Kevin Lauziere presented an overview of the typical processes involved in biologics manufacturing from development to commercial scale. He discussed the key stages including cell culture, seed bioreactors, production bioreactors, harvest and clarification, chromatography, filtration and virus inactivation. He emphasized important considerations at each stage such as equipment sizing, automation, parallel processing and process controls. The goal was to provide an understanding of how process development influences commercial scale equipment design and operations.

1. Welcome
Tracking Single-Use & Scale-Up Best Practices
[Webinar Series]
Webinar #2: An Overview of Biologics Manufacturing Processes and Things to
Consider from Development to Commercial Scale

2. An Overview of Biologics Manufacturing
Processes and Things to Consider from
Development to Commercial Scale
Kevin Lauziere

3. About Your Presenter – Kevin Lauziere
• Degree in Biochemistry from Boston College, and has worked for a
number of companies in the Boston area including Genzyme, BASF
Bioresearch Center, Abbott Bioresearch Center and Bristol-Myers
Squibb.
• Worked in the process development area early in career designing
methods for purifying proteins to be scaled up to the
manufacturing level.
• Currently a consultant with over 27 years in the industry currently
working with The Quantic Group, Ltd.
• Over the years he has contributed to the design, start-up and
validation of two commercial manufacturing facilities working with
both internal and external engineering resources.

4. Outline
• Overview of a typical biologics manufacturing process
that uses mammalians cells for protein expression
• Discuss the importance of process development and the
influence this has on commercial scale equipment design
and operations
• Discuss how automation can be used for running some
process operations and the pros and cons of using it

5. Overview of a typical biologics
manufacturing process
Ion Exchange
Chromatography
Hydrophobic
Interaction
Chromatography
Viral
Filtration
UF/DF
Fine Purification
Formulation
Bulk Fill
Affinity
Chromatography
Product Capture
low pH Viral
inactivation
Spinner or
Shake
Flask
Cell
bank
vial
Seed Bioreactors
Production
Bioreactor
Depth
Filtration
Cell Culture
Clarified
Harvest
Centrifugation
Clarification

6. Overview of a typical biologics
manufacturing process
• Vial Thaw and Seed Train
• Seed Bioreactor
• Production Bioreactor
• Harvest/Clarification
• Chromatography
• Tangential Flow Filtration
• Virus Filtration/Inactivation
• Formulation and Bulk Fill
• Sampling and Testing of Process Intermediates

7. Vial Thaw and Seed Train
• Typically a single vial of cryogenically preserved cells is
thawed to start the seed train for a mammalian cell
culture process. Vial thaw can be accomplished:
• At room temperature
• In a water bath
• In a heating block
• In an incubator
– Resuspension process
• Typically use an enriched medium to dilute any cryopreservatives
• Transfer thawed cells into different volumes to target a specific
range for initial cell density
• Viability ranges vary with each process

8. Vial Thaw and Seed Train
• The number of stages in a seed train vary with every
process
– It may utilize T-flasks, shake flasks, spinners and or wave bags
– Volume of the culture varies and this puts demands on the
design and footprint of the manufacturing area
• Considerations include:
– Number of containers at each stage
– Are there multiple trains running in parallel
– Equipment needed (biosafety cabinets, incubators)
– Utilities
– Classification of the rooms
– Open or closed processing

9. Seed Bioreactor
• Seed Bioreactor
– Used for cell expansion –
goal to obtain sufficient
number of cells at a target
cell density to seed the
production bioreactor

10. Seed Bioreactor
• Seed Bioreactor
– Size and number
– Method of inoculation
– Area classification
– Parallel trains – add process robustness and provide backup in
the event of a lost seed bioreactor
– Requirements for additions
– Process control strategy
• pH
• Dissolved Oxygen
• Agitation
• Temperature
– Automation and process monitoring

11. Production Bioreactor
• Size
• Area classification
– Closed processing
• Parallel trains
• Requirements for additions
• Process control strategy
– pH
– Dissolved Oxygen
– Agitation
– Temperature

12. Production Bioreactor
• Harvest criteria
– Viability
– Titer
– Duration
• Automation and process monitoring
– Glucose
– Lactate
– pCO2

13. Harvest/Clarification
• Initial crude clarification – separates cells and cellular
debris from harvest broth
• Method
– Centrifugation
– Depth Filtration
– Microfiltration
– Combination of these
(i.e. centrifugation and
depth filtration)
• Area classification
• Redundant equipment
• Automation

14. Chromatography
• Number of steps
– Resin chemistry
– Order of steps
• Loading conditions
• Volume of eluates
– Volume measurement
• Scale of columns
– Single or multiple cycles per lot
• Based on amount of product and column capacity
– Resin cost is also considered
– Buffer volumes (1x buffers or concentrates)
• Dilution strategy
– Flowrate ratio
– Conductivity setpoint

15. Chromatography
• Eluate vessel size and capability
– Process sampling
– Mixing
– Temperature control
– Process intermediate manipulations
– Filtration (In-line or post processing)
• Area classification
• Processing temperature
– Column operation temperature versus hold temperature for
process intermediates
• Redundant equipment
• Automation

16. Tangential Flow Filtration
• System size
– Volume
• Starting volume
• Final volume
– Processing time
• Amount of membrane surface area
• Pump sizing
– Operating conditions
• Transmembrane pressure
• Crossflow rate
– Amount of product
• Final concentration
• System hold up

17. Tangential Flow Filtration
• Diafiltration
– Number of volumes exchanged (5X, 8X, 10X)
– Concentration at which diafiltration is performed
– Solubility (Isoelectric point)
• Area classification
• Processing temperature
• Automation

18. Virus Filtration/Inactivation
• Filtration
– Membrane surface area
– Pore size
– Filter material compatibility
– Physical room change during the process
– Volume
– Processing time
• Product concentration

19. Virus Filtration/Inactivation
• Inactivation by pH adjustment
– Product degradation
– Duration of hold
– Volume
– Strength of acid and base used for adjustment
– Rate of addition
• Inactivation by detergent
– Volume
– Duration of hold
– Removal of detergent
• Area classification
• Processing temperature
• Automation

20. Formulation and Bulk Fill
• Environment
– Open or closed processing
– Temperature
• Containers
– Material of construction
– Leachables
– Container closure
– Labeling
– Volume
– Source and Condition

21. Formulation and Bulk Fill
• Open fill
– Performed in a biosafety cabinet (Grade A, Class 100)
– Grade B, Class 1000 room
• Rooms like this are expensive to build
• Closed fill
– Performed in a Grade C, Class 10,000 area where all
connections are closed
• Sampling strategy
– Number of samples
– Container type

22. Formulation and Bulk Fill
• Bulk fill room
– Single product use
– Multi-product use
• Scheduling
• Room Turnover Process
• Shared Equipment
• Shared Personnel
• Temperature
– Product stability
– Type of label
– Operational considerations
• Affect on staff and their ability to perform tasks

23. Thank You
Questions?

24. #1 Tracking Single-Use : An Introduction to Single-Use Manufacturing Systems
Including a Survey of Technology, New Developments and Economic and Operational
Issues
Tuesday, January 19, 2016 / 1pm-2pm (ET)
Presenter: Geoff Hodge
Next Up: High Level Recombinant Protein Production in Insect Cell Culture
Tuesday, January 26, 2016 / 1-2pm (ET)
Presenter: Dr. Kamal Rashid
#2 An Overview of Biologics Manufacturing Processes and Things to Consider from
Development to Commercial Scale
Monday, January 25, 2016 / 1-2pm (ET)
Presenter: Kevin Lauziere