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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