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Improving Gas Liquid Mass Transfer and

Scale-Up for Single-Use Bioreactors

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Introduction
Goal is to improve sparging system

What is good sparging?

Efficiency
O2 delivery vs. CO2 stripping
Shear effects
Foam generation

How does it scale?

Agitation
Relative gas entrance distribution
Liquid column height
Gas partial pressure concentration
Gas flow rate
Pore material & design

What is gained?
Better cell culture environment
Lower operating cost
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Figure 1: Sintered PVDF Micro Sparger

What is kLa?
Comes from simplified gas liquid film theory equation:
NL = kLa(CLi CL )
kL is the transfer coefficient
a is the area of flux
CLi is the interface concentration
CL is the liquid bulk concentration

NL is the rate of transfer

Liquid

Driving factors
Differential partial pressure
Surface area available for transfer vs.
volume
Gas bubble liquid film thickness

Gas bubble liquid film resistivity

Figure 2: Simplified Mass Transfer Diagram

Measuring O2 and CO2 Transfer


Measure kLa via Dynamic Method1
Commonly used for measuring O2
delivery performance

O2

CO2

100

% Gas
Saturation 80
60

Can assess CO2 stripping as well


Repeatable, consistent and inexpensive

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20
0

O2 vs. CO2 considerations

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83

85

87

89

Minutes
Figure 3: CO2 Stripping Data Vertically Mirrored On O2

Solubility
Operating partial pressure

Note: CO2 sensor limits exceeded causing signal saturation near 20%.

Table 1: Driving Partial Pressure Delta Available to Strip CO2 and Add O2 in a Typical Animal Cell Culture Bioreactor (in atm
assuming 1 atm ambient pressure) and Corresponding Liters of Dissolved Gas in DI H2O at 37C2,3
Reactor dissolved
O2 set point
30% air saturation
50% air saturation

CO2 stripping partial

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O2 delivery partial pressure delta

Liters dissolved gas

pressure delta with air

sparging with air

sparging with O2

CO2

O2

0.06

0.147

0.937

47.4

3.3

0.06

0.11

0.89

47.4

5.6

CO2 Stripping vs. O2 Delivery Ratio


CO2 is produced by cells and is added to control pH
At some point, CO2 production becomes greater than stripping

Why do we care?
Dissolved CO2 Carbonic acid
Lowers vessel pH
Base is added to regulate pH
Vessel osmolality increased

CO2 ~ transparent to cell membrane


Lowers cell pH
Cells expend energy to handle excess CO2

CO2 buildup negatively affects cell growth and product yield

Current Typical Sparger Design


Micro sparger
Sintered (polymer, metal, glass/ceramic)
Generates a bell curve of small bubbles
(typically < 1 mm)

Variable
Membrane/mesh (polymer, metal)
Can generate small or large bubbles
depending on mesh density and material

Macro sparger
Open pipe (polymer, metal)

Drilled hole; disc, bar, ring (metal)

Figure 4: Various Sparger Designs

Table 2: Generalized Typical Sparger Characteristics


Sparger Type
Ave. bubble dia. (mm)
Size distribution
Pre-distribution
Efficiency
Flow rate
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Sintered
<1
wide bell
low
high
low

Membrane
< 1 to > 3
narrow to wide bell
low
low to high
low to high

Open Pipe
>2
wide bell
low
low
mid to high

Drilled Hole
>1
uniform to wide bell
low to high
low to mid
mid to high

Why Dual Sparger Configurations?


A single sparger cannot be tuned efficiently for all environments
Different reactor size (scaling the same geometry the same behavior)
Different liquid column height
Different agitation behavior
Different proximity to sparger and bulk liquid

Different cell culture/cell lines/ processes


Different mass transfer demands
Different media and solution behavior

Time
Bulk solution changes during culture operation

Dual spargers allow adaptive load balancing between micro and macro
for best CO2 stripping verses O2 kLa
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General Bubble Behavior

Figure 5: Trending With Respect to Bubble Diameter4,5


Note: Approximation assumes laminar free-rise bubble behavior

Small bubble
High surface/vol. + high residence time = high
efficiency (generally high kLa)

PROs

CONs

Increased risk of cell damage


Increased risk of foam issues
Increased risk of gas hold up
Poor CO2 stripping vs. O2 delivery ratio
Low gas throughput supported

Large bubble

High CO2 stripping vs. O2 delivery ratio


Reduced foam issues
High gas throughput supported
Reduced risk of cell damage (unless
excessive entrance velocity)6

Lower efficiency (can lead to very poor kLa)


Tall column heights and high agitation
can mitigate

Chaos and Randomized Bubble Size Generation


Chaos = wide bell curve of bubble size

Plateau
Region

0.6

12

0.5

10

0.4

0.3

0.2

0.1

0
0

kLa ratio (1 = 2.4 mm dia.)

Normalized Distribution

Reduced effectiveness
Skewed CO2 stripping / O2 delivery ratio
kLa performance after volume and kLa
skewing applied
Original bubble size standard deviation
Volume skew (4/3*pi*d/2^3)
kLa Skew 7.954*d^2.48 + diminishing
returns near 0.078 mm Diameter

Bubble Diameter (mm)

Figure 6: kLa and Volume Shifts of Bubble Diameter Bell Curve at 250L With Target Ave. Bubble Dia. of 2.4mm

Note: Area under the green curve is reduced over 20%; additionally skewing based on actual data, bell curve having normal distribution is
an assumption purely for demonstrative purpose

Sintered Micro Sparger = typically below plateau region


Bubble sizes generated from a sintered sparger produce a wide bell curve
Bell curve sits below equilibrium threshold
Increased risk of foam stability, cell damage, gas hold-up issues
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Cell Damage From Sparge Systems


Mechanical cell damage is relative to culture sensitivity
Small bubble size (sub 0.5 mm)
High entrainment and high shear when bursting per volume of gas

High gas entrance velocity (> 30 m/s)


Direct shear damage at high gas entrance velocities6

Impeller shear
Sparge systems dependent on high agitation may require damaging impeller
speeds

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Macro Sparger: Stainless vs. Polymer


Surface energy
Stainless steel high energy material; favors water over air7
Pores release bubbles more readily

Exhibit additional release behaviors earlier


Water layer on pore can shrink the functional size
o

Native polyethylene polymers are low energy; favors air (contact angle > 90 )8
Pores will hold onto a bubble more aggressively

Same size pores in polyethylene spargers generate larger bubbles than in metal

Pre-distribution
Stainless vessels = wide separation between pores
SUS vessels = sparge mechanism in small area
Results in loss of efficiency

Agitation and velocity dependence (chaos by design)


Typical stainless macro spargers are dependent on one or both:
High gas velocities
Impeller to break up large bubbles

Matching sparger pore size and quantity from stainless steel to single-use may
not give equivalent results
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Initial Investigation
Single pore analysis using existing film disc tooling
Laser-generated pores from 0.02 to 6mm diameter
Study behavior with precision mass flow controller (MFC) and high speed
camera
Determine relative bubble size generated by a given pore size

Observed pore behavior w/ respect to bubble generation (3 phases)


Pulse modulated (release single bubbles, ~same size)
Bubble size increase (bubble size increases notably with flow rate)

Chaos (jetting, tearing, shattering, wide bell curve of sizes)

Term the flow limit of pulse modulated behavior as pore saturation

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Initial Investigation Results


Micro sparger
Micro spargers in drilled hole form are cost-prohibitive
Small pore size = low flow rate
Require > 3000 pores for a 250L reactor = $$$

Macro sparger
Initial testing with two boundaries
Mid laser capability 0.175mm pore diameter
Pore saturation estimate (0.1vvm, 500 pores)

Results shows behavior consistent with single pore study


kLa per hour 20

Pulse
modulated

Pore
saturation

Bubble size
increase

0.05

0.1

Chaos
begins

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12
8
4
0
0

0.15
DHS AIR VVM

0.2

0.25

Figure 7: 250L Drilled Hole Sparger 500 Pores 0.203mm dia. O2 kLa, 139rpm, 6.67cm dia. Disk
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0.3

Column Height Analysis


Frit demonstrates ~100% efficiency (bubbles equilibrate, scale with vvm)
Table 3: kLa Scaling With Liquid Column Height Using Sintered Polymer Frit: Pre-Saturation Flow Rates
Vessel
Vol (L)
50
250
2000

VVM
0.005
0.005
0.005

O2 Delivery
kLa
kLa/cm
9.91
0.190
9.56
0.105
9.77
0.055

CO2 Stripping
kLa
kLa/cm
1
0.019
.96
0.011
.89
0.005

%CO2/O2
kLa
10.1%
10.0%
9.1%

Note: Frit slight CO2 stripping reduction with larger vessels likely attributed to buildup in headspace and hold-up gasses

0.178mm pore size CO2 partial pressure equilibrates faster than O2


Table 4: kLa Scaling With Liquid Column Height Using 0.178mm Pore DHS: Pre-Saturation Flow Rates
Vessel
Vol (L)
50
250
1000

VVM
0.0125
0.0125
0.0125

O2 Delivery
kLa
kLa/cm
2.05
0.039
3.54
0.039
5.71
0.040

CO2 Stripping
kLa
kLa/cm
1.06
0.020
1.59
0.017
2.08
0.015

%CO2/O2
kLa
51.4%
44.8%
36.5%

0.838mm pore size shows bubbles not approaching equilibration (scale with column ht.)
Table 5: kLa Scaling With Liquid Column Height Using 0.838mm Pore DHS: Pre-Saturation Flow Rates
Vessel Vol
(L)
1000
2000

VVM
0.05
0.05

O2 Delivery
kLa
kLa/cm
7.49
0.053
9.98
0.056

CO2 Stripping
kLa
kLa/cm
3.95
0.028
4.82
0.027

%CO2/O2
kLa
52.7%
48.3%

Note: 2000L vessel gets slight efficiency bump from using 2 discs instead of one; better pre-distribution

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Refined Analysis
Pre-distribution study performed
Size discs as large as possible

Initial results = refined targets


Achieve ~50% CO2 stripping / O2 delivery kLa ratio

Figure 8. Drilled Hole Sparger Disc


Representation

Culture tests show ideal ratio is ~33%

Achieve O2 delivery kLa > 10 (0.1vvm & ~29 W/m3 agitation)


Refine laser approach
250L 30m s threshold

pore saturation

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kLa 1/hr

15
10

Pulse Modulated

Bubble Size

Chaos

5
0
0

0.05

0.1

0.15

0.2

VVM

Figure 9: 250L Drilled Hole Sparger 760 Pores 0.233mm Dia. kLa
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0.25

0.3

Operation in 3 Phases: Pulse Modulated 5 sLPM

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Operation in 3 Phases: Pulse Modulated 25 sLPM

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Operation in 3 Phases: Bubble Size Inc. 40 sLPM

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Operation in 3 Phases: Bubble Size Inc. 55 sLPM

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Operation in 3 Phases: Start of Chaos 60 sLPM

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Results Characterization
Reduced or eliminated base
consumption in test vessels

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175
140
105
70

20
15
10

Not dependent on agitation bubble


breakup

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36

33

30

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kLa Ratio

0.6
0.5
0.4
0.3
0.2
0.1
0

180
135
90
45
0 3 6
9 12 15
18 21 24
0
27 30 33
36 39
Micro Sparger sLPM

Macro Sparger sLPM

0.7

Uniform bubble size

Refined control methods should


further increase benefits

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Micro Sparger sLPM

Consistent character

Trivial gas velocities

12

0
9

0
6

35
3

5
0

kLa 1/hrs

Increased protein yield in 250L side by


side (not yet statistically significant)

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Macro Sparger sLPM

Promising performance

Figures 10 and 11: 2000L Drilled Hole Sparger O2 kLa


Performance Map and CO2/O2 Stripping / Delivery kLa Ratio Map

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Realized Performance Improvement


CO2 Stripping kLa
DHS

kLa per hour

Open pipe

O2 Delivery kLa Performance


frit
DHS

6.0

Open pipe

frit

kLa per hour 30

5.0

25

4.0

20

3.0

15

2.0

10

1.0

0.0

0
0

0.01

0.02

0.03

0.04

0.05

0.01

VVM

0.02
VVM

0.03

0.04

Figures 12 and 13: 250L Frit, DHS, Open Pipe Comparison

DHS: Linear response, close to


theoretical max CO2 stripping
Frit: Limited flow capacity
Open pipe: Nonlinear and weak

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DHS: Linear response, good slope


Frit: Excellent efficiency
Open pipe: Nonlinear and weak

0.05

Realized Performance Improvement


CO2 Stripping / O2 Delivery Ratio
O2

kLa 1/hr 14

CO2

12
10
8
6
4
2
0
0

0.01

0.02

0.03

VVM

Figure 14: 250L Frit, DHS, Open Pipe Comparison

Key takeaways:
Linear correlation, ratio near 50%
DHS O2 delivery increase is not excessive
Open pipe has similar ratio but lacks effectiveness
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0.04

0.05

Bibliography & Calculations


1)

Pauline M. Doran. Bioproces Engineering Principles. Elsevier Science & Technology Books, 1995,
ISBN:0122208552
(chapter 9 is of primary focus), linearizing equation used for data sets is:
Data used for calculations is from 20 to 80% of air saturation values
Delay applied between sparge up and down settings to reduce holdup-gas influence
O2 delivery kLa sparge down with pure N2, sparge up with air
CO2 stripping kLa sparge down with pure CO2, sparge up with air
Finesse TruFluor DO, Polestar DO (ppm class), CO2 sensors used to measure dissolved gas

concentration

Typical ambient atmospheric pressure of 865mbar, data normalized against lab pressure changes and
drift prior to kLa calculations

sensor

Test data clearly shows that CO2 sensor data, when mirrored, under the noted conditions, is identical to O 2
sensor data. Further CO2 sensing after confirming this fact has been carried out using O 2 sensors only.

2)

Estimates based on Henrys constants. NIST Chemistry Webbook (2005). Retrieved September 25, 2013 from
http://webbook.nist.gov/chemistry/

3)

Henrys constant values were adjusted by temperature dependence according to: Francis L. Smith and Allan H.
Harvey (September 2007). Avoid Common Pitfalls When Using Henry's Law. CEP (Chemical Engineering
Progress). ISSN 0360-7275 in conjunction with enthalpy of solution values from CRC Handbook of Chemistry and
Physics 87th Ed.

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Bibliography & Calculations (Continued)


4)

Scaling estimates are taken from the following equation simplifications:

A general equation for sphere terminal velocity is:


Term. Velocity = sqrt[(2massgravity_accelleration) / (fluid_densitycross_section_areadrag_coeff)]
Since we are looking at relative terminal velocity differences we will assume that gravitation acceleration, and fluid density are reasonably constant.
This leaves:
Relative Terminal Velocity = sqrt [mass / cross_section_areadrag_coeff]

In this case the mass*g is buoyancy and directly proportional to volume, substituting volume of a sphere and area of a circle and simplifying constants
leads to:
Relative Terminal Velocity = sqrt [ Bubble_diameter / Drag_coeff ]
The drag coefficient is:
Drag_coeff = (2Drag_Force) / (Fluid_densityvelocity2Cross_section_area)

Drag force assuming approximate laminar flow is a constant multiplies by velocity and fluid density is assumed constant leading to:
Relative Terminal Velocity = sqrt [ Bubble_diameter^2Relative Terminal Velocity ]
Further simplification leads to:
Relative Terminal Velocity = Bubble_diameter^2

And so an increase in bubble diameter yields roughly a squared increase in averaged vertical terminal velocity.

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Bibliography & Calculations (Continued)


5)

In practice actual bubble size impact scaling is further modified by changes in partial pressure equilibrium during
bubble rise, deformation of assumed bubble sphere, differences in single pore testing measured bubble diameter
vs. actual bubble diameter influenced by vessel agitation mechanism, and possible other unknown factors.
The following figure shows actual kLa values (adjusted for column height ratio differences) and normalized with
respect to 250L vessel data. Estimated kLa performance ratios based from single pore bubble size data analysis
and equations predicting cubic scaling effect. We can see in practice that actual scaling was closer to an exponent
of ~2.5 though much of the skewing from predicted appears to occur in the smaller bubble size estimates. Further
analysis of bubble size behavior in actual vessel must be carried out to help determine principle source of
inaccuracy.

kLa ratio w respect to 250L


2.5
2
1.5

Estimated kLa w bub sz


Actual kLa w bub sz

actual CO2 kLa w bub sz

0.5
y = 13.876x-3
0
1.5

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y = 8.387x-2.416
2.5

y = 7.954x-2.476
3.5

Bibliography & Calculations (Continued)


6)

Ying Zhu et all. NS0 Cell Damage by High Gas Velocity Sparging in Protein-Free and Cholesterol-Free Cultures.
Biotechnology and Engineering, 2008, DOI 10.1002/bit.21950

7)

Sachiro Kakinoki et all. Surface Modification of SUS 316L Stainless Steel with Tartaric Acid Derivative-Crosslinked
Human Serum Albumin Matrices. The Open Biotechnology Journal, 2008, 2, 143-147

8)

Accu Dyne Test Data: http://www.accudynetest.com/polytable_03.html?sortby=contact_angle as of 2013

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