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A Chromatography Process from Lab to Production Scale: A Column Packing Study
Wei Guo, B Thiyagarajan and Nandu Deorkar
Mallinckrodt Baker Inc. / Tyco Healthcare
November 2006
Trademarks are owned by Mallinckrodt Baker, Inc. unless otherwise noted.
Dimensions of Chromatographic Processes
Resolution
Recovery
Capacity
Speed
Key Attributes of Ideal Chromatographic Media
Physical and mechanical propertieso Simple column packing procedureo Stable column bed under various conditions
Chemical stabilityo Cleaning in place and sterilization using strong reagentso Column regeneration
Backbone and functional groups o Minimum irreversible adsorptiono Selectivity and consistent performance
0.15 (Weak anion)0.25 (strong anion)
Mixed mode- Primarily strong anion exchanger with anion exchange sites
PolyQUAT
0.15-0.25 (strong cation)0.15-0.25 (weak cation)0.05-0.12 (anion)
Mixed mode- Primarily Strong cation exchanger with weak cation and anion exchange sites
PolyCSx
0.15-0.25 (cation)0.05-0.12 (anion)
Mixed mode- Primarily weak cation exchanger with weak anion exchange sites
PolyABx
0.3-0.4 (anion)Weak anion exchangerPolyPEI
Ion Exchange Capacity, meq/ml
FunctionalityName
Novel BAKERBONDTM Polymeric Chromatographic Media
Schematic Ligand Structure of BAKERBOND PolyABx
Weak anion exchangerSecondary amine –NH-Tertiary amine –N=
Weak cation exchangeCarboxyl -COOH
FunctionalityGroup
Objectives
Investigate lab to process scale column packing and performance
Develop robust column packing protocol
Correlate column integrity and separation performance
Characteristics of a Well Packed Column
Constant bed volume under different flow rates
Reproducible separation performance
Optimal flow distribution across the column
Acceptable efficiency and asymmetry for chosen application
Common Packing Methods
Flow packing Pressure packing Suction packing Axial compression packingIn situ packing (slurry transfer) Combination of some methods aboveDry packing
BAKERBOND PolyABxPressure Flow Profile
Column size: 165 X 90 mm ID and 155 X 450 mm ID
0
10
20
30
40
50
60
70
80
0 100 200 300 400 500 600
Linear Velocity (cm/hr)
Pre
ssur
e D
rop
(psi
)
Column Packing Instrumentation
Column Packing Parameter
Slurry and packing solutiono Slurry and pack in 50 mM sodium chlorideo 2 M sodium chloride / pack with 1 M NaClo Slurry in sterilant buffer (sodium acetate and
benzyl alcohol)/ pack with 1 M NaCl
Slurry ratio (20-60%)Packing flow rate
Slurry Transfer and Flow Packing Procedure for BAKERBOND
PolyABx ColumnMeasure amount of BAKERBOND PolyABx-35Desired packed column volume = 1.05 X settled BAKERBOND PolyABx volumeMake a 50% slurry in packing buffer2 M sodium chloride Transfer the slurry to the empty column with diaphragm pump at 100 to 700 cm/hrPack with 1 M NaCl at 100 to 700 cm/hr for 2 to 3 column volume
Slurry Transfer and Flow Packing Procedure for BAKERBOND
PolyABx Column (Continued)
Lower down the upper adapter near to the top of bedPack again with 1 M NaCl at 100 to 700 cm/hr for 2 to 3 column volume or till bed height is constant Lower down the upper flow adaptor to the top of bed and lock it in place
Particle Size Distribution over Repeated Use
-1
4
9
14
19
24
0.01 0.1 1 10 100 1000 10000
Diameter (micron)
Freq
uenc
e (%
) Initial polymer
Ratio 1:2
Ratio 1:4
Comparison at 80 Cycles
Particle Size Distribution BAKERBOND PolyABxAfter Slurry Transfer of BAKERBOND PolyABx to Column (50% and 25% slurry)
Particle Size Distribution over Repeated Use
Initial BAKERBOND PolyABx
BAKERBOND PolyABx after 6 cycles of packing and unpacking cycles.
Column Integrity Testing Protocol
Equilibrate column with 2 to 5 column volumes of 1.0 M NaCl Load 0.5% to 2.0% column volumes of 2.0 M NaCl / 0.4% acetone solutionElute column with 2 column volumes of 1.0 M NaCl at 20 to 100 cm/hrDetermine asymmetry and efficiency
Lab Scale Packed Column Performance
1.02108204
1.18107903
1.11105512
1.1493911
AsymmetryEfficiencyPack
XK16 column (120X16 mm ID)
Efficiency (Ave.): 10388 (1s 675)Asymmetry (Ave.): 1.11 (1s 0.07)
c:\UNICORN\Local\fil\reason06\result\xk column packing\!efficiency01009\efficiency01001.res efficiency01001:1_Cond efficiency01(1144421389)001:1_Cond2 efficiency01(1144426518)001:1_Cond efficiency01(1144432952)001:1_Cond
4.0
6.0
8.0
10.0
mS/cm
4.0
6.0
8.0
10.0
mS/cm
0.0 10.0 20.0 30.0 40.0 ml
Process Scale Column Packing
Uniformity in slurry and ease processing Left- Slurry in the column before packing Right: After 2-3 column volume packing buffer flow at 100 cm/hour
Slurry height: 30 cm Bed height: 15.5 cm
Packed Column Integrity Test
125 X 450 mm ID; Flow rate: 22 cm/hr
1.1813926Conductivity
AsymmetryEfficiencyDetection
Evaluation of Process Column
Eastern River column (120 X450 mm ID), column packed using 2 M NaCl slurry. Sample: 1% CV 2 M NaCl
Column Performance by Conductivity
0
5000
10000
15000
20000
2 4 6 8 10 12 14
Test Number
Effic
ienc
y (p
late
/m)
0
0.5
1
1.5
2
Asy
mm
etry
Efficiency
Assymetry Efficiency (Ave.): 13522 (1s 585)Asymmetry (Ave.): 1.28 (1s 0.09)
Packing BAKERBOND PolyABx from Different Slurry Solutions
------- 50% Slurry in 2 M NaCl ------- 50% Slurry in initial sterilant buffer
Eastern River Column125 X 450 mm ID
Comparison of Process and Lab Scale Columns
15,677Lysozyme
23,023Cytochrome C
9,888IgG
efficiencySample
Process Column (140 X 90 mm ID)Lab Column (100 X 10 mm ID)
18,544Lysozyme
22,589IgG
efficiencySample
Properties of BAKERBOND PolyABx-35 allow easy transfer from lab to process scaleDeveloped column packing procedure can be used for packing lab scale to process scale columnHigh mechanical strength permit easy and fast column packing
No defining or buffer exchange requiredSlurry transfer at high flow ratePacking at high flow rate/pressureLinear pressure flow profile
The packed column provide reproducible and consistent protein separation performance from lab scale to process scale
Conclusion
Acknowledgements
• Charles R Deily• Bob Buss• Audrey Parker• Joe Mladosich• Charles Keller