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Ben Madsen, Jordan Cobia,

Paula Decaria, Nephi Jones R&D - SUT

High Density Culture Strategies for Improved Scalability with Single-Use Systems

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Abstract

Current study shows

• Integration of

• Thermo Scientific HyPerforma Single-Use

Bioreactor (SUB)

• XCellTM ATF6 Single-Use System

• The S.U.B. was able to support high-density

cultures

• >40E06 cells/mL

• Comparison with

• XCellTM ATF6 Stainless Steel System

• Scale-up criteria for both S.U.B. and ATF

• The data show that the S.U.B. and ATF can

be used

• in a high-density seed train

• or as a production vessel system.

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• The industry is moving toward high-density

and high-productivity cultures with a desire to

reduce

• processing times

• hardware footprint

• Applications include:

• perfusion

• concentrated fed-batch

• intensified seed trains

INTRODUCTION

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• Used in conjunction to explore the capabilities

of the bioreactor and controller in achieving

high cell density cultures

INTRODUCTION

• HyPerforma S.U.B.

• Repligen ATF6 perfusion system

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MATERIALS AND METHODS

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Table 1. Culture components and cells

Components/

Conditions Stainless Steel ATF Single-Use ATF

Cell Line

CHO-DP12

(ATCC# 1934)

Adapted to LONG®R3IGF-I

Media Composition

CD OptiCHO media

+ 100 ng/mL LR3

+ 4 mM GlutaMAX

Base 1.0N NaOH N/A

Antifoam 10,000 ppm Antifoam C as needed

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MATERIALS AND METHODS

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Table 2. Bioreactor conditions

Components/

Conditions Stainless Steel ATF Single-Use ATF

Bioreactor Thermo Fisher

Bioreactor W. Vol. 40L

Temp 37°C

pH 7 (CO2/1N NaOH) 7.0 ± 0.2 (CO2)

Agitation 172 rpm (20 W/m3)

DO Set point

50%

(Pure O2 through standard

drilled hole sparger)

30%

(Pure O2 through standard

drilled hole sparger)

Air Headspace sweep

at 3 L/min Air

Antifoam dosing

20 ppm/day

(Based on Visual

Observation)

7 ppm/day (Controlled via

foam sensor)

Glucose Feed No glucose feeds

Seeding Density ~0.4E6 Cells/mL ~0.4E6 Cells/mL

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Drilled-Hole Sparger

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ATF conditions

Components/ Conditions

Stainless Steel ATF Single-Use ATF

ATF System ATF 6 ATF 6 SU

ATF Filter F6:RF02PES

0.2 µmPES HF Filter SA 2.53 m2

Perfusion rate

Day 0-3: None Day 0-3: None Day 3-4: 1 BRV

(40L/day) Day 3-5: 1 BRV (40L/day) Day 4-5: 1.5 BRV

(60L/Day) Day 5-end: 2 BRV

(80L/Day) Day 5-end: 2 BRV

(80L/Day) Flux 1.67 LMH 1.67 LMH

ATF Rate 12 LPM 19.2 LPM Shear Rate 1415 s-1 2264 s-1

Cell Bleed Constant

Target Cell Density 40E06 cells/mL

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MATERIALS AND METHODS

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Figure 1. Viability and viable cell density

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Figure 2. IgG production

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Figure 3. Total cumulative protein vs. IVCC

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Figure 4. Oxygen flow rates and viable cell densities

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• ATF Shear Rate: 2021 s-1

• SUB kLa: 60 h-1 (using O2)

Theoretical evaluation of scale-up criteria for S.U.B. and ATF

S.U.B.

Total / Working

Volume

ATF

System

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Available Filters

(m2)

ATF Flow Rate

(L/min) LMH filtrate

1” Connection

Tubing Δp (psi)

DHS Flow at 0.1

VVM (L/min)

kLa (hr-1)

(using O2 gas)

50L / 40L ATF4/6 0.8/2.5 8.4 or 14.9 2.5 or 1.25 <0.01 5 58

100L / 100L ATF6 2.5 14.9 2.5 <0.01 10 59

250L / 200L ATF6 4.2 25.1 3.0 0.02 25 60

500L / 500L ATF10 11 68.4 2.84 0.07 50 68

1000L / 1000L ATF10 11 68.4 5.7 0.25 100 67

2000L / 2000L 2 ea ATF10 2 ea @ 11 68.4 5.7 0.25 200 76

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• Stable cell density of >40E06 cells/mL among all cell runs with a 20-day culture using the ATF SU

at 2 VVD.

• Growth limits due to glucose consumption; cell growth independent of bioreactor and ATF

performance.

• Protein production similar among cell runs, indicating equivalent performance.

• Drilled-hole sparger in the 50L S.U.B. capable of meeting oxygen demand as a stand-alone

sparger.

• System scale-up based on achievable ATF shear and flux rates is shown.

Conclusions

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FUTURE STUDIES

• Added glucose feed to determine maximum

cell density achievable in 50L S.U.B.

• Characterization of high-density culture

solutions and the effect on sparging dynamics

and mass transfer.

• Use high-density culture to seed larger vessel

at higher seed density (>10X seed density) to

show feasibility of high-concentration seed

train.

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Questions

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