Wolf-Dieter Deckwer

Membrane adsorbers for selective removal of bacterial endotoxin

Surface-modified flat-sheet microfiltration membranes were functionalised with poly-L-lysine, polymyxin B, poly(ethyleneimine), L-histidine, histamine, alpha-amylase and DEAE as well as deoxycholate. Their suitability to remove endotoxin from both buffers and protein solutions was examined using bovine serum albumin, murine IgG1 and lysozyme as model proteins. In protein-free solutions reduction from 6000 EU/ml to <0.1 EU/ml was achieved with all applied ligands; only alpha-amylase as well as L-histidine and histamine, when immobilized via the non-ionic spacer bisoxirane, exhibited low clearance factors at neutral pH. The adsorption of endotoxin is mainly ruled by electrostatic interaction forces. Thus in multi-component systems, such as endotoxin-contaminated protein solutions, competing interactions take place: acidic proteins compete with endotoxin for binding sites at the membrane adsorbers, basic proteins compete with the ligands for endotoxin and act as endotoxin carriers. With properly chosen conditions the membrane adsorbers presented here show exceptional effectiveness also in the presence of proteins. They are generally superior to functionalised Sepharose chromatographic sorbents and allow fast processing. They may contribute to reduce the risks in the application of parenterals and diagnostics.

Endotoxin removal with poly(ethyleneimine)-immobilized adsorbers : Sepharose 4B versus flat sheet and hollow fibre membranes

Poly(ethyleneimine) was immobilized on poly(vinyl alcohol)-coated nylon flat sheet membranes, poly(vinyl alcohol) and poly(ethylenevinyl alcohol) hollow fibre membranes as well as Sepharose 4B. The resulting poly(ethyleneimine)-immobilized adsorbers were used for removal of E. coli derived endotoxin from buffers and bovine serum albumin solutions. The efficiency of poly(ethyleneimine) proved to be constant over a wide pH range, including phosphate buffered saline. The performance depended upon the matrix type employed: endotoxin clearance factors varied from 100 to 120,000 in protein-free solutions and 40 to 33,000 in solutions of bovine serum albumin using 6000 EU/ml as feed concentration. The best adsorber was the flat sheet membrane-immobilized poly(ethyleneimine), followed by the hollow fibre-immobilized poly(ethyleneimine) and poly(ethyleneimine)-Sepharose. The factors influencing endotoxin clearance were the mass transport (convective systems were superior to the diffusive system), the chemical composition and the surface structure of the underlying matrix.

The Use of Hydrocyclones for Mammalian Cell Retention in Perfusion Bioreactors

A new type of hydrocyclone was designed and used in continuous mammalian cell cultivation processes. The apparatus gave cell separation efficiencies as high as 99%. Due to the fact that a 5-L-bioreactor was used, a sub-optimal configuration of overflow and underflow diameters was used in order to obtain high cell viabilities combined with separation efficiencies suitable for cell retention. Viability was always above 90% during the 23 days run. The addition of 0.1% Pluronic F68 was found to be necessary for guaranteeing high growth rates and productivity.

Separation of Mammalian Cells Using a Specially Designed Rotating Disc Filter

Through continuous fresh nutrient supply and metabolite removal, combined with cell retendon in the bioreactor, perfusion cultures allow high cell concentrations and product titres to be achieved at relatively high medium throughput rates, resulting in high productivities [1]. However, a critical technical question that remains in perfusion processes is the cell retention device to be employed [2]. The use of membrane-based filtration devices for mammalian cell separation presents several advantages, such as a cell-free permeate and a straightforward scale-up. However, membrane fouling is still a major concern, specially during long-term perfusion cultivations. Optimisation of mammalian cell filtration requires a careful adjustment of the shear field in the filter module to allow minimisation of both fouling and cell damage. In rotating disc filters, the relative motion between membrane and rotor allows creating a high shear stress at the membrane surface, while decoupling shear rates from transmembrane pressure and flow rate through the system [3].