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General Guidance for Estimating Media Requirements for PuraBead XL Chromatography Adsorbents

Many of the chromatography adsorbents used in process-scale applications for purification of biopharmaceuticals exhibit varying degrees of compression. The observed compression is the volume reduction of packed bed compared to the volume of the gravity settled adsorbent. This compression is obtained when fluid occupying the interstitial space is expelled by means of a mechanical force without causing unduly deformation of the beads. The compression is dependent on a number of factors including the bed height, the flow rate, pressure, temperature, viscosity, pH and ionic strength of the mobile phase buffers as well column packing techniques.

While for smaller diameter laboratory columns, the walls of the column provides bed support which allows higher flow rates, this ‘’wall support’’ effect diminishes as the column diameter increases. Thus, the maximum attainable flow rates decreases as the increasing column diameter and this effect diminishes beyond column diameter of 10cm. The maximum flow is also inversely proportional to the height of packed bed, with lower flow velocities obtained with increasing bed height.

Therefore, for adsorbent volume requirement for scale-up it is important to carry out some basic process development to examine the effects of resin volume on column stability, resin compression in a 10cm diameter column. This is critical for achieving the target compression to avoid over compression and headspace formation in process scale stainless steel columns.

Due to the complex nature of flow induced mechanical compression, the most direct method for determining the amount of resin required and the desired compression ratio is the use the flow consolidation method which takes into consideration the hydrodynamic properties of compressible media and the relationship between the bed height and flow (1).

1. Resin Compression Determination Method

For resin compression determination, ProMetic BioSciences uses a 100mm I.D. column. Resins are exchanged either into water or 0.1M NaCl. The resin bed is settled which can be aided by allowing the column to drain under gravity flow. Preservative solution is replaced with appropriate packing solution under gravity flow and the bed is re-suspended by reverse flow and allowed to settle once more in the packing solution. After resin bed has settled, the head plate is lowered to approximately 5cm above the settled bed and connected to a primed pump. Flow is started by starting the pump and pressure-flow data is collected for incremental increase in the mobile phase velocity. At each increment, the bed height and pressure is allowed to stabilise and recorded. These measurements are continued to the limit pressure of 3bar.

The settled bed height L0 is obtained from the plot of bed-height against flow velocity at the y axis-intercept, which represents the bed height at zero flow (2). Percent compression factor is obtained from the following equation:

% compression = (L0 – LP/ L0) x 100

Where:

L0 = the bed height at zero flow

LP = the bed height of the packed column at the limit pressure

For chromatography adsorbents based on PuraBead XL, compression factor of 20% is recommended. Typically, 55% gel slurry is used to pack process scale stainless steel columns.

Calculation of total column packed bed volume volume:

Packed bed volume = 3.14 x (I.D./2)2 x h

Example:

Column I.D = 10cm

bed height = 20cm

percent compression = 20cm

Packed bed volume = 3.14 x (10/2)2 x 20 = 1570 mL

Calculation of settled gel required to a given packed bed volume:

Settled bed volume = 100 x LP/(100 - %compression) 

Settled bed volume = (100 x 1570)/(100 – 20) = 1962.5 mL

Calculation of amount 55% slurry required to pack the column:

References

1 Tapia, J.O., Escobar, C. & Weidner, J., (2009), Chromatography Optimization Strategy. BioPharm. International, March 2009, 46 – 57

2. Keener, R.N., Fernandez, E.J., Maneval, J.E. & Hart, R.A. (2008), Advancement in the modelling of pressure-flow for guidance of development and scale-up of commercial-scale biopharmaceutical chromatography. Journal of Chromatography A. 1190, 127 - 140

A useful reference for predication of scale-up:

Tran, R., Joseph, J.R., Sinclair, A.R., Bracewell, D., Zhou, Y. & Tichner-Hooker, N.J. (2007), A Framework for Prediction of Scale-up When Using Compressible Chromatography Packings. Biotechnol. Prog. 23, 413 - 422