educational web seminar “scaling up cell culture:...

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Educational Web Seminar

“Scaling up Cell Culture: Application of Closed Cell Culture Systems in Clinical Research”

Thursday, May 8, 201411:00 AM 11:00 AM -- 12:15 PM ET12:15 PM ET

Adrian Gee, MI Biol, PhDProfessor, Departments of Medicine and Pediatrics

Center for Cell and Gene TherapyBaylor College of Medicine

Juan Vera, MDProfessor, Department of MedicineCenter for Cell and Gene Therapy

Baylor College of MedicineBaylor College of Medicine

Darin Sumstad, CLS (ASCP)Lead Medical Technologist

Fairview Clinical Cell Therapy LaboratoryUniversity of Minnesota

Today’s web seminar presentation slides are available publicly at www.pactgroup.net

CE Credit and certificates of attendance provided upon request

The Accreditation Council for Continuing Medical Education (ACCME) is the governing body thataccredits AABB to provide continuing medical education credits for physicians. In accordance with theACCME Standards for Commercial Support, AABB implemented mechanisms, prior to the planning andimplementation of this CME/CEU activity, to identify and resolve conflicts of interest for all individualsin a position to control content of this CME/CEU activity.

Faculty Disclosure Nature of Relationship Manufacturer/ProviderAdrian Gee, MI Biol, PhD None Speaker N/A

Juan Vera, MD Yes Scientific Advisor Wilson Wolf ManufacturingJuan Vera, MD Yes g

Darin Sumstad, CLS (ASCP) None Speaker N/A

Debbie Wood None Planning Committee PACT Staff N/A

David Styers None Planning Committee PACT Staff N/A

Karin Quinnan None Planning Committee PACT Staff N/A

Laarni Ibenana None Planning Committee PACT Staff N/A

Holly Baughman None Planning Committee PACT Staff N/A

Sharon Moffett None AABB Staff N/A

Jared Case None AABB Staff N/A

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In this web seminar, speakers will discuss best practices for producing sufficient numbers of cells required for clinical use. Significant advantages of closed cell culture systems over the commonly used traditional open systems will be highlightedcommonly used traditional open systems will be highlighted. Speakers will share their clinical research results generated

through the application of these culture techniques.

Inclusion of companies in this web seminar does not indicate endorsement by either the speakers or PACT nor is it meant to implyspeakers or PACT, nor is it meant to imply that their products or services are superior

to those of other companies.

Mesenchymal Stromal CellsLarge-scale Culture

Adrian GeeCenter for Cell & Gene Therapy

Baylor College of MedicineHouston, Texas

Hanley et al: Efficient manufacturing of therapeutic mesenchymal stromal cells with the use of the Quantum Cell Expansion System

Cytotherapy in Press

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Human Stem Cell

Adult Stem Cell

Pancreatic

Adult Somatic Stem Cells

Gut

Eye

Mesenchymal

Muscle

Tendon

LigamentMarrow Stroma

Pancreas Islets

RetinaLimbal Stem cells

Neuronal

Epidermal

Hematopoietic

Hepatic

Cartilage

Fat

FatCord Blood

MarrowApheresis

Cord Blood

Bone Marrow

Peripheral Blood

Clinical Trials using MSCwww.ClinicalTrials.gov: 352 studies on Mesenchymal stem cells

• Osteoarthritis• Severe Brain Injury (Adipose)• Ischemic Stroke (BM)

• Ischemic cardiomyopathy (BM)

• Multiple sclerosis (Adipose, BM, Cord blood)

• Systemic sclerosis• Liver cirrhosis• Lateral epicondylitis (Adipose)

• Crohn’s disease (BM, Adipose)

• Pulmonary fibrosis (BM)

• Parkinson’s disease (BM)

• Acute respiratory distress (Adipose)• Myocardial infarction• Liver failure• Cleft lip and palate• Cartilage defects (BM)Lateral epicondylitis (Adipose)

• Hereditary ataxia• Rheumatoid arthritis (Cord Blood)• Osteoarthritis (BM)

• Ulcerative colitis (Cord blood)• Type 1 diabetes (Cord Blood)• Type 2 diabetes• Spinal Cord Injury (BM)

• Critical limb ischemia in diabetes (Adipose)• Tibial & Femoral fractures• Cerebral artery infarcts (BM)

Cartilage defects (BM)

• Retinitis pigmentosa (BM)

• Degenerative disc disease• Cerebellar ataxia (Adipose)• Ulcerative colitis (Adipose)• Lupus nephritis• Mental retardation• Muscular dystrophy (Cord Blood)• Amyotrophic lateral sclerosis • Chronic wound healing• Emphysema

Other uses for MSCs

Adult Somatic Stem Cells

Muscle

Tendon

Ligament

Cartilage

Fat

“Other”

Mesenchymal

P ti f G HD i h t l i l

Immunosuppressive Effect

Combat GvHDCondition Recipients for Transplant

• Prevention of GvHD in hematological malignancies

• Prevention of subclinical rejection in organ transplant

• Treatment of chronic GvHD

• Promotion of engraftment in unrelated BMT

• Treatment of steroid refractory GvHD

• Poor graft function

• Co‐infusion with cord blood stem cells

• Induction of renal transplant tolerance

• Co‐infusion in mismatched mini‐transplants

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Aim

• Generation of allogeneic MSC for treatment of stroke

• FDA wanted us to use a method in clinical trials – Ed Horwitz, CHOP,

• Used platelet lysate instead of serum

• Wanted to try to close up system

• Looked at methods that would do that

Traditional Culture Method

• Automated bioreactor

• Closed system

• Whole marrow as starting material

Terumo Quantum Bioreactor

material

• Cells must meet all criteria for MSC

• Cleared for IND manufacturing

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Terumo Quantum Bioreactor

• Stand‐alone device• Automated , hollow‐fiber bioreactor system

Hanley et al: Efficient manufacturing of therapeutic mesenchymal stromal cells with the use of the Quantum Cell Expansion System Cytotherapy in Press

Quantum Manufacturing Procedure

Wash out non‐

adherent cells @ 24‐

48hr

10‐14 days 7‐10 days

Flow rate starts @ 0.1ml/min.

Monitor lactate & glucose If >lactate 4mM then double feeding rate, until reaches 0.4ml/min and lactate is at

4mM

Flow rate starts @ 0.1ml/min.

Monitor lactate & glucose If >lactate 4.5mM then

double feeding rate, until reaches 1.6ml/min and

lactate is at 5mM


0200400600800

10001200

0 10 20 30 40

MSC Line A

0200400600800

10001200

0 10 20 30 40

MSC Line B

Quantum

Flasks

Cells x 106

Expansion of MSCs in the Quantum and Flasks

0 10 20 30 40

‐300

200

700

1200

0 10 20 30 40

MSC Line C

0 10 20 30 40

0

500

1000

1500

0 10 20 30 40

MSC Line D

DaysDays

Cells x 106


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Cell Doubling Times

FlasksQuantum

MSC A MSC B MSC C MSC D Mean


MSC Phenotype & Viability

Flasks (n=3) Quantum (n=4)


CFU‐F Recovery


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MSC Differentiation Potential

Cells tested for ability to differentiate into:‐

• Adipogenic cells

O t i ll

From: Research Center for Molecular Medicine, Debrecen, Hungaryhttp://rcmm.dote.hu/research‐groups/oxidative‐sress‐and‐adp‐ribosylation/

• Osteogenic cells

• Chondrogenic cells

All three cell types seen

T cells suppression by MSC

Proliferation 100

75

50

FlasksQuantum

1:0 1:1 1:05 1:0.1 1:0.05 Unstim

% CD4 T Cell 50

25

0


T‐175 Flask BioreactorPer Flask/Expansion Set

Seeding Cells 2 3

Exchanging Media 2 1

Cell Dissociation 6 3

T l 10 7

Open events using Flasks versus the Bioreactor

Total 10 7

Per Donor (340 Flasks)

340 Flasks 3,400 7

Number of Donors / Expansion Sets

16 Donors* 19 Expansion Sets

(1 donor)

Total 54,400 133


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Considerations

• Growth assessment is indirect – lactate production

• Disposable must be pre‐coated with fibronectin

• Can be accomplished in 4 hours

• Cost of disposablep

• Offset by cost of labor

• Cost of bioreactor

• Offset by multiple applications (retrovirus manufacturing)

Regulatory Considerations

• Cleared by FDA for use in stroke Phase 1 trial

• Selling point – closed system

• Comparability of cells from flasks and bi tbioreactor

• Issues regarding allogeneic “cell bank”

• Number of patients to be treated

• Degree of testing required

Collaborators

• Patrick Hanley – Children’s National Medical Center, Washington D.C.

• Zhuyong Mei – CAGT

• April Durett – CAGT

• Graca Cabreira‐Harrison Texas Heart InstituteGraca Cabreira Harrison – Texas Heart Institute

• Mariola Klis, Wei Li, Yali Zhou – CAGT

• Peiman Hematti & Debra Bloom – University of Wisconsin

• Sean Savitz et al. ‐ University of Texas Health Science Center

• Brent Rice – Terumo BCT

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Optimizing culture of suspension cells using the G‐Rex

• Prolonged culture period • Extensive manipulation ‐risk of contamination

Limitations of conventional suspension cell culture methods

risk of contamination• Labor intensive• Require highly trained personnel

• Excessive use of reagents

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Limited volume of media and gas exchange

Conventional Cultureware

Wilson Wolf ManufacturingGas Permeable Devices (G‐Rex)

• Gas permeable membrane allows exchange of CO2 and O2

• Supports cell growth with large volumes of media

• Reduces feeding frequency and manipulation

• No rocking or stirringVera JF et al. JIT. 2010

G‐Rex 100 (100cm2)

SA: 100 cm2 Vol: 500 ml

SA: 100 cm2 Vol: 2000 ml

G‐Rex 100

G‐Rex 100L

SA: 10 cm2 Vol: 40 ml

G‐Rex 10

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G‐Rex vs Conventional cultureware

12

15

sion

0

3

6

9

G‐Rex Plate

Fold expan

Questions poll #1 Have you heard about the G‐Rex?

Have you used the G‐Rex?

•Yes •No

•Yes•No

What cells have you expanded in the G‐Rex •T cells•Regulatory T cells•NK cells•Cell lines•Murine cells

Questions poll #2

What applications do you use your cells for?

•Preclinical/Basic research •Clinical•Both

How many cells do you require for your application?

•5x10e8 •5x10e8 to 1x10e10•1x10e10

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1st – What is the optimal seeding density?

2nd – What is the optimal volume of media to use?

3rd – How can cell expansion be monitored?

1st – What is the optimal seeding density?

2nd – What is the optimal volume of media to use


Low seeding density results in greater fold expansion

75

95

115

on

‐5

15

35

55

1.00E+06 5.00E+05 2.50E+05 1.25E+05 6.50E+04

Cells/cm2 (x 106)

Fold Expansio

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Low cell density = Higher cell expansion

rs

Max cell density 10E+07 cells/cm2

or t

Optimal seeding density

Time

Cell numbe

Op

timal

tim

e fo

cultu

re h

arve

st

1st – What is the optimal seeding density = 1.2E+05 cells/cm2

2nd – What is the optimal volume of media to use?


10ml of media/cm2 resulted in the maximum cell expansion

2 (x 106)

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12

15

Volume of media/cm2

Cells/cm

0

3

6

0.5 2 5 10 15 20

14

Addition of media at the culture onset resulted in short culture period

10

12

14

16 10mL/cm2 (2.5mL*4)

10mL/cm2 (5mL*2)

10mL/cm2

(x 106)

0

2

4

6

8

0 3 6 9 13 16 20 24

Cells/cm

2

Time in culture


2nd – What is the optimal volume of media = 10ml/cm2


Inverse correlation between cell number and glucose concentration

10

12

14

200

250

300GlucoseCells

g/dl] C

e

0

2

4

6

8

0

50

100

150

Day 0 Day 3 Day 6 Day 9

Glucose [m

g ells/cm2

Time in culture

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Glucose assessment can be used to predict cell output

er (x 10

6)

80

100

120

140 Hemocytometer

Flow

Formula

Cell numbe

Days in culture

0

20

40

60

80

0 4 8 12


2nd – What is the optimal volume of media = 10ml/cm2

3rd – How to monitor cell expansion = Glucose consumption

Are this observations reproducible?

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Day 0

•Celgene

Multi center PACT study

Combination of optimal culture conditions using the G‐Rex

•25E+06 cells•1 L of media

G‐Rex 100M

•Celgene •City of Hope •CAGT

Combination of optimal culture conditions using the G‐Rex

Day 0 Day 12

•25E+06 cells•1 L of media

G‐Rex 100M G‐Rex 100M

•1.4E+09 cells

Multi center study of optimal G‐Rex culture conditions

100

1000

10000CelGeneBaylorCity of Hope

er (x 106)

1

10

100

Day 0 Day 12

Cell numbe

Days in cultureBajgain et al. Mol Therapy – Methods and Development, 2014 In press

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The culture conditions in the G‐Rex are linear scalable

x 106)

1000

10000 G‐Rex 5

G‐Rex 100

G‐Rex 500

Cell number (x

0.1

1

10

100

Day 0 Day 10

Bajgain et al. Mol Therapy – Methods and Development, 2014 In press

What about the cell harvest?

GatheRex device

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GatheRex device

Questions poll #3

How would you like to see the G‐Rex develop?

• Closed system devices• Larger capacity• Multiwell platformp

Conclusions •G‐Rex provides a simple yet highly efficient platform for the expansion of suspension cells

Optimal G‐Rex conditions:•Seeding density: 1.25E+05 cells/cm2

•Media volume: 10mL of media/cm2

•Simple culture assessment: Glucose•Over 100 fold expansion in 10 days of culture•No feeding/manipulation required•Robust and easily scalable•Validated by multi‐center study•GatheRex/semi‐automatic harvest process

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AcknowledgementCenter for Cell and Gene Therapy

Pradip BajgainLara Brenner Roopa MucharlaUsanarat AnurtahapanNatasha LaptevaAdrian P. Gee

Wilson Wolf Manufacturing

John WilsonDan Welch

Celgene Cellular Therapeutics

Bitao LiangXi h LAdrian P. Gee

Helen HeslopAnn LeenCliona RooneyMalcolm Brenner

University of Wisconsin ‐Madison

John CentanniKyle Ripple

Xiaohua Lu

City of Hope

Christine HallDavid HsuLarry Couture

Scaling Up Cell Culture: Application of Closed Cell Culture Systems in Clinical ResearchPACT Webinar - May 8, 2014

D i S t d CLS T h i l L dDarin Sumstad, CLS-Technical Lead

MSC’s Glioblastoma UCB T-Regulatory Cells

Disclaimer This discussion will be based on the

implementation of the GE WAVE bioreactor system in our production processes. The speaker does NOT endorse the specific purchase of this orendorse the specific purchase of this or any other equipment discussed.

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Objectives Discuss WAVE platform Outline techniques utilized for WAVE

implementation into existing academic cGMP production lab

Discuss supporting equipment used to Discuss supporting equipment used to make this process easier.

Discuss “lessons learned” and solutions to production bottlenecks.

Questions?

History Why evaluate a WAVE BioReactor?

Current processing workflow will allow for expansions in excess of 20 billion cells - the ‘old fashioned’ way○ High sterility risk

E t l l b i t○ Extremely labor intense○ PI’s always want more…..

Incoming external projects already established on platform

History

vs

Provided by

Space saving, processing time, and a decrease in contamination risk make bioreactors an attractive alternative to traditional culture flasks.

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History Evaluate Current Production Processes

Traditional T-Flasks, Cell Factories, Culture Bags○ Open System

Internal processes developed to ‘close’ as much as possible (Rigging sets etc )possible (Rigging sets, etc.)

○ Equipment Standard – CO2 incubator, PVC tube sealer, Sterile

connecting device (SCD)

○ Media Requirements Low/Medium – Max required ~ 50L

Getting Started

Provided by

Getting Started

Provided by

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Getting Started New processing workflows would be

required to accommodate our evolution into large scale bioreactor processing Equipment management

Media preparation management

Fluid transfer management

Training

Getting Started

Materials Compatibility Can already stocked items be used for the

culture?○ Review supplied production procedure and/or

provide researcher with a spreadsheet template to complete.

○ Compare with available materials and re-distribute for acceptability approval

Not all materials will be available in-house

Materials CompatibilityMaterials Compatibility

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Materials CompatibilityMaterials Compatibility

Media Management Base Media Configurations

Traditionally offered in bottles – not ideal for closed system processing

Aseptically transferring from hard to soft wall vessel is challengingvessel is challenging

Media Management Base Media Configurations

Most media manufacturer’s will ‘custom fill’ into bags, but this requires a substantial initial investment of media (300 - 400L) to be economically viable○ If custom fill is an option for institution, media

preparation / storage of large volumes of media may be difficultdifficult

○ High risk – Premature trial closure – large monetary value tied up in

media – that will eventually expire! Storage – Manufacturer may not be willing to store

- Current capacity to hold on-site- Additional capital equipment investment- Institutional liability – power loss, equipment failure, etc.

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Equipment Equipment Requirements

Bioreactor○ Multiple required – coverage required for

downtime

Tube Sealer / SCD larger tubing not Tube Sealer / SCD – larger tubing not compatible with current models

Accessory equipment○ Uninterrupted power supply(UPS), real-time

equipment monitoring, CO2 supply access

EquipmentLarge volume cell harvest

• Additional equipment is required for the efficient processing of final cultured product.

• Traditional centrifuge not optimal

• CellSaver 5+, Cobe 2991, LOVO etcLOVO, etc.

• Validation required

EquipmentCobe 2991 Cell Saver LOVO

Max Process Volume Unlimited* Unlimited* 5L*

Observed NC Recoveries > 70% > 80% > 95%

Process Rate 125 ml/min** 250 ml/min 150 ml/min

Min Final Product Volume (mL) 50 70 50

*Theoretical / > 10L processed on LOVO with similar results

**3 min centrifugation time

Optimization work required - dependent on cell types / load concentration

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Equipment BioProfile 400*

pH, pO2, pCO2, Na+, K+, Gluc, Lac, NH4+,

Glu, Gln, Osm

*Information obtained from Nova Biomedical

Equipment Vi-Cell XR*

Automation of the standard trypan blue assay

% Viability

Total cell concentration

Total viable cell concentration

Mean cell size

*Information obtained from Beckman Coulter

Final Result - Optimized Procedure

Initial cell culture protocol

Optimized cell culture protocol

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Lessons Learned Historical bottleneck = generating cells

shift production bottleneck to final formulation and media management (including waste)

Immediate need for media companies to Immediate need for media companies to supply base media in bags as a catalog item (2L / 5L / etc.)

Budget for supporting equipment

Special Thanks!Dr. Dave McKennaDiane KadidloLisa VanOrsowSheryl AdamsNancy BostromNancy ColeyAnh Do

Cell Therapy Clinical Laboratory / MCT Support Staff / PACT / GE

Anh DoLeyla HassanJulie LatourLien LeStacy LinnMichelle LucioKristen ReynaMolly RicciCindy Stanaway

Past and Present Collaborators

“Scaling Up Cell Culture: “Scaling Up Cell Culture: Application of Closed Cell Culture Application of Closed Cell Culture

Systems in Clinical Research”Systems in Clinical Research”

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Speaker Contact EmailSpeaker Contact Email

Adrian [email protected]

Juan VeraJuan [email protected]

Darin [email protected]

WebWeb Seminar Presentation SlideSeminar Presentation Slidess

Today’s web seminar presentation slides and presentation slides from previous web seminars are available publicly atweb seminars are available publicly at

www.pactgroup.net www.pactgroup.net

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CE CreditCE CreditPhysiciansThis activity has been planned and implemented in accordance with the Essential Areas and Policies ofthe Accreditation Council for Continuing Medical Education (ACCME) through the joint sponsorship ofAABB and PACT. AABB is accredited by the ACCME to provide continuing medical education forphysicians (Provider number 0000381). AABB designates this educational activity for a maximum of 1hour of AMA PRA Category 1 credit™ toward the AMA Physicians Recognition Award. Each physicianshould claim those credits that he/she actually spent in the activity.

California Clinical Laboratory PersonnelAABB is an approved, accrediting agency for continuing education for California-licensed clinicallaboratory personnel. This event has been approved for a maximum of 1.0 contact hours. AABB’saccrediting agency number is 0011. California clinical laboratory personnel must provide a personalsignature and other required information on the attendance log.

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General AttendeesAdministrators, nurses (other than California-licensed nurses), clinical laboratory personnel (otherthan California- and Florida-licensed personnel), and other health-care professionals may receive acertificate of attendance.

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CE CreditCE Credit

Sign and fax roster to 240-306-2527

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Note: Please complete within 48 hrs of the web seminar

(Survey link above is embedded in the reminder email sent 05/07/14)

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