Abstract
A capture step was developed using the expanded bed adsorption technology to separate a protein of interest on a cation exchanger from a crude Escherichia coli homogenate. This method was developed in bench-top scale using a STREAMLINE 25 column (Amersham Pharmacia Biotech, Sweden) and STREAMLINE SP. The development was based on earlier experiments performed in a packed bed column (SP-Sepharose FF) to investigate the conditions for sample application, wash and elution. The packed bed method was transformed into an expanded bed method by slightly modifying the wash procedure and cleaning in place (CIP). This method was then scaled-up to pilot scale and used for production of the fusion protein according to cGMP.
The yield over the step in pilot scale was 70-85% compared with only 30-50% in small scale. Pressure build-up, attachment of biomass to the adsorbent and collapses of the expanded bed were phenomena seen in small scale but not in pilot scale. The scale-up of the step significantly improved the performance of the step.
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Abbreviations
- CIP:
-
clean in place
- GMP:
-
good manufacturing procedure
- ECP:
-
Escherichia coli protein(s)
- CV:
-
column volume
- WFI:
-
water for injection
- QC:
-
quality control
- DTT:
-
dithiothreitol
- ELISA:
-
enzyme linked immuno sorbent assay
- CFU:
-
colony forming unit
References
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© 1999 Springer Science+Business Media Dordrecht
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Brobjer, M. (1999). Development and scale up of a capture step (expanded bed chromatography) for a fusion protein expressed intracellularly in Escherichia coli . In: Mattiasson, B. (eds) Expanded Bed Chromatography. Springer, Dordrecht. https://doi.org/10.1007/978-94-017-1519-5_24
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DOI: https://doi.org/10.1007/978-94-017-1519-5_24
Publisher Name: Springer, Dordrecht
Print ISBN: 978-90-481-5380-0
Online ISBN: 978-94-017-1519-5
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