title: Solid-phase PEGylation of recombinant interferon alpha-2a for site-specific modification: process performance, characterization, and in vitro bioactivity.
abstract: 'Solid-phase' PEGylation, in which a conjugation reaction attaches proteins to a solid matrix, has distinct advantages over the conventional, solution-phase process. We report a case study in which recombinant interferon (rhIFN) alpha-2a was adsorbed to a cation-exchange resin and PEGylated at the N-terminus by 5, 10, and 20 kDa mPEG aldehydes through reductive alkylation. After PEGylation, a salt gradient elution efficiently purified the mono-PEGylate of unwanted species such as unmodified IFN and unreacted PEG. Mono-PEGylation and purification were integrated into a single, chromatographic step. Depending on the molecular weight of the mPEG aldehyde, the mono-PEGylation yield ranged from 50 to 65%. Major problems associated with the solution-phase process such as random or uncontrollable multi-PEGylation and post-PEGylation purification difficulties were overcome. N-terminus sequencing and MALDI-TOF mass spectrophometry confirmed that the PEG molecule was conjugated only to the N-terminus. A cell proliferation study indicated reduced antiviral activity of the mono-PEGylate compared to that of the unmodified IFN. As higher molecular weight PEG was conjugated, in vitro bioactivity and antibody binding activity, as measured by a surface plasmon resonance biosensor, decreased. Nevertheless, trypsin resistance and thermal stability were considerably improved .
authors.affiliation: Bioprocessing Research Laboratory, Department of Chemical Engineering, Hanyang University, Ansan, Korea.
authors.forename: Byung Kook
authors.initials: BK
authors.lastname: Lee
authors.forename: Jin Sook
authors.initials: JS
authors.lastname: Kwon
authors.forename: Hyung Jin
authors.initials: HJ
authors.lastname: Kim
authors.forename: Shuichi
authors.initials: S
authors.lastname: Yamamoto
authors.forename: E K
authors.initials: EK
authors.lastname: Lee
mesh_terms: D049109:Cell Proliferation / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000737:chemistry* / Q000494:pharmacology*; D013058:Mass Spectrometry; D011092:Polyethylene Glycols / Q000737:chemistry*; D011489:Protein Denaturation; D011994:Recombinant Proteins; D012680:Sensitivity and Specificity; D012996:Solutions / Q000737:chemistry; D013696:Temperature
chemical_list: D000077190:Interferon alpha-2; D016898:Interferon-alpha; D011994:Recombinant Proteins; D012996:Solutions; D011092:Polyethylene Glycols