Protein sorption and recovery by hydrogels using principles of aqueous two‐phase extraction

Abstract

Use of the thermodynamic principles of aqueous two-phase extraction (ATPE) to drive protein into a crosslinked gel is developed as a protein isolation and separation technique, and as a protein loading technique for drug delivery applications. A PEG/dextran gel system was chosen as a model system because PEG/dextran systems are widely used in aqueous two-phase extraction and dextran gels (Sephadex(R)) are common chromatographic media. The effects of polymer concentrations and molecular weights, salts, and pH on the partitioning of ovalbumin matched ATPE heuristics and data trends. Gel partition coefficients (Cgel/Csolution) increased with increasing PEG molecular weight and concentration and decreasing dextran concentration (increased gel swelling). The addition of PEG to the buffer solution yielded partition coefficients more than an order of magnitude greater than those obtained in systems with buffer alone, or added salt. A combined salt/PEG system yielded an additional order of magnitude increase. For example, when ovalbumin solution (2.3 mg/mL) was equilibrated with Sephadex(R) G-50 at pH 6.75, the partition coefficients were 0.13 in buffer, 0.11 in buffer with 0.22M KI, 2.3 in 12 wt% PEG-10,000 and 32.0 in 12 wt% PEG-10, 000 with 0.22M KI. The effect of anions and cations as well as ionic strength and pH on the partitioning of ovalbumin also matched ATPE heuristics. Using the heuristics established above, partition coefficients as high as 80 for bovine serum albumin and protein recoveries over 90% were achieved. In addition, the wide range of partition coefficients that were obtained for different proteins suggests the potential of the technique for separating proteins. Also, ovalbumin sorption capacities in dextran were as high as 450 mg/g dry polymer, and the sorption isotherms were linear over a broad protein concentration range.

Recovery and separation of cell lysate proteins using hydrogels guided by aqueous two‐phase extraction principles

“…Since the heuristics of ATPE and results from our previous work with pure protein solutions indicate that increasing the concentration and molecular weight of PEG will increase the partitioning of proteins into the dextran phase, a 12 wt% PEG-10,000 solution was used. The salts potassium iodide and tetrabutylammonium¯uoride (Bu 4 NF) were used since they should have among the strongest eects on protein partitioning but aect positively and negatively charged proteins in opposite manners, according to the virtual partition coecient concept of Johansson (1974) and our previous results (Gehrke et al, 1998a). Based on this work, KI should favor the partitioning of negatively charged proteins into the dextran phase while Bu 4 NF should favor the partitioning of positively charged proteins into the dextran.…”

“…The enhancement ratios of KI/PEG and Bu 4 NF/PEG were both over 12. Since total loading is proportional to the swelling degree, as well as the partition coecient, minimizing gel deswelling in the loading solution would increase total loading even further (Gehrke et al, 1998a). The level of deswelling can be anticipated from the slope of the tie lines on the polymer/polymer/water phase diagram, and the deswelling is not always as large as it is in the case of PEG solution/dextran gel (Gehrke et al, 1995;Putka, 1998).…”

“…In previous publications the use of the thermodynamic principles of aqueous two-phase extraction (ATPE) to extract proteins from a solution into a hydrogel phase by adding salts and polymers to the solution was called`s olution-controlled gel sorption'' (SCGS) (Gehrke et al, 1991(Gehrke et al, , 1998a. This concept was proposed for use as a protein isolation and separation technique.…”

“…The concept of SCGS has been demonstrated in PEG/dextran systems using model proteins (Gehrke et al, 1991(Gehrke et al, , 1995(Gehrke et al, , 1998a. Increasing the nominal molecular weight of PEG in increments up to 10,000 steadily increased the partition coecient (K = concentration of protein in gel/concentration of protein in solution) of cytochrome c and ovalbumin.…”

Characteristics of protein partitioning in an aqueous micellar‐gel system

“…The partitioning of globular hydrophilic proteins has been studied using various systems. Gehrke et al (1991Gehrke et al ( , 1998 studied ovalbumin partitioning for different conditions in a two-phase system consisting of an aqueous PEG/dextran solution and a Sephadex G-50 gel. Other systems for protein and biomolecular partitioning include aqueous nonionic micellar two-phase systems (Kamei et al, 2002;Liu et al, 1998;Nikas et al, 1992;Rangel-Yagui et al, 2003) and membranes and fibrous gels (Lazzara et al, 2000;White and Deen, 2001).…”