1. Gene Therapy Production Facility Considerations
Robert Sausville
Center for Biologics Evaluation and Research
Office of Compliance and
Biologics Quality
Division of Case Management
11/15/2018

2. Overview
General Considerations
Special Considerations
Biosafety Levels
Air Quality
Environmental Monitoring
Validation
Qualification
Miscellaneous

3. Overview New manufacturing areas retrofits of existing facilities
located within existing facilities
Designed to minimize potential for contamination
between products
between early and late stages of production
Operations should be well controlled
Personnel should be appropriately trained
someone responsible for facility operations, equipment validation and maintenance, record keeping

4. General Considerations
Good techniques and appropriate equipment used to minimize exposure to infectious agents
Strict adherence to standard practices and techniques (consistent manufacture of product)
Safety equipment (primary barrier)
biological safety cabinet
closed containers
other designs to minimize aerosols
Facility design (secondary barrier)
amount of protection depends on the nature of the infectious agent and production associated risks (aerosols)

5. General Considerations cont.
As the risk for aerosol transmission increases, higher levels of primary containment and multiple secondary barriers may become necessary

6. General Considerations cont.
Facility designed for aseptic processing
Smooth surfaces, seamless tile, etc.
Hard, easily cleanable and impervious finishes
Non shedding ceilings
HEPA filtered air from seperate air handling unit
separation of production from other areas of the facility (often a hospital)
Phase 1/II
Class 100,000: general manufacturing areas (10,000 III)
Class 100: all open manipulations

7. General Considerations cont.
Separate entrance/gowning area
Air locks (containment)
No personnel access between vector production and transduction areas
Room(s) for support areas
buffer media prep, glass/equipment wash
Storage areas
raw materials, cell banks
adjacent to production if possible to minimize traffic in and out of the facility

8. General Considerations cont.
Material and personnel flows designed to maximize efficiency and minimize mix-ups
unidirectional flows where possible
also controlled by procedures or temporally
Emergency power for critical systems (UPS)

9. General Considerations; Control of the Facility
Production area(s) should be separate from other activities (research, testing)
Potential cross-contamination should be minimized
between process steps, and
other production activities
Access into the production area should be limited
Equipment used in production should not be shared with non-production activities
Environmental monitoring and product testing should be performed to ensure adequate control of the process/area
Spill/accident procedures should be in place

10. Special Considerations; Multi-use scenarios
Concurrent vs. campaign production will impact design considerations
Personnel work on one cell line at a time
Procedures in place to prevent cross contamination
Appropriate changeover procedures
Adequate segregation of concurrent activities
color coded labeling
bar coded
The sponsor is responsible for assuring that the contract manufacturer has all the procedures in place

11. Special Considerations Cont.
Potential Routes of Cross Contamination
Centrifuges (generation of aerosols)
one sample processed at a time
cleaning/sanitization of centrifuges between “lots”
Pipettors
effective cleaning procedures
filters
Incubators

12. Appropriate BioSafety Level
In general, BioSafety Level 2
Transduction
Higher risk, BioSafety Level 3
Vector Preparation
Defined in CDC-NIH publication Biosafety in Microbiological and Biomedical Laboratories

13. BioSafety Level 2 Transduction and non-viral vector preparation
access limited
personnel trained in handling pathogenic agents
infectious wastes are decontaminated before disposal
gowning required (lab coat, hair cover)
gloves should also be worn for aseptic manipulations
Class I or II Biosafety Cabinets to be used:
for procedures potentially creating aerosols
aerosol generation should be minimized
with high concentrations or large volumes of infectious agents

14. BioSafety Level 3
Viral Vector prep areas, higher levels of containment
Negative pressure or “sink” for containment
All activities with infectious materials are conducted in biological safety cabinets
Class I, II or III may be used
Passage between two sets of doors is a basic requirement
An autoclave for decontaminating waste is available
preferably in the laboratory
Ducted exhaust provided
not recirculated
may be discharged to the outside without being filtered

15. BioSafety Level 4 Class III biosafety cabinets
or personnel in suits with life support systems
All materials must be autoclaved before leaving BSL4 area
Exhaust air HEPA filtered

16. Air Quality
Recommend that production areas receive single pass air (no recirculation) for vector production
Dedicated air handler where possible
Segregating air supply from rest of facility important, (hospital setting)
HEPA-filtered air
Objective: to meet Class 100,000 specifications for Phase I/II
In-line vs. terminal

17. Air Quality cont.
Air pressure differentials between areas should be balanced to maintain cleanliness or containment
Positive (aseptic processing)
Negative (containment) for steps needing greater than BSL-2
Open steps in biosafety cabinets (Class II)
Quality monitored to assure facility is acceptable for production

18. Environmental Monitoring
Testing of surfaces and for viable and non-viable particulates
Demonstrate facility under control
as part of validation
routine monitoring program
Frequency and intensity dependent on how close to GMPs the facility will operate
specifications based on desired Class

19. Environmental Monitoring cont.
Viable particulates
active air monitoring devices (slit to agar, centrifugal samplers)
settling plates (passive, less desirable)
Non-viable particulates
particle counters
room classifications, certification of biosafety cabinets
Surface “contact” plates or swabs
monitor cleaning efficacy and personnel asepsis

20. NASA Standards; Viable Air Particulates
Room classification, defined by Federal Standard 209E, determined by non-viable particulate monitoring under dynamic conditions.
Class 100,000
2.5 CFU/ ft3
Class 10,000
0.5 CFU/ ft3
Class 100
0.1 CFU/ ft3

21. Settling Plates Exposure times
Class 100,000
1 CFU/ 9cm plate
0.11 hours
2 CFU/ 9cm plate
0.21 hours
1 CFU/ 14cm plate
0.04 hours
2 CFU/ 14 cm plate
0.09 hours
Class 100
1 CFU/ 9cm plate
2.65 hours
2 CFU/ 9 cm plate
5.31 hours
1 CFU/ 14 cm plate
1.10 hours
2 CFU/ 14 cm plate
2.21 hours

22. Validation
“Establishing documented evidence which provides a high degree of assurance that a specific process will consistently produce a product meeting its predetermined specifications and quality attributes.”

23. Validation “Sliding Scale” approach for clinical manufacturing
Facilities involved in clinical manufacturing should be in compliance with the concepts of cGMPs
Do not expect full validation in early stages (may not have 3 repetitive runs, worst case configuration, etc.)
facility supplying clinical material to other institutions implies that it meets cGMPs and should approach what is expected for commercial facilities
Phase 3 material should be manufactured at close to full cGMP

24. “Sliding Scale” Approach, An Example
Autoclave used to prepare sterile materials
Early (I/II)
demonstrate proper cycle achieved
monitor temperature, pressure, and time
use of biological indicators for verification
loads not well defined

25. Autoclave cont. Middle (II/III)
temperature mapping done to determine cold/hot spots
biological indicators placed to verify cycle at problems points
loads are somewhat defined

26. Autoclave cont. Late (III+)
lethality of cycle determined at monitored points
loads are well defined and standardized
each load configuration has been mapped or worst-case load has been validated
Another example
Sanitizer effectiveness
Phase I/II supported by literature
Phase III supported by validation

27. Process Validation Sterilization Decontamination Aseptic Processing
Cleaning
Inactivation/removal of adventitious agents and other contaminants

28. Equipment Qualification
Program in place to demonstrate that equipment operates as expected
Should include periodic monitoring

29. Equipment cont. Air handlers Biological safety cabinets pressures
filter integrity
airflows; velocities
leak testing

30. Equipment cont. Incubators uniform temperature carbon dioxide filters
Centrifuges
speed
vacuum (ultras)
temperature

31. Equipment cont. Autoclaves temperature pressure kill cycle
Lyophilizers
shelf temperature
vacuum

32. Raw Materials
Critical raw materials - established criteria for acceptance from vendors:
sterility
adventitious agents
activity/purity
Vendor’s Certificate of Analysis
identity test where possible
Inventory Control
proper storage
FIFO

33. Water Should meet Water for Injection (WFI) specifications :
microbial <10 CFU/100ml
endotoxin <0.25 EU/ml
chemical tests per USP
WFI for all product contact surfaces and formulations
May be purchased

34. Personnel Designated person in charge of facility Responsible for:
limiting access
training
maintenance/safe operations
writing and enforcing procedures
Production personnel are trained (periodic retraining)
Appropriately gowned for production step

35. Final Thoughts Design facility for worst-case = maximum flexibility
Consider filing a Master File for facilities handling several IND products
Meet with FDA to discuss your Phase I/II (or III!) facility