On-line characterization of polyethylene glycol-modified proteins

Kunitani, Michael; Dollinger, Gavin; Johnson, D.; Kresin, Lilia

doi:10.1016/0021-9673(91)85014-7

Cited by 53 publications

(37 citation statements)

References 21 publications

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“…Combination of several methods is necessitated for the compositional analysis of the PEGylation preparation, since the peaks are sometimes overlapped in the HPLC mode and the bands are smeared in the SDS-PAGE mode [12].…”

Section: Resultsmentioning

confidence: 99%

“…The assumptions made were that the higher molecular weight gel band ladders represented one molar incremental additions of PEG to the protein [12,23]. Figure 3 shows the result of SDS-PAGE for PEGylated HSA.…”

Section: Sds-page Characterization Of Peg-hsa Conjugatesmentioning

confidence: 99%

“…If the size ratio of two PEGylated species differing in one PEG chain is small, they could not be well separated by this method [11]. In some cases, the HPLC peaks may be broad and overlapping due to heterogeneity of the products with varied PEG number and position of attachment [12].…”

Section: Introductionmentioning

confidence: 99%

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Native PAGE eliminates the problem of PEG–SDS interaction in SDS‐PAGE and provides an alternative to HPLC in characterization of protein PEGylation

Zheng

2007

Electrophoresis

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PEGylation of proteins has become an increasingly important technology in recent years. However, determination and characterization of the PEGylation products are problematic especially for the reaction mixture containing various modified proteins, unreacted PEG, and unmodified protein. A comparative study was carried out with two HPLC methods and two electrophoresis methods for characterization of the reaction mixture in PEGylation of HSA with PEG 5000, 10000, and 20000. RP-HPLC fails to give the correct information about the reaction of PEG 20000. Size-exclusion HPLC (SE-HPLC) produced very poor resolution on the PEG 5000 reaction. SDS-PAGE can run multiple samples of all PEGylation but the bands were smeared or broadened probably due to the interaction between PEG and SDS. On the other hand, native PAGE eliminates the problem of PEG-SDS interaction and provides better resolutions for all samples. Various PEGylated products and unmodified protein migrate differentially in native PAGE under nondenatured conditions. The results demonstrated that native PAGE could be a good alternative to HPLC and SDS-PAGE for the analysis of PEG-protein conjugates especially for characterization of the PEGylation mixture.

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Section: Resultsmentioning

confidence: 99%

Section: Sds-page Characterization Of Peg-hsa Conjugatesmentioning

confidence: 99%

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Native PAGE eliminates the problem of PEG–SDS interaction in SDS‐PAGE and provides an alternative to HPLC in characterization of protein PEGylation

Zheng

2007

Electrophoresis

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“…However, the distribution coefficient in SEC is not governed by molecular weight but, more generally, by the hydrodynamic volume or viscosity radius, R h , of the molecule (18). A universal calibration method, though not in common use for proteins, can be produced for a given SEC media for globular proteins and flexible polymers such as PEG in terms of K av versus viscosity radius (19).…”

Section: Apparatus and Materialsmentioning

confidence: 99%

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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“…5 Abbreviations used: SOD, superoxide dismutase; PEG, polyethylene glycol 5000; TNBS, trinitrobenzene sulfonic acid; CE, capillary A variety of analytical techniques have been used as zone electrophoresis; MALDI-MS, matrix-assisted laser desorption/ alternatives to the TNBS procedure to characterize the ionization-mass spectrometry; FTIR, Fourier-transform infrared conjugation of PEG to proteins. These include HPLC spectroscopy; GPC, gel-permeation chromatography; m-PEG, me- (8), size-exclusion chromatography (9), electrophoresis thoxy PEG; ATR, attenuated total internal reflectance; PEG-S, PEGsuccinate; RSD, relative standard deviation.…”

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confidence: 99%

Comparison of Results of Various Methods Used to Determine the Extent of Modification of Methoxy Polyethylene Glycol 5000-Modified Bovine Cupri-Zinc Superoxide Dismutase

Bullock

Chowdhury

Severdia

et al. 1997

Analytical Biochemistry

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On-line characterization of polyethylene glycol-modified proteins

Cited by 53 publications

References 21 publications

Native PAGE eliminates the problem of PEG–SDS interaction in SDS‐PAGE and provides an alternative to HPLC in characterization of protein PEGylation

Native PAGE eliminates the problem of PEG–SDS interaction in SDS‐PAGE and provides an alternative to HPLC in characterization of protein PEGylation

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

Comparison of Results of Various Methods Used to Determine the Extent of Modification of Methoxy Polyethylene Glycol 5000-Modified Bovine Cupri-Zinc Superoxide Dismutase

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