Characterization of the Degradation Products of a Color-Changed Monoclonal Antibody: Tryptophan-Derived Chromophores

Abstract

We describe the characterization of degradation products responsible for color change in near UV-visible light-irradiated and heat-stressed monoclonal antibody (mAb) drug product in liquid formulation. The treated samples were characterized using reversed-phase HPLC and size-exclusion HPLC with absorption spectroscopy. Both methods showed color change was due to chromophores formed on the mAb but not associated with the formulation excipients in both light-irradiated and heat-stressed mAb samples. These chromophores were further located by a new peptide mapping methodology with a combination of mass spectrometry and absorption spectroscopy. Mass spectrometry identified the major tryptophan oxidation products as kynurenine (Kyn), N-formylkynurenine (NFK), and hydroxytryptophan (OH-Trp). The absorption spectra showed that each of the tryptophan oxidation products exhibited a distinct absorption band above 280 nm shifted to the longer wavelengths in the order of OH-Trp < NFK < Kyn. The Kyn-containing peptide was detected by absorption at 420 nm. No new absorption bands were observed for either methionine or histidine oxidation products. This confirmed that tryptophan oxidation products, but not methionine and histidine oxidation products, were responsible for the color change. It is worth noting that a new oxidation product with the loss of hydrogen (2 Da mass decrease) for Trp-107 of the heavy chain was identified in the heat-stressed mAb sample. This oxidized tryptophan residue exhibited a distinct absorption band at the maximum absorbance wavelength 335 nm, which is responsible for the color change to yellow. This study showed that the new peptide mapping methodology with a combination of mass spectrometry and absorption spectroscopy is useful to identify tryptophan oxidation products as chromophores responsible for color change in stressed mAb drug product.

Developability Assessment During the Selection of Novel Therapeutic Antibodies

“…Reactive oxygen species responsible for oxidation of therapeutic proteins may originate from light exposure, reactive oxygen species formed in combination with surfactants such as polysorbate, and transition metals, for example, from the cell culture medium or the equipment used for bioproduction . Besides inactivation, products formed during oxidation of proteins may lead to the formation of noncovalent or covalent aggregates or colored by‐products …”

“…80,81 Besides inactivation, products formed during oxidation of proteins may lead to the formation of noncovalent or covalent aggregates 80,82 or colored by-products. 83 Solvent exposure has been discussed as a prerequisite for Trp oxidation, but the rates of oxidation and the products formed indicate that additional structural features are involved in defining oxidation hotspots. 80,82 Exposed Trp residues are rather common in antibody CDRs where they strongly contribute to affinity and specificity of antigen binding.…”

In vitro and in vivo modifications of recombinant and human IgG antibodies

“…92 Oxidation of Trp to form various products during exposure to light or heat causes changes to the color of mAb products. 98 Metalcatalyzed oxidation can also lead to oxidative carbonylation of Arg, Pro, Lys and Thr, especially when those residues are located on the surface of the molecules. 99 The observation of oxidative carbonylation on mAbs in the unstressed drug substance indicates that such reactions can occur during manufacturing because of product contact with metal surface.…”

Impact of cell culture on recombinant monoclonal antibody product heterogeneity

“…Coloration of recombinant monoclonal antibody drug substances has turned out to be a common quality attribute, especially for high concentration solutions. Oxidation of tryptophan residues, the presence of advanced glycation end products (AGEs), and association of recombinant monoclonal antibodies with B‐vitamins, their degradation products or B‐vitamin‐mediated reaction products have been identified to contribute to the color. For example, oxidation of tryptophan is the major source of the yellow color in one case, but the formation of AGEs is the major source of yellow color in a different case .…”