Large scale containment

GMO and Biosafety
Laboratory
Production

The Bible

NIH Guidelines
“… any rDNA experiment ... must be submitted to NIH
or another Federal Agency that has jurisdiction for
review and approval.”
rDNA Project
Application
Risk assessment
Go / No go
Exemption:
Specific host/vector
systems
Exception?

Pathogen Classes (NIH)
Risk
Group
RG I
RG II
RG III
RG IV
Definition
Agents not associated with disease in
healthy adult
Agents associated with human disease,
rarely serious; preventive or therapeutic
intervention is available
Agents associated with serious or lethal
disease; preventive or therapeutic
intervention may be available; high
individual risk, low community risk
Agents that are likely to cause serious or
lethal disease; preventive or therapeutic
intervention usually not available; high
individual risk, high community risk
Examples
E. Coli K12,
Asporogenic B.
subtilis,
AAV type 1-4
B. Pertussis
H. Influenzae
Dengue, YF 17D ,
Rabies, Hep AE
Y. Pestis
Brucella, JE, YF WT
HIV, HTLV
No bacteria
Ebola, Marburg,
Lassa, H. simiae

Laboratory Handling of Agents
• General rule
• RG I agents BL1 containment (≈ no containment)
• RG II agents BL2 containment
• RG III agents BL3 containment
• RG IV agents BL4 containment

Genetic Manipulation of Pathogens
• General rule
• rDNA into RG II agents BL2 containment
• rDNA into RG III agents BL3 containment
• rDNA into RG IV agents BL4 containment
BUT
• Rec strain more hazardous than parent strain
higher containment level
• Rec strain attenuated or having irreversibly lost
virulence factors may qualify for a reduction of
containment compared to the RG of the parent strain

Containment Measures
• Biological containment
Host-vector systems designed to minimize:
• Survival of the vector in its host outside the laboratory
• Transmission of the vector to other non-laboratory hosts
• Physical containment
• BL1 BL4
• Large scale containment
• “BL+”
• Containment for animal testing facilities

Host-Vector Systems
HV-1
• Moderate level of
biological containment
• E. Coli K12 with nonconjugative
plasmid or
variants of bacteriophages
such as λ. No conjugative
plasmids nor generalized
transducing phages
• Other hosts comparable to
E.coli K12
HV-2
• High level of biological
containment
• Escape of DNA via survival
or transmission to other
organisms

Physical Containment

BL1
• Practices
• Lab access restricted
• Work surfaces decontaminated every day and after any
spillage
• Contaminated liquid/solid waste decontaminated before
disposal (transfer to distant decon site in leak-proof
container)
• No mouth pipetting
• No smoking, eating, drinking, no food storage in lab
• Hand wash procedures
• Avoid aerosols
• Protective clothes & change rooms
• Insect and rodent control program in place

• Facilities - equipment
BL1
• Lab design to permit easy cleaning
• Benchtop impervious to water, acids, alkalis, organic
solvents, moderate heat
• Sturdy lab furniture, space between benches & cabinets
for easy cleaning
• Hand wash sink
• Screens if windows can be opened

BL2
• Practices: BL1 +
• Training and information of personnel
• Specific requirements: immunizations
• Serological monitoring of personnel, if appropriate
• Hazard warning sign identifying pathogen and name + tel
of prioncipal investigator
• Dedicated protective clothes & change rooms
• Gloves to avoid skin contact with rDNA molecules and for
handling experimental animals
• Use of sharps restricted to the strict minimum,
autoclaveable sharp disposal container
• Spill accidents reporting to the NCA
• Biosafety manual

BL2
• Facilities - equipment: BL1 +
• Windows sealed
• Biological safety cabinets (cat. I or II)
• Other appropriate personal protective or physical
containment whenconducting procedures with high
potential of creating aerosols (grinding, centrifuging, sonic
disruption…)
• Centrifugation in the open lab with sealed safety cups,
opening under biological safety cabinet

• Practices: BL2 +
BL3
• Lab doors closed when work in progress
• All activities in biosafety cabinet
• No operation in open vessels conducted on benches
• Use of plastic-backed paper toweling for easy cleaning
• Dedicated protective clothes & change rooms,
decontaminated prioe to laundering or disposal
• Gowns including surgical masks
• Respirator in rooms containing experimental animals
• HEPA filtration of vacuum lines

BL3
• Facilities - equipment: BL2 +
• Biological safety cabinets (cat. I, II or III)
• Lab separated from areas of unrestricted traffic flow
• Passage through a set of two doors
• Double-door clothe change room with airlock
• Shower optional
• Wall, floor, ceiling impervious to water, acids, alkalis,
organic solvents, moderate heat
• Thru-wall pipework, cables etc… sealed
• Foot-, elbow-operated or automatic hand wash station
• Windows closed and sealed
• Self-closing access doors
• Decon autoclave preferably in the lab

BL3
• Facilities - equipment: BL2 + (contd.)
• Ducted exhaust air ventilation system creating directional
airflow that draws air into the lab from the entry area
• Exhaust air from the lab room may be dischared to the
outside withoout being filtered
• Exhaust air away from air intakes and not re-circulated
• HEPA-filtered air from biosafety cabinets Cat. I or II is
dischared directly through the builmding exhaust system;
in this case, interference with the lab air balance must be
avoided
• HEPA-filtered air from biosafety cabinets Cat. I or II may
be recirculated within the lab provided the cabinet is tested
and certified at least every 12 months

BL4
• BL4 units (4 total in the world) are for lab scale
operations with the most dangerous viruses (Ebola,
Lassa, Marburg…)
• Agents mostly lethal, no prevention, no cure
• NIH guidelines provide the requirements for lab scale
BL4 containment. Large scale operations under this
type of environment are not considered.

Large Scale
Physical Containment

Large Scale
• History (EU)
• Large scale (>5 or 10 L) required an increase by one
stage of the containment level
e.g. B. pertussis (BL2) BL3 at large scale
• Appendix K (NIH): BLx BLx-Large Scale
• BL1-Large Scale recommended for production of viable
organisms containing rDNA that require BL1 at lab scale
• BL2-Large Scale recommended for production of viable
organisms containing rDNA that require BL2 at lab scale
• BL3-Large Scale recommended for production of viable
organisms containing rDNA that require BL3 at lab scale
• No provisions for production of viable organisms
containing rDNA that require BL4 at lab scale

Large Scale: Principles
• Primary containment
• Closed systems: fermentor, bioreactor, Westfalia
separator…, CIP-SIP
• Open system in an enclosure: centrifuge, filtration
system in biosafety cabinet…
• Secondary containment
• Controlled area design: airlocks, change rooms, negative
pressure in controlled area, self closing doors, dedicated
decontamination autoclave, exhaust air handling…

BL1-Large Scale
• = BL1 +:
• Reporting of spills and accidents resulting in overt
exposure of organisms reported to Lab Director; medical
evaluation, surveillance and treatment of staff as
appropriate
• Anything >10 L must be in a closed system; smaller
cultures (seeding etc…) can be handled outside a closed
system provided all BL1 physical containment
requirements are met
• Culture fluid (including samples…) shall not be removed
from a closed system unless inactivated by a validated
inactivation procedure
• Sample collection from-, addition of materials to a closed
system conducted to minimize aerosol & contamination

BL1-Large Scale (contd.)
• Exhaust gases removed from a closed system or other
primary containment equipment treated by filters
equivalent to HEPA filters or other processes
(incineration…) to minimize the release of viable
organisms
• No opening of closed system until after sterilization
• Emergency plan: procedures to handle large losses of
culture on an emergency basis

BL2-Large Scale
• = BL1-Large Scale +:
• Anything >10 L must be in a closed system; smaller
cultures (seeding etc…) can be handled outside a closed
system provided all BL2 physical containment
requirements are met
• Rotating seals of a closed system designed to prevent
leakage of viable organisms (e.g. double mechanichal
seal with steam circulation)
• Sensing devices to monitor integrity of containment
during operations
• Closed system integrity testing: prior to usage and after
replacement or modification of containment features;
Procedures for testing appropriate to the equipment
design (e.g. supplier’s specs);

BL2-Large Scale (contd.)
• All operations, interventions and testing recorded
• Biosafety sign posted on each closed system and
primary containment equipment when in use

• = BL2-Large Scale +:
BL3-Large Scale
• Anything >10 L must be in a closed system; smaller
cultures (seeding etc…) can be handled outside a closed
system provided all BL3 physical containment
requirements are met

BL3-Large Scale (contd.)
• Specific lab requirements: closed systems and
primary containment equipment located in a
dedicated area which:
• Has a separate entry area: double doored space such as
an airlock & change room
• Surfaces of walls, ceilings and floors permit easy
decontamination
• Sealed thru-wall tubing, cables etc…
• Hand washing facility (foot-, elbow- or automatic
operation) near each major work area and near each
primary exit
• Shower facility in close proximity to the controlled area

BL3-Large Scale (contd.)
• Controlled area designed to preclude release of fluids
outside the controlled area in case of accidental spill
• Decontamination procedure in place
• Ventilation controlling air movement; movement must be
from areas of lower contamination potential to areas of
higher contamination potential
• If the ventilation system provides positive pressure
supply air, the system must operate in a way that
prevents the reversal of air movement
• Exhaust air from controlled area is not recirculated to
other areas of the facility
• Exhaust air must be treated (HEPA-filtered, thermal
oxidation…) to prevent the release of viable organism

BL3-Large Scale (contd.)
• Personnel operational procedures
• Entry exclusively through entry area
• City clothes exchanged with work garments such as
jump suits, lab coats, pants & shirts, head cover, shoe
covers
• On exit from controlled area, work clothing is stored in a
separate locker than the entry locker; clothing must be
decontaminated before laundering.
• Entry restricted to authorized personnel, no authorization
under 18 years of age
• No animals or plants in controlled area
• Access doors kept closed except as strictly necessary;
service doors sealed & locked during operations
• Hand wash upon exiting

What Category is
our Bp Strain?

Wild type B. pertussis
ptx (S1…S5)
Cm R
pSS4245::Gen R
ptx promoter
I-SceI gene
I-SceI site
Cointegrate (Str R, Cm R )
Cm R
Str R
Str R active in B. pertussis, NOT in E. coli
Conjugative transfer, Str selection,
ptx repression
ptx induction
DSB
Wild type (~50%)
Resolution
Cm R (~50%)
Cm R
ptx (S2…S5)

B. Pertussis (Cm R ) Cm R ptx (S2…S5)
S1*
pSS4245::S1*
ptx promoter
I-SceI gene
I-SceI site
Cointegrate (Str R , Cm R )
Str R active in B. pertussis, NOT in E. coli
Conjugative transfer, Str selection,
ptx repression
Str R
ptx induction
DSB
Cm R
Cm R (~50%)
Resolution
S1*, Cm S (~50%)
ptx (S1*…S5)

• NIH scope
Our rec. B. pertussis strain?
• Irreversible loss of active pertussis toxin, an
important virulence factor
• However, B. pertussis produces several other toxic
and non-toxic virulence factors which are still
present: dermonecrotic toxin, tracheal cytotoxin,
adenylate cyclase/hemolysin, FHA, pertactin…
Bp 9K-129G remains a RG II pathogen
Production in BL2-Large Scale

Our rec. B. pertussis strain?
• EU scope
• Probably not subject to EU directive on the contained
use of GMO:
“Self-cloning consisting in the removal of nucleic acid
sequences from a cell of an organism which may or
may not be followed by reinsertion of all or part of
that nucleic acid (or a synthetic equivalent) with or
without prior enzymic or mechanical steps into cells
of the same species…”
Bp 9K-129G has to be treated as wt B. pertussis
Production in BL2-Large Scale

Our Pilot Plant?
• General industry trend
• Do more than strictly required:
- guidelines are always evolving
- modifying an operating plant is difficult and costly
• Necessity to combine:
- GMP (protect the product)
- Biosafety (protect staff and environment)
Our Ayuthaya facility is destined to a RG II agent, thus
requiring BL2-LS but is designed to qualify to BL3-LS