Evaluation the effects of flow rate, pressure and bed height on packing efficiency of chromatography media

Mahdi Karimi,1 Mohammad malekdoost,2 Neda abadian,3,*

1. Biopharmaceutical Research Center, AryoGen Pharmed Inc., Alborz University of Medical Sciences, Karaj, Iran
2. Biopharmaceutical Research Center, AryoGen Pharmed Inc., Alborz University of Medical Sciences, Karaj, Iran
3. Biopharmaceutical Research Center, AryoGen Pharmed Inc., Alborz University of Medical Sciences, Karaj, Iran

Abstract


Introduction

Since chromatography is an important and global unit operation in downstream bioprocessing, the preparation and qualification of packed media are critical steps to ensure purification process robustness and final product safety. before starting the purification process, column efficiency evaluation plays a central role in the qualification and monitoring of packed chromatography media. moreover, the column efficacy can also be used in-between runs to check the bed integrity changes. this study describes the investigation of packing efficiency of chromatography columns to meet the need of robustness and accurate packing method of various media in biopharmaceutical industry.

Methods

In this study, different media including mabselect sure lx (ge healthcare, usa), capto adhere (ge healthcare, usa), sp sepharose ff (ge healthcare, usa), q sepharose ff (ge healthcare, usa), toyopearl butyl-650m (tosoh, japan) and sephadex g25 coarse (ge healthcare, usa) were prepared and packed with distinctive bed height of 28, 30, 28, 5, 10 and 33 cm respectively for scale-down analysis. in order to evaluate the effects of different flow rates on packing efficiency, each experiment was performed by applying 4 cv of equilibration solution in defined flow rates. depending on the chemistry of the chromatography media, the equilibration solution contained ultra-pure water (upw) or 0.4 m nacl. next, 0.01 cv of sample containing 1-2 % v/v acetone or 0.8 m nacl was injected to the column followed by 2 cv of equilibration solution in order to elute the sample. the results were evaluated to determine the column packing efficiency parameters such as height equivalent theoretical plates (hetp), asymmetry factor (as), h factor, peak retention and pressure-flow characteristics. in the following, to evaluate the pressure effect on packing efficiency, two different system pressure, 0.25 and 0.05 mpa, were operated and column efficacy parameters were assessed subsequently. moreover, to determine the effect of bed height, three different heights of 3, 5 and 10 cm in q sepharose fast flow resin were investigated.

Results

The results showed that in mabselect sure lx, capto adhere and toyopearl butyl-650m media, with increasing of flow rates from 200 to 440 cm/h with intervals of 30 cm/h, as were improved 33 %, 55 % and 15 % respectively. moreover, in sp sepharose ff, q sepharose ff, and sephadex g25 coarse media with increasing the flow rates from 100 to 280 cm/h with intervals of 30 cm/h, the efficacy parameters weren’t changed. in addition, the results of packing procedure with different system pressures displayed that the column efficacy between two methods didn’t have any significant variations. moreover, with increasing of bed height from 3 to 10 cm in q sepharose ff, the as parameter was improved from 2 to 1.6.

Conclusion

The results of this study showed that the effect of flow rate on packing procedure depends on media characteristics. the efficacy parameters are improved significantly with increasing the flow rates in mabselect sure lx, capto adhere and toyopearl butyl-650m media but this approach doesn’t have any impact on packing of sp sepharose ff, q sepharose ff, and sephadex g25 media. in addition, the results cleared that although the pressure has no effect on packing procedure but the increasing of bed height may has positive effect on packing procedure.

Keywords

Asymmetry, chromatography media, flow rate, packing efficiency