Separation of oxidized and deamidated human growth hormone variants by isocratic reversed-phase high-performance liquid chromatography.
Reversed-phase high-performance liquid chromatography (RP-HPLC) was utilized for the separation of recombinant human growth hormone (hGH) variants on a C18 silica column at 55 degrees C using an isocratic mobile phase which contained 27% 1-propanol in a 25 mM potassium phosphate buffer, pH 6.5. Three of the obtained peaks were characterized by tryptic mapping and mass spectrometry; two of the peaks were found to contain oxidized hGH (dioxy Met14/Met125 and Met125 sulfoxide) while the third contained a deamidated form (Asn149-->Asp149 or Asn152-->Asp152). Compared to the European Pharmacopoeia RP-HPLC method of hGH analysis, this new method gives two additional peaks and a 50% reduction in the analysis time.
Karlsson
G
G
Pharmacia and Upjohn, Stockholm, Sweden.
Gellerfors
P
P
Persson
A
A
Norén
B
B
Edlund
P O
PO
Sandberg
C
C
Birnbaum
S
S
D000577:Amides / Q000737:chemistry; D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D013006:Growth Hormone / Q000737:chemistry / Q000302:isolation & purification*; D006801:Humans; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010449:Peptide Mapping; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013056:Spectrophotometry, Ultraviolet; D014357:Trypsin / Q000737:chemistry
D000577:Amides; D013006:Growth Hormone; D014357:Trypsin