Effect Of PEG Molecular Weight Research Articles

Effects on peptide binding affinity for TNFα by PEGylation and conjugation to hyaluronic acid

Conjugation of cytokine-neutralizing monoclonal antibodies (mAb) to hyaluronic acid (HA) having Mw of 1.6MDa was previously shown to be an effective strategy for localized delivery to sites of inflammation. Despite the disparity in size of the mAb and HA, the mAb–HA conjugate was found bind tumor necrosis factor-α (TNFα) as strongly as the non-conjugated antibody, suggesting conjugation to this charged polysaccharide can provide an alternative to poly(ethylene glycol) (PEG) conjugation, which has been shown to reduce binding interactions for many proteins. To explore conjugation chemistries more systematically, we report a study on a model peptide inhibitor of tumor necrosis factor-α to investigate the effects of site-specific conjugation to HA and PEG. We compared the binding affinities of a variety of WP9QY peptide–polymer conjugates for TNFα in order to examine the effects of PEG molecular weight as well as the effects of PEG versus functionalized hyaluronic acid (HA) conjugation. The results indicate that the binding affinity of the PEG conjugates decreases in comparing PEG with mass 2k, 10k, and 30k, which was attributed to PEG shrouding of the peptide, while conjugation to a 66kDa HA chain preserved peptide binding affinity. We attribute this difference to the increased solubility of HA compared to PEG, potentially due to the carboxylic acid functional groups. In addition, the results demonstrate that conjugation to HA via a short PEG linker significantly enhances the association rate kon, which may reflect an increased peptide accessibility. By balancing both the advantages associated with the PEG conjugates and with the HA conjugates, the HA–PEG2k–WP9QY conjugate was able to improve the binding affinity of the peptide for TNFα by a factor of two. Optimization of polymer chemistry could be used to improve delivery of protein therapeutics for localized and systemic administration.

Liquid–liquid equilibrium of aqueous two-phase systems containing poly(ethylene glycol) of different molecular weights and several ammonium salts at 298.15 K

Liquid–liquid equilibrium data of aqueous two-phase systems (ATPSs) formed by poly(ethylene glycol) (PEG) of different molecular weights (1000, 2000, 6000)+ammonium carbonate ((NH4)2CO3), ammonium tartrate ((NH4)2C4H4O6), diammonium hydrogen phosphate ((NH4)2HPO4), ammonium sulfate ((NH4)2SO4)+water were determined at 298.15K, respectively. The binodal data were correlated by using a four-parameter equation. The effect of PEG molecular weight on binodal curves was investigated, and it was found that the biphasic region expanded with an increase in PEG molecular weight. Meanwhile, it was also observed that the ability of the investigated salts for phase separation follows the order: (NH4)2HPO4>(NH4)2SO4>(NH4)2C4H4O6>(NH4)2CO3. Furthermore, a relatively simply two parameter equation, Othmer–Tobias and Bancroft equations was successfully used to correlate the tie-line data.

Surface modification of commercial polyamide desalination membranes using poly(ethylene glycol) diglycidyl ether to enhance membrane fouling resistance

To improve fouling resistance, polyamide reverse osmosis (XLE) and nanofiltration (NF90) membranes were modified by grafting poly(ethylene glycol) (PEG) diglycidyl ether (PEGDE) to their top surfaces from aqueous solution. The effect of PEG molecular weight (200 vs. 1000) and treatment solution concentration (1% (w/w) vs. 15% (w/w)) on water flux and NaCl rejection was measured. PEGDE grafting density as well as surface properties of modified and unmodified membranes, including charge, hydrophilicity and roughness, were measured and compared. The fouling resistance of modified membranes to charged surfactants (i.e., sodium dodecyl sulfate (SDS) and dodecyltrimethylammonium bromide (DTAB)) and emulsions of n-decane and these charged surfactants was compared to that of unmodified membranes. In general, modified membranes exhibited improved fouling resistance and an improved ability to be cleaned after fouling compared to unmodified membranes. Fouling resistance increased with increasing PEG molecular weight, but showed little dependence on treatment solution concentration, suggesting that further improvements in membrane fouling resistance might be obtained by using lower concentrations of higher molecular weight PEG for surface modification.

Improving glutathione extraction from crude yeast extracts by optimizing aqueous two-phase system composition and operation conditions

PEG-Dextran and PEG-salt aqueous two-phase systems (ATPS) have been applied to separate glutathione (GSH) from crude yeast extracts. Single-factor experiments were carried out to determine the important factors influencing the partition coefficient and extraction yield. The effect of PEG molecular weight, phase-forming components, PEG and Dextran concentration, pH value, and temperature on the GSH partitioning behavior in ATPS was investigated. Three factors, Dextran concentration, pH value, and temperature, were confirmed to have significant influence on the partition coefficient and extraction yield. These factors were further analyzed with the aid of central composite rotatable design and response surface methodology. The optimal conditions for GSH extraction in the PEGDextran system were determined, including PEG molecular weight 6,000, 10% PEG concentration, 14% Dextran concentration, pH 5.2, and temperature 32 °C. A high extraction yield (83.55%) of GSH from crude yeast extracts was achieved under these optimized conditions. This work is very helpful for developing one efficient and cost-effective process for the separation and purification of GSH from yeast broths.

INITIAL DROPLETS FORMATION AND GROWTH PROCESS OF ACRYLAMIDE DURING AQUEOUS TWO-PHASE POLYMERIZATION IN AQUEOUS POLY(ETHYLENE GLYCOL) SOLUTION

In order to explore the droplet formation process of the aqueous two-phase polymerization of acrylamide(AM) in aqueous poly(ethylene glycol)(PEG) solution,dynamic light scattering(DLS) was employed to investigate the appearance,the size and size distribution of droplets in the initial stage of polymerization.The effects of PEG molecular weight and concentration on the droplets size and distribution were studied.It was found that the droplets stability in aqueous PEG solution decreased,and the droplets aggregated with each other to form larger and more uniform droplets with decreasing PEG molecular weight or PEG concentration.The morphology of the droplets rich in polyacrylamide(PAM) was observed with transmission electron microscopy(TEM).The droplets shape in the initial stage of polymerization is spherical but polydisperse.However,some oval droplets formed in the product,while the conversion was still very low,which indicated some coalescence of droplets in this stage.As the polymerization proceeds,the droplet size increased rapidly and the size distribution of droplets become uniform.These results were well in agreement with the DLS results.With using the spectrophotometer,the phase separation point was determined where the transparence of reaction solution declined suddenly.The effect of various parameters on the critical conversion of phase separation was measured by bromating method.It decreased with the increase of PEG molecular weight or PEG concentration.With raising temperature from 30℃ to 70℃,the critical conversion decreased at first,and then turned to increase when the temperature was at about 50℃.The critical molecular weight was also systemically investigated by gel permeation chromatogram(GPC).It could be seen that the evolution of critical molecular weight corresponded well with that of critical conversion at various reaction conditions.On the basis of above mentioned results,an initial droplet formation and growth mechanism of AM aqueous two-phase polymerization in aqueous PEG solutions was proposed.As the reaction starts,primary radicals,generated by decomposition of the initiator,grow in the continuous phase by the addition of monomer units until they reach their critical chain length.Then these polymer radicals precipitate and aggregate to form nuclei.The nuclei are not stable,and they will immediately aggregate with each other to form tiny droplets.Then monomer and initiator diffuse into the droplets rapidly,and polymerization takes place in both the continuous phase and the dispersed phase(inside the droplets).In the earlier stage,the PAM droplets are stable and grow mainly by polymerization inside the droplet.Because new smaller droplets generate from continuous phase at the same time,the droplet size distribution becomes wide.However,these droplets become unstable with the polymerization and aggregate with each other to make the droplet size distribution become narrow again.

Bovine serum albumin partitioning in polyethylene glycol (PEG)/potassium citrate aqueous two-phase systems

The extraction and back-extraction of bovine serum albumin (BSA) have been studied by liquid–liquid extraction with poly(ethylene glycol) (PEG)/potassium citrate aqueous two-phase system (ATPS). In this work, the ATPS was examined with regard to the effects of PEG molecular weight (PEG 1000, 2000, 4000 and 6000), PEG and potassium citrate concentration, BSA concentration ( C BSA) and pH on BSA partition. The pH was found to have significant effects on BSA partition with low molecular weight PEG 1000. The yield of the BSA, 99%, was obtained in the top phase under the following conditions: 19% (w/w) PEG 1000, 20% (w/w) potassium citrate and 0.75 mg/g C BSA at pH 7.0 and 30 °C. BSA can be re-extracted to a new citrate phase by decreasing the pH of the system with a 92% yield. The back-extraction not only separates the BSA from the polymer, but also allows the polymer to be recycled. The global yield ( Y e + Y be) is up to 91%.

Effect of PEG molecular weight on Bottom-up Filling of Copper Electrodeposition for PCB

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Formowanie wlokien z terpoli(estro-etero-estrow) metoda przedzenia ze stopu lub elektroprzedzenia z roztworu

Novel terpoly(ester-ether-ester)s (TEEE) built of hard segments, formed by poly(butylene terephthalate) (PBT), and soft blocks built of dilinoleic acid (DLA) residues and poly(ethylene glycol) (PEG) of different molecular weights (Table 1) were prepared. The fibers were prepared from the materials obtained using the methods of melt spinning or electrospinning from the solution (Fig. 1 and 5). The effects of PEG molecular weight and addition of organic thermal stabilizer (a-tocopherol, VE) as well as of addition of inorganic modifier (hydroxyapatite, HAP) on melt flow rate (MFR) (Fig. 2) and on mechanical (Fig. 3) and fiber-forming properties of TEEE were studied. Hybrid microfibers were prepared by melt spinning from the system containing HAP nanocrystalline ceramics. It was found that this ceramics thermally stabilized the material similarly as VE did. Terpolymers prepared from PEG of higher molecular weight (PEG 4600 ) showed very good mechanical properties and fibers prepared exhibited excellent elastic properties (high values of elastic recovery, Table 2).

Protein Resistance of PEG-Functionalized Dendronized Surfaces: Effect of PEG Molecular Weight and Dendron Generation

Dendronized surfaces were prepared by chemisorption of poly(ethylene glycol) monothiol (HS-PEG650-OH) onto gold-coated silicon wafers followed by functionalization of the PEG terminal OH group with aliphatic polyester dendrons, generation 1−4, using divergent dendron growth. PEG monomethyl ether (PEG-OMe) chains of various molecular weight (MW) were covalently attached to the peripheral hydroxyl groups of the dendronized surfaces via EDC coupling and investigated for protein adsorption. Protein adsorption studies were carried out using fibrinogen (Fg) and lysozyme (Lys) as model proteins from phosphate buffered saline (PBS) (Fg, Lys) and plasma (Fg). In the first part of this study, the effect of functionalization of the peripheral hydroxyl groups with PEG-OMe oligomers (Mn = 350 Da) on protein adsorption was investigated. Results showed that adsorption of both Fg and Lys was reduced when dendronized surfaces were grafted with PEG-OMe oligomers. To investigate the effect of molecular weight on protein adsorption, PEG-OMe chains of greater length (750, 2000, and 5000 Da) were coupled to first generation dendronized surfaces (Au-G1(OH)). Results showed that protein adsorption decreased with increasing PEG-OMe MW up to 2000 Da. To further investigate the effect of dendron generation on protein resistance, dendronized surfaces of generation 1−4 were coupled with PEG2000-acid. Subsequent protein studies showed a decrease in Fg and Lys adsorption with increasing dendron generation.

Effect of PEG molecular weight and linking chemistry on the biological activity and thermal stability of PEGylated trypsin

PEGylated proteins are routinely used as therapeutics, but systematic studies of the effect of PEG molecular weight and linking chemistry on the biological activity and particularly the thermal stability of the conjugated protein are rarely made. Here, activated monomethoxypolyethylene glycol (mPEG)s (Mw 1100, 2000 and 5000 g/mol) were prepared using succinic anhydride (SA), cyanuric chloride (CC) or tosyl chloride (TC) and used to synthesise a library of trypsin conjugates. The enzyme activity ( K M, V max and K cat) of native trypsin and the mPEG-modified trypsin conjugates was compared using N-benzoyl- l-arginine p-nitroanilide (BAPNA) as a substrate, and their thermal stability determined using both BAPNA and N-α-benzoyl- l-arginine ethyl ester hydrochloride (BAEE) as substrates to measure amidase and esterase activity respectively. The effect of conjugate chemistry on trypsin autolysis was also examined at 40 °C. PEG-trypsin conjugates containing the higher molecular weight of mPEG (5000 g/mol) were more stable than free trypsin, and the conjugate containing CC-mPEG 5000 g/mol had the best thermal stability.

Hydrophilic and flexible polyurethane foams using sodium alginate as polyol: Effects of PEG molecular weight and cross-linking agent content on water absorbency

Hydrophilic and flexible polyurethane foams were prepared using sodium alginate as a polyol, and characterized by optical microscopy, FT-IR spectroscopy, density measurements, volume swelling, and water absorbency. Optical microscopy revealed that the resulting cells were closed with round and elongated shapes. FT-IR confirmed that the urethane linkages were formed between the isocyanate and sodium-alginate. As an indirect measurement of porosity, the apparent density indicated an initial decrease followed by an increase with increasing glycerin content. The volume-swelling ratio was initially constant, followed by a gradual decrease with glycerin content. The volume swelling ratio increased with PEG molecular weight. The water absorbency initially increased, followed by a decrease with increasing glycerin content. The correlation-ships between water absorbency, density, and volume-swelling ratio indicated that the absorbency was predominantly influenced by density when the PEG molecular weight was low and was greatly affected by the volume-swelling ratio when the PEG molecular weight was relatively high.

High-pressure CO2-enhanced polymer interdiffusion and dissolution studied with in situ ATR-FTIR spectroscopic imaging

A novel application of attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopic imaging to study polymer interdiffusion and dissolution under high-pressure or supercritical carbon dioxide environments has been demonstrated. Miscible systems of polyvinylpyrrolidone (PVP) and poly(ethylene glycol) (PEG) of different molecular weights have been chosen for this investigation. These systems were subjected to a controlled pressure of CO2 and the interfacial area of contact between the two polymers was studied by ATR-FTIR imaging in situ. Using this spectroscopic imaging approach, the phenomenon of polymer interdiffusion enhanced by CO2 dissolved in both polymers was investigated as a function of time. The evolution of spatially resolved images as a function of time was studied with FTIR imaging and the corresponding concentration profiles for both polymers were obtained. The chemical specificity of FTIR imaging also allowed us to measure the amount of CO2 dissolved in each domain of the polymer system. Effects of PEG molecular weight and pressure of CO2 on the mechanism and the rate of polymer interdiffusion was investigated. This approach has not only shown the ability to visualise the process of interdiffusion but also demonstrated the ability of high-pressure CO2 to ‘tune’ the rate of interdiffusion, this information is important for a better understanding of CO2-induced mixing of polymeric materials.

Effects of Poly(ethylene glycol) (PEG) Concentration on the Permeability of PEG-Grafted Liposomes

Poly(ethylene glycol)-grafted liposomes (PEG-liposomes) were prepared from dipalmitoylphosphatidylcholine (DPPC) with various amounts of distearoyl-N-monomethoxy poly(ethylene glycol)-succinyl-phosphatidylethanolamines (DSPE-PEG) with PEG molecular weights of 1000, 2000, 3000 and 5000. The effects of DSPE-PEG concentration on the permeability of PEG-liposomes were investigated using carboxyfluorescein (CF). In the gel state, the CF leakage from PEG-liposomes was decreased with increasing mole fractions of DSPE-PEG for all PEG molecular weights. In the liquid-crystalline state, the CF leakage from PEG-liposomes containing DSPE-PEG1000 gradually increased with increasing mole fractions of DSPE-PEG, while that of PEG-liposomes whose molecular weight in PEG units was above 2000 rapidly decreased by the addition of DSPE-PEG. Furthermore, no effect of PEG molecular weight on CF leakage was observed. The relationship between the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) (or 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH)) and the mole fraction of DSPE-PEG for PEG-liposomes was also investigated. No significant changes in fluorescence polarization of DPH for liposomal bilayer membranes was observed in the gel and liquid-crystalline states due to the addition of DSPE-PEG, while that of TMA-DPH was decreased compared with that of liposomes without DSPE-PEG in both states.

Physicochemical and biological characterization of polyethylenimine-graft-poly(ethylene glycol) block copolymers as a delivery system for oligonucleotides and ribozymes.

Two different series of polyethylenimine (PEI) block copolymers grafted with linear poly(ethylene glycol) (PEG) were investigated as delivery systems for oligodeoxynucleotides (ODN) and ribozymes. The resulting interpolyelectrolyte complexes were characterized with respect to their physicochemical properties, protection efficiency against enzymatic degradation, complement activation, and biological activity under in vitro conditions. The effect of PEG molecular weight and the graft density of PEG blocks on complex characteristics was studied with two different series of block copolymers. The resulting ODN complexes were characterized by photon correlation spectroscopy (PCS) and laser Doppler anemometry (LDA) to determine complex size and zeta potential. Electrophoresis was performed to study the protective effects of the different block copolymers against enzymatic degradation of ODN. Intact ODN was quantified via densitometric analysis. Ribozymes, a particularly unstable type of oligonucleotides, were used to examine the influence of block copolymer structure on biological activity. The stabilization of ribozymes was also characterized in a cell culture model. Within the first series of block copolymers, the grafted PEG chains (5 kDa) had marginal influence on the complex size. Two grafted PEG chains were sufficient to achieve a neutral zeta potential. Within the second series, size and zeta potential increased with an increasing number of PEG chains. A high number of short PEG chains resulted in a decrease in complex size to values comparable to that of the homopolymer PEI 25 kDa and a neutral zeta potential, indicating a complete shielding of the charges. Complement activation decreased with an increasing number of short PEG 550 Da chains. Ribozyme complexes with PEG-PEI block copolymers achieved a 50% down-regulation of the target mRNA. This effect demonstrated an efficient stabilization and biological activity of the ribozyme, which was comparable to that of PEI 25 kDa. PEGylated PEI block copolymers represent a promising new class of drug delivery systems for ODN and ribozymes with increased biocompatibility and physical stability.

PEGylation of growth hormone-releasing hormone (GRF) analogues

Synthetically produced GRF 1–29 (Sermorelin) has an amino acid composition identical to the N-terminal 29 amino acids sequence of the natural hypothalamic GHRH 1–44 ( Figure 1). It maintains bioactivity in vitro and is almost equally effective in eliciting secretion of endogenous growth hormone in vivo. The main drawbacks associated with the pharmaceutical use of hGRF 1–29 relate to its short half-life in plasma, about 10–20 min in humans, which is caused mostly by renal ultrafiltration and enzymatic degradation at the N terminus. PEGylation has been considered as one valid approach to obtain more stable forms of the peptide, with a longer in vivo half-life and ultimately with increased pharmacodynamic response along the somatotropic axis (endogenous GH, IGF-1 levels). Different PEGylated GRF conjugates were obtained and their bioactivity was tested in vitro and in vivo by monitoring endogenous growth hormone (GH) serum levels after intravenous (i.v.) injection in rats, and intravenous and subcutaneous (s.c.) injection in pigs. It was found that GRF–PEG conjugates are able to bind and activate the human GRF receptor, although with different potency. The effect of PEG molecular weight, number of PEG chains bound and position of PEGylation site on GRF activity were investigated. Mono-PEGylated isomers with a PEG 5000 polymer chain linked to Lys 12 or Lys 21 residues, showed high biological activity in vitro, which is similar to that of hGRF 1–29,and a higher pharmacodynamic response as compared to unmodified GRF molecule.

Impact of Molecular Weight on Miscibility and Interdiffusion between Poly(N‐vinyl pyrrolidone) and Poly(ethylene glycol)

AbstractMiscibility and mutual diffusion between poly(N‐vinylpyrrolidone) (PVP) and short‐chain poly(ethylene glycol)s (PEGs) of different molecular weight were studied by optical wedge microinterferometry (WMI). The PVP Mn varied from 1 300 to 360 000 g · mol−1, whereas that of PEG ranged from 200 to 6 000 g · mol−1. Photographs of interference patterns measured at λ = 546 nm with an optical microscope were used to observe the PVP/PEG interface. PVP blends with short‐chain PEG fractions, ranging in molecular weight from 200 to 1 500 g · mol−1, were found to be fully miscible above the temperatures of PEG fusion, whereas an increase in the molecular weight of up to 3 000 g · mol−1 renders the two polymers completely immiscible. Successive photographs of interference patterns (the interferogram of the contact interface between PEG6000 (left) and PVP is shown in the Figure) were used to determine composition‐distance profiles and to calculate the composition‐dependent mutual diffusion coefficient in miscible PVP/PEG blends. It was shown that the effects of PVP molecular weight are in reasonable agreement with those observed for other compatible polymer systems. In contrast to PVP behavior, the effects of PEG molecular weight indicate appreciable contribution of hydrogen bonding of terminal hydroxyl groups in short PEG chains to the miscibility and diffusivity behavior in the PVP/PEG system. The difference in the effects of PEG and PVP chain lengths is ascribed to hydrogen bonding between PEG terminal hydroxyl groups with carbonyl side groups of repeat units in PVP macromolecules and ether oxygens in the PEG chains. Interferogram of the contact interface between PEG 6000 (left) and PVP. Magnification: 60‐fold.magnified imageInterferogram of the contact interface between PEG 6000 (left) and PVP. Magnification: 60‐fold.

Rice straw as a support for immobilization of microbial lipase.

Candida rugosa lipase was covalently immobilized on rice straw activated with glutaraldehyde using poly(ethylene glycol) (PEG) as the stabilizing agent. The effects of PEG molecular weight and enzyme loading were studied according to a full 2(2) factorial design. Higher immobilization yields (>70%) were attained when the lipase loading was 95 units/mg of dry support, independent of PEG molecular weight. All derivatives showed high hydrolytic and synthetic activities. This work provides preliminary results on the use of agricultural residues as a support matrix for immobilizing lipase and on the application of the resulting derivatives to butyl butyrate synthesis as a study model.

Effect of PEG molecular weight on the tensile toughness of starch/PCL/PEG blends

The blend of a gelatinized starch and poly(e-caprolactone) (PCL) was prepared and the effect of starch gelatinization on the mechanical properties of the blend was studied. The gelatinization of starch resulted in good dispersion of the starch in the PCL matrix and a higher modulus and strength of the blend. The mechanical properties of the starch/PCL/poly(ethylene glycol) (PEG) blends were also investigated. From the change of the toughness of the blends with the PEG molecular weight, it was found that the blend containing PEG of molecular weight 3400 shows the highest tensile toughness. It was also found from the SEM images that the blend containing PEG of molecular weight 3400 had the smallest domain size of the starch dispersion phases, which implies that PEG of the proper molecular weight could effectively stabilize the interface of the starch/PCL blend. The PEG of the proper molecular weight seems to locate mainly at the interface between the starch and PCL phases and to interact with both the starch phase and the PCL phase. The interactions between starch and PEG and between PCL and PEG in the blend were studied using DSC and FTIR techniques. © 2000 John Wiley & Sons, Inc. J Appl Polym Sci 77: 2049–2056, 2000

Tumor accumulation of poly(ethylene glycol) with different molecular weights after intravenous injection

Body distribution of poly(ethylene glycol) (PEG) with different molecular weights (MWs) was investigated after intravenous injection into mice with a tumor mass of their back subcutis and footpad to evaluate the effect of PEG molecular weight on the tumor accumulation. PEG did not exhibit specific affinity for any organs and PEG with higher MW tended to remain in the blood circulation of tumor-bearing mice longer than PEG with lower MW, similarly to normal mice. PEG accumulated at the tumor tissue to a significantly higher extent than at the normal tissue, irrespective of the molecular weight and the tumor loading site. Such high tumor accumulation of PEG increased with an increase in the size of the tumor mass, leveling off at a certain size. This size dependence was in accord with that of the vasculature volume in the tumor tissue. Because PEG extravasates to the interstitial space of the tumor, the tumor accumulation seems to be governed mainly by the level of hyperpermeable vasculature. The PEG molecules accumulated at the tumor tissue were retained longer than those at the normal tissue. The tumor accumulation of PEG became maximal at MW around 200,000. A pharmacokinetic study demonstrated that the accumulation rate of PEG at the tumor tissue was higher than that at the normal tissue and decreased with increasing molecular weight. On the other hand, the higher the molecular weight of PEG, the longer the retention time in the blood circulation. It is likely that the balance between these two factors, the accumulation rate of PEG and the blood retention, results in maximal accumulation of PEG with a medium molecular weight.

The application of aqueous two-phase systems to the purification of pharmaceutical proteins from transgenic sheep milk.

Transgenic sheep milk containing the protein human alpha 1-Antitrypsin (AAT) was partitioned in Poly(ethylene glycol) (PEG)-Sulphate and PEG-Phosphate biphasic systems. Individual partition coefficients for AAT and some of the milk proteins were determined in these systems. The effects of PEG molecular weight, pH and the inclusion of NaCl on the partitioning of the proteins were also studied. It was found that increasing the concentration of NaCl and decreasing the molecular weight of the PEG resulted in an increase of the partition coefficients of the proteins to the upper (PEG) phase. This partitioning effect was greater for the more hydrophobic proteins and particularly in systems having a pH close to the isoelectric point of the protein. Solubilities of the proteins in increasing concentrations of ammonium sulphate were measured in order to investigate the effects of hydrophobic and electrostatic interactions on the partitioning of these proteins in aqueous two-phase systems. Those proteins that precipitated at low levels of ammonium sulphate showed an increase in partition coefficient at low concentrations of NaCl, or they were precipitated at the interface of the phase at low concentrations of NaCl. Proteins that had low salting out constants in ammonium sulphate solutions were relatively unaffected by NaCl in ATPS. It is probable however that conformational changes and the state of aggregation of proteins are also important and should be invoked in describing the partitioning behavior observed for beta-Lg for example. Comparison of theoretical and experimental values for AAT yield and purity showed clearly that partition coefficients are influenced by the degree of purity and values obtained with purified standards are not necessarily the same as for the same protein present in a complex mixture. Under the most favourable conditions using a 4% w/w loading of transgenic ovine milk, we obtained a 91% yield of AAT in the PEG phase with a purity of 73%.