The recovery of cutinase of Fusarium solani pisi produced by the yeast Saccharomyces cerevisiae was studied in a fluidised bed adsorption system directly integrated with a productive fermenter (so-called direct product sequestration; DPS). The relative efficiency of this system was compared with the one of a conventional purification process by discrete sequences of fermentation, broth clarification, ultrafiltration and fixed bed anion exchange chromatography. By direct product sequestration of the extracellular heterologous cutinase it was possible, through only one unit operation: (i) to perform broth clarification, (ii) to obtain a high cutinase concentration factor, and (iii) to recover cutinase with a specific activity that equalled that obtained with the conventional purification process. It was also possible (iv) to substantially reduce the total process time, (v) to improve the overall yield, and (vi) to increase cutinase productivity. Furthermore, the procedure outlined is suitable for large scale bioprocess exploitation.

In this study we detail the rational design of new chromatographic adsorbents tailored for the capture of plasmid DNA. Features present on current chromatographic supports that can significantly enhance plasmid binding capacity have been identified in packed bed chromatography experiments and blueprints for improved expanded bed adsorbents have been put forward. The characterisation and testing of small (20-40 microm) high density (>3.7 g cm(-3)) pellicular expanded bed materials functionalised with various anion exchange structures is presented. In studies with calf thymus DNA, dynamic binding capacities of 1.2 and 3.4 mg ml(-1) were recorded for prototype diethylaminoethyl-and polyethylene imine-linked adsorbents which were respectively 25 and 70 fold higher than those of equivalently derivatised commercial expanded bed materials. The prototype polyethylene imine-coupled material exhibited severe sensitivity to inter-particle bridging by nucleic acid polymers, gave low DNA recoveries (<37%) and proved difficult to regenerate. In contrast, few operational difficulties were experienced with the diethylaminoethyl-linked prototype adsorbent and successful high capacity (>0.8 mg ml(-1)) capture of plasmid DNA from crude neutralised E. coli lysate was demonstrated.

Improved specificity and binding affinity by molecularly imprinted polymers is possible by development of novel functional materials. Furthermore, increasing the cross-link density of imprinted polymers by using cross-linking functional groups was anticipated to improve polymer molecular recognition. A novel cross-linking monomer derived from an L-aspartic acid precursor was synthesized and employed in molecularly imprinted polymers to mimic more closely the scaffolding of proteins, and thus provide more protein-like selectivity. Chromatographic results revealed a more than 7-fold improvement in polymers imprinted using the new monomer versus a traditionally formulated polymer imprinted with methacrylic acid as the functional monomer.

The isolation and preliminary characterization of a trypsin-like protease with high activity at 20 degrees C is described. This protease was isolated from Antarctic krill (Euphasia superba) by a two-step chromatography process and the use of zymogram analyses. The protease has a molecular weight of 30 kDa and a pI of 4.1. Its specific activity at 20 degrees C on BAPNA is 0.5 U/mg.

Expanded bed adsorption (EBA) is an integrated technology for the primary recovery of proteins from crude feedstock. Interactions between solid matter in the feed suspension and fluidised adsorbent particles influence bed stability and therefore have a significant impact on protein adsorption in expanded beds. In order to design efficient and reliable EBA processes a strategy is needed, which allows to find operating conditions, where these adverse events do not take place. In this paper a methodological approach is presented, which allows systematic characterisation and minimisation of cell/adsorbent interactions with as little experimental effort as possible. Adsorption of BSA to the anion exchanger Streamline Q XL from a suspension containing S. cerevisiae cells was chosen as a model system with a strong affinity of the biomass towards the stationary phase. Finite bath biomass adsorption experiments were developed as an initial screening method to estimate a potential interference. The adhesiveness of S. cerevisiae to the anion exchanger could be reduced significantly by increasing the conductivity of the feedstock. A biomass pulse response method was used to find optimal operation conditions showing no cell/adsorbent interactions. A good correlation was found between the finite bath test and the pulse experiment for a variety of suspensions (intact yeast cells, E. coli homogenate and hybridoma cells) and adsorbents (Streamline Q XL, DEAE and SP), which allows to predict cell/adsorbent interactions in expanded beds just from finite bath adsorption tests. Under the optimised operating conditions obtained using the prior methods, the stability of the expanded bed was investigated during fluidisation in biomass containing feedstock (up to 15% yeast on wet weight basis) employing residence time distribution analysis and evaluation by an advanced model. Based on these studies threshold values were defined for the individual experiments, which have to be achieved in order to obtain an efficient EBA process. Breakthrough experiments were conducted to characterise the efficiency of BSA adsorption from S. cerevisiae suspensions in EBA mode under varying operating conditions. This allowed to correlate the stability of the expanded bed with its sorption efficiency and therefore could be used to verify the threshold values defined. The approach presented in this work provides a fast and simple way to minimise cell/adsorbent interactions and to define a window of operation for protein purification using EBA.

Retention and manipulation of microbial cells through exploitation of ultrasonic forces has been reported as a novel cell immobilisation technique. The spatial ordering of yeast cells, within suspensions subjected to an ultrasonic standing wave field, was analysed for the first time. A technique, based on 'freezing' the spatial arrangement using polymer gelation was developed. The resultant gel was then sectioned and examined using microscopic techniques. Light Microscopy confirmed the presence of specific regions in the ultrasonic field, where the cells are organised into bands corresponding to the standing waves' pressure nodal planes. Computer Image Analysis measurement of several physical parameters associated with this cell distribution matched the values derived from the theoretical model. The spatial cell-cell re-arrangement within each band and uneven distribution along the nodal planes have been analysed by Scanning Electron Microscopy. These results complement the ongoing study of the process of immobilisation of microbial cells by ultrasound standing waves.

A robust new adsorptive separation technique specifically designed for direct product capture from crude bioprocess feedstreams is introduced and compared with the current bench mark technique, expanded bed adsorption. The method employs product adsorption onto sub-micron sized non-porous superparamagnetic supports followed by rapid separation of the 'loaded' adsorbents from the feedstock using high gradient magnetic separation technology. For the recovery of Savinase from a cell-free Bacillus clausii fermentation liquor using bacitracin-linked adsorbents, the integrated magnetic separation system exhibited substantially enhanced productivity over expanded bed adsorption when operated at processing velocities greater than 48 m h(-1). Use of the bacitracin-linked magnetic supports for a single cycle of batch adsorption and subsequent capture by high gradient magnetic separation at a processing rate of 12 m h(-1) resulted in a 2.2-fold higher productivity relative to expanded bed adsorption, while an increase in adsorbent collection rate to 72 m h(-1) raised the productivity to 10.7 times that of expanded bed adsorption. When the number of batch adsorption cycles was then increased to three, significant drops in both magnetic adsorbent consumption (3.6 fold) and filter volume required (1.3 fold) could be achieved at the expense of a reduction in productivity from 10.7 to 4.4 times that of expanded bed adsorption.

The adsorption and desorption pattern of alkaline protease was studied using different aliphatic and aromatic hydrophobic ligands. Overall, higher adsorption was obtained on ligands coupled to 6% cross-linked gel than the 4% gel. The highest adsorption was obtained on butyl (94%) and phenyl (98.4%) of 6% cross-linked gel. The adsorption was dependent on concentration and nature of the ligand. In a single-step operation, almost 20-fold purification with 40% yield of the enzyme was obtained using all the optimized experimental parameters.

Molecularly imprinted polymeric membranes have been emerged since 1990. Among various kinds of molecular imprinting studies, the application of molecular imprinting to membrane separation is still a novel investigation. In the present review paper, molecularly imprinted polymeric membranes are summarized and examined. The application of molecular imprinting to membrane separation shortly leads to high performance separation membranes.

To avoid the intrinsic problem of aggregation associated with the traditional solution-phase refolding process, we proposed a solid-phase refolding method integrated with the expanded bed adsorption chromatography. The model protein was a fusion protein of recombinant human growth hormone and a glutathione S-transferase fragment. It was demonstrated that the inclusion body proteins in the cell homogenate could be directly refolded with higher yield. To verify the applicability of this method, we have tested with success three types of the starting materials, i.e., rhGH monomer, inclusion bodies containing the fusion protein, and the E. coli cell homogenate. This direct refolding process could reduce the number of the renaturation steps required and allow the refolding at a higher concentration, approximately 2 mg fusion protein per ml resin.