Hans-Olof Johansson

Driving forces for phase separation and partitioning in aqueous two-phase systems

A set of simple analytical equations, derived from the Flory-Huggins theory, are used to identify the dominant driving forces for phase separation and solute (e.g., protein) partitioning, in the absence and presence of added electrolyte, in every general class of aqueous two-phase systems. The resulting model appears to capture the basic nature of two-phase systems and all trends observed experimentally. Case studies are used to identify fundamental differences in and the magnitudes of enthalpic and entropic contributions to partitioning in polymer-polymer (e.g., PEG-dextran), polymer-salt, and thermoseparating polymer-water (e.g., UCON-water) two-phase systems. The model therefore provides practitioners with a better understanding of partition systems, and industry with a simple, fundamental tool for selecting an appropriate two-phase system for a particular separation.

Effects of salts and the surface hydrophobicity of proteins on partitioning in aqueous two-phase systems containing thermoseparating ethylene oxide-propylene oxide copolymers

The partitioning of five well-characterised model proteins, bovine serum albumin (BSA), lysozyme, [beta ]-lactoglobulin A, myoglobin and cytochrome c, in aqueous two-phase systems has been studied. As top phase polymers PEG (polyethylene glycol, 100% EO) and the thermoseparating ethylene oxide (EO)-propylene oxide (PO) random copolymers, Ucon 50-HB-5100 (50% EO, 50% PO) and EO30PO70 (30% EO, 70% PO), respectively, were used. The top phase polymers are increasing in hydrophobicity with increasing content of PO. Reppal PES 200 (hydroxypropyl starch) was used as the bottom phase polymer. Phase diagrams for Reppal PES 200-PEG and Reppal PES 200-EO30PO70 two-phase systems were determined. The partitioning of four salts with different hydrophobicity, and also the effect of the salts on protein partitioning in these systems, was studied. It was found that the partitioning of the salts followed the Hofmeister series. The partitioning of proteins with low surface hydrophobicity, myoglobin and cytochrome c, was little affected by hydrophobic polymers and salts. However, the partitioning of a protein with higher surface hydrophobicity, lysozyme, was strongly affected when polymer hydrophobicity was increased and a hydrophobic counterion was used. A protein with a relatively hydrophobic surface can be partitioned to a phase containing a thermoseparating EO-PO copolymer by using a hydrophobic counterion. The partitioning of lysozyme and cytochrome c in the polymer-water system formed after temperature-induced phase separation was also examined. Both proteins partitioned exclusively to the water phase. A separation of the protein and polymer was obtained by temperature-induced phase separation on the isolated phase containing the EO-PO copolymer. The partitioning data also indicated that the hydroxypropyl starch polymer had a weak negative charge. (Less)

Thermoseparating water/polymer system: A novel one-polymer aqueous two-phase system for protein purification

In this study we show that proteins can be partitioned and separated in a novel aqueous two-phase system composed of only one polymer in water solution. This system represents an attractive alternative to traditional two-phase systems which uses either two polymers (e.g., PEG/dextran) or one polymer in high-salt concentration (e.g., PEG/salt). The polymer in the new system is a linear random copolymer composed of ethylene oxide and propylene oxide groups which has been hydrophobically modified with myristyl groups (C(14)H(29)) at both ends (HM-EOPO). This polymer thermoseparates in water, with a cloud point at 14 degrees C. The HM-EOPO polymer forms an aqueous two-phase system with a top phase composed of almost 100% water and a bottom phase composed of 5-9% HM-EOPO in water when separated at 17-30 degrees C. The copolymer is self-associating and forms micellar-like structures with a CMC at 12 microM (0.01%). The partitioning behavior of three proteins (lysozyme, bovine serum albumin, and apolipoprotein A-1) in water/HM-EOPO two-phase systems has been studied, as well as the effect of various ions, pH, and temperature on protein partitioning. The amphiphilic protein apolipoprotein A-1 was strongly partitioned to the HM-EOPO-rich phase within a broad-temperature range. The partitioning of hydrophobic proteins can be directed with addition of salt. Below the isoelectric point (pI) BSA was partitioned to the HM-EOPO-rich phase and above the pI to the water phase when NaClO(4)was added to the system. Lysozyme was directed to the HM-EOPO phase with NaClO(4), and to the water phase with Na-phosphate. The possibility to direct protein partitioning between water and copolymer phases shows that this system can be used for protein separations. This was tested on purification of apolipoprotein A-1 from human plasma and Escherichia coli extract. Apolipoprotein A-1 could be recovered in the HM-EOPO-rich phase and the majority of contaminating proteins in the water phase. By adding a new water/buffer phase at higher pH and with 100 mM NaClO(4), and raising the temperature for separation, the apolipoprotein A-1 could be back-extracted from the HM-EOPO phase into the new water phase. This novel system has a strong potential for use in biotechnical extractions as it uses only one polymer and can be operated at moderate temperatures and salt concentrations and furthermore, the copolymer can be recovered.

Purification of protein and recycling of polymers in a new aqueous two-phase system using two thermoseparating polymers

In this study we present a new aqueous two-phase system where both polymers are thermoseparating. In this system it is possible to recycle both polymers by temperature induced phase separation, which is an improvement of the aqueous two-phase system previously reported where one of the polymers was thermoseparating and the other polymer was dextran or a starch derivative. The polymers used in this work are EO50PO50, a random copolymer of 50% ethylene oxide (EO) and 50% propylene oxide (PO), and a hydrophobically modified random copolymer of EO and PO with aliphatic C14H29-groups coupled to each end of the polymer (HM-EOPO). In water solution both polymers will phase separate above a critical temperature (cloud point for EO50PO50 50 degrees C, HM-EOPO, 14 degrees C) and this will for both polymers lead to formation of an upper water phase and a lower polymer enriched phase. When EO50PO50 and HM-EOPO are mixed in water, the solution will separate in two phases above a certain concentration i.e. an aqueous two-phase system is formed analogous to poly(ethylene glycol) (PEG)/dextran system. The partitioning of three proteins, bovine serum albumin, lysozyme and apolipoprotein A-1, has been studied in the EO50PO50/HM-EOPO system and how the partitioning is affected by salt additions. Protein partitioning is affected by salts in similar way as in traditional PEG/dextran system. Recombinant apolipoprotein A-1 has been purified from a cell free E. coli fermentation solution. Protein concentrations of 20 and 63 mg/ml were used, and the target protein could be concentrated in the HM-EOPO phase with purification factors of 6.6 and 7.3 giving the yields 66 and 45%, respectively. Recycling of both copolymers by thermoseparation was investigated. In protein free systems 73 and 97.5% of the EO50PO50 and HM-EOPO polymer could be recycled respectively. Both polymers were recycled after aqueous two-phase extraction of apolipoprotein A-1 from a cell free E. coli fermentation solution. Apolipoprotein A-1 was extracted to the HM-EOPO phase with contaminating proteins in the EO50PO50 phase. The yield (78%) and purification factor (5.5) of apolipoprotein A-1 was constant during three polymer recyclings. This new phase system based on two thermoseparating polymers is of great interest in large scale extractions where polymer recycling is of increasing importance.

Aqueous polymer two-phase systems formed by new thermoseparating polymers

A set of new polymers that can be used as phase forming components in aqueous two-phase systems is presented. All polymers studied have thermoseparating properties i.e. form one separate polymer enriched phase and one aqueous solution when heated above the critical temperature. This property makes the polymers attractive alternatives to the polymers used in traditional aqueous two-phase systems such as poly(ethylene glycol) (PEG) and dextran. The thermal phase separation simplifies recycling of the polymers, thus making the aqueous two-phase systems more cost efficient and suitable for use in large scale. Thermoseparating polymers studied have been copolymers of ethylene oxide and propylene oxide (EO-PO), poly (N-isopropylacrylamide) (poly-NIPAM), poly vinyl caprolactam (poly-VCL) and copolymers of N-isopropylacrylamide and vinyl caprolactam with vinyl imidazole (poly(NIPAM-VI) and poly(VCL-VI), respectively). In addition, the copolymer poly(NIPAM-VI) has the property to be uncharged at pH above 7.0 and positively charged at lower pH. This allows the partitioning of protein to be directed by changing the pH in the system instead of the traditional addition of salt to direct the partitioning. Hydrophobically modified EO-PO copolymer (HM-(EO-PO)) with alkyl groups (C14) at both ends forms two-phase system with for example poly(NIPAM-VI). The phase diagram for poly(NIPAM-VI)/HM-(EO-PO) was determined and the model proteins lysozyme and BSA were partitioned in this system. For BSA in poly(NIPAM-VI)/HM-(EO-PO) system a change in pH from 8.0 to 5.4 results in a change of partition coefficient from K=0.8 to K=5.1, i.e. BSA could be transferred from the HM-(EO-PO) phase to the poly(NIPAM-VI) phase. BSA partitioning in poly(NIPAM-VI)/HM-(EO-PO) system allows quantitative BSA recovery, and recoveries of poly(NIPAM-VI) and HM-(EO-PO) were 53% and 92%, respectively, after the thermoseparation step.

Temperature-induced phase partitioning of peptides in water solutions of ethylene oxide and propylene oxide random copolymers

A thermoseparating random copolymer (Ucon 50-HB-5100) composed of (50%) ethylene oxide and (50%) propylene oxide has been used to form an aqueous two-phase system by heating the polymer-water solution above the cloud point of the copolymer. In the formed two-phase system a water rich top phase is in equilibrium with an aqueous polymer rich bottom phase. The partitioning of amino acids and peptides in this aqueous two-phase system has been studied. Hydrophobic peptides (containing aromatic amino acids) were strongly partitioned to the polymer rich phase, while hydrophilic peptides were enriched in the water rich phase. The effect of temperature on the partitioning was investigated and a decreased partitioning to the polymer rich phase was obtained upon temperature increase. The effect of two salts (NaClO4 and Na2SO4) on the partitioning of a positively charged polypeptide, poly(Lys, Trp), was very strong. With NaClO4 the polypeptide was quantitatively partitioned to the polymer rich phase while with Na2SO4 the polypeptide was partitioned to the water rich phase. Model calculations based on a modified Flory-Huggins theory have been performed to better understand the experimental behavior.

Multimodal chromatography: An efficient tool in downstream processing of proteins.

Chromatography has become an indispensable tool for the purification of proteins. Since the regulatory demands on protein purity are expected to become stricter, the need for generating improved resins for chromatographic separations has increased. More advanced scientific investigations of protein structure/function relationships, in particular, have also been a driving force for generating more sophisticated chromatographic materials for protein separations. As a consequence, the development of alternative chromatographic strategies has been very rapid during the past decade and several new ligands have been designed and explored both in the laboratory and in large‐scale industrial settings. This review describes some of these efforts using multimodal chromatography, where two or more physicochemical properties are used to enhance the specificity of the interactions between the protein and the ligand on the chromatographic matrix. In addition to experimental studies, computer modeling of ligand‐protein binding has improved the design of ligands for protein recognition. The use of descriptors as well as in silico docking methods have been implemented to design multimodal resins in several instances.

Protein partitioning in thermoseparating systems of a charged hydrophobically modified ethylene oxide polymer

The phase behavior of a thermoseparating cationic hydrophobically modified ethylene oxide polymer (HM-EO) containing tertiary amines has been investigated at different pH, salt and sodium dodecyl sulfate (SDS) concentrations, in order to find a water/HM-EO two-phase system suitable for protein partitioning. The used polymer forms micellar aggregates that can be charged. By changing pH and SDS concentrations the netcharge of the SDS/HM-EO aggregate can be shifted from positive to negative. Bovine serum albumin (BSA) and lysozyme were partitioned in the thermoseparated two-phase systems of the cationic polymer at different pH, salt and SDS concentrations. The dominant attractive interactions between the polymer aggregates and the studied proteins were shown to be of electrostatic (Coulomb) nature rather than hydrophobic interaction. At low ionic strength the positively charged polymeric aggregates attracted negatively charged BSA and repelled positively charged lysozyme. Upon addition of SDS the negatively charged aggregates attracted lysozyme and repelled BSA. Thus, it was possible to direct proteins with different charges to the polymeric phase and redirect them to a polymer-depleted phase by changing the netcharge of the polymeric aggregates. The effect of different salts on the partitioning of BSA in a system of slightly positively charged HM-EO was studied. NaCl and KBr have a significant effect on driving the BSA to the polymer-depleted phase, whereas KF and K2SO4 have a smaller effect on the partitioning. The cloud point temperature of the charged polymer decreased upon addition of SDS near the isoelectric molar ratio of SDS to polymer and also upon salt addition. In the latter case the decrease was smaller than expected from model calculations based on Flory-Huggins theory, which were performed for a charged thermoseparating polymer at different charges and salt concentrations.

Plasmid DNA partitioning and separation using poly(ethylene glycol)/poly(acrylate)/salt aqueous two-phase systems.

Phase diagrams of poly(ethylene glycol)/polyacrylate/Na(2)SO(4) systems have been investigated with respect to polymer size and pH. Plasmid DNA from Escherichia coli can depending on pH and polymer molecular weight be directed to a poly(ethylene glycol) or to a polyacrylate-rich phase in an aqueous two-phase system formed by these polymers. Bovine serum albumin (BSA) and E. coli homogenate proteins can be directed opposite to the plasmid partitioning in these systems. Two bioseparation processes have been developed where in the final step the pDNA is partitioned to a salt-rich phase giving a total process yield of 60-70%. In one of them the pDNA is partitioned between the polyacrylate and PEG-phases in order to remove proteins. In a more simplified process the plasmid is partitioned to a PEG-phase and back-extracted into a Na(2)SO(4)-rich phase. The novel polyacrylate/PEG system allows a strong change of the partitioning between the phases with relatively small changes in composition or pH.

Macromolecular crowding and its consequences.

Incompatible pairs of polymers separate into two phases in aqueous solution above a few percentage points total concentration. Protein pairs can also produce phase separation, but at somewhat higher concentrations. In this chapter, we explore the effect of high background concentrations of macromolecules on phase separation of pairs of species which would not be at sufficiently high concentration to separate in the absence of the uninvolved species. Effects produced by such high background concentrations are known as macromolecular crowding. Dramatic enhancements in various association reactions due to crowding have been predicted and observed but its effects on phase separation in biological mixtures typical of the cytoplasm have not been examined. Here, we describe a calculation based on the Flory-Huggins treatment of concentrated polymer solutions that sheds some light on this issue. We find that a background of 20 wt % of a high molecular weight species greatly reduces the concentrations needed to produce phase separation in a mixture of two incompatible macromolecules if one is more hydrophobic than the other. Given the high total concentration of macromolecules in cytoplasm, it is perhaps surprising that phases have not been observed. This issue is discussed and some explanations offered.