title: Photosensitizers form in histidine buffer and mediate the photodegradation of a monoclonal antibody.
abstract: Fluorescent light (FL) photodegradation of a monoclonal antibody (mAb) formulated in histidine buffer is mediated by histidine-derived photosensitizers that accumulate and greatly increase with light exposure. Histidine-derived photosensitizers are the primary mediators of Trp photooxidation. FL-photodegradation requires light exposure and is pH dependent. It is significantly reduced or eliminated by buffer exchanges, by oxygen depletion, or at pH values greater than 7. Antibody-fragment MS ion counts reveal that oxidation of a single light chain Trp in CDR1 correlates with binding loss. Multiple heavy chain methionines oxidize, but poorly correlate with binding loss. Photosensitizers extracted from photo-aged histidine buffer are potent mediators of FL-photodegradation including oxidation and, to a lesser degree, fragmentation and aggregation of the mAb. These photosensitizers absorb visible light and have neutral mass of 187.1- 386.1 Da. They are also fluorescent with ex/em at 360/450 nm. When spiked into histidine or MES buffered mAb formulations they produce a concentration dependent and pronounced increase in FL-photodegradation; however, no oxidation or loss of antibody function occurs in the dark and hydrogen peroxide does not oxidize Trp. The major component is consistent with histidine oxidation to 6a-hydroxy-2-oxo-octahydro-pyrollo[2,3-d]imidazole-5-carboxylic acid. Photosensitizer levels measured in the formulation prior to light exposure, are linearly related to the FL-photodegradation observed and can predict degradation in photostability testing.
authors.affiliation: Department of Analytical Development, FibroGen Inc., San Francisco, California 94158, USA. sstroop@fibrogen.com
authors.forename: Steven D
authors.initials: SD
authors.lastname: Stroop
authors.forename: David M
authors.initials: DM
authors.lastname: Conca
authors.forename: Robert P
authors.initials: RP
authors.lastname: Lundgard
authors.forename: Mark E
authors.initials: ME
authors.lastname: Renz
authors.forename: Lynn M
authors.initials: LM
authors.lastname: Peabody
authors.forename: Scott D
authors.initials: SD
authors.lastname: Leigh
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000378:metabolism*; D002021:Buffers; D019075:Electrophoresis, Capillary; D005453:Fluorescence; D006639:Histidine / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000737:chemistry / Q000378:metabolism*; D008027:Light; D010084:Oxidation-Reduction; D010782:Photolysis; D017319:Photosensitizing Agents / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D002021:Buffers; D007074:Immunoglobulin G; D017319:Photosensitizing Agents; D006639:Histidine