The Use of Disposable Technologies in Antibody Manufacturing Processes

The Use of Disposable Technologies in Antibody Manufacturing Processes Tim Matthews and Brad Wolk Process Development Engineering Genentech, 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 Feed Sample Vial Spinners Batch Re-feed Inoculum Scale-up Production Harvesting Operations Harvested Culture Fluid 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

Purification Applications Buffers / Sanitization solutions Feed Sample Load Conditioning Protein Load Chromatography and Tangential Flow Filtration Applications Protein Pools / Bulk Drug Substance Buffer preparation (dissolution) in disposable containers Sterile storage of buffers and protein pools in disposable containers Sterile sampling Aseptic / sterile connecting devices

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) 120 100 80 60 40 20 0 2.0E-03 1.8E-03 1.6E-03 1.4E-03 1.2E-03 1.0E-03 8.0E-04 Extracted TOC (PPM) from Bioprocess Bags. Bags stored at 40 degrees Celcius for 180 Days. Water 0.1M Sodium Hydroxide 0.1M Phosphoric Acid 20X PBS EVA ULDPE #1 ULDPE #2 LLDPE Glass Solution Flux (cm/day) Through Bag Films at 40 o C Water 0.1M Sodium Hydroxide 0.1M Phosphoric Acid 20X PBS 50% Methanol 6.0E-04 4.0E-04 2.0E-04 0.0E+00 EVA ULDPE #1 ULDPE #2 LLPE

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

Wave Biotech Inflated plastic bag forms a disposable cultivation chamber wave motion Rocking Motion Cell culture media Rocking Motion wave motion Wave action creates large turbulent surface for oxygen transfer Wave action sweeps up cells and prevents settling Courtesy of

Oxygen Mass Transfer (k L a) in Wave Bags kla(hr-1) 70 60 50 40 30 20 10 0 10-L Wave Bag 30% Volume 4 degrees 7 degrees 10 degrees 10 15 20 25 30 35 Rock Rate (rock/min) kla (hr-1) 35 30 25 20 15 10 5 0 10-L Wave Bag 50% Volume 4 Degrees 7 Degrees 10 Degrees 10 15 20 25 30 35 Rock Rate (rock/min) 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

Wave System1000 Evaluated at Genentech for production of monoclonal antibodies in CHO 500 liters maximum working volume System allows temperature, ph, and dissolved oxygen control Courtesy of

Monoclonal Antibody Production in CHO using Wave System 1000 Bioreactor 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 %Viable 0 1 2 3 4 5 6 7 8 9 101112131415 Run Time (d) 400L Stirred Tank #1 400L Stirred Tank #2 400L Stirred Tank #3 400L Wave Bag 4L Wave Bag Satellite Packed Cell Volume Fraction Antibody Titer 400L Stirred Tank #1 400L Stirred Tank #2 400L Stirred Tank #3 400L Wave Bag 4L Wave Bag Satellite 400L Stirred Tank #1 400L Stirred Tank #2 400L Stirred Tank #3 400L Wave Bag 4L Wave Bag Satellite 0 1 2 3 4 5 6 7 8 9 10111213 14 15 Run Time (d) 0 1 2 3 4 5 6 7 8 9 10111213 14 15 Run Time (d)

Media / Buffer bag configuration up to 2500 liters* Vent filter for repeated use *Bags >500L benefit from suspension systems Fill Line Sterile Inlet Filter Media /Buffer Bag Flush Bag Sample bags Drain Line to Chrom Skid; 1.5 Triclamp / Disposable connector

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 200 Liters 500 Liters Photograph Courtesy of Stedim, LLC website

Bags with In-Line Dilution Blender Buffer concentrates Check Mix Filter Water Accept/Reject Column 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

Inline Dilution of Buffer Concentrates with Bags Buffer Prep Filter Buffer Hold Filter Process Prep Normal- Strength Buffer Conc. Prep Filter Conc. Hold Water Filter Process Prep Concentrated Buffer Conc. Bag Water Filter Process Buy Concentrated Buffer

Protein Pools in Bags Recirculation Mixing Example 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 OD (Normalized) 6 5 4 3 2 1 Flowmeter Injection Port OD Meter Peristalic Pump 1000L Buffer Bag Mixing Studies - 10 LPM (Combined Runs) - 051205 1000L Buffer Bag water / acetone water / high density dye 1M Na2SO4 / acetone 0 0 2 4 6 8 10 12 Time (min)

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 cgmp production 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