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

• 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
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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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Background – Cholesterol
• 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)
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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 6
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

• 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
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Discovery of Negative Interaction

● Cholesterol-Lipid Concentrate
▲ EX-CYTE
 Fetal Bovine Serum
Solid lines = Wave Bioreactor
Dotted lines = Shake-flask

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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
MPM + CLC

● Control Wave bioreactor

 Time = 0hr flask
▲ Time = 4hr flask
 Time = 7.5hr flask
 Time = 22hr flask

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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

2L Wave Cultivation Cell Growth in Cell Growth in

Condition (MPM with Standard Process Value Changed Value Wave Control Shake-
CLC) Bioreactor 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

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* 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

Cholesterol-Lipid Conc. (CLC) Methyl-β-Cyclodextrin (mβCD)

Solid lines = SF with coupons

Dotted lines = SF without coupons

Cholesterol (synthetic)
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 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

▲ Cholesterol
 mβCD
 mβCD-cholesterol complex
 No additive
Solid lines = Wave Bioreactor
Dotted lines = Shake-flask

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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

Weight Ratio Molar Ratio Presence of

Medium Cell Growth in
Cholesterol to Cholesterol to Wave LLDPE
Formulation Flask
mβCD mβCD Coupons
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

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Proof of Concept in 2L Wave Bioreactors

3mg/L cholesterol 1.2mg/L cholesterol

1:21M ratio cholesterol:mβCD 1:0.6M ratio cholesterol:mβCD

1.8mg/L cholesterol 2.4mg/L cholesterol

1:0.6M ratio cholesterol:mβCD 1:0.6M ratio cholesterol:mβCD

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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

Cholesterol-Lipid Conc. (CLC) Methyl-β-Cyclodextrin (mβCD)

Solid lines = SF with coupons

Dotted lines = SF without coupons

Cholesterol (synthetic)
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Identification of Problem - Viability

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