Photodegradation of Human Growth Hormone: A Novel Backbone Cleavage between Glu-88 and Pro-89

Abstract

The exposure of protein pharmaceuticals to light can cause loss of potency, oxidation, structural changes and aggregation. To elucidate the chemical pathways of photodegradation, we irradiated human growth hormone (hGH) at λ = 254 nm, λ ≈ 265-340 nm, and λ ≈ 295-340 nm (using the spectral cutoff of borosilicate glass) and analyzed the products by mass spectrometry. By means of LC-MS/MS analysis, we observed an unusual peptide backbone cleavage between Glu-88 and Pro-89. The crystal structure of hGH indicates that these residues are in proximity to Trp-86, which likely mediates this backbone cleavage. The two cleavage fragments observed by MS/MS analysis indicate the loss of CO from the amide bond and replacement of the Glu-C(═ O)Pro bond with a Glu-H bond, accompanied by double bond formation on proline. The reaction is oxygen-independent and likely involves hydrogen transfer to the Cα of Glu-88. To probe the influence of the protein fold, we irradiated hGH in its unfolded state, in 1:1 (v/v) acetonitrile/water, and also the isolated tryptic peptide Ile-78-Arg-90, which contains the Glu-88-Pro-89 sequence. In both cases, the cleavage between Glu-88 and Pro-89 was largely suppressed, while other cleavage pathways became dominant, notably between Gln-84 and Ser-85, as well as Ser-85 and Trp-86.

Thiyl Radical Reactions in the Chemical Degradation of Pharmaceutical Proteins

“…Human growth hormone (hGH) belongs to the class of four-helix bundle proteins [72] and is used for the treatment of pediatric hypopituitary dwarfism [73], as well as children [73] and adults [74] with hGH deficiencies. HGH is sensitive to deamidation [73,75,76], N-terminal truncation [77], oxidation [4,7,73,75,76,78,79,80,81], aggregation [73], and photo-degradation [82,83,84]. The structures of a trisulfide [76,85,86,87] and a thioether [88] variant, originating from the biosynthetic pathway, have been characterized by mass spectrometry.…”

The Effects of Light-Accelerated Degradation on the Aggregation of Marketed Therapeutic Monoclonal Antibodies Evaluated by Size-Exclusion Chromatography With Diode Array Detection

“…Exposure to light has been shown to cause decay in various medical fields. Photodegradation by UV irradiation, for example, caused unusual backbone cleavage in the human growth hormone 18 and a very recent study detected several chemical changes in the murine growth hormone also induced by UV irradiation. 19 These changes included photo-oxidation products such as methionine sulfoxide, ether cross-linking between Cys and Ser residues, thioether crosslinking between Cys residues, the transformation of Cys into Ala, and various hydrolytic fragments.…”

Photo-Oxidation of Therapeutic Protein Formulations: From Radical Formation to Analytical Techniques

“…Fragmentation processes on the backbone of mAbs induced by light are less often documented for protein drugs than side chain modifications [169,178,181]. However, some ROS are known to fragment the backbone [189] as a result of their conformational flexibility and solvent conditions [180]. Especially fragmentation around the hinge region has been documented [190,191].…”