Journal of Chromatography B (v.807, #1)

FM iii (iii).

Preface by Charles A Haynes (1).

The practical application of aqueous two-phase systems (ATPS) to process development has been exploited for several years for the recovery of biological products. Unfortunately, this has not resulted in an extensive presence of the technique in commercial processes. Some of the main identified reasons for such situation involve the full understanding of the mechanism governing phase formation and the behaviour of solute partitioning in ATPS processes, the cost of phase forming polymers and the necessary extended time to understand the technique for process development. In this review paper, some of the practical disadvantages attributed to ATPS are addressed. The practical approach exploited to design ATPS processes, the application to achieve process integration, the increasing use for the recovery of high-value products and the recent development of alternative low cost ATPS, are discussed. It is proposed that the potential trend in the application of ATPS processes for the recovery of biological products will involve the recovery of high-value bio-particulate products with medical applications. This proposed trend in the application of ATPS will address the urgent need to rapidly and economically bring new biopharmaceutical products to market using scaleable and efficient bioprocess technology.
Keywords: Reviews; Aqueous two-phase systems; Partitioning; Process integration; Proteins;

Aqueous two-phase systems are composed of aqueous solutions of either two water-soluble polymers, usually polyethylene glycol (PEG) and dextran (Dx), or a polymer and a salt, usually PEG and phosphate or sulfate. Partitioning of proteins in such systems provides a powerful method for separating and purifying mixtures of biomolecules by extraction. If one of the phase forming polymers is a crosslinked gel, then the solution-controlled gel sorption may be considered as a modification of aqueous two-phase extraction. Since PEG/dextran systems are widely used in aqueous two-phase extraction and dextran gels (Sephadex) are common chromatographic media, we choose a PEG/dextran gel system as a model system in this study. The partitioning behavior of pure bovine serum albumin (BSA) in PEG/dextran gel systems is investigated to see the effects of variations in PEG and NaCl concentrations on the partition coefficient K. By making use of the Box–Wilson experimental design, K is shown to be maximized at 9.8 (%, w/w) PEG and 0.2 M NaCl concentrations, respectively, as 182.
Keywords: Box–Wilson experimental design; Aqueous two-phase system; Polyethylene glycol; Sephadex; Bovine serum albumin;

Protein denaturation and aggregation are well-known problems in the pharmaceutical industry. As the protein aggregates, it loses its biological activity and creates problems in its administration to patients. In this paper, we explore the use of aqueous two-phase systems, capillary zone electrophoresis, and dynamic light scattering for the monitoring of protein denaturation and aggregation. Our studies focus on human IgG and HSA. Capillary zone electrophoresis was used to monitor changes in the charge to size ratio of the proteins upon denaturation and dynamic light scattering was used to detect the presence of any aggregates and to monitor the size of the proteins. The information obtained from aqueous two-phase partitioning is similar to that obtained from capillary zone electrophoresis. The simplicity of aqueous two-phase system and its low cost (compared to the other analytical techniques) suggest that it can be routinely used for the quality control of some pharmaceutical preparations.
Keywords: Dynamic light scattering; Aqueous two-phase systems; Partitioning; Aggregation; Denaturation; Immunoglobulin G;

The hydrophobic contributions of 17 individual peptides, fused to the N-terminal of Bacillus stearothermophilus lactate dehydrogenase (LDH) were studied by hydrophobic interaction chromatography (HIC) and aqueous two-phase system (ATPS). The constructs were sequenced from a protein library designed with a five-amino acid randomised region in the N-terminal of an LDH protein. The 17 LDH variants and an LDH control lacking the randomised region were expressed in Escherichia coli. HIC and ATPS behaviour of the proteins indicated significant differences in protein hydrophobicity, even though the modifications caused only 1% increase in protein molecular weight and 2% variation in isoelectric points. HIC and ATPS results correlated well (R 2=0.89). Protein expression was clearly affected by N-terminal modification, but there was no evidence that the modification affected protein activity. A GluAsnAlaAspVal modification resulted in increased protein expression. In most cases, HIC and ATPS results compared favourably with those predicted on the basis of 34 amino acid residue hydrophobicity scales; assuming exposure of tag residues to solution. Exceptions included LeuAlaGlyValIle and LeuTyrGlyCysIle modifications, which were predicted, assuming full solution exposure, to be more hydrophobic than observed.
Keywords: Hydrophobicity; Aqueous two-phase systems; Partitioning; Escherichia coli; Fusion proteins; Lactate dehydrogenase; Peptides;

A process for the primary recovery of B-phycoerythrin from Porphyridium cruentum exploiting aqueous two-phase systems (ATPS) was developed in order to reduce the number of unit operations and benefit from an increased yield of the protein product. The evaluation of system parameters such as poly(ethylene glycol) (PEG) molecular mass, concentration of PEG as well as salt, system pH and volume ratio was carried out to determine under which conditions the B-phycoerythrin and contaminants concentrate to opposite phases. PEG 1450-phosphate ATPS proved to be suitable for the recovery of B-phycoerythrin because the target protein concentrated to the top phase whilst the protein contaminants and cell debris concentrated in the bottom phase. An extraction ATPS stage comprising volume ratio (Vr) equal to 1.0, PEG 1450 24.9% (w/w), phosphate 12.6% (w/w) and system pH of 8.0 allowed B-phycoerythrin recovery with a purity of 2.9 (estimated as the relation of the 545–280 nm absorbances). The use of ATPS resulted in a primary recovery process that produced a protein purity of 2.9±0.2 and an overall product yield of 77.0% (w/w). The results reported demonstrated the practical implementation of ATPS for the design of a primary recovery process as a first step for the commercial purification of B-phycoerythrin produced by P. cruentum.
Keywords: Bioprocess intensification; Aqueous two-phase systems; Porphyridium cruentum; B-phycoerythrin;

Surfactant-aided size exclusion chromatography by D.A Horneman; M Wolbers; M Zomerdijk; M Ottens; J.T.F Keurentjes; L.A.M van der Wielen (39-45).
The flexibility and selectivity of size exclusion chromatography (SEC) for protein purification can be modified by adding non-ionic micelle-forming surfactants to the mobile phase. The micelles exclude proteins from a liquid phase similar to the exclusion effect of the polymer fibers of the size exclusion resin. This surfactant-aided size exclusion chromatography technology (SASEC) is demonstrated on the separation of two model proteins; bovine serum albumin (BSA) and myoglobin (Myo). The effect of the added surfactants on the distribution behavior of the proteins is predicted adequately by a size exclusion model presented in this paper.
Keywords: Micelle-forming surfactants; Bovine serum albumin; Myoglobin;

Optimized extraction by cetyl trimethyl ammonium bromide reversed micelles of xylose reductase and xylitol dehydrogenase from Candida guilliermondii homogenate by Ely Vieira Cortez; Adalberto Pessoa; Maria das Graças de Almeida Felipe; Inês Conceição Roberto; Michele Vitolo (47-54).
The intracellular enzymes xylose reductase (XR, EC 1.1.1.21) and xylitol dehydrogenase (XD, EC 1.1.1.9) from Candida guilliermondii, grown in sugar cane bagasse hydrolysate, were separated by reversed micelles of cetyl trimethyl ammonium bromide (CTAB) cationic surfactant. An experimental design was employed to optimize the extraction conditions of both enzymes. Under these conditions (temperature=5 °C, hexanol: isooctane proportion = 5% (v/v), butanol concentation=22%, surfactant concentration = 0.15 M, pH=7.0 and electrical conductivity=14  mS cm−1) recovery values of about 100 and 80% were achieved for the enzymes XR and XD, respectively. The purity of XR and XD increased 5.6- and 1.8-fold, respectively. The extraction process caused some structural modifications in the enzymes molecules, as evidenced by the alteration of K M values determined before and after extraction, either in regard to the substrate (up 35% for XR and down 48% for XD) or cofactor (down 29% for XR and up 11% for XD). However, the average variation of V max values for both enzymes was not higher than 7%, indicating that the modified affinity of enzymes for their respective substrates and cofactors, as consequence of structural modifications suffered by them during the extraction, are compensated in some extension. This study demonstrated that liquid–liquid extraction by CTAB reversed micelles is an efficient process to separate the enzymes XR and XD present in the cell extract, and simultaneously increase the enzymatic activity and the purity of both enzymes produced by C. guilliermondii.
Keywords: Reversed micelles; Candida guilliermondii; Xylitol dehydrogenase; Xylose reductase; Enzymes;

Liquid–liquid extraction of xylitol dehydrogenase from Candida guilliermondii homogenate by reversed micelles by Ely Vieira Cortez; Adalberto Pessoa; Maria das Graças de Almeida Felipe; Inês Conceição Roberto; Michele Vitolo (55-60).
The intracellular enzyme xylitol dehydrogenase (XD, EC 1.1.1.9) from Candida guilliermondii, grown in sugarcane bagasse hydrolysate, was separated by reversed micelles of BDBAC [N-benzyl-N-dodecyl-N-bis (2-hydroxyethyl) ammonium chloride] cationic surfactant. An experimental design was employed to evaluate the influence of the following factors on the enzyme separation: temperature, co-solvent concentration and surfactant concentration. The results showed that just the temperature did not show significant effect on XD recovery. A model was used to represent the activity recovery and fit the experimental data. Under optimized conditions, the recovery of total activity was about 121%, and the purity increased 2.3-fold.
Keywords: Candida guilliermondii; Xylitol dehydrogenase;

Bromelain partitioning in two-phase aqueous systems containing PEO–PPO–PEO block copolymers by Ana Paula Brescancini Rabelo; Elias Basile Tambourgi; Adalberto Pessoa (61-68).
Bromelain is an enzymatic complex obtained from pineapple (Ananas comosus) fruits and stem. Thermoseparation of bromelain by poly(ethylene oxide) (PEO)– poly(propylene oxide) (PPO)– poly(ethylene oxide) (PEO) block copolymers aqueous solutions was studied. Triblock copolymers with different EO percentages and different molecular mass were evaluated. Copolymer solutions at different pH values, buffer concentrations and copolymer concentrations were investigated. It was found that cloud point temperature increases as a function of %EO and decreases with copolymer molecular mass, copolymer concentration and buffer concentration. The results showed that all the studied factors influenced enzyme partition. The best conditions were copolymer with 10% EO and molecular mass of 2000 g/mol, temperature of 25 °C, copolymer concentration of 5% (w/w), pH 6.0 and salt concentration of 15 mM. Enzyme activity recovery around 79.5%, purification factor around 1.25 and activity partition coefficient around 1.4 were obtained.
Keywords: Partitioning; Aqueous two-phase systems; Enzyme purification; Thermoseparation; Bromelain;

Purification of a recombinant, thermostable α-amylase (MJA1) from the hyperthermophile, Methanococcus jannaschii, was investigated in the ethylene oxide–propylene oxide random copolymer (PEO–PPO)/(NH4)2SO4, and poly(ethylene glycol) (PEG)/(NH4)2SO4 aqueous two-phase systems. MJA1 partitioned in the top polymer-rich phase, while the remainder of proteins partitioned in the bottom salt-rich phase. It was found that enzyme recovery of up to 90% with a purification factor of 3.31 was achieved using a single aqueous two-phase extraction step. In addition, the partition behavior of pure amyloglucosidase in polymer/salt aqueous two-phase systems was also evaluated. All of the studied enzymes partitioned unevenly in these polymer/salt systems. This work is the first reported application of thermoseparating polymer aqueous two-phase systems for the purification of extremophile enzymes.
Keywords: Aqueous two-phase systems; Methanococcus jannaschii; Partitioning; Thermoseparation; α-Amylase; Amyloglucosidase;

Mass transfer in aqueous two-phases system packed column by L Igarashi; T.G Kieckbusch; T.T Franco (75-80).
The behavior of xylanase extraction in a packed column using polyethylene glycol (PEG) 4000 and dipotassium phosphate was studied. The possibility of using the packed column in continuous operations for enzyme extraction was studied since the previous work had only addressed the semi-continuous extraction of enzyme. The influence of several kinds of packings, Raschig rings, glass spheres and polystyrene rings were studied as well the superficial velocity ratio of the salt and the PEG phases. Packed column showed a good efficiency of overall mass transfer coefficient, around three times higher than sieve plate column, for xylanase extraction. The best selectivity was obtained with the polystyrene ring where 94% of xylanase was recovery to the polymeric whereas just 3% of contaminant was recovery to this phase. The residence time distribution was adjusted by the Model of Reactors in Series.
Keywords: Aqueous two-phase systems; Mass transfer; Xylanase;

Effect of electrostatic energy on partitioning of proteins in aqueous two-phase systems by A. Olivera-Nappa; G. Lagomarsino; B.A. Andrews; J.A. Asenjo (81-86).
An attempt has been made to adopt a different approach to evaluate the effect of a protein’s charge on its partitioning behaviour in PEG/salt aqueous two-phase systems (ATPS). This has been done using a computer methodology (DelPhi) that allows the calculation of the electrostatic solvation energy that charged proteins present in a particular media such as aqueous polymer–salt systems. This calculation was done for the protein in each of the phases and a correlation was investigated that related the electrostatic energy difference of the protein in each of the phases and its partition coefficient in ATPS. Such correlation resulted in a statistical model that also included the effect of molecular weight and a shape factor at each particular pH. A global correlation which included the effect of pH was also found. All the correlations were statistically evaluated and gave good results.
Keywords: Aqueous two-phase systems; Partitioning; Electrostatic energy; Proteins;

A novel inexpensive affinity purification technology is described based on recombinant expression in Escherichia coli of the polypeptide or protein target fused through its N-terminus to TmXyn10ACBM9-2 (CBM9), the C-terminal family 9 carbohydrate-binding module of xylanase 10A from Thermotoga maritima. Measured association constants (K a) for adsorption of CBM9 to insoluble allomorphs of cellulose are between 2×105 and 8×106  M−1. CBM9 also binds a range of soluble sugars, including glucose. As a result, a 1 M glucose solution is effective in eluting CBM9 and CBM9-tagged fusion proteins from a very inexpensive commercially-available cellulose-based capture column. A processing site is encoded at the C-terminus of the tag to facilitate its rapid and quantitative removal by Factor Xa to recover the desired target protein sequence following affinity purification. Fusion of the CBM9 affinity tag to the N-terminus of green fluorescent protein (GFP) from the jellyfish, Aquorin victoria, is shown to yield >200 mg l−1 of expressed soluble fusion protein that can be affinity separated from clarified cell lysate to a purity of >95% at a yield of 86%.
Keywords: Purification; Escherichia coli; Polypeptides; Xylanase 10A; Carbohydrates;

Decontamination of surfaces by lysozyme encapsulated in reverse micelles by Ondrea Bermudez; Daniel Forciniti (95-103).
Cells and enzymes can be used to decontaminate soil, water supplies, personal equipment, weapons and hospital equipment that have been exposed to bacteria, toxins or viruses. One of the problems associated with the use of microorganisms and enzymes for decontamination purposes is that the presence of water is not acceptable for some applications such as electronic equipment. One way of circumventing this problem is to allow the enzyme to distribute between a water phase and an organic phase-containing surfactant and then use the encapsulated enzyme in reverse micelles directly into the device to be clean. Reverse micelles were used to deliver the enzyme (lysozyme) to the cell–surface interface. They serve as a way to increase the local concentration of lysozyme and decrease the amount of water delivered. Specifically, we explored the lysis by free lysozyme and lysozyme encapsulated in reverse micelles of Klebsiella pneumoniae and Staphylococcus epidermidis attached to steel, glass, and hydroxyapatite. These two bacteria have been selected because they are known to be pathogenic and because of their differences in cell wall structure. Lysozyme was added to the surfaces in either reverse micelles or as a free solution and was tested under conditions of stirring and no stirring. Stirring was implemented to study the interplay between mass transfer limitations and surface roughness. We have shown that free lysozyme or lysozyme encapsulated in reverse micelles is capable of decontaminating surfaces of different texture. Lysis of the cells is slower when the encapsulated enzyme is used but lysis is more complete.
Keywords: Reversed micelles; Enzyme kinetics; Scintillation counting; Dynamic light scattering; Lysozyme;

Recovery in aqueous two-phase systems of lutein produced by the green microalga Chlorella protothecoides by Mayra Cisneros; Jorge Benavides; Carmen H Brenes; Marco Rito-Palomares (105-110).
In this study the potential use of aqueous two-phase system (ATPS) to establish a viable process for the recovery of lutein from the green microalga Chlorella protothecoides is evaluated. The partitioning behaviour of lutein, a representative model of natural compounds of commercial interest, was investigated in a polyethylene glycol (PEG)-phosphate system. An evaluation of system parameters including PEG molecular mass, the concentrations of PEG, phosphate and product concentration was conducted, to estimate conditions under which lutein partitions preferentially to the top phase whilst cell debris partition to the opposite phase. The necessary addition of ethanol to the ATPS for the dissolution of lutein affected the phase formation and such effect was evaluated using the change in the volume ratio produced. ATPS extraction comprising Vr=1.0, PEG 8000 22.9% (w/w) and phosphate 10.3% (w/w), pH 7.0 provided the conditions for the concentration of lutein into the upper phase and the cell debris preferentially to the bottom phase. The use of ATPS resulted in a primary recovery process to obtain lutein with an overall product yield of 81.0±2.8%. The findings reported here demonstrate the potential of ATPS for the further development of a prototype process to recover lutein from C. protothecoides as a first step for the generic application of this technique.
Keywords: Aqueous two-phase systems; Chlorella protothecoides; Lutein;

Quantification of solid cell material by detection of membrane-associated proteins and peptidoglycan by P. van Hee; A.P.J. Middelberg; R.G.J.M. van der Lans; L.A.M.van der Wielen (111-119).
Quantification of solid cell material (cell debris) is necessary for the optimisation of the efficiency of bioseparations. Cell debris can be quantified by detection of a component present in the cell wall that can act as a marker for cell debris. Membrane-associated proteins have previously been used as a marker for cell debris. This marker was quantified by SDS–PAGE with densiometry. In this paper cell debris quantification methods are presented that are faster and more accurate, i.e. membrane-associated protein quantification with the Protein 50 Labchip® of Agilent Technologies, or that make use of peptidoglycan as marker for cell debris, i.e. a spectrophotometric muramic acid assay.
Keywords: Cell debris; Escherichia coli; Pseudomonas putida; Acetaldehyde; Muramic acid; Peptidoglycan;

Polyelectrolyte complexes as a tool for purification of plasmid DNA by Per-Olof Wahlund; Per-Erik Gustavsson; Vladimir A Izumrudov; Per-Olof Larsson; Igor Yu Galaev (121-127).
The demand for highly purified plasmids in gene therapy and plasmid-based vaccines requires large-scale production of pharmaceutical-grade plasmid. Plasmid DNA was selectively precipitated from a clarified alkaline lysate using the polycation poly(N,N′-dimethyldiallylammonium) chloride which formed insoluble polyelectrolyte complex (PEC) with the plasmid DNA. Soluble PECs of DNA with polycations have earlier been used for cell transformation, but now the focus has been on insoluble PECs. Both DNA and RNA form stable PECs with synthetic polycations. However, it was possible to find a range of salt concentration where plasmid DNA was quantitatively precipitated whereas RNA remained in solution. The precipitated plasmid DNA was resolubilised at high salt concentration and the polycation was removed by gel-filtration.
Keywords: Purification; Plasmid DNA; Polyelectrolyte complexes;

Characterization of supermacroporous monolithic polyacrylamide based matrices designed for chromatography of bioparticles by Fatima M Plieva; Irina N Savina; Sahar Deraz; Jonatan Andersson; Igor Yu Galaev; Bo Mattiasson (129-137).
Supermacroporous monolithic acrylamide (AAm)-based cryogels were prepared by radical cryo-polymerizaton (polymerization in the moderately frozen system) of AAm with functional monomers and cross-linker N,N′-methylene-bis-acrylamide (MBAAm). Electron microscopy studies revealed supermacroporous structure of the developed cryogels with pore size of 5–100 μm. Cryogel porosity depended on cryo-polymerization conditions. More than 90% of the monolithic bed volume is the interconnected supermacropores filled with water and less than 10% of the monolithic volume is pore walls. The total protein binding capacity (lysozyme in the case of immobilized metal affinity chromatography (IMAC) column and bovine serum albumin (BSA) in the case of anion-exchange (AE) column) was independent of the flow rates till 600 cm/h. Chromatographic behavior of E. coli cells when a cell suspension was applied to ion-exchange cryogel columns depended on both the density of functional ligand and the porosity of the cryogel.
Keywords: Monolithic column; Bioparticles; Cryogel; Supermacroporous polyacrylamide;

Aqueous two-phase systems (ATPS) were applied for extraction of small molecules (polycetides) – retamycin, an anthracyclin, and two red pigments, rubropunctamin and monascorubramin – from the whole culture media of Streptomyces olindensis and Monascus purpureus. ATPS allows, in one step, the separation of the small hydrophobic molecules in the PEG rich phase, from the filamentous microorganisms, which remains in the salt phase. Through experimental designs, the main variables and their levels were defined, as follows: for retamycin extraction, PEG 6000 (10%, w/w), phosphate at 20% (w/w) and pH 6.0 led to the higher partition coefficient, K r = 8.2, and yield = 91.3%; for red pigments, the statistical analysis indicate PEG 6000 (20%, w/w) and phosphate at 15% (w/w), for a high partition coefficient, (K pig = 113 and 150).
Keywords: Aqueous two-phase system; PEG/salt; Polycetides; Clarification;

Application of polyethylene glycol-based aqueous biphasic reactive extraction to the catalytic oxidation of cyclic olefins by Ji Chen; Scott K Spear; Jonathan G Huddleston; John D Holbrey; Robin D Rogers (145-149).
Glutaric acid and 1,2,3,4-butanetetracarboxylic acid (BTCA) have been synthesized by sodium tungstate catalyzed oxidation of the cyclic olefins: cyclopentene and 1,2,3,6-tetrahydrophthalic anhydride (THPA), using hydrogen peroxide in a polyethylene glycol (PEG)-2000/NaHSO4 aqueous biphasic system (PEG-ABS). The production of glutaric acid and BTCA was found to increase from the monophasic to the biphasic regimes, and was found to be greatest at short tie-line lengths (TLLs), close to the system’s critical point, yielding glutaric acid and BTCA in 73.1 and 82.5% yield, respectively. The results imply that mutual mixing or contact of the components is important, because the product dicarboxylic acids were found to increase from the monophasic side to the critical point and decrease from the critical point to more divergent regimes. The two reactions were compared with adipic acid synthesis from cyclohexene in terms of the cyclic olefin structure, and the partitioning of the dicarboxylic acid product in the ABS.
Keywords: Aqueous two-phase systems; Catalysis; Oxidation; Cyclic olefins;

Effects of speciation on partitioning of iodine in aqueous biphasic systems and onto ABEC® resins by Scott T. Griffin; Scott K. Spear; Robin D. Rogers (151-156).
Polyethylene glycol (PEG)-aqueous biphasic systems (ABS) and PEG-grafted aqueous biphasic extraction chromatographic (ABEC) resins have been shown to remove inorganic species from environmental and nuclear wastes. The partitioning behavior of several iodide species (iodide, iodine, triiodide, iodate, and 4-iodo-2,6-dimethylphenol (I-DMP)) have been studied for PEG (MW 2000)-salt systems and ABEC resins. Iodide partitioning to PEG-rich phases or onto ABEC resins can be enhanced by derivatization with 2,6-dimethylphenol to form 4-iodo-2,6-dimethylphenol or by addition of I2 to form triiodide. Conversely, iodide partitioning to the PEG-rich phase or onto ABEC resins is reduced by oxidation of iodide to IO3 . Partitioning studies of iodide, iodate, and iodine in a PEG-ABS are compared to results using ABEC resins.
Keywords: Partitioning; Aqueous two-phase systems; Iodide; Iodine; Iodate; Triiodide;

The dynamic viscosities of aqueous polyethylene glycol, aqueous bovine serum albumin, and polyethylene glycol–bovine serum albumin–water solutions were measured at temperatures of 15, 20, 25, 30 and 35 °C. To estimate the viscosity values of polyethylene glycol–bovine serum albumin–water solutions, a one parameter Grunberg-like model which was satisfactorily used earlier by the present author for polyethylene glycol-dextran–water solutions was employed. The disposable parameter a for our temperature range was estimated as 3.71. The relative errors varying from 0.29 to 18.98 in absolute value indicates that the Grunberg-like model works perfectly for polymer-protein solutions as well.
Keywords: Grunberg equation; Viscosity; Aqueous two-phase systems; Polyethylene glycol; Bovine serum albumin;

Alloimmunization to donor blood group antigens remains a significant problem in transfusion medicine. To attenuate the risk of alloimmunization, we have pioneered the membrane grafting of methoxypoly(ethylene glycol) (mPEG) to produce immunocamouflaged red blood cells (RBC). Grafting of the mPEG was accomplished using cyanuric chloride activated mPEG (CmPEG; M r=5000), benzotriazole carbonate methoxyPEG (BTCmPEG; M r=5000 or 20,000); or N-hydroxysuccinimidyl ester of mPEG propionic acid (SPAmPEG; M r=2000, 5000, or 20,000). Because of the heterogeneity of grafting, a crucial tool in developing the stealth RBC is an ability to purify the modified RBC from unmodified (immunogenic) donor cells. As demonstrated, a (5, 4) dextran:PEG aqueous two-phase polymer partitioning system cleanly separated the immunologically silent mPEG-grafted human RBC from control or lightly modified cells. Cell mixing experiments employing varying ratios of mPEG-modified and control RBC confirmed the purification efficacy of the phase partitioning system. Proportional changes in PEG-rich phase partitioning were achieved by increasing either the quantity of surface mPEG or the mPEG molecular weight. The biological viability of purified mPEG-RBC (BTCmPEG; M r=20,000) was demonstrated by their normal in vivo survival at immunoprotective grafting concentrations (≤2 mmol/L). The effective immunocamouflaging of RBC antigens coupled with efficient purification of the immunocamouflaged population provides encouragement for the further development of the stealth erythrocyte.
Keywords: Purification; Aqueous two-phase systems; Partitioning; Methoxypoly(ethylene glycol);