osteogenesis Kit A10072 gentamicin (optional)

StemPro Adipogenesis Kit A10070 gentamicin (optional)

StemPro Chondrogenesis Kit A10071 gentamicin (optional)

MSC Media System Summary

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Increasing yield through scale up technologies

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Process considerations and control

Bioprocess• Connect biology and engineering • Establish process, manufacturing, scale and

drive COGs

What are the critical considerations?• Quality of supporting materials and reagents• Process choices• Development & regulatory challenges

Biology En

Bioprocess

Scalable & robust processes should be selected with thoughts of commercialization to avoid costly change control in the future

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Lot-size Challenges

Planar Cultures

Packed Bed / Suspension

Cultures

Product Doses

Rowley et al., Bioprocess International, March 2012

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Increasing Yield to Meet Demand

Monoclonal Antibody Production• Substantial increase in yield over the last 20 years

Dos

es p

er L

ot

0

200

400

600

800

1000

1200

Control ProcessOptimized Process

Time (Days)0 2 4 6 8 10 12 14 16

Via

ble

Cel

l Den

sity

(VC

/ml)

0.0

5.0e+6

1.0e+7

1.5e+7

2.0e+7

2.5e+7

IgG

Tite

r (m

g/L)

0

500

1000

1500

2000

2500

3000

3500

Control VCDOptimized VCDControl TiterOptimized Titer

Allogeneic Cellular Therapy • Similar progression needed for CT

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Culture Media Design Strategy

Mix Empirical and Focused Methods:

• DOE

• Metabolite analysis

• Cytokine/growth factor pathway

• Characterization checkpoints

• Gene expression analysis

• High throughput studiesCheckpoint

0

1

2

3

4

5

6

7

8

9

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Fold In

crease

DOE Medium VariantMedium

Cel

l Gro

wth

Checkpoint

Checkpoint

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• StemPro MSC SFM XenoFree• Solohill™ plastic microcarriers coated with CELLStart™ CTS™•Incorporation of partial medium exchanges • Yield of 1.2×108 MSCs (~1.5-2.0×105 cells/ml)• A completely xeno-free microcarrier-based culture system was successfully implemented for the expansion of human MSC.

A dos Santos et al., Tissue Eng. Part C, 2011

Scalability

BM MSCASC

0.0E+00

5.0E+04

1.0E+05

1.5E+05

2.0E+05

2.5E+05

0 2 4 6 8 10 12 14

Cel

ls /

mL

Time (days)

(n=2)

Cel

l Den

sity

(cel

ls/m

l)

Cel

l Den

sity

(cel

ls/m

l)

100ml spinner flask 1L bioreactorA B

(n=2)

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• Fed-batch approach showed comparability to partial medium exchange

• Used metabolite and spent media data from previous bioreactor run to design a nutrient feed.

• Tested fed-batch approach based on calculated consumption rates

0.0E+00

5.0E+04

1.0E+05

1.5E+05

2.0E+05

2.5E+05

0 1 2 3 4 5 6 7 8

Cell Den

sity (cells/m

l)

Time (days)

Fed‐batch

25% 2 days

25% daily25% Exchange every other day

25% Exchange daily

Medium Optimization: Nutrient Feed Development

2014 Jan 14. doi: 10.1002/bit.25187. [Epub ahead of print]

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MSC Colony Number Greater Under Hypoxic Conditions (P2 culture)

Grown under hypoxic conditions.

Grown under normal oxygen conditions.

Greater MSC colony formation following

isolation and expansion under hypoxic

environment vs. normal oxygen

Greater MSC colony formation following

expansion under hypoxic environment vs.

normal oxygen

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CTS platform & advantages

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CTS Brand – Translational tools

FDA DMF or 510(k) ISO & GMP

manufacturing Animal origin-free or

xeno-free Certificates of Origin Certificates of Analysis Adventitious agent

testing Sterility testing Mycoplasma testing Endotoxin testing

29 | Life Technologies Proprietary & Confidential |

Cell Therapy Systems (CTS) Regulatory Considerations

• Customers in Cell Therapy space need access to tools and reagents that allow them to transition from research applications into clinical applications

• Current regulatory guidelines are not “black-and-white” and are evolving as the field emerges

• The goal of the CTS product line provide qualifying reagents as developers prepare to move their concepts into the clinic

• What CTS offers• High quality materials• Unified documentation for CTS productsCertificate of Analysis > Certificate of OriginAnimal origin designation> Adventitious agent testingProduct reserves (lot specific) > Raw material alertsDrug/Device Master File (FDA) for relevant products

• Technical, regulatory, and web-based support• Dedicated support team

• Media, Buffers, Growth Factors, Enzymes, Supplements, Devices

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Process Check for cGMP Reagents

Reagent & media labeling• 510K cleared media products, Drug Master File• Animal Origin Free, Xeno-Free, IVD• For manufacturing of cell-based products (CTS)

Quality systems under which reagents are manufactured

• (21 CFR Part 820)• ISO 13485:2003 Certification

Raw Material Sourcing• Audits• Paper trail/validated vendors• Retest

Documentation to support traceability, quality, and intended use

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Thermo Fisher Cell Therapy

Enabling rapid progressfrom cell to market

Provider of stem cell and cell engineering tools for development

Provider of workflow solutions for isolation and expansion of cells

Custom solutions and scalable manufacturing from clinical to commercial stage

Used in a broad range of cell therapy applications where consistency, quality and safety are critical

Consultative partner providing deep domain knowledge and experience Scalable and reliable solutions for

cell therapy research and production

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Immune Cells for Therapy

Four major types :CIK, T cells, Dendritic cells and Natural killer (NK) cells

• Cytokine-induced killer (CIK) cell: Cytokine-induced killer (CIK) cells are CD3-and CD56-positive, non-major histocompatibility complex (MHC)-restricted. When reintroduced back to patients after autologous stem cell transplantation, CIK cells may recognize and kill tumor cells associated with minimal residual disease (MRD). (21 clinical trials*)

• Natural killer (NK) cell: A population of activated, immortalized, interleukin-2 (IL-2)-dependent, cytotoxicnatural killer (NK) cells with potential antitumor activity. (273 clinical trials* )

• Dendritic cell (DC): Immune cells that process antigen material and present it on the surface to other cells of the immune system. They act as messengers between the innate and adaptive immunity.(403 clinical trials* )

• T Cells : T cells or T lymphocytes belong to a group of white blood cells known as lymphocytes, and play a central role in cell-mediated immunity (5427 clinical trials* )

*Clinical trial data ( www.clinicaltrials.gov) – US centric

Pre-Clinical and Cinical studies with CIK cells

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T Cell Workflow for Immunotherapy

34

CD3/CD28 activated T Cells are typically expanded in media supplemented with human serum.

In order to make a commercial-scale process possible, a GMP, regulatory-compliant, scalable substitute is needed.

Serum-free Process for T Cell Expansion

35

Normalized T cell growth (seven donors)

DOE media variant performance

Medium 2

Medium 3

Medium 4

Medium 5

Medium 6

Medium 7

Medium 8

Medium 9

Medium 10

0 20 40 60 80 100 120


36

A GMP xeno-free serum replacement supplement was developed

Control + 5% HS

Control + 10% SRS-XF

Control + 5% SRS-XF

Control + 2% SRS-XF

Control (no additions)

Growth Kinetics from One Representative Donor

T cells expanded in control medium supplemented with human serum or SRS-XF show similar growth kinetics and total fold expansion after 2 weeks in culture.

2-5% SRS-XF support expansion of CD4 and CD8 T cell subsets when expanded in OpTmizer CTS

OpT2% HS

N=4

OpT10% SRS

N=4

OpT5% SRS

N=4

OpT2.5% SRS

N=4

OpTN=4

OpT2% HS

N=4

OpT10% SRS

N=4

OpT5% SRS

N=4

OpT2.5% SRS

N=4

OpTN=4


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CTS Immune Cell SR Benefits

Security of Supply

Traceable

Saves time and cost

Immune Cell Qualified

Scalable process

Flexible

Regulatory compliant & commercial

path

Superior performance

42%

25%

5% AB serum5% SRS-XF

Intracellular IFN production

Superior Intracellular IFNproduction

For in vitro diagnostic use

Qualified suppliers Multiple GMP sites

Eliminate need to qualify serum

Similar performance with reduced level

Bead based QC Assay

COO, COA, Donor Testing, DMF

Can replace human serum or FBS and be used with different media

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T Cell Workflow for Immunotherapy

Blood

Collection

Cells Activated Cell Expansion

Bead Removal and Formulation

Infusion

AIM V CTSOpTmizer™ CTS

IL-2, IL-7 CTS,IL-4 CTS

DynaMag™CTS

DynaMagCTS

DPBS CTSDynabeads™

CD3/CD28 CTS

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CIK Clinical Immunotherapy Workflow

What’s needed for CIK cultures ?What’s needed for CIK cultures ?

• Medium

• Culture bag/culture vessel

• IL-2, IFN- γ, CD3

• Human AB serum

• CD3, CD4, CD8, CD56 for FACS

• Ficoll-Paque, centrifuge tubes (fresh PBMNC prep)

• Medium

• Culture bag/culture vessel

• IL-2, IFN- γ, CD3

• Human AB serum

• CD3, CD4, CD8, CD56 for FACS

• Ficoll-Paque, centrifuge tubes (fresh PBMNC prep)

PatientPatient

PBMNC isolationPBMNC isolation

Peripheral bloodCollection

Peripheral bloodCollection

Harvest, Rinse, Verify CIK & Infuse into Patient

Harvest, Rinse, Verify CIK & Infuse into Patient

Expansion in culture bag

Expansion in culture bag

Cells stimulated with IFN-γ, CD3 antibody

Cultured for 14-21 days

40

BioProcess Optimization for CTL Production

Vera et al ; 2010 J ImmunotherapyWilson-Wolf

41

Input-Output Control Design for DC Cultures

CD-206+ (DC)CD-86+ (DC)CD-80+ (DC)CD-14+ (Monocyte)

*In order to be a DC all three receptors must be expressed

42

Media Systems for Immune Cells

Cell type TxTime Media Vol/tx Substrate Cytokines/Abs Harvest

Cell No.Release assay

CIK 3-4 wk RPMI 1640 4L Retronectin (for Takara SOP), CD3 ab

- IL-2, IFNg- CD3, CD4, CD8 CD56,

All - FACS- Possible functional assay

NK 3-4 wk RPMI 1640 4L Feeder cells - IL-2, IL-15- CD3, CD4, CD8, CD56

All - LDH assay- FACS- Possible functional assay

DC 10 d RPMI 1640 or AIM-V

3L N/A - GM-CSF, IL-4, TNFa- CD1a, CD40, CD80, CD83, CD86, HLA-DR

2e6-8 - FACS- Possible functional assay

T cell (multiple subtypes incl Treg)

10 d to 4 wk

RPMI 1640, AIM-V or OpTmizer

3-10 L CD3 ab - IL-2- CD3, CD4, CD8, CD25, CD28(?), CD45, CD56

3e8-10 - FACS- Possible functional assay

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Summary: T Cells/CIK

• A GMP compatible serum free and xenofree culture media and a bioprocess optimization for human T Cells was developed efficiently to meet autologus therapy demands of T cells.

• CIK cells can be cultured efficiently for adoptive immunotherapy needs and holds a greater promise for future.

• Media and process development for scalable approaches can take a great idea and transform it into a cost effective and efficient therapy.

44

State-of-the-art Manufacturing Facilities

Auckland and Christchurch, New Zealand Newcastle, Australia

• Sera• Protein products• GMP 21 CFR 820

Cramlington, UK• Single-Use technologies• Class 10,000/ISO 7

clean room

Logan, Utah• Single-Use technologies• Class 10,000/ISO 7

clean room

Grand Island, NY• Cell culture media, reagents• Sera • ISO 13485• GMP 21 CFR 820

Bedford, MA• Chromatography resins• ISO 13485

Naarden, The Netherlands

• Affinity ligands

Paisley, Scotland• Cell culture media, reagents• ISO 13485• GMP 21 CFR 820

Warrington, UK• Analytics kits

Rochester and Fairport• ISO 9001:1994 • FDA Registered Facility

Miami, OK• ISO 9001:2008

Newport, UK• ISO 9001:2008

Roskilde, Denmark• ISO 13485:2003• ISO 9001:2008• FDA Registered Facility

Suzhou, China• ISO 13485• ISO 9001• ISO 14001

Oslo & Lillestrøm, Norway• Dynabeads

45

Acknowledgments

Instituto Superior TecnicoCláudia Lobato da Silva Francisco dos SantosPedro Z. AndradeManuel M. AbecasisJoaquim M.S. Cabral

Andy CampbellYuan WenShayne BoucherSandy KuligowskiEric RoosAngel Rohena-VarelaMelanie AndolinaKaroline SchjetneBrian NewsomMohan VemuriMark PowersTanja AarvakStephen GorfienScott JacobiaGrethe Økern

© Thermo Fisher Scientific Inc. All rights reserved. Solohill is a registered trademark of Solohill Engineering, Inc.

gmp reagents for regenerative medicine: process research ... · 7/15/2008 · • optimized mix of...

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