UV photodegradation of murine growth hormone: chemical analysis and immunogenicity consequences.
During manufacturing, therapeutic proteins may be exposed to ultraviolet (UV) radiation. Such exposure is of concern because UV radiation may cause photooxidative damage to proteins, which in turn could lead to physical changes such as aggregation and enhanced immunogenicity. We exposed murine growth hormone (mGH) to controlled doses of UV radiation, and examined the resulting chemical, physical and immunogenic changes in the protein. mGH chemical structure was analyzed by mass spectrometry after UV irradiation. Photooxidation products detected by mass spectrometry included methionine sulfoxide formed at Met[127] and Met[149] residues, and, tentatively assigned by MS/MS analysis, ether cross-links between original Ser[78] and Cys[188], and Cys[206] and Ser[213], and a thioether cross-link between Cys[17] and Cys[78] residues, transformation of Cys[189] into Ala, and various hydrolytic fragments. Physical damage to UV-irradiated mGH was monitored by infrared spectrometry, chromatographic analyses, and particle counting by micro-flow imaging. UV radiation caused mGH to aggregate, forming insoluble microparticles containing mGH with non-native secondary structure. When administered subcutaneously to Balb/c or Nude Balb/c mice, UV-irradiated mGH provoked antibodies that cross-reacted with unmodified mGH in a fashion consistent with a T-cell dependent immune response. In wildtype Balb/c mice, titers for anti-mGH IgG1 antibodies increased with increasing UV radiation doses.
University of Colorado at Boulder, Department of Chemical and Biological Engineering, United States.
Amber Haynes
AH
Fradkin
University of Kansas, Department of Pharmaceutical Chemistry, United States.
Olivier
O
Mozziconacci
University of Kansas, Department of Pharmaceutical Chemistry, United States.
Christian
C
Schöneich
University of Colorado, Denver, Department of Pharmaceutical Sciences, United States.
John F
JF
Carpenter
University of Colorado at Boulder, Department of Chemical and Biological Engineering, United States. Electronic address: Theodore.randolph@colorado.edu.
Theodore W
TW
Randolph
D000818:Animals; D000906:Antibodies / Q000097:blood; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D056148:Chromatography, Reverse-Phase; D003429:Cross Reactions; D004307:Dose-Response Relationship, Radiation; D004355:Drug Stability; D013006:Growth Hormone / Q000008:administration & dosage / Q000737:chemistry / Q000187:drug effects* / Q000276:immunology; D007279:Injections, Subcutaneous; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D010782:Photolysis; D011487:Protein Conformation; D011489:Protein Denaturation; D012995:Solubility; D021241:Spectrometry, Mass, Electrospray Ionization; D013055:Spectrophotometry, Infrared; D013601:T-Lymphocytes / Q000187:drug effects / Q000276:immunology; D053719:Tandem Mass Spectrometry; D013678:Technology, Pharmaceutical / Q000379:methods; D014466:Ultraviolet Rays
D000906:Antibodies; D013006:Growth Hormone
Growth hormone; Immunogenicity; Mass spectroscopy; Photoirradiation; Protein aggregation; Subvisible particles