Solid-phase PEGylation of recombinant interferon alpha-2a for site-specific modification: process performance, characterization, and in vitro bioactivity.
'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 .
Lee
Byung Kook
BK
Bioprocessing Research Laboratory, Department of Chemical Engineering, Hanyang University, Ansan, Korea.
Kwon
Jin Sook
JS
Kim
Hyung Jin
HJ
Yamamoto
Shuichi
S
Lee
E K
EK
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
D000077190:Interferon alpha-2; D016898:Interferon-alpha; D011994:Recombinant Proteins; D012996:Solutions; D011092:Polyethylene Glycols