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Equipment Challenges
Heater
– Problem: As perfusion ramped up, difficulty maintaining temperature at set point – Investigation:
Checked PID parameters and RTD probe Increased set point to compensate Following run, removed heating blanket cover to ensure blanket contact with vessel; tested electrical wiring
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SUB History at Centocor
Centocor has three sizes of Hyclone SUBs
– 50 L – 250 L – 1000 L
Centocor has SUBs in two facilities
– Development Pilot Plant – Small Scale Clinical Pilot Plant
25.0
3 L Glass Run 2
30.0
100 L Stainless Steel
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Process Challenges
DO
– – – – Problem: Day 6: DO reading drifting down Investigation: Actual DO verified by offline BGA sampling Resolution: Probe grounding modified Effects: DO variations may have led to drop in viability
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Perfusion
DEFINITION: Continuous bioreactor operation with cell retention to achieve high cell density cultures
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Perfusion
Advantages of Perfusion Smaller reactors needed Healthier culture with less wastes leads to enhanced product quality Higher cell densities Less frequent bioreactor turnaround Clean harvest stream for DSP
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ATF Technology
22”
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Hyclone SUB Setup
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Hyclone SUB Setup
22”
ATF hookup
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SUB with ATF Setup
To cold room
Outline
Background on Perfusion Comparison of Disposable vs. Conventional Reactor Performance Process and Equipment Challenges090706 Bhatia
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Project Motivation
50 L SUB 100 L Stainless Steel 3 L Glass Run 1 3 L Glass Run 2
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Run Data Comparisons
Titers
Titers 0.0
5.0
50 L SUB
10.0
15.0 Time (days)
20.0
3 L Glass Run 1
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Run Data Comparisons
Viable Cell Density
25.0 VCD (e6 cells/mL) 20.0 15.0 10.0 5.0 0.0 0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 Time (days)
7
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ATF Technology
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ATF Technology
Driving Pressure (DP):
– Pushes up the ATF diaphragm from the bottom of the pump housing, maintains the movement of diaphragm in the pump head, and drives the cell culture from the pump head back to the bioreactor.
– Resolution: SUB BPC manufacturing size error led to poor contact between heating blanket and process fluid – Effects: Temperature stayed within recommended operating parameters
All SUBs thus far have been used for fed-batch only However, the next several products in Centocor’s pipeline are all perfusion processes
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DP = Head Space Pressure + Hydrostatic Pressure + Dynamic Pressure Drop in Piping
0
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ATF Technology
Characteristics of ATF-4
– – – – – – – Filter surface area: 0.42 m2 Filtration capacity: 30 L/day Pump displacement volume: 380 mL Flow rate: 3.8 L/min Number of fibers/cartridge: 520 Lumen void volume: 160 mL Lumen cross sectional area: 4.1 cm2
50L SUB 100 L Stainless Steel 3 L Glass Run 1 3 L Glass Run 2
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Run Data Comparisons
Viability
100.0 90.0 80.0 % Viability 70.0 60.0 50.0 40.0 30.0 20.0 0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 Time (days)
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Process Conditions
pH Control: Sodium Carbonate and Carbon Dioxide – Set point: 7.4 DO Control: Air sparge and Oxygen sparge – Set point: 40% Perfusion Profile: – Day 2: 0.2 bioreactor volumes/day – Day 3: 0.4 bioreactor volumes/day – Day 4: 0.8 bioreactor volumes/day – Day 5 and on: 1.0 bioreactor volumes/day
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Equipment Challenges
Exhaust filter
– Problem: On day 28, exhaust filter clogged leading to pressure buildup in BPC – Investigation:
No foam problems Temporary venting through overlay filter Following run, filter examined for visual failure Early in run, filter heater not grounded
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Equipment Challenges
Agitator
– Problem: Periodic grinding noises generated during operations – Investigation: Removed shaft and inspected for wear following run; small particles had rubbed off – Resolution: Reported to Hyclone for further investigation – Effects: None
To demonstrate an established perfusion process in a single-use bioreactor (SUB) To compare disposable bioreactor perfusion data with conventional (glass and stainless steel) data at different scales