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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 Figure 1: Sintered PVDF Micro Sparger

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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
Liquid
• NL is the rate of transfer

• 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

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Measuring O2 and CO2 Transfer
• Measure kLa via “Dynamic Method”1 O2 CO2
100
• Commonly used for measuring O2 % Gas
Saturation 80
delivery performance
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• Can assess CO2 stripping as well
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• Repeatable, consistent and inexpensive
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0
• O2 vs. CO2 considerations 81 83 85 87 89 91
Minutes
• Solubility Figure 3: CO2 Stripping Data Vertically Mirrored On O2
• 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 37ºC2,3

Reactor dissolved CO2 stripping partial O2 delivery partial pressure delta Liters dissolved gas
O2 set point pressure delta with air sparging with air sparging with O2 CO2 O2

0.06 0.147 0.937 47.4 3.3


30% air saturation
0.06 0.11 0.89 47.4 5.6
50% air saturation

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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

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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 Sintered Membrane Open Pipe Drilled Hole
Ave. bubble dia. (mm) <1 < 1 to > 3 >2 >1
Size distribution wide bell narrow to wide bell wide bell uniform to wide bell
Pre-distribution low low low low to high
Efficiency high low to high low low to mid
Flow rate low low to high mid to high mid to high

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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 Large bubble

• High surface/vol. + high residence time = high • High CO2 stripping vs. O2 delivery ratio
efficiency (generally high kLa) • Reduced foam issues
PROs • High gas throughput supported
• Reduced risk of cell damage (unless
excessive entrance velocity)6
• Increased risk of cell damage
• Increased risk of foam issues
• Increased risk of gas hold up • Lower efficiency (can lead to very poor kLa)
CONs
• Poor CO2 stripping vs. O2 delivery ratio • Tall column heights and high agitation
• Low gas throughput supported can mitigate

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Chaos and Randomized Bubble Size Generation
• Chaos = wide bell curve of bubble size
• Reduced effectiveness
• Skewed CO2 stripping / O2 delivery ratio
Plateau
0.6 12
Normalized Distribution

Region

kLa ratio (1 = 2.4 mm dia.)


0.5 10
kLa performance after volume and kLa
0.4 8 skewing applied
0.3 6 Original bubble size standard deviation

0.2 4
Volume skew (4/3*pi*d/2^3)
0.1 2
0 0 kLa Skew 7.954*d^2.48 + diminishing
0 1 2 3 4 5 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 Pore Bubble size Chaos
modulated saturation increase begins
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0
0 0.05 0.1 0.15 0.2 0.25 0.3
DHS AIR VVM

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

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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 O2 Delivery CO2 Stripping %CO2/O2
Vol (L) VVM kLa kLa/cm kLa kLa/cm kLa
50 0.005 9.91 0.190 1 0.019 10.1%
250 0.005 9.56 0.105 .96 0.011 10.0%
2000 0.005 9.77 0.055 .89 0.005 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 O2 Delivery CO2 Stripping %CO2/O2
Vol (L) VVM kLa kLa/cm kLa kLa/cm kLa
50 0.0125 2.05 0.039 1.06 0.020 51.4%
250 0.0125 3.54 0.039 1.59 0.017 44.8%
1000 0.0125 5.71 0.040 2.08 0.015 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 O2 Delivery CO2 Stripping %CO2/O2
(L) VVM kLa kLa/cm kLa kLa/cm kLa
1000 0.05 7.49 0.053 3.95 0.028 52.7%
2000 0.05 9.98 0.056 4.82 0.027 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

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Pulse Modulated Bubble Size Chaos
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0
0 0.05 0.1 0.15 0.2 0.25 0.3
VVM
Figure 9: 250L Drilled Hole Sparger 760 Pores 0.233mm Dia. kLa

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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
• Promising performance
• Reduced or eliminated base

Macro Sparger sLPM


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consumption in test vessels 25
175
20
• Increased protein yield in 250L side by 15
140
105

kLa 1/hrs
side (not yet statistically significant) 10 70
5 35
0 0

0
3
6
9
12
15
18
21
24
27
30
33
36
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Micro Sparger sLPM
• Consistent character
• Not dependent on agitation bubble
breakup 0.7
0.6
• Uniform bubble size 0.5

Macro Sparger sLPM


0.4
• Trivial gas velocities
kLa Ratio
0.3
0.2 180
0.1 135
90
0
0 3 6 45
• Refined control methods should 9 12 15
18 21 24
27 30 33 0
36 39
further increase benefits Micro Sparger sLPM

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 O2 Delivery kLa Performance
DHS Open pipe frit
kLa per hour DHS Open pipe frit
6.0
kLa per hour 30
5.0
25
4.0
20
3.0 15
2.0 10
1.0 5
0.0 0
0 0.01 0.02 0.03 0.04 0.05 0 0.01 0.02 0.03 0.04 0.05
VVM VVM

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

• DHS: Linear response, close to • DHS: Linear response, good slope


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

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Realized Performance Improvement
CO2 Stripping / O2 Delivery Ratio
O2 CO2
kLa 1/hr 14

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10
8
6
4
2
0
0 0.01 0.02 0.03 0.04 0.05
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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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 sensor
drift prior to kLa calculations

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 Henry’s constants. NIST Chemistry Webbook (2005). Retrieved September 25, 2013 from
http://webbook.nist.gov/chemistry/
3) Henry’s 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[(2∙mass∙gravity_accelleration) / (fluid_density∙cross_section_area∙drag_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_area∙drag_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 = (2∙Drag_Force) / (Fluid_density∙velocity2∙Cross_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^2∙Relative 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

1.5
Estimated kLa w bub sz
Actual kLa w bub sz
1
actual CO2 kLa w bub sz

0.5

y = 13.876x-3 y = 7.954x-2.476
y = 8.387x-2.416
0
1.5 2 2.5 3 3.5

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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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