Perspectives on:
Chromatography as an
Integrated Process
Al Williams:
Applications Manager
GE Bioscience
Williams;
November 2006
Williams;
November 2006
Large Scale Planning
Plant Design
Facility drawing – 3D
Williams;
November 2006
Suite Design
General layout
Buffer tank outside clean room
System
In-line dilution
≥ 1,5 * column diameter
System
In-line dilution
System
In-line dilution
D
D
O
O
Maintenance area
Column
2m
Crane
Column
< 1m
Column
< 1m
O
R
R
Pack
station
Transfer panel
Feed
tank
Williams;
November 2006
O
Pack
station
Detail Oriented
Williams;
November 2006
Ergonomics & Work Flow
•
•
•
•
EHS, Safety
FDA/EPA/OSHA///// compliance
SOP Writing
Validation Master Plan
Williams;
November 2006
The True Challange
Williams;
November 2006
Selectivity
Resolution =
Efficiency
Williams;
November 2006
Selectivity: Peak Position
Williams;
November 2006
Efficiency
0
0
Williams;
November 2006
200
20
400
40
60
Time (min)
600
80
Efficiency: Peak Width
Efficiency is a measure of the peak width - W1/2
Concentration
N = 5.54 (Vr/ W1/2)2
N = Plates
N/L = Plates / Meter
H = HETP = Meters/Plate
Hred = Beads / Plate
100% Max
W1/2
50% Max
Williams;
November 2006
Time/Volume
Vr
Efficiency
Asymmetry
Good Asymmetry
low selectivity
high efficiency
low efficiency
high selectivity
high efficiency
low efficiency
Williams;
November 2006
Poor Asymmetry
Column Edge Seal Designs
Williams;
November 2006
Bu
The Problem in Designing
Chromatography Processes
Pr
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mp
Sa
rs
ffe
nk
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T
Meth
yo
a
L
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t
u
P ack
Pu
s?
p
m
ess
ure
?
s?
od ?
w
F lo
+
Column
Williams;
November 2006
=
+
Media
?
Operator
?
Result
What Do We Need?
•
•
•
•
•
Basic Knowledge of Application
Reasonable Expectation
Knowledge of the Media
Packing Method
Test Method
• Equipment Expectations
Williams;
November 2006
Constant Pressure or Constant Flow
Packing Methods
Add Slurry
Compress
Set Adaptor
Start Flow
Consolidate
3
2
1
Williams;
November 2006
During the Pack
• Settling
• Convective Force
• Viscous Drag
Williams;
November 2006
Determine Pressure Flow
Profiles
Flow
Flow
Flow
Flow
bkc
Bed
Height
Velocity
Williams;
November 2006
Pressure
Determine Pack & Run
Regimes
Determine the Compression Factor (Cf) = Vgs / Vc
Vcrit
Packing Velocity
Packing Pressure
Velocity
V gs
Run
Run
Bed
Volume
Gap
Vc
Williams;
November 2006
∆P
∆P max
Facility Design Criteria
From Pressure Flow Data
• Pump/Drain Size
• Piping/Tank Sizes
• Column
Specifications
Williams;
November 2006
• WorkFlow/Ergonomics
• Weight issues
• Material Handling
Issues
• Spare Parts Storage
Optimize Compression Factor
HETP
Af
1.20
Velocity
Williams;
November 2006
1.10
1.15
Pressure
1.25
Examine Wall Effects at L
•
Pressure/flow curves in columns with different diameters
80
bkc
I.D. cm
10
Velocity (cm/h)
70
60
20
30
40
60
80
100
50
40
30
20
10
Unstable
0
0
1
2
3
4
5
Pressure (bar)
See: Stickel, J. & Fotopoulos, A., Biotechnol. Prog 2001, 17, 744 - 51.
Williams;
November 2006
6
Wall Effects
None
Large
Small
Williams;
November 2006
Wall Effects
1000 Bead Widths
Williams;
November 2006
Stickel Model
Stickel J. & Fotopoulos A, Biotechnol. Prog 2001, 17, 744-751
Stickel Plot for Q Sepharose Big Beads
200000
160000
µ critLo
120000
80000
40000
y = 4901.5x + 33055
Cf = 1.14
0
Williams;
November 2006
0
5
10
15
Lo/D
20
25
30
Now and in the Near future
Sepharose™ 4 Fast Flow
(High porosity)
MPa
Sepharose 6 Fast Flow
(Medium porosity)
8.0
7.0
6.0
5.0
4.0
3.0
2.0
1.0
0.0
Increasing flow rate
Williams;
November 2006
Other Facility Concerns
•
•
•
•
•
•
Packing and or Unpacking Tanks
Media; Batch Tailing & Used Storage
IBC For Used resin and Tailings
Cleaning IBCs, Washer Compatibility
Resin Lifetime
Disposal
Williams;
November 2006
Avoid System Effects
•
•
•
•
•
External Volumes Vext
Gradient Delay Volume
Mixing Efficiency
Wall Effects
System Pressures
Williams;
November 2006
Gradient Types
%B
‡ Continuous gradient
‡ Step gradient
‡ Complex gradient
Volume
Williams;
November 2006
Gradient Shape / Volume Effects
AU
%B
1
3
4 5
2
AU
7
6
%B
1
7
2
AU
3
4
5
6
%B
1
2
3+4
5+6
7
Column volume
Williams;
November 2006
Effects of Gradient Slope
• Decreasing Slope
– Increases Peak Volume
– Decreases Retention
Volume
– Increases Resolution
– Increases Time
• Selectivity Increases
• Efficiency Decreases
Williams;
November 2006
• Increasing Slope
– Decreases Peak Volume
– Increases Retention
Volume
– Decrease Resolution
– Decreases Time
• Selectivity Decreases
• Efficiency Increases
External Volume (Vext)
A
B
Product
SAMPLE
Williams;
November 2006
Monitors
Waste
External Volume Effects
SP Sepharose HP
I.D.= 2.6 cm, L= 17 cm
20000
18000
Pre-Column
Efficincy (N/m)
16000
Plug Top
Post Column
Plug Bottom
RTD TOP
14000
"RTD Bottom
12000
10000
8000
6000
4000
2000
0
0
2
3
4
Vext (%Vc)
Williams;
November 2006
5
10
15
Gradient Delay Volume
A
B
S
Williams;
November 2006
Monitors
System Wall Effects
Step
Pulse
Williams;
November 2006
System Effects
100 % B
100 % A
Volume
Williams;
November 2006
Asymmetric Mixing Effects
Mixer or
Air Trap
A
F/ µ
t
Τ
Î
∆Ρ
Vext
Vg
B
S
Williams;
November 2006
Monitors
System Effects
Symmetric
Williams;
November 2006
Asymmetric
Well Designed Response
Williams;
November 2006
Other System Effects
• Flow Deviations
– Pulsation
– Siphoning
• Inlet Pressure Changes
• Outlet Pressure Changes
Williams;
November 2006
Designing To System Effects
•
•
•
•
•
•
•
Keep Vext and Vg << Vc
Properly Size Mixers and Air Traps
Keep Piping Runs Short
Avoid Unnecessary Plumbing
Keep Post Column Pressures Low
Use Proper I.D. & Keep I.D. Constant
Use Pre-column Feedback Controls
Williams;
November 2006
Distribution Effects
Wall Effect
Jetting
Williams;
November 2006
Bubbles
Even
Distribution Effects
Williams;
November 2006
2003 Lab Visit & Dye Test
MPLC1600 HP Base Matrix L=15
Amgen Methodology
Williams;
November 2006
2003 Lab Visit & Dye Test
MPLC1600 HP Base Matrix L=15
Our Methodology
Williams;
November 2006
2003 Lab Visit & Dye Test
MPLC1600 HP Base Matrix L=15
Wall Effect
Williams;
November 2006
Screen Retainer Bolts
Good Distribution Systems
• Asymmetry independent of Vs
• Asymmetry independent of Flow / ∆P
Williams;
November 2006
Examine Test Conditions
Distribution Analysis
Van Deemter Analysis
5600
1.5
5400
7000
1.5
1.4
1.4
6500
4800
Efficiency
Efficiency
5000
Asymmetry
Asymmetry
5200
1.3
6000
1.2
5500
1.1
1.3
1.2
1.1
4600
5000
1
1
4400
0.9
4200
4000
0.8
5
15
25
Velocity
Williams;
November 2006
35
45
55
4500
0.9
4000
0.8
0
0.5
1
1.5
2
2.5
3
Sample Volume (%Vc)
3.5
Distribution Analysis
1.5
End Cell Volume
1.4
6500
1.3
6000
1.2
5500
1.1
5000
1
4500
0.9
4000
0.8
0
0.5
1
1.5
Vs (%Vc)
Williams;
November 2006
2
2.5
3
3.5
Asymmetry
Efficiency (N/L)
7000
Distribution Effects
Van Deemter Analysis
5600
1.5
1.4
5200
1.3
5000
1.2
4800
1.1
4600
1
4400
0.9
4200
4000
0.8
5
Williams;
November 2006
15
25
35
Velocity (cm/h)
45
55
Asymmetry
Efficiency (N/L)
5400
Distribution Analysis
Sepharose HP L=15 cm w/ NaCl
7000
2.2
6000
2
5000
1.8
N/m
1.6
As
4000
1.4
3000
1.2
2000
1
0
Williams;
November 2006
5
10
Sample Volume (% of Cv)
15
Af
N/m
1M ID Column
Distribution Effects
Van Deemter Analysis
1.8
5600
Prototype
5400
1.6
5000
1.4
4800
1.2
4600
4400
1
4200
0.8
4000
5
Williams;
November 2006
15
25
35
Velocity (cm/h)
45
55
Asymmetry
Efficiency (N/L)
5200
Distribution Effects
Williams;
November 2006
Q Sepharose 6 FF, I.D. 100 cm, L=20 cm
Sample Blue Dextran 1 mg/ml, Vs= 0.5L
CFV 1000 Dye Test
•
•
•
•
•
•
•
•
•
Superdex 200 PG
N/L = 10,000 Af = 1.45
CF = 1.16
L = 30 Vc= 236L
Flow 16 cm/hr
Sample: 1 mg/ml Phenol red in
10 mm NaOH
Mobile Phase 10 mM NaOH
100 mM NaCl
Vs = 1% Vc
5 injects 50 L apart via Index
Column as Superloop
Williams;
November 2006
Harvesting The Bed 1
• Pump off Top Adaptor
– Close MPb
– Flow in MPt
Williams;
November 2006
Harvesting The Bed 2
• Suck Off Excess
Liquid From Bed with
Pack Station
Williams;
November 2006
Top of the Bed
• Bed Expanded to
31.5 cm
• Last inject tailing to
surface
• Non Specific
Absorbtion on Top
Williams;
November 2006
Non Uniform Axial Expansion of
Bed
5
4
3
2
1
Williams;
November 2006
Harvesting The Bed 3
• Excavate sides
• Remove Tube
Williams;
November 2006
Cross Section
• Bed shaped about 20
cm from each outer
edge
Williams;
November 2006
Cross Section
• Bed Exposed to 28
cm from each outer
edge
Williams;
November 2006
Harvesting the bed 4
• Bed Reflected @ 8
cm from Center
• Bed Reflected @ 6
cm from Center
Williams;
November 2006
Harvesting The Bed 5
• 4 cm from
center
• 2 cm from
center
Williams;
November 2006
Harvesting The Bed 6
• 1 cm from
center
• Center
Williams;
November 2006
Harvesting The Bed 7
• Clear Zone About 4
cm in diameter 2 cm
high
Williams;
November 2006
Think Now; Column & System
• Pulse or Breakthrough
• Inline Dilution
– Reduce tank or bag size Facility Space
– Reduce Number of Buffers
– Reduce QC QA and prep time Costs
• Risks to Process
– Not Precise
– Not Accurate
– Must Validate
Williams;
November 2006
Continuous Just-in-time Buffer
Preparation
Benefits
•Eliminate buffer storage vessels
-free up valuable real estate
-reduce capital and equipment maintenance costs
•Automated and controlled blending
-reduce buffer preparation time
-record and log all parameters with the production run data
-reduce QC testing
-scalable, consistent performance
Williams;
November 2006
Just-in-time Buffer Blending and
Dilution Systems
Set Gradient Criteria to Application
Prove performance
-accuracy
-time to reach set point
-scalable
-dilution
Williams;
November 2006
+/- 1%
<30 seconds (waste)
mls/minute to
hundreds of liters/min
up to 20X
Just-in-time Buffer Blending and
Dilution Systems
•
•
Programming
Control & Operation:
– Recipe built into the chromatography system
-Mixing parameters are programmed as part of the chromatography run
-Buffers are mixed in line and arrive at the column ‘Just-In-Time’
Williams;
November 2006
Just-in-time Buffer Blending and
Dilution Systems
•
EQUIPMENT DESIGN
•
-2 pumps for dilution only
•
-3 pumps for simultaneous gradient and/or dilution
• Switch Valve Based Systems
• Pressure / Flow Feed Back Controlled Metering Valves
• Other mixed technologies
Williams;
November 2006
Just-in-time Buffer Blending and
Dilution Systems
•
CONTROL METHOD
•
Flow Control OR
-One flow meter per pump
meters
-Mixing control based on flow
Williams;
November 2006
Flow-Conductivity Control
-One conductivity monitor and one or more flow
-Mixing control based primarily on conductivity
Williams;
November 2006
Williams;
November 2006
Dilution test Apr 19, 2006
Test 1000 l/h
Ratio 5.0
4.0
UV
compensated
Dilution and Gradient
Grad Setp
Calc
Read
Eq %
Error
Read
Eq %
Error
5%
0.0208
0.0214
5.16%
0.16%
0.0211
0.0214
5.06%
0.06%
10%
0.0415
0.0424
10.22%
0.22%
0.0423
0.0424
10.03%
0.03%
25%
0.1038
0.1066
25.68%
0.68%
0.1057
0.1066
25.22%
0.22%
50%
0.2075
0.2123
51.15%
1.15%
0.2114
0.2123
50.22%
0.22%
75%
0.3113
0.3176
76.52%
1.52%
0.3170
0.3176
75.14%
0.14%
90%
0.3735
0.3809
91.77%
1.77%
0.3804
0.3809
90.11%
0.11%
95%
0.3943
0.4014
96.71%
1.71%
0.4016
0.4014
94.96%
-0.04%
100%
0.4151
0.4227
101.84%
1.84%
0.4227
0.4227
100.00
%
0.00%
UV max
peak
2.07525
calculated from the 60% point got from the UV linearity test done Apr18, 2006
(60% values)/0.6
Williams;
November 2006
Calc
Dilution test Apr 18, 2006
Test performed 300 l/h
Ratio 5.0
4.0
Dilution and Gradient
Grad Setp
Calc
Read
UV compensated
Eq %
Error
Calc
Read
Eq %
Error
5%
0.0208
0.0227
5.47%
0.47%
0.0210
0.0227
5.39%
0.39%
10%
0.0415
0.0442
10.65%
0.65%
0.0421
0.0442
10.50%
0.50%
25%
0.1038
0.1085
26.14%
1.14%
0.1052
0.1085
25.78%
0.78%
50%
0.2075
0.2149
51.78%
1.78%
0.2105
0.2149
51.06%
1.06%
75%
0.3113
0.3194
76.95%
1.95%
0.3157
0.3194
75.89%
0.89%
90%
0.3735
0.3805
91.68%
1.68%
0.3788
0.3805
90.40%
0.40%
95%
0.3943
0.4006
96.52%
1.52%
0.3999
0.4006
95.18%
0.18%
100%
0.4151
0.4209
101.41%
1.41%
0.4209
0.4209
100.00%
0.00%
UV max peak
2.07525
calculated from the 60% point got from the UV linearity test done Apr18, 2006
(60% values)/0.6
Williams;
November 2006
Variables To Consider
•
•
•
•
2 Pump Dilution System....7000 LPH
Williams;
November 2006
Viscosity
Temperature
Miscible/Gas
Usable Flow
Rate Range
• Inlet /Outlet
Pressure Effects
• Stock Solution
Reproducibility
Just-in-time Buffer Dilution
Conductivity Feedback Control
Error = +/- .01%
Buffer A:
Buffer B:
Calculated values Example:
10% solution value = (29.6)(10%)
Equivalent % =
Williams;
November 2006
Read Value
Calculated 100%
DI Water
NaCl at 29.6 mS/cm
Large media handling
Concept description
9 Media shipped either in standard pack size or
in column equivalent volume
9 Tools for removing ethanol
9 Tools incl. methods for fast and easy remixing
of chromatographic resin
9 Convenient transfer to feed tank
9 Easy packing of column via automated pack
station
Williams;
November 2006
Large media handling
Solutions – shipping containers
Williams;
November 2006
Large media handling
Solutions – stirrer stand
Williams;
November 2006
Large media handling
Solutions – media wand
Williams;
November 2006
Large media handling
Solutions – feed tank
Williams;
November 2006
Ethanol Calcs for Virgin Resin
• For Agarose based Media From GE
– Slurry% as Supplied = 70
– % Ethanol as Supplied = 18 – 20%
– Matrix Volume = 8 – 10%
– Targeted Overage of Fill = 2%
– Nominal Media Volume = Vgs
Williams;
November 2006
How Much Ethanol as supplied
5 L Container
Vgs = 5 x 1.02 = 5.1L as a 70% Slurry
Matrix Volume = Vgs x 0.1 = 0.51L
Vt = (5.1 / .7 ) - 0.51 = 7.3 – 0.51 L
= 6.8L
Volume Ethanol = 6.8 L x .2 = 1.36L
Gallon Proof = 1.36 L / 3.85 L/g = .35
or ca, 0.7 Gallon Proof / L media supplied
Williams;
November 2006
Decanting Removes ?
• 5L Container
Vt = 7.3 L
Vgs = 5.1 L
Decantation removes = 7.3 – 5.1 = 2.2 L
2.2 L @ 20% = 0.44 L @ 100% Ethanol
0.44 / 1.36 ~ 32% removed
Refill with WFI to same level = 14% Ethanol
Williams;
November 2006