review of single use technologies in biomanu in bioprocessing. ... wave biotech rocking ......

Review of Single Use Manufacturing TechnologiesCopyright ©2007 Xcellerex

Review of Single Use Technologies inBioManufacturing

Parrish M. Galliher

Breakthroughs in Bioprocessing


Outline

• Introduction – Strategic Outlook forBioPharma

• Advantages and Limitations of Single UseSystems

• Integration/Scale Up Challenges ofDisposables

• Single Use Bioreactor Data

• Conclusions


BioPharma Strategy - New ParadigmIndustry Growth

• 15% annual avg.

• >15 approved mAbs

• >150 mAbs in clinic

• expanding pipelines

Smaller Markets

• Fewer blockbusters

• Personalized medicine

• Genetic screening

New Technology

• Better process yields

• Potent compounds

• Drug delivery

Cost Pressures

• Health care reform

• Pricing controls

• Biosimilar drugs

Capacitydemands

Smaller R&Dbudgets

Smaller batchsizes

.

Need for more efficient PD & Mfg.fast, flexible and inexpensive manufacturing capacity


Xcellerex’s Technology will be Commercial ScaleFuture Quantity Per Drug Demand for Biologics is decreasing

Time

Mark

et

Dem

an

dper

Dru

g

Niche markets

Improved potency

Improving yields

Traditional Platform Scale – 2,000L to 20,000L

Xcellerex XDR Platform Scale – 200L to 2,000L


Advantages of Single Use SystemsReductions in:

– Cleaning

– Sterilization

– Engineering cost

– Equipment lead time

– Utility requirements

– Validation

– Quality / Regulatory burden

– Space

– Labor

– Waste generation

Improvements in:

– Manufacturing quality

– Capital investment

– Facility buildout time

– Cycle time

– Flexibility

– Environmental impact

– COGS


Limitation of Single Use Systems

• Mixing/Buffer/Media prep – Rate of liquid transfers

• Bioreaction – Lab to commercial scalability within thesame reactor design/configuration

• Cell Harvest/TFF - Recirculating processes that requirehigh pressure and high flow rates

• Disposable Chromatography is still TBD

• Non-standard, multiple connection options

• Disposable sensors are limited


Technology Survey

Products and vendors listed in the followingslides are provided for reference and do notconstitute a complete list or an endorsementof any specific vendor or product


• Bioprocess bags• Cell culture bioreactors• Separations (Cfg., TFF, rotary drum, filters)

– Harvest– Virus removal / sterilization– Concentration / buffer exchange

• Purification – membranes• Tubing welders / connectors / sealers• Integrating stainless and disposables connectors• Sensors

Enabling Single Use Technologies


Applications: Media, buffer, product processing,formulation

Capacity: 10 L to 10,000 L

Vendors/Types: Hyclone MixTainer, LevTech/Sartoriuslevitated prop tank, Wave FlexMixer, Xcellerex XDMstirred tank

Integration Challenges: powder addition, connectors

Scale Up Challenges: powerful mixing, bags that flex toachieve mixing rely heavily on bag seam strength anddurability

Mixing Systems


XDM-100 and 200 Disposable Tank Mixer


Invitrogen/Xcellerex XDM Commercial Disposable

Mixers for powdered media mixing and delivery

Powdered media/buffer mixing

XDM-200XDM-100

Powdered media/buffer mixing


Applications: culture media and buffers, product,samples or waste.

Capacity: 10 mL to 2500 L (up to 10,000 L)

Vendors: HyClone, Stedim, TC Tech, Charter, Newport:USP Class VI tested, gamma irradiated

Types: Monolayer, multilayer, LDPE, EVA, etc.

Integration Challenge: connectors not common

Scale Up Challenge: bag seam weld strength, rate offluid transfers, robust, cheap tubing >1” diameter

BioProcess Bags/Tank Liners


Application: Culture of eukaryotic cells

Capacity: ~10 mL – ~10 L / 25K cm2

Vendors: Corning, Nunc, Wave, Bellco

Types: TC flasks, rollers, spinners, shake flasks, hollowfibers, expanded T-flasks (Cell Factory, Cell Cube), novelbioreactors (Wave, BelloCell)

Integration challenges: tubing/connectors sizes andcompatibility

Scale Up Challenge: model system that scales to 10,000L

Small Scale Cell Culture Bioreactors


Applications: Culture of mammalian, insect or plant cells insuspension. (Many vendors supply large hollow fiber systemsfor anchorage dependent cell culture).

Capacity: 1 L – 200 L

Vendors: HyClone SUB stirred tank, Wave Biotech rockingsystem, Xcellerex XDR stirred tank bioreactor

Integration Challenge: liners, connectors, sensors, filters,controllers

Scale Up Challenge: model system that scales to 10,000L

Mid Scale Cell Culture Bioreactors


XcellerexXDR-200workingvolumedisposablestirred tankreactor


Application: Culture of mammalian, bacterial, yeast,insect or plant cells in suspension.

Capacity: 200-1,000L working volume

Vendors: Wave Biotech 500L rocker, HyClone SUBstirred tank, Xcellerex 1,000L stirred tank bioreactor

Integration Challenge: liners, connectors, sensors,filters, controllers

Scale Up Challenge: avoid stressing bag seams, smallscale modeling system that scales to 10,000L

Large Scale Cell Culture Bioreactors


Xcellerex 1,000L (wv) Disposable Stirred TankReactor - XDR™


Application: Separation of cells from growth mediumduring perfusion or for terminal cell harvest.

Capacity: Up to 100-200 L/hr

Vendors/Types: Kendro (centrifuge), Steadfast (rotarydrum filter), Spectrum and GE (recirc. hollow fiber),Millipore POD system (dead end), Cuno depth filtration.All product contact surfaces disposable

Integration Challenges: connectors

Scale Up Challenges: recirculating systems: disposabletubing not amenable to high flow rates and pressures

Cell Harvest


Application: Clarification / sterilization of media, buffersand process intermediates, cell harvest, and removal ofparticulates.

Capacity: Syringe filters, 30” capsules, flat membrane

generally available, (larger by custom order)

Vendors/Types: Millipore POD, Pall, Sartorius, Meissner,

Cuno – larger capsules coming available, many available

pre-sterilized and integrity tested.

Integration challenges: connector compatibility

Scale Up challenges: >1000L capacity is lacking

Dead End/Depth Filtration


Application: Perfusion, cell harvest, purification,concentration, and formulation / buffer exchange.

Capacity: Up to 5.6 m2

Vendors/Types: Spectrum HF, GE hollow fiber

Integration: disposable pump integration that is durableyet disposable

Scale Up Challenge: recirculating systems: disposable

tubing not amenable to high flows/pressures

Tangential Flow Filtration


Application: Mechanical reduction of viral load bynanofiltration.

Capacity: 15 - 200 L/hr. (depending on pore size, filter

medium & process stream)

Vendors/Types: Millipore dead end, Pall dead end,

Asahi-Kasei


Scale Up Challenges: larger scale requires more area

Virus Reduction


Application: Flow-through removal of contaminants,bind-and-elute purification of small or dilute processstreams.

Capacity: 20 L/min., 5g DNA binding capacity

Vendors/Types: Pall, Millipore and Sartoriusfunctionalized filter membranes.

Integration Challenges: connectors, area

Scale Up Challenges: less binding capacity compared tochromatography resins in general

Purification – Membranes


Applications: Aseptic filling into vials

Capacity: Clinical to commercial {?}

Vendors/Types: Millipore Acerta bag based fillingsystem, MedInstill injection filling/laser sealing


Scale Up Challenges: not clear yet

Vial Filling


SensorsApplications: Process wide

Capacity: N/A

Vendors/Types: Wave Biotech, (pH, DO2),Flourometrix and PreSens optical sensors,microprobes

Integration Challenges: Cytoxicity, irradiatability,fit up into bags, tubing, dead zone elimination,signal response time

Scale Up Challenges: stability, non-fouling,validatable


Applications: Aseptic / sterile connections

Capacity: 1/4” to 3/4” OD tubing

Vendors/Types : CPT (C-Flex), Pall ACDs, BioQuate,Millipore. CPT connector is similar to a tubing welder.Pall & BioQuate connectors are similar to a quick-connectwhich can make sterile connections in a non-sterileenvironment.

Integration Challenges: no one system connects all andthey are one time use (versus welders that can reweld)

Scale Up Challenges: connectors >1” diameter

Sterile Tubing Connectors


Application: Aseptic / sterile connections

Capacity: 1/4” to 3/4” OD tubing

Vendors/Types: Terumo, Wave, Sebra. Several deviceshave been validated by the vendor and/or biopharmmanufacturers. Can be used on PVC and EVA (Sebra), orTygon, C-flex and Pharmed (Terumo, Wave) tubing.

Integration Challenges: no one system welds all

Scale Up Challenges: welding tubing >1” diameter

Connectors - Tubing Welders


Application: SIP-able connections between disposabletubing to stainless steel valves.

Capacity: 1/2” ID x 3/4” OD

Vendors: Millipore, Colder, TC Tech

Comments: Can be added as option to tubing onbioprocess bags for sterile transfer to or from stainlesssteel systems.

Integration Challenges: no one connects all

Scale Up Challenges: none

SIP Tubing Connectors


Application: Disposable filter capsule capable ofwithstanding temperature and pressure required for steam-in-place.

Capacity: 5” housing

Vendors/Types: Pall - Can house vent or liquidsterilizing filters or virus removal filters.

Integration Challenges: connections

Scale Up Challenges: larger size not available

SIP Filter Capsules


Applications: Used in place of sampling ports onstainless steel tanks.

Capacity: 20 mL - 1 L sample bags

Vendors/Types: NovAseptic - sheathed cannula, tubingset and bag as a pre-sterilized, closed system. Outside ofsheath sterilized during vessel SIP. Can load multiplecannulae into single septum sample port.

Integration Challenges: none

Scale Up Challenges: none

Sampling System


Technology Trends• Standard connectors

• Non-recirculating TFF

• Integration of existing stainless todisposable

• Development of new enabling technology

– Sensors

– Chromatography (scaleable and economical)

• Fully disposable biomanufacturing >1,000L

• Yeast, bacteria, fungal systems coming!


Types of Cells Grown in XDRsCell type/product Mode scale of runs done L (wv) 2007 planned runs

Hybridoma/mab fedbatch 2x 200 - GMP 200 - GMP

CHO/mab-fusion batch 2x200, 2 x 1,000L 1,000LCHO/fus. protein fed batch 2x 200, 3 x 500 GMP 200, 500 - GMP

Insect S2/vaccine fed batch 200 200 - GMP

Insect SF9/vaccine fed batch 200, 2,000

Human/fus. protein perfusion 200 1,000

Yeast (Sacc.) fed-batch 150 1,000

E. Coli fed-batch 200

Vero/vaccine batch 200, 1,000


GMP XDR-200 and 1000Systems


BioNet DeltaV GMP Process Controller


XDR-1000 DO Control, fusionprotein

DO

0

10

20

30

40

50

60

70

80

90

100

Time

5/14

/067:

21

5/14

/0620

:55

5/15

/0610

:29

5/16

/060:

03

5/16

/0613

:36

5/17

/063:

12

5/17

/0616

:44

5/18

/066:

18

5/18

/0619

:52

5/19

/069:

26

DO


XDR-1000 pH control, fusion proteinpH

6.5

6.6

6.7

6.8

6.9

7

7.1

7.2

7.3

7.4

7.5

Tim

e

5/1

4/0

61

:33

5/1

4/0

69

:18

5/1

4/0

61

7:0

6

5/1

5/0

60

:51

5/1

5/0

68

:37

5/1

5/0

61

6:2

4

5/1

6/0

60

:08

5/1

6/0

67

:56

5/1

6/0

61

5:4

1

5/1

6/0

62

3:2

7

5/1

7/0

67

:13

5/1

7/0

61

4:5

9

5/1

7/0

62

2:4

6

5/1

8/0

66

:32

5/1

8/0

61

4:1

8

5/1

8/0

62

2:0

4

5/1

9/0

65

:50

5/1

9/0

61

3:3

4

pH

pH controlimplemented


XDR-1000 Temp Control878-03 1000L Bioreactor Run, Temperatures, 3/11/2006

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

4:00 8:00 12:00 16:00 20:00 0:00 4:00 8:00 12:00

Time

Te

mp

era

ture

InD

eg

ree

sC

Bioreactor Temp

Ambient Temp


XDR-1000 Mass TransferKLa measurements, single sparger


XDR-1000L KLa hr-1 vs RPM and

Sparge Rate

0

2

4

6

8

10

12

0.0 1.0 2.0 3.0 4.0 5.0 6.0

Air Sparge Rate, SLPM

KL

ah

r-1 50 RPM

75 RPM

100 RPM

(Multiple spargers)


XDR-200 Scalability ComparisonCell Density

Production Viable Cell Density

0.00E+00

2.00E+06

4.00E+06

6.00E+06

8.00E+06

1.00E+07

1.20E+07

1.40E+07

1.60E+07

1.80E+07

2.00E+07

0 24 48 72 96 120 144 168 192 216 240 264 288 312 336 360 384 408 432

Batch Age (hrs)

Via

ble

Ce

llD

en

sit

y(c

ell

s/m

L)

3000LengineeringGMP1

GMP2

GMP3

300L

XDR200

XDR200-2

-1

Feeds run XDR-200-2

Feeds run XDR-200


XDR-200 Comparison, ViabilityProduction % Viability

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

0 24 48 72 96 120 144 168 192 216 240 264 288 312 336 360 384 408 432

Batch Age (hrs)

%V

iab

ilit

y

3000L engGMP1GMP2GMP3300LXDR200XDR200-2

-1


XDR-200 Comparison TiterProduction Titer

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

1.2

1.3

1.4

1.5

120 144 168 192 216 240 264 288 312 336

Age (hrs)

Tit

er

(mg

/mL

)

3000L engGMP1GMP2GMP3300LXDR200XDR200-2


XDR-200 Comparison, pCO2Production pCO2 (mmHg)

0

20

40

60

80

100

120

0 24 48 72 96 120 144 168 192 216 240 264 288 312 336

Age (hours)

pC

02

(mm

Hg

)

3000L eng

GMP1

GMP2

GMP3

300L

XDR200

XDR2002-2


XDR-200 CHO PerfusionCulture - Viable Cell Density

XDR200 vs 10L BR ( Viable Cell Density)

0.00

2.00

4.00

6.00

8.00

10.00

12.00

-5.0 0.0 5.0 10.0 15.0 20.0 25.0

Days in Production

Via

ble

Ce

llD

en

sit

y(e

6c

ell

s/m

l)

XDR200

10L BR

Perfusionrate = 1 VVD


XDR-200 CHO PerfusionCulture - % Viability

XDR200 vs 10L BR (Viability)

60.0

65.0

70.0

75.0

80.0

85.0

90.0

95.0

100.0

-5.0 0.0 5.0 10.0 15.0 20.0 25.0

Days in Production

Via

bil

ity

(%)

XDR200

10L BR


XDR-200 Insect Cells S2- fed batch

XDR-200 Growth Curves Vials #21

0.00E+00

5.00E+06

1.00E+07

1.50E+07

2.00E+07

2.50E+07

3.00E+07

3.50E+07

4.00E+07

4.50E+07

5.00E+07

0 48 96 144 192

Time in XDR-200 (h)

Ce

llD

en

sit

y

80

100

#21-1A Viable cells/ml

#21-1A Viability (%)

Induce with 0.2 M CuSO4

Added Glucose

AddedGlucose

Added Antifoam and Glucose

Added Antifoam

Added Antifoam

Highest celldensity

achieved instainless steel

bioreactors was32 x E6 cell/ml


XDR-200 Insect Cells S2- fed batch - pCO2

pO2 and pCO2

0

50

100

150

200

0 120 240 360 480 600 720

Time (h)

pO

2(m

mH

g)

0

50

100

150

200

pC

O2

(mm

Hg

)

pO2 (mm Hg)

pCO2 (mm Hg)


XDR-200 Yeast - S. cerevisiaeRun 130-999 - XDR-200: S. cerevisiae fermentation

0.200

5.200

10.200

15.200

20.200

25.200

30.200

35.200

0:00:0

0

8:15:0

0

9:45:0

0

12:45:0

0

15:45:0

0

18:45:0

0

20:45:0

0

23:15:

00

24:30:0

0

26:15:0

0

27:30:0

0

28:2

4:00

30:27:0

0

32:15:0

0

34:15:0

0

35:30:0

0

38:15:0

0

41:15:0

0

43:1

5:00

45:00:0

0

46:15:

00

47:30:0

0

48:15:0

0

49:48:0

0

50:37:

00

EFT (hr)

OD

60

0

0.000

0.500

1.000

1.500

2.000

2.500

3.000

3.500

4.000

g/L

Density EtOHGlucose 2 per. Mov. Avg. (Glucose)Poly. (Density)


XDR-1000 Bioreactor Runs

CHO Cell Line

Viable Cell Density

0.00E+00

1.00E+06

2.00E+06

3.00E+06

4.00E+06

5.00E+06

6.00E+06

0 25 52 74 97 122 145 168 195 217 239

Hours

Via

ble

Cell/m

L Run#2

Run#1

XDR-1000, CHO, fusion mAbviable cell density


XDR-1000 Bioreactor Runs

CHO Cell Line

Cell Viability

0.0%

10.0%

20.0%

30.0%

40.0%

50.0%

60.0%

70.0%

80.0%

90.0%

100.0%

0 25 52 74 97 122 145 168 195 217 239

Hours

%V

iab

le Run#2

Run#1

XDR-1000, CHO, engineered mAbcell viability


Progress for Single Use Systems

• Wide acceptance of bioprocess bags

• Single use bioreactors are scalable andperformance is comparable to SS bioreactors

• 1,000L (wv) stirred tank bioreactor breakthroughopens large scale/commercial applications

• Single pass cell clarification/removal (POD)simplifies 1° recovery

• Membranes for purification improving


Summary - Challenges for Disposables

• Mixing/Buffer/Media prep – Rate of liquid transfers

• Bioreaction – Lab to commercial scalability within the samereactor design/configuration

• Cell Harvest/TFF - Recirculating processes that require highpressure and high flow rates

• Membrane Purification – capacity and DNA/virus clearance

• Disposable Chromatography is still TBD

• Non-standard, multiple connection options

• Disposable sensors are limited

• Plastic durability and weld strength needed for scale up


Acknowledgements

• Geoff Hodge

• Dan Mardirosian

• Patrick Guertin

• Michael Fisher

• Keith Kropp

• Pat Puma

review of single use technologies in biomanu in bioprocessing. ... wave biotech rocking ......

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