Characterization and stability of N-terminally PEGylated rhG-CSF.
OBJECTIVE
The liquid stability of rhG-CSF was investigated after polyethylene glycol (PEG) with an average molecular weight of 6000 daltons was covalently attached to the N-terminal methionine residue.


METHODS
The conjugation methods chosen for modifying the N-terminal residue were alkylation and acylation. The N-terminally PEGylated rhG-CSF conjugates were purified by cation exchange chromatography. The physical characterization methods of SDS-PAGE, endoproteinase peptide mapping, circular dichroism and in-vivo bioassay were used to test for differences between the PEG-rhG-CSF molecules.


RESULTS
Physical characterization indicated no apparent differences in the rhG-CSF molecules that were conjugated with either method. Stability, in liquid at elevated temperatures, of these conjugated molecules indicated that the primary pathway of degradation was aggregation. Conjugation through alkylation offered the distinct advantage of decreasing, by approximately 5 times, the amount of aggregation present as compared to acylation.


CONCLUSIONS
We suggest, that the increased stability observed for the molecules utilizing the alkylation conjugation method may be due to the preservation of charge on the alpha amino group of rhG-CSF.
Kinstler
O B
OB
Department of Pharmaceutics and Drug Delivery, Amgen Incorporated, Thousand Oaks, California 91320, USA.
Brems
D N
DN
Lauren
S L
SL
Paige
A G
AG
Hamburger
J B
JB
Treuheit
M J
MJ
D016179:Granulocyte Colony-Stimulating Factor / Q000031:analogs & derivatives / Q000737:chemistry*; D011092:Polyethylene Glycols; D011994:Recombinant Proteins
D011994:Recombinant Proteins; D016179:Granulocyte Colony-Stimulating Factor; D011092:Polyethylene Glycols