To overcome these disadvantages, process specialists have been focusing increasing attention on the developing area of membrane-based
chromatography. Membrane adsorbers, as they are often known, work on exactly the same principles as resin-based chromatography
systems, with the ligand (e.g., quaternary ammonium) being bound to a support medium. In principle, any ligand that can be
bound to a chromatography resin can be bound to a support medium, in this case a membrane.
One major difference between a membrane support and a resin support is porosity. Membrane adsorbers in ion-exchange membranes
typically have a pore size of >3 μm. This nearly eliminates the diffusion limitation, improving flow rates and chromatographic
kinetics. The near absence of pore diffusion means that with membrane-based ion-exchange modules, there is a significant level
of flexibility in flow rate selection.
Figure 1
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The open structure of the membrane is obvious when visually compared with a conventional bead of approximately 90 μm in size
(Figure 1).
Because membrane chromatography capsules are single-use, they are simple to use. Once the module is installed and flushed
with buffer, processing can begin. Once processing is completed, the module is removed and disposed of, without the need for
washing, eluting, cleaning, or regenerating. This yields a significant reduction in processing time and buffer volumes.
With the increasing emphasis on reducing development times and costs, membrane chromatography products are being used in more
and more cGMP processes.
APPLICATION OF MEMBRANE CHROMATOGRAPHY FOR PROTEIN PURIFICATION
The UK contract manufacturer Avecia Biotechnology successfully applied membrane adsorption in a cGMP process involving the
manufacture of B2365, a recombinant protein produced in E. coli. The B2365 protein is initially expressed as a fusion protein and purified from the bacterial lysate by affinity chromatography.
The fusion protein is then immobilized onto glutathione sepharose affinity chromatography media. The column is subsequently
washed to remove the bulk of the impurities. Once washed, the immobilized glutathione-S-transferase (GST)-B2365 protein is
cleaved from the column using a GST-3C protease, which also binds to the column, allowing the cleaved B2365 molecule to be
washed from the column, leaving both the GST tag and GST-3C protease immobilized on the column. Although B2365 is of high
purity after this single step, it is then further purified in a polishing chromatography step to remove free GST that may
have leached from the column.
BACKGROUND
Previously, Avecia had purchased the GST-3C enzyme from an outside vendor, but to reduce overhead costs and to be able to
certify the origins of the protease as non-animal-derived and free of transmissible spongiform encephalopathies (TSE), an
internal project arose with the remit of producing 400 g of GST-3C protease for subsequent B2365 manufacture. GST-3C protease
is a genetically engineered fusion protein expressed in E. coli, consisting of Mr 20,000 human rhinovirus 3C protease coupled with a Mr 26,000 GST tag. This protease was specifically designed to facilitate removal of the protease by allowing simultaneous protease
immobilization and cleavage of GST fusion proteins on glutathione sepharose chromatography media. GST-3C protease specifically
cleaves between the Gln and Gly residues of the recognition sequence of LeuGluValLeuPheGln/GlyPro.
Exploiting the GST tag for purification using glutathione sepharose affinity chromatography resulted in rapid purification
and high purity levels after a single purification step. This constituted a quick and easy purification strategy, but analysis
of the column eluate indicated, not unexpectedly, that the endotoxin level was extremely high.
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