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The physical behavior of the binary phase systems of the non-ionic polyoxyethylene detergent Agrimul NRE 1205 and water was investigated. This technical detergent can be used for the large-scale recovery of biomolecules in detergent based aqueous two-phase systems. The phase diagram was determined. It shows significant and unexpected differences to highly purified detergents. Very similar to neat detergents the phase diagram can be influenced by auxiliary chemicals thus shifting the entire phase diagram in general to lower temperatures. This was demonstrated by lowering the cloud-point by various additions. The concentration factor, as an important parameter of a first capture step in purification was investigated and modeled. Auxiliary chemicals, temperature change and change in detergent concentration also influence the viscosity and density of the phases. These experimental data are shown. They can help to explain the separation behavior of proteins. In large-scale separations aqueous two-phase systems are separated using disc-stack centrifuges. It is demonstrated that this is not a feasible method for detergent-based aqueous two-phase extraction and the physical reason is presented.

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.

The parameters important for an optimisation of cloud point extraction in technical scale were investigated using a genetically engineered fusion protein derived from endoglucanase I expressed in Trichoderma reesei and the nonionic polyoxyethylene Agrimul NRE 1205. The key parameters are temperature, detergent concentration, and additional salts. These parameters are interdependent, thus there is an optimum in the partition coefficient with respect to detergent concentration and a maximum for the partition coefficient and the yield with respect to temperature. These results were confirmed for the detergent C12E5 to demonstrate that these optima are due to the nature of polyoxyethylenes. Cloud point extraction was found to be only slightly affected by pH. In the case studied extraction of whole broth is favourable for a high yield and partition coefficient, since fusion protein adhering to the cells can be solubilized. However some loss of detergent which remains in the fungal biomass was observed.

Detergent-based aqueous two-phase systems have the advantage to require only one auxiliary chemical to induce phase separation above the cloud point. In a systematic study the efficiency of tryptophan-rich peptide tags was investigated to enhance the partitioning of an enzyme to the detergent-rich phase using cutinase as an example. Up to 90% enzyme activity could be extracted in a single step from whole broth of recombinant Saccharomyces cerevisiae expressing cutinase variants carrying a (WP)4 tag. In contrast, the extraction yield of wild type cutinase was 2-3% only. The detergent concentration and the temperature are the main parameters to optimize the extraction yield. Considering availability, extraction yields, and price the detergent Agrimul NRE 1205 served best for enzyme recovery.

Expanded bed adsorption (EBA) is an integrated technology for the primary recovery of proteins from unclarified feedstock. A method is presented which allows a qualitative and quantitative understanding of the main mechanisms governing the interaction of biomass with fluidized resins. A pulse response technique was used to determine the adsorption of various cell types (yeast, Gram positive and Gram negative bacteria, mammalian cells and yeast homogenate) to a range of commercially available matrices for EBA. Cells and cell debris were found to interact with the ligands of agarose based resins mainly by electrostatic forces. From the adsorbents investigated the anion exchange matrix showed the most severe interactions, while cation exchange and affinity adsorbents appeared to be less affected. Within the range of biologic systems under study E. coli cells had the lowest tendency of binding to all matrices while hybridoma cells attached to all the adsorbents except the protein A affinity matrix. The method presented may be employed for screening of suitable biomass/adsorbent combinations, which yield a robust and reliable initial capture step by expanded bed adsorption from unclarified feedstock.

The performance of a Graesser Raining Bucket Contactor in an extraction using aqueous two-phase systems was characterized by axial mixing coefficients and counter current mass transfer for whey protein purification. The influence of rotor speed, phase ratio and phase velocity on the salt phase axial mixing coefficients was determined applying a dispersion model to the residence time distribution experiments. When dissolving whey powder in the salt-rich phase of the system, alpha-lactalbumin was extracted to the polyethylene-glycol-rich phase, whereas beta-lactoglobulin predominantly remained in the bottom phase, making the process suitable for removal of the major allergen beta-lactoglobulin. The mass transfer in the phase system was determined from the steady state data of alpha-lactalbumin extraction. On the basis of a diffusion model, the efficiency of the counter current extraction under different process conditions was calculated from results of the mass transfer experiments.

The application of an aqueous two-phase system is described for the extraction of DesPro(2)-Val15-Leu17-aprotinin from yeast culture supernatant, using native chymotrypsin as affinity ligand. The interaction is driven by hydrophobic forces and leads to the accumulation of the aprotinin-chymotrypsin complex in the salt-rich (bottom) phase of a polyethyleglycol/salt system. The complex may be dissociated at low pH values. The separation of the recombinant aprotinin and the protease required chromatographic processes, which proved difficult to interface with the affinity extraction.

A purification method for formate dehydrogenase from Candida boidinii has been designed by using a sequence of membrane-based adsorbents. The membranes carrying ion-exchange and dye ligands (Sartobind) are general-purpose adsorbents for proteins. The membranes were used in an on/off mode. A sequence of cation exchange, dye-ligand and anion exchange allows to purify the enzyme as judged by SDS-PAGE and assay of specific activity. The sequence of ligands and the buffer conditions were chosen in such a way that the fraction eluted from one membrane could be directly loaded on the next membrane. Thereby an integrated version of MDLC was realized with membrane-based adsorbents. The excellent scaleability from micro- to laboratory scale (factor 40) indicates that the method could afford a general approach to develop purification trains since the micro-scale allows for a rapid screening of adsorbent types and sequences.

Cholesterol oxidase from various bacterial sources (membrane-bound and extracellular) was studied in Triton X-114R solutions above the cloud point. The influence of temperature, salt, enzyme concentration and source, and pH on phase equilibrium and enzyme partitioning was investigated in this detergent-based aqueous two-phase system. The method combines remarkable recovery (over 70% and 90% in the detergent-rich phase for the extracellular and membrane-bound forms, respectively) and 10 to 20-fold concentration of the enzyme in just one purification step. The results from cholesterol oxidase are compared with other proteins, both hydrophobic and hydrophilic. The system shows considerable promise for selectively partitioning proteins based on their surface hydrophobicity.

We report the partition coefficients of lysozyme, chymotrypsinogen-A, albumin and catalase in sixty four Polyethyleneglycol/Dextran/Water systems at 4, 25 and 40 degrees C. We found that the partition coefficients of the four proteins generally increase with increasing temperature. The influence of temperature on the partition coefficient seems to be highly dependent on the kind of protein which is partitioned and on the total polymer concentration, but does not, in general, depend on the molecular weight of the polymers. The partition coefficients of small and hydrophilic proteins like lysozyme and chymotrypsinogen-A are only slightly affected by changes in temperature, while the partition coefficients of bigger and more hydrophobic proteins like albumin and catalase are strongly affected by changes in temperature. The results suggest the incorporation of attractive forces (possible electrostatic) into a model previously reported by us.

Aqueous two-phase systems form the basis for an extraction technology of proteins. Trends in the development of the technology are reviewed and discussed with regard to future applications.