Analytical partitioning of poly(ethylene glycol)-modified proteins

Delgado, Cristina; Malmsten, Martin; Alstine, James M. Van

doi:10.1016/s0378-4347(96)00522-1

Cited by 32 publications

(25 citation statements)

References 34 publications

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“…Also, the viscosity radius of a protein conjugated with distinct PEG spheroids would tend to be increasingly independent of N above a value of 2 or 3, since each additional PEG group added would likely be contained within the swept volume of a rotating protein molecule with a PEG spheroid grafted on opposite sides. Tables 1 to 3 Partition of proteins such as BSA and lactalbumin into aqueous phases rich in PEG is well documented (31,34,35) particularly in the presence other polymers such as dextran. We considered the possibility that the Superdex media, being dextran based, might provide a unique partitioning environment that caused the PEG layer to preferentially associate with the protein rather than the matrix.…”

Section: Modelmentioning

confidence: 99%

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Prediction of the Viscosity Radius and the Size Exclusion Chromatography Behavior of PEGylated Proteins

2004

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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-toprotein 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 PEGproteins produced using three native proteins, α-lactalbumin (14.2 kDa MW), β−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 PEGproteins 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.

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Section: Modelmentioning

confidence: 99%

“…Instead, it is possible to speculate on a variety of favourable PEG to protein interactions such as hydrogen bonding, divalent cation chelation, hydrophobic interactions, and so on (29,31,33).…”

Section: Modelmentioning

confidence: 99%

Prediction of the Viscosity Radius and the Size Exclusion Chromatography Behavior of PEGylated Proteins

2004

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“…For example the log of the partition coefficient (K) of PEG-modified proteins between the two aqueous phases in a PEG-dextran two polymer, or PEG-salt two-phase system increases directly with degree of PEGylation of the protein (Delgado et al, 1997;Harris and Zalipsky, 1997;Karr et al, 1986). This is fortuitous as cell debris, endotoxin, some proteins and other contaminants often partition in favour of the non-PEG-rich phase.…”

Section: General Considerationsmentioning

confidence: 99%

“…PEG-proteins will vary in properties between those which may be modified slightly (e. g. low N and small MW PEGs) to those more PEG-like in character. Surface property variation may be related to the weight fraction of PEG in the PEG-protein conjugate (Delgado et al, 1997) while size and other properties are related to volume fraction (keeping in mind the large viscosity radius contribution of the PEG) (Clark et al, 1996;Fee and Van Alstine, 2004). Thus approaches related to fractionation on the basis of size, charge, and hydrophobicity may be successful in terms of separating PEG-proteins from other reaction product mixture components.…”

Section: General Considerationsmentioning

confidence: 99%

“…Second, conjugation to amino acid residues that alter their charge nature (e. g. convert amine groups to amides) or take on charge at certain pH values alter this potential charge and will affect the isoelectric point (pI). Third, protein surface localised PEGs may hydrogen bond with acidic or other groups and raise their pKa (Delgado et al, 1997). Azarkan et al (1996) used thiol PEGylation as a strategy to purify chymopapain for x-ray crystallography and found that PEGylated species bound more weakly to a cation exchanger.…”

Section: Charged-based Separationsmentioning

confidence: 99%

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PEG-proteins: Reaction engineering and separation issues

Fee

Alstine²

2006

Chemical Engineering Science

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Poly(ethylene glycol)-conjugated (or PEGylated) proteins are an increasingly important class of therapeutic proteins that offer improved in vivo circulation half lives over their corresponding native forms. Their production involves covalent attachment of one or more poly(ethylene glycol) molecules to a native protein, followed by purification. Because of the extremely high costs involved in producing native therapeutic proteins it is important that subsequent PEGylation processes are as efficient as possible. In this paper, reaction engineering and purification issues for PEGylated proteins are reviewed. Paramount considerations for PEGylation reactions are specificity with respect to the conjugation site and overall yield. Batch PEGylation reaction methods are discussed, along with innovative methods using packed bed or "on-column" approaches to improve specificity and yield. Purification methods are currently dominated by ion exchange and size exclusion chromatography. Other methods in common use for protein separations, including hydrophobic interaction chromatography, affinity chromatography and membrane separations, are rarely used in PEGylated protein purification schemes. A better understanding of the effects of PEGylation on the physicochemical properties of proteins (isoelectric point, surface charge density and distribution, molecular size and relative hydrophobicity) and interactions between PEGylated proteins and surfaces is needed for the future development of optimal purification processes and media.

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Purification of PEGylated Proteins

Fee

Alstine²

2011

Methods of Biochemical Analysis

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Analytical partitioning of poly(ethylene glycol)-modified proteins

Cited by 32 publications

References 34 publications

Prediction of the Viscosity Radius and the Size Exclusion Chromatography Behavior of PEGylated Proteins

Prediction of the Viscosity Radius and the Size Exclusion Chromatography Behavior of PEGylated Proteins

PEG-proteins: Reaction engineering and separation issues

Purification of PEGylated Proteins

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