Post on 28-Nov-2014
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Cholesterol Delivery to NS0 Cells: Challenges and Solutions in Disposable Bioreactors
BioEdge Consulting, LLC
Outline
• Background
• Process Development Project
• Discovery of Negative Interaction
• Investigational Experiments
• Development of Solution
• Recommendations
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Background – Wave Bioreactors
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• Disposable bioreactor with wave-like motion to mix and aerate
• Working volumes from 1L to 500L
• Constructed of three layers with a LLDPE contact surface
• Offers facility flexibility and reduces capital costs
• Quicker turnaround due to avoidance of many SIP/CIP procedures
Background – NS0 Cells
• NS0 and CHO: common mammalian cell lines for mAb production
• NS0 cell line derived from IgG-producing murine myeloma cells
• Glutamine synthetase (GS) selection system to increase recombinant protein expression
• NS0 cells are typically cholesterol auxotrophic; require exogenous source
• Cholesterol-independent cell line at Merck
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• Cholesterol is a hydrophobic molecule with a hydrophilic head found in the lipid bilayer of the cell membrane
• Needs a carrier molecule to dissolve in aqueous solutions
– Albumin protein in Fetal Bovine Serum (FBS)
– Fatty acids in Serologicals EX-CYTE (animal sourced)
– mβCD in Invitrogen Cholesterol-Lipid Concentrate (CLC, non-animal sourced)
Background – Cholesterol
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Background – mβCD
• Methyl-beta-cyclodextrin (mβCD) is a ring-like oligosaccharide with a hydrophilic exterior and a hydrophobic interior cavity
• Encapsulates and solubilizes hydrophobic molecules in aqueous solutions
• Routinely used in food and medical industries, safe for human consumption
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Process Development Project
• Production of a therapeutic mAb in GS-NS0
• Scaled-up seed train into Wave bioreactors
• Used cholesterol-dependent NS0 cell line due to concerns of product quality impact
• Goal to switch cholesterol source from FBS to CLC to avoid animal-sourced components
• Planned initial Wave experiment comparing FBS, EX-CYTE, and CLC
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Discovery of Negative Interaction
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• Experiment involved three 2L Waves containing Merck Proprietary Medium (MPM)1. FBS (Hyclone, 5% vol/vol)
2. EX-CYTE (Serologicals, mfr. instructions)
3. 250x CLC (Invitrogen, mfr. instructions)
• Each Wave inoculated at ~0.15x106vc/ml with subsequent sterile removal of 50ml aliquot for shake-flask
• Cultivated in 5% CO2 for five days
Discovery of Negative Interaction
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● Cholesterol-Lipid Concentrate
▲ EX-CYTE
Fetal Bovine SerumSolid lines = Wave BioreactorDotted lines = Shake-flask
Investigation – Time of Interaction
• Interaction between cholesterol source, Wave Bioreactor, and NS0 cells
• Natural cell death is slower: toxic phenomena
• Next question: When does it happen?
• Experiment #2:
– Wave with MPM+CLC inoculated as before, with control flask drawn 0hr
– Additional aliquots drawn at 4hr, 7.5hr, and 22hr post-inoculation and transferred to flasks
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Investigation – Time of Interaction
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● Control Wave bioreactor Time = 0hr flask▲ Time = 4hr flask Time = 7.5hr flask Time = 22hr flask
MPM + CLC
Investigation – Cultivation Conditions
• Cells removed at 4hr dropped to 20% viability, but increased to 70% by d7 → recovery possible
• Irreversible cell damage occurred between 4 and 7.5hr post-inoculation
• Next question: Are cells more sensitive to cultivation conditions in presence of CLC?
• Experiment #3:
– Same 2L Waves, MPM, and CLC concentration
– Vary CO2 percentage and flow (pH control)
– Vary Wave platform rocking rate and angle
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Investigation – Cultivation Conditions
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2L Wave Cultivation
Condition (MPM with
CLC)
Standard Process Value Changed Value
Cell Growth in
Wave
Bioreactor
Cell Growth in
Control Shake-
Flask
CO2 Delivery Initial Fill, 5% CO2 Initial Fill, 0.1% CO2 No Yes
CO2 Delivery Initial Fill, 5% CO2 Continuous, 5% CO2 No Yes
CO2 Delivery Initial Fill, 5% CO2 Continuous, Variable CO2* No Yes
Rocking Rate 17 rpm Static No Yes
Rocking Rate 17 rpm 4 rpm No Yes
Rocking Rate 17 rpm 9 rpm No Yes
Rocking Rate 17 rpm 25 rpm No Yes
Rocking Angle 8º 4º No Yes
Rocking Rate & Angle 17 rpm, 8º 4 rpm, 4º No Yes
* To maintain pH 7.15
Investigation – Chemical Conditions
• In all cultivation conditions, cells grew normally in flasks and died rapidly in Waves
• Next question: Is it the chemical conditions? Literature shows mβCD can extract cholesterol from membranes
• Experiment #4:
– Include mβCD and cholesterol separately
– Use cholesterol-independent cells to test if toxicity occurs, not just cholesterol depletion
– Decouple physical environment by placing “coupons” of Wave LLDPE in PC flasks
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Investigation – Chemical Conditions
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Solid lines = SF with couponsDotted lines = SF without coupons
Cholesterol-Lipid Conc. (CLC) Methyl-β-Cyclodextrin (mβCD)
Cholesterol (synthetic)
Identification of Problem
• Rapid cell death in CLC flask w/ coupons, but not in control flask, points to interaction between LLDPE surface and CLC
• Normal growth in both cholesterol flasks, with and without coupons, rules out influence
• Poor growth in mβCD flask shows carrier molecule is likely the culprit
• Experiment #5
– Confirm above results in 2L Wave Bioreactors
– Include Waves with cholesterol-mβCD complex (literature ratio) and no cholesterol additive
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Identification of Problem - Confirmation
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▲ Cholesterol mβCD mβCD-cholesterol complex No additiveSolid lines = Wave BioreactorDotted lines = Shake-flask
Mechanism
• Since cyclodextrins are known to extract cholesterol from membranes, propose that mβCD in excess depletes cells of membrane-bound cholesterol
– This depletion likely occurs in all vessels, but in equilibrium with reverse reaction
– LLDPE may irreversibly entrap cholesterol-mβCD complexes until cell membranes depleted
– Other papers propose ink-bottle-like pores in LLDPE surface
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Proposed Three-Way Interaction
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Development of a Solution
• Experiment #6:
–Use cholesterol-dependent NS0 cells
–Minimize excess free mβCD by lowering ratio of cholesterol to mβCD, but still keep cholesterol soluble
–Reduce overall concentration of cholesterol close to minimum needed by cells
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Development of a Solution
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Medium
Formulation
Weight Ratio
Cholesterol to
mβCD
Molar Ratio
Cholesterol to
mβCD
Presence of
Wave LLDPE
Coupons
Cell Growth in
Flask
CLC n/a n/a Yes No
2mg/L chol-mβCD 1:70 1:21 Yes No
2mg/L chol-mβCD 1:45 1:13 Yes No
2mg/L chol-mβCD 1:25 1:7.3 Yes No
3mg/L chol-mβCD 1:2 1:0.6 Yes Yes
3mg/L chol-mβCD 1:2 1:0.6 No Yes
Proof of Concept in 2L Wave Bioreactors
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3mg/L cholesterol1:21M ratio cholesterol:mβCD
1.2mg/L cholesterol1:0.6M ratio cholesterol:mβCD
1.8mg/L cholesterol1:0.6M ratio cholesterol:mβCD
2.4mg/L cholesterol1:0.6M ratio cholesterol:mβCD
Recommendations
• Use a cholesterol-independent cell line when possible, but confirm there is no impact on product quality
• GE now makes Wave disposable bioreactors in EVA and nylon/EVOH, although need to be tested for each process
• With LLDPE bioreactors, use excess of cholesterol to cyclodextrin in medium (1:0.6)
• GE now recommends pretreatment of Wave bags with 5x lipid supplement overnight (procedure 28-9308-85AA)
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Conclusions
• A “roadblock” interaction was observed in Waves using CLC in place of FBS
• A series of investigative experiments were performed to determine root cause
• Proposed root cause was depletion of membrane cholesterol and irreversible entrapment of cholesterol-mβCD complex on the LLDPE surface; caused by excess mβCD
• A reduced molar ratio of 1:0.6 cholesterol to mβCD overcame negative interaction (2.4mg/L cholesterol optimal)
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Backup Slides
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Discovery of Negative Interaction - Viability
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Investigation – Time of Interaction - Viability
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Investigation – Chemical Conditions - Viability
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Solid lines = SF with couponsDotted lines = SF without coupons
Cholesterol-Lipid Conc. (CLC) Methyl-β-Cyclodextrin (mβCD)
Cholesterol (synthetic)
Identification of Problem - Viability
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