Packing and Quality Control of a Chromatography Column

Josephine Hui-Chung Cheng

Asia Technology & Tranining Center Merck Ltd., Taiwan, Oct. 2nd, 2009!

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Outline

•! Introduction

•! Packing techniques!

•! Quality control of packed columns

•! Troubleshooting

•! Conclusions!

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Fermenter Centrifuge Concentration

pH/Salt Adjustment Formulation,

Sterile Filtration

Dia-Filtration pH

Adjustment

Dia- Filtration

$ $ $ $ $

$ $

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For cost efficient separations we need to

•! Maximize throughput

•! Minimize pressure drop

•! Maintain a stable packed bed

•! Produce an efficient column!

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•! In a well-packed column the density of the packed resin is homogeneous with a controlled interstitial volume (space between the particles).!

schematic cross-section

How is the resin arranged in a efficient column?

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Introduction

•! Irregularities in packing cause

- uneven flow within the bed

- band broadening

- zone mixing

- changes in flow rate

!

! subsequent loss of product yield and quality!

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Points to consider

Column

Packing method Resin

•! Rigid vs. non-rigid materials

•! Back pressure of the process

•! Predictable scale-up

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Common Packing Techniques

•! Dynamic Axial Compression (DAC)

- Achieved by axially compressing the media slurry

•! Flow Packing

- Uses constant pressure or flow rate

•! Stall Packing

- Packing by pumping media into the column via nozzles!

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Dynamic Axial Compression

Step 1b Step 2

In brief

Step 1a / 1b: Pump slurry into column filled with buffer

Step 2: Lower plunger to final bed height

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Flow Packing

Step 1 Step 2a Step 2b

In brief

Step 1: Transfer slurry into empty column and resuspend homogeneity before lowering the plunger into the slurry

Step 2a / 2b: Immediately apply flow to pack the resin

Step 3: Lower plunger to final bed height

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Pressure Flow Course during Flow Packing

A) Packing with constant flow B) Packing with constant pressure

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Quality control of packed beds

•! Diagnositic tools

–! Apperance (e.g. discolouration etc.)

–! Plate numbers / HETP, Asymmetry (Efficiency)

–! pressure vs flow curve

•! Checking time

–! After packing

–! After / before each separation run

–! At regular intervals

•! Parameters to be looked at.

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Test Systems

tracer eluent detection

water 150 mM NaCl conductivity

1M NaCl 150 mM NaCl conductivity

1 % - 3 % acetone 150 mM NaCl UV at 280 nm

sample volume:

•! rule of thumb: 1 % - 2 % of packed bed volume

•! keep thickness of sample layer constant to compare with different scales (height, diameter)

Possible interaction of sample with resin may influence test results.

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Plate Number & Asymmetry

As – asymmetry ; As > 1 = tailing

As < 1 = fronting

HETP (height equivalent of theoretical plates) (m)

dp – mean particle diameter (m)

h – reduced bed height (-)

L – bed height (m)

N – number of theoretical plates (-)

peak width at

half peak

height

elution volume Ve

A und B at 1/10 peak height

volume

U V -

A b s o

r b t i o

n

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Pressure flow curve for 10x20 cm column flow packed with Fractogel® EMD SO3

- (M)

maximum operating pressure "

1.9 bar @ 270 cm/h

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Some parameters influencing column performance

•! Resin nature

•! Compression

•! Mobile phase

•! Slurry concentration

•! Reslurry

•! Slurry settling time

•! Packing flow rate

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Pressure Flow Curve Non-rigid materials

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Pressure Flow Curve Semi-rigid materials

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Effect of surface chemistry on P vs F curve

Pressure flow behavior of Fractogel® AEX resins

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Impact of compression on PvsF

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Pressure flow curves of Fractogel® EMD SO3

- (M) in different mobile phases

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Slurry concentration effect

Slurry concentration Plates As

55% 734 1.03

60% 721 1.01

70% 680 1.18

80% 551 1.37

89% 532 1.73

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Impact of re-slurrying resin on HETP

Ve (L)

Fractogel® cation exchanger

Column: 46 x 20 cm, 20% compression

Running Buffer: 0.15 M NaCl

Resin reslurried in column and packed

by axial compression with inlet open

N/m = 2633, As = 1.4, H = 038 cm

Resin settled overnight followed by axial compression with inlet open

N/m = 1817, As = 2.59 H = 0.055

co

nd

ucti

vit

y (

mS

)

Q = 144 cm/hr, PB = 5 psig

Sample = 330 mL, DI Water

conditioned for 30 min at 30 psig

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Slurry settling times

Settling Time (min) Plates As

0.75 670 1.33

0.75 670 1.23

2 652 1.03

2 659 1.18

3 709 1.19

3 760 1.15

6 648 1.24

6 731 1.19

Mean 687 1.19

STD 41.15 0.085

Err% of Mean 6% 7%

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Packing flow rate

Initial packing flow rate (mL/min)

Initial linear packing flow

(cm/hr) Total Plates As

600 458 702 1.18

450 344 710 1.15

393 300 695 1.08

331 250 560 1.4

202 153 535 1.34

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Principles in Packing!

•! Handle resin gently (especially during preparation of resin)

•! Work quickly (avoid sedimentation)

•! Use a slurry concentration 3 70 %

•! Avoid entry of air

•! Pack at initial flow rate 4 300 cm/h

•! Pack the resin to 25% compression (15% for lab scale)

•! Avoid temperature fluctuation

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Trouble Shooting

symptom cause

double peak •! blocked frit

fronting •! bed is packed too hard (over-compressed)

•! defect in packed bed (cracks, cavities)

tailing •! bed is not packed hard enough ( under-compressed)

•! defect in packed bed (gap, cavities)

•! extra system volumes (zone-spreading by test setup, e.g. air trap)

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Trouble Shooting

symptom cause

increase in pressure •! insufficient clean in place procedure (CIP)

•! deterioration of resin (aging)

instable bed

(re-compression)

•! high viscosity of sample

•! bed is not packed hard enough ( under compressed)

•! blocked / contaminated resin

•! alteration of matrix properties (after high CIP cycle number)

•! gel bed exposed to high flow / pressure

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Wrong procedure leads to

•! fingering that is definitely the start point (76 cm column) of column problems

•! followed by channelling

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Gap Formation

The column is packed not hard enough (under-compressed).

A recompression of the packed bed may occur at high flow rate / pressure.

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Air trapped in the top filter causes resin to pack against the head during axial compression

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Success Story

900 mm l. x 16mm i.d.

PBV: 180ml

90 cm l. x 90 cm i.d.

PBV: 570 L

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Conclusions

•! Packing should not be overlooked at process development scale!

•! Slurry packing followed by continuous axial compression with the outlet open is the preferred method in columns equipped with hydraulics.

•! Slurry packing followed by flow packing/ axial compression method with the outlet open is best for “pack in place” columns with no hydraulic.

•! Packing should be performed at the maximum flow rate possible (> 300 cm/hr Fractogel® media) and should be applied immediately and maintained as long as possible (given pressure limits of the system) until the bed has reached a stable bed height.

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Conclusions

•! “Semi-rigid Materials” allow for shift of critical flow rate to higher values as compared to “Non-rigid Materials”

•! Letting the slurry settle for up to 6 minutes after having made a homogeneous slurry appears to have no effect.

•! Fractogel® resins should be packed to 25% compression for optimum results.

•! The pressure flow behavior of Fractogel® resins is chemistry dependant.

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Asia Technology & Training Center at Taipei/ Taiwan

- column packing area!

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Asia Technology & Training Center at Taipei/ Taiwan

- bio-purification area!

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Technical support and service (off- and on-site support) - Answers to customer requests (e.g. application guidance, product properties) - On-site customer support (e.g. column packing, process optimisation) - Troubleshooting

- Technical workshops (public class & custom program) - Technical product positioning/ sample pervision

Training classes (selection of topics) - Biochromatography: From Resin Selection to Column Packing. - Purification strategies for Biomolecules from Small to Large scale - Purification Strategies for Antibodies.

- Hands-on trainings in chromatography column packing

Services to AAA customers

Asia Technology & Training Center - ATTC activities!

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Ion exchange

media

Hydrophobic

media

Affinity

media

Size exclusion

media

Fractogel®

– EMD TMAE Hicap

– EMD TMAE

– EMD DEAE

– EMD DMAE

– EMD SO-3

– EMD SE Hicap

– EMD COO-

Fractogel®

– EMD TA

– EMD Chelate

– EMD Amino

– EMD Epoxy

Fractogel®

– EMD BioSEC

Fractogel®

– EMD Propyl

– EMD Phenyl

... an excellent investment for your process economy!

Fractogel# resins Product portfolio

ATTC service mail:

ATTC.Service@merck.com.tw

Thank you very much for your

attention!