Isoform separation and structural identification of mono-PEGylated recombinant human growth hormone (PEG-rhGH) with pH gradient chromatography.
Human growth hormone plays an essential role in the treatment of dwarfism diseases, but it is limited in its short circulating half-life. Nowadays, some manufacturers are trying to take advantage of polyethylene glycol (PEG) conjugated with recombinant human growth hormone (rhGH) to improve its half-life and efficacy. However, the modified products are heterogeneous mixtures composed of reaction products with different modification sites. It is generally known as a challenging task to separate and characterize a PEGylated product, especially for its positional isoforms. In this study, cation exchange high performance liquid chromatograph (IEC-HPLC) based on a pH gradient separation method was presented to separate five position isomers of rhGH conjugated with a 40-kDa branched PEG N-hydroxysuccinimidyl (NHS) functional group. Then Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALD-TOF MS) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) indicated that each of five materials collected by IEC-HPLC was conjugated with only one branched PEG chain. Furthermore, rhGH and PEG-rhGH were digested by trypsin and peptides were collected by reversed phase high performance liquid chromatography (RP-HPLC). Following MALDI-TOF MS, PEG modification sites were determined through comparative analysis of peptide mapping between PEG-rhGH and rhGH. Finally, biological activities of those positional isomers were performed in vivo and very small variations were observed. This method was shown to be suitable for heterogeneity analysis of PEGylated biopharmaceutical products.
MOE Key Laboratory of Systems Bioengineering, Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China; Collaborative Innovation Center of Chemical Science and Engineering, Tianjin 300072, China.
Xiufeng
X
Qin
National Institutes for Food and Drug Control, Beijing 100050, China.
Jing
J
Li
MOE Key Laboratory of Systems Bioengineering, Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China; Collaborative Innovation Center of Chemical Science and Engineering, Tianjin 300072, China.
Yong
Y
Li
MOE Key Laboratory of Systems Bioengineering, Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China; Collaborative Innovation Center of Chemical Science and Engineering, Tianjin 300072, China.
Yiru
Y
Gan
MOE Key Laboratory of Systems Bioengineering, Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China; Collaborative Innovation Center of Chemical Science and Engineering, Tianjin 300072, China. Electronic address: huang@tju.edu.cn.
He
H
Huang
National Institutes for Food and Drug Control, Beijing 100050, China. Electronic address: liangchenggang@nifdc.org.cn.
Chenggang
C
Liang
D002853:Chromatography, Liquid / Q000379:methods*; D019382:Human Growth Hormone / Q000031:analogs & derivatives* / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007536:Isomerism; D011092:Polyethylene Glycols / Q000032:analysis / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism
C562190:polyethylene glycol-recombinant human growth hormone; D019382:Human Growth Hormone; D011092:Polyethylene Glycols
Biological activity; PEG-rhGH; PEGylation site; Positional isomer; pH gradient chromatography