Positional isomers of monopegylated interferon alpha-2a: isolation, characterization, and biological activity

The success of recombinant interferon alpha in the clinic in part is limited by two properties of the protein: short serum half-life and immunogenicity. To improve these properties, interferon alpha-2a was conjugated with polyethylene glycol (PEG-5000). A homogeneous preparation of monopegylated int...

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Bibliographic Details
Published in:Analytical biochemistry 1997-05, Vol.247 (2), p.434-440
Main Authors: Monkarsh, S P, Ma, Y, Aglione, A, Bailon, P, Ciolek, D, DeBarbieri, B, Graves, M C, Hollfelder, K, Michel, H, Palleroni, A, Porter, J E, Russoman, E, Roy, S, Pan, Y C
Format: Article
Language:English
Subjects:
Animals
Antiviral Agents - chemistry
Antiviral Agents - isolation & purification
Antiviral Agents - pharmacology
Cell Division - drug effects
Cell Line
Chromatography, High Pressure Liquid - methods
Chromatography, Ion Exchange - methods
Cytopathogenic Effect, Viral - drug effects
Dogs
Humans
Interferon-alpha - chemistry
Interferon-alpha - isolation & purification
Interferon-alpha - pharmacology
Isomerism
Molecular Structure
Polyethylene Glycols - chemistry
Polyethylene Glycols - isolation & purification
Polyethylene Glycols - pharmacology
Recombinant Proteins
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Summary:The success of recombinant interferon alpha in the clinic in part is limited by two properties of the protein: short serum half-life and immunogenicity. To improve these properties, interferon alpha-2a was conjugated with polyethylene glycol (PEG-5000). A homogeneous preparation of monopegylated interferon alpha-2a was subjected to vigorous analytical and activity characterization. A newly developed ampholyte-free chromatofocussing-like cation-exchange HPLC method utilizing a sulfopropyl resin was used to separate the monopegylated protein into 11 species. Peptide mapping, sequencing, and mass spectrometric analyses indicated that these species are positional isomers where each isomer represents a single polymer molecule conjugated to one specific lysine residue. No species with a modification at the amino terminus was observed. All 11 isomers show antiviral and antiproliferative activities in the same range as the parent monopegylated interferon alpha-2a.
ISSN:0003-2697
DOI:10.1006/abio.1997.2128