Carboxyalkylated histidine is a pH-dependent product of pegylation with SC-PEG.
OBJECTIVE
Pegylation of therapeutic protein usually results in a mixture of monopegylated proteins with differing sites of modification. With rh-interferon-alpha2A pegylation, we have found that this heterogeneity includes two classes of pegylation site chemistry, the relative proportions of which can be adjusted by reaction pH.


METHODS
The effect of pegylation reaction pH on the relative proportion of three peaks produced was investigated. Products were purified and characterized by peptide mapping, chemical stability to neutral hydroxylamine, and biologic activity.


RESULTS
Reactions at basic pH levels produced a mixture of products pegylated at lysine residues as has been observed elsewhere. However, the dominant product of reactions at mildly acidic levels of pH showed distinct chemistry and higher cytopathic effect activity. The primary site of modification at this pH was His34. We developed a quantitative assay using sensitivity to neutral hydroxylamine to measure the proportion of urethane bonds involving carboxyalkylated histidines. This assay showed that histidine was pegylated preferentially at low pH levels with another protein, rh-Interleukin-10.


CONCLUSIONS
Reaction pH can be used to select the preferred pegylation site chemistry.
Wylie
D C
DC
Department of Biotechnology Development, Schering-Plough Research Institute, Union, New Jersey 07083, USA. david.wylie@spcorp.com
Voloch
M
M
Lee
S
S
Liu
Y H
YH
Cannon-Carlson
S
S
Cutler
C
C
Pramanik
B
B
D000478:Alkylation; D000595:Amino Acid Sequence; D001681:Biological Assay; D018188:Cardiovirus Infections / Q000188:drug therapy / Q000473:pathology; D004686:Encephalomyocarditis virus; D006639:Histidine / Q000737:chemistry*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D019811:Hydroxylamine / Q000737:chemistry; D016899:Interferon-beta / Q000737:chemistry / Q000494:pharmacology; D016753:Interleukin-10 / Q000737:chemistry; D007536:Isomerism; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification; D011092:Polyethylene Glycols / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D013388:Succinimides / Q000737:chemistry*
D010455:Peptides; D011994:Recombinant Proteins; D013388:Succinimides; D016753:Interleukin-10; D019811:Hydroxylamine; D011092:Polyethylene Glycols; D006639:Histidine; D016899:Interferon-beta; C050761:polyethylene glycol bis(succinimidyl succinate)