Purification of pegylated proteins.

Separation of PEGylated proteins is challenging because PEG itself is a relatively inert, neutral, hydrophilic polymer and the starting point for PEGylation is a pure protein. Thus, other than molecular weight and size, differences in the physicochemical properties typically used to fractionate proteins may be slight between different PEGylated forms of a protein. The usual properties of electrostatic charge and molecular weight (size) form the basis of the most commonly used separation techniques, particularly IEC, SEC, and ultrafiltration. The main effect of PEGylation on ion-exchange separations is to shield the electrostatic charges on the protein surface and to reduce the strength of interactions with higher PEG chain molecular weight or higher PEGylation extent. Thus, ion exchange can be used very effectively to separate on the basis of PEGylation extent for low extents, but as N increases, the effectiveness of separation rapidly diminishes. Separation of positional isomers is possible by RPC or ion exchange at analytical scale, but it is problematic at the preparative scale due to the small size of the differences in electrostatic interactions between isomers. PEGylation imparts significant changes in molecular weight with each chain added to a protein and there are corresponding increases in molecular size, so SEC and ultrafiltration (and dialysis) are effective methods for separating native and PEGylated proteins. However, the relative size difference between variants differing in PEGylation extent by one adduct reduces with N, so that efficient SEC separation between PEGylated species differing by one PEG chain is not likely to be economic at the preparative scale for N > 3. This holds true even for PEG proteins produced with large PEG polymers (Mr > or =20 kDa). For small PEG polymers (Mr = 2 kDa), only native and PEGylated species can be separated effectively. At the analytical scale, with proper calibration, SEC can provide valuable information on PEGylation extent. Membranes can be used to reduce the concentration of smaller molecular weight species by dialysis but cannot fully remove them, and an operational trade-off between purity and yield is required. Gel electrophoresis can confirm PEGylation reactions have proceeded and indicate the relative purity of products, but its use to confirm PEGylation extent requires further research. The main drawback of separations based solely upon molecular size is that they cannot differentiate between positional isomers. Capillary electrophoresis is an exception, quantitatively combining any or all of size, shape, conformational freedom, and small differences in protein surface properties to separate by both PEGylation extent and positional isomerism. Relative hydrophobicity is a useful property for analytical separations using RPC, but HIC, which is used routinely for production-scale purification of proteins, does not appear to be particularly useful for separation of PEGylated species.

Novel in situ polymerized coatings for hydrophobic interaction chromatography media.

Hydrophobic interaction chromatography (HIC) and other capture media are typically produced by grafting different ligands to base matrices at defined surface densities. This often complicates media production. An alternative approach to media involving in situ radical initiated polymerization was used to graft polymer coatings directly at Sepharose(R) polymeric base matrices. This method appears suitable for producing many different chromatography media on a variety of base matrices. In the present study, it also favorably increased the solution pressure-flow properties of a Sepharose base matrix used to produce HIC media. A wide range of HIC media could be produced by simply varying the reaction ratio of butyl vinyl ether, and hydroxybutyl vinyl ether. The new HIC media was evaluated using five test proteins (bovine serum albumin, ribonuclease A, alpha-chymotrypsinogen A, myoglobin and alpha-lactalbumin). The media exhibited classic HIC behavior, predictably controlled hydrophobicity, plus good protein selectivity, capacity (70mgprotein/ml gel) and often total protein recovery. By modifying the degree of matrix hydrophobicity, we could also reduce effects of protein denaturation often seen with conventional HIC and observed as multiple peaks in the chromatograms. Separation of crude protein extracts from Eschericha coli, expressing a green fluorescent protein (GFPuv) and, a more hydrophobic, recombinantly-modified, tyrosine-tagged green fluorescent protein (YPYPY-GFPuv), was also performed. These proteins were very stable, exhibited significantly different retention times, and could be used to study the ability of the media to work with complex protein mixtures. Such GFP mutants appear ideal for characterizing the performance of chromatographic media.

Partitioning in aqueous two-phase systems: Analysis of strengths, weaknesses, opportunities and threats.

For half a century aqueous two-phase systems (ATPSs) have been applied for the extraction and purification of biomolecules. In spite of their simplicity, selectivity, and relatively low cost they have not been significantly employed for industrial scale bioprocessing. Recently their ability to be readily scaled and interface easily in single-use, flexible biomanufacturing has led to industrial re-evaluation of ATPSs. The purpose of this review is to perform a SWOT analysis that includes a discussion of: (i) strengths of ATPS partitioning as an effective and simple platform for biomolecule purification; (ii) weaknesses of ATPS partitioning in regard to intrinsic problems and possible solutions; (iii) opportunities related to biotechnological challenges that ATPS partitioning may solve; and (iv) threats related to alternative techniques that may compete with ATPS in performance, economic benefits, scale up and reliability. This approach provides insight into the current status of ATPS as a bioprocessing technique and it can be concluded that most of the perceived weakness towards industrial implementation have now been largely overcome, thus paving the way for opportunities in fermentation feed clarification, integration in multi-stage operations and in single-step purification processes.

Ion exchange chromatography of antibody fragments.

Effects of pH and conductivity on the ion exchange chromatographic purification of an antigen-binding antibody fragment (Fab) of pI 8.0 were investigated. Normal sulfopropyl (SP) group modified agarose particles (SP Sepharosetrade mark Fast Flow) and dextran modified particles (SP Sepharose XL) were studied. Chromatographic measurements including adsorption isotherms and dynamic breakthrough binding capacities, were complemented with laser scanning confocal microscopy. As expected static equilibrium and dynamic binding capacities were generally reduced by increasing mobile phase conductivity (1-25 mS/cm). However at pH 4 on SP Sepharose XL, Fab dynamic binding capacity increased from 130 to 160 (mg/mL media) as mobile phase conductivity changed from 1 to 5 mS/cm. Decreasing protein net charge by increasing pH from 4 to 5 at 1.3 mS/cm caused dynamic binding capacity to increase from 130 to 180 mg/mL. Confocal scanning laser microscopy studies indicate such increases were due to faster intra-particle mass transport and hence greater utilization of the media's available binding capacity. Such results are in agreement with recent studies related to ion exchange of whole antibody molecules under similar conditions.

Modeling of protein interactions with surface-grafted charged polymers. Correlations between statistical molecular modeling and a mean field approach.

Ion exchange media involving charge groups attached to flexible polymers are widely used for protein purification. Such media often provide enhanced target protein purity and yield. Yet, little is understood about protein interaction with such media at the molecular level, or how different media architectures might affect separation performance. To gain a better understanding of such adsorptive systems, statistical mechanical perturbation calculations, utilizing a Debye-Hückel potential, were performed on surface-grafted charged polymers and their interaction with model proteins. The studied systems were weakly charged, and the polymers were linear and relatively short (degree of polymerization is 30). Segment distributions from the surface were also determined. The interaction of spherical model protein particles of 12-30 A radius were investigated with respect to polymer grafting density, distance from matrix surface, protein charge, and ionic strength. The partitioning coefficient of the model proteins was determined for different distances from the surface. An empirical mean field theory that scales the entropy of the protein with the square of the protein radius correlates well to Monte Carlo statistical modeling results. Upon adsorption to the polymer layers, the model proteins exhibit a critical surface charge density that is proportional to the ionic strength, independent of the grafting density, and appears to be a fundamental determinant of protein adsorption. Partitioning of protein-like nanoparticles to the charged polymer surface is only favored above the particle critical charge density.

Prediction of the viscosity radius and the size exclusion chromatography behavior of PEGylated proteins.

Size exclusion chromatography (SEC) was used to determine the viscosity radii of equivalent spheres for proteins covalently grafted with poly(ethylene glycol) (PEG). The viscosity radius of such PEGylated proteins was found to depend on the molecular weight of the native protein and the total weight of grafted PEG but not on PEG molecular weight, or PEG-to-protein molar grafting ratio. Results suggest grafted PEG's form a dynamic layer over the surface of proteins. The geometry of this layer results in a surface area-to-volume ratio approximately equal to that of a randomly coiled PEG molecule of equivalent total molecular weight. Two simple methods are given to predict the viscosity radius of PEGylated proteins. Both methods accurately predicted (3% absolute error) the viscosity radii of various PEG-proteins produced using three native proteins, alpha-lactalbumin (14.2 kDa MW), beta-lactoglobulin dimer (37.4 kDa MW), and bovine serum albumin (66.7 kDa MW), three PEG reagents (2400, 5600, and 22500 MW), and molar grafting ratios of 0 to 8. Accurate viscosity radius prediction allows calculation of the distribution coefficient, K(av), for PEG-proteins in SEC. The suitability of a given SEC step for the analytical or preparative fractionation of different PEGylated protein mixtures may therefore be assessed mathematically. The methods and results offer insight to several factors related to the production, purification, and uses of PEGylated proteins.