TITLE

The Separation of Recombinant Human Growth Hormone Variants by UHPLC

AUTHOR(S)
Karlsson, Göran; Eriksson, Kathryn; Persson, Annevi; Månsson, Håkan; Söderholm, Susanne
PUB. DATE
November 2013
SOURCE
Journal of Chromatographic Science;Nov2013, Vol. 51 Issue 10, p943
SOURCE TYPE
Academic Journal
DOC. TYPE
Article
ABSTRACT
Reversed-phase ultra-high-performance liquid chromatography (RP-UHPLC) was used for the separation of recombinant human growth hormone (rhGH) variants. A bridged ethylene hybrid silica C18 column was used at 37°C. The composition and temperature of the mobile phase were optimized for the separation. An isocratic elution, with approximately 46% acetonitrile in 25 mM potassium borate buffer (pH 8.5), was found to give superior selectivity in comparison with commonly used mobile phases. The method separated eight rhGH variants: (i) di-oxy Met14/Met125 sulfoxide, (ii) Met125 sulfoxide, (iii) Met14 sulfoxide, (iv) mono-deamidated (Asn149 → Asp149 or Asn152 → Asp152), (v) di-deamidated (Asn149 → Asp149 and Asn152 → Asp152), (vi) clip (Thr142-Tyr143), (vii) desPhe1 and (viii) trisulfide (Cys182-SSS-Cys189) from each other and from the native rhGH. Characterization of the purified variants was conducted by liquid chromatography–mass spectrometry tryptic mapping. The novel mobile phase, in combination with the UHPLC system, generated a significantly higher resolution than previously reported reversed-phase LC methods, including pharmacopoeal methods, for analyzing rhGH.
ACCESSION #
91961992

 

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