The Use of Disposable Technologies in Antibody ... · The Use of Disposable Technologies in...

The Use of Disposable Technologies in Antibody Manufacturing Processes

Tim Matthews and Brad Wolk

Process Development EngineeringGenentech, Inc.

South San Francisco, CA

Outline• Rationale for using disposable technologies• Cell Culture applications (bag focus)• Purification applications (bag focus)• Qualification strategy• Case studies

– Disposable bioreactors– Media / Buffer storage in bags– Protein pools in bags– Protein bulk drug substance in bags

• Conclusions

Rationale for Using Disposable Systems

• Technologies can offer economic advantages in:– Operating expense– Capital expenditure– CIP / SIP– Cleaning validation (between runs, between products)– Maintenance (tank inspection, corrosion, etc.)– Installation / implementation time

• Improved aseptic / sterile processing and sampling• Increase plant capacity (turnaround time, space

efficiency)• Consider up-charge on raw materials and disposal costs

Cell Culture Applications

• Disposable Bioreactors up to 500L (or greater) operating volume• Media & nutrient preparation (dissolution) in disposable containers• Sterile storage of media and nutrients in disposable containers up to

2500L• Sterile storage of harvested cell culture fluid in disposable containers

up to 2500L• Sterile sampling• Sterile / Aseptic connecting devices• Harvesting using Disposable Systems – disposable depth filters

Spinners Batch Re-feed Inoculum

Scale-upProduction Harvesting

OperationsHarvested

Culture Fluid

Vial

SampleFeed

Purification Applications

• Buffer preparation (dissolution) in disposable containers• Sterile storage of buffers and protein pools in disposable

containers• Sterile sampling• Aseptic / sterile connecting devices

Protein Load

Load Conditioning

Buffers / Sanitization

solutions

Chromatography and Tangential Flow Filtration Applications

Protein Pools / Bulk Drug Substance

SampleFeed

Qualification of Disposable Systems (Bags)

• General Considerations:– Extractables / Leachables– Vapor Transmission– Integrity / Sterility Validation Testing

• Application Specific:– Sampling– Disposable bioreactors– Buffer / media storage– Protein pools / Bulk Drug Substance storage

• Incorporate new technologies early in development:– R&D production / GLP Toxicology– Early Stage Clinical Production– Late Stage Clinical / Marketed Production

Extractables and Vapor Transmission from Bioprocess Bags using Model Solvents

• Model solvents chosen to mimic aggressive process conditions

• The complete bags were tested as one unit (ports, fittings, tubing, & film)

• High storage temp and hold duration

• Small bags used (maximize surface area to volume)

• Measure extractables– GC/MS, HNMR, TOC,

etc.• Measure weight loss of

liquid (solvent transport through film)

Extracted TOC (PPM) from Bioprocess Bags. Bags stored at 40 degrees Celcius for 180 Days.

0

20

40

60

80

100

120

EVA ULDPE #1 ULDPE #2 LLDPE Glass

Water0.1M Sodium Hydroxide0.1M Phosphoric Acid20X PBS

Solution Flux (cm/day) Through Bag Films at 40oC

0.0E+00

2.0E-04

4.0E-04

6.0E-04

8.0E-04

1.0E-03

1.2E-03

1.4E-03

1.6E-03

1.8E-03

2.0E-03

EVA ULDPE #1 ULDPE #2 LLPE

Water0.1M Sodium Hydroxide0.1M Phosphoric Acid20X PBS50% Methanol

Case Studies Using Disposable Systems

• Disposable bioreactors• Media / buffer bags &

bag containment systems

• Protein Pools in bags• Bulk drug substance in

bags– Shippable bags– Freezable bags

BAG FEATURES

•Sterile

•Mixing

•Sensors (pH, DO, Temp)

•Integrated Volume Measurement

•Sampling

•Transportable

Disposable Bioreactor Technology Choices*

• HyNetics• HyClone• ATMI• LevTech• Xcellerex• Stedim/Applikon• Wave Biotech

– Wave Reactor– FlexMixer

– LevTechATMIHyClone

*Any of these systems could be suitable for solid-liquid dissolution (media or buffer prep) or protein pools

Rocking Motion wave motion

Inflated plastic bag forms a disposable cultivation chamber

Rocking Motion wave motion

Wave action sweeps up cells and prevents settling

Wave action creates large turbulent surface for oxygen transfer

Cell culture media

Courtesy of

Wave Biotech

Oxygen Mass Transfer (kLa) in Wave Bags

• Tests performed with water• kLa increases with angle and rock rate• Operating at 30-50% volume can still achieve kLa’s over

20 h-1

10-L Wave Bag 30% Volume

010203040506070

10 15 20 25 30 35Rock Rate (rock/min)

kLa(

hr-1

)

4 degrees7 degrees10 degrees

10-L Wave Bag 50% Volume

05

101520253035

10 15 20 25 30 35Rock Rate (rock/min)

kLa

(hr-1

)

4 Degrees

7 Degrees

10 Degrees

Wave System1000

Courtesy of

• Evaluated at Genentech for production of monoclonal antibodies in CHO

• 500 liters maximum working volume

• System allows temperature, pH, and dissolved oxygen control

Monoclonal Antibody Production in CHO using Wave System 1000 Bioreactor

%Viable

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Run Time (d)

400L Stirred Tank #1



400L Wave Bag

4L Wave Bag Satellite

Packed Cell Volume Fraction

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Run Time (d)




400L Wave Bag


Antibody Titer

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Run Time (d)




400L Wave Bag


• Wave compared side by side with standard 400 liter stirred tank reactor

• High density CHO culture• Operated under Temp, pH, and DO

control.• Wave system showed excellent cell

growth and antibody production characteristics

• Analyses have shown comparable product and impurity profiles


BAG FEATURES

•Sterile

•Mixing



•Sampling

•Transportable





Media / Buffer bag configuration up to 2500 liters*

Vent filter for repeated use

Drain Line to ChromSkid; 1.5”Triclamp / Disposable connector

Fill Line

Sterile Inlet FilterSample bags

Flush Bag

Media /Buffer Bag

*Bags >500L benefit from suspension systems

Buffer Bag Holders• There are several “off

the shelf” choices for basic bag holders

• Containment up to 2500 liters

• Smaller systems are stackable

• Bottom feed for easier access when stacked

• Bottom drain for better liquid recovery & priming

500 Liters200 Liters

Photograph Courtesy of Stedim, LLC website

Bags with In-Line Dilution• Most buffers can be

concentrated >>10-fold• Buffer concentration

allows bags to be used even at very large scale production

• Bags offer corrosion resistance

• Use concentrated buffers in bags then dilute, in-line, to target concentration

• Deliver diluted buffer to unit operations

Buffer concentrates

Blender

Water

ColumnMix

Check

Accept/Reject

Filter

Buffer Prep Filter Buffer Hold Filter Process

Conc. Bag Water Filter Process

Conc. Prep Filter Conc. Hold Water Filter Process

Prep Concentrated Buffer

Buy Concentrated Buffer

Prep Normal-Strength Buffer

Inline Dilution of Buffer Concentrates with Bags






BAG FEATURES

•Sterile

•Mixing



•Sampling

•Transportable

Protein Pools in Bags – Recirculation Mixing Example

PeristalicPump

Flowmeter OD Meter

Injection Port 1000L Buffer Bag• Various mixing technologies

are available: See Disposable Bioreactor technologies

• Allows bags to be used for intermediate protein pool storage

• Liquid – Liquid mixing strategy using bags

• Not as useful for solid – liquid mixing (dissolution).

• Use of standard bag holder and peristaltic pump

• Slight modification to bag to facilitate sterile recirculation path (Aseptic connecting devices / tubing welders)

• Satisfactory mixing was demonstrated even with high viscosity and density differences

1000L Buffer Bag Mixing Studies - 10 LPM (Combined Runs) - 051205

0

1

2

3

4

5

6

0 2 4 6 8 10 12

Time (min)

OD

(Nor

mal

ized

)water / acetone

water / high density dye

1M Na2SO4 / acetone






BAG FEATURES

•Sterile

•Mixing



•Sampling

•Transportation

Bulk Drug Substance in Bags• Bags can be used for longer-term storage of bulks in

bags if product stability permits• Product can be held as liquid (scales up to ~500

liters) or frozen in bags (at smaller scales: up to about 20 liter per bag currently)

• Great care must be taken to ensure bag integrity is maintained

• Convenient format that allows low-cost storage alternative to stainless steel or higher-alloy tanks

• Useful format for shipping to filling sites• Product stability testing must be performed on a

case-by-case basis

Bags for Liquid Bulk Shipment• Reduce dependence on

stainless or higher alloy tanks for storage of protein solutions

• Heavily used for shipment of WFI, media, buffers, serum.

• 500L is typically maximum shipping volume

• Shipping containers have to be economical but extremely robust

• Bag integrity must be maintained – compare to standard tanks

• Economic evaluation should be performed to determine “acceptable” failure rate 1:1,000? 1:10,000?

200L Commercial System

200L System in Envirotainer at Genentech

Freezable Bags for Bulk Storage• Bags can be used for

frozen storage of protein bulks

• Specialty films available• Special containment

systems to protect bag and tubing must be used

• Freezing and thawing rate can be difficult to control

• More useful with small bulks (<20 liters)

• There are commercially available systems for this application

Conclusions• Disposable systems offer many advantages in the

production of monoclonal antibodies in cell culture, purification, and bulk storage areas.

• Product offerings are becoming more comprehensive and can penetrate many areas of clinical and commercial manufacturing facilities

• Current disposable technologies (bags, fittings, filters, connecting devices) are validate-able for use in cGMPproduction of parenteral products

• Disposable probes for bioreactors (pH, DO) need improvement to make useful for the rigors of production environments

• Genentech anticipates future production facilities will be designed around the concept of disposable technologies

Acknowledgements• IBC Coordinators• Genentech

– Donna Giandomenico– Bryan Bean– Kellen Mazzarella– Cell Culture Pilot Plant

• HyClone– Jerry Branscomb– Stephen Twigg

• Stedim– Brent Bushnell– Dave Belomy– Bob Smith-McCollum

• Wave Biotech– Vijay Singh– Scott Westerhout

• XCellerex– Parrish Galliher– Michael Fisher– Susan Dexter

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