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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