Light-Induced Histidine Adducts to an IgG1 Molecule Via Oxidized Histidine Residue and the Potential Impact of Polysorbate-20 Concentration.

PURPOSE: Histidine (His) undergoes light-induced reactions such as oxidation, crosslinking and addition. These reactions are initiated by singlet oxygen (1O2) to generate His photo-oxidation products, which are subject to nucleophilic attack by a non-oxidized His residue from another protein or by nucleophilic buffer components such as Tris and His. This report aims to identify light-induced His-adducts to a monoclonal antibody (mAb-1) due to the reaction of His molecules in the buffer with the photooxidized His residues under ICH light conditions. Since polysorbate-20 (PS-20) is a commonly used excipient in biotherapeutics formulation, it is also important to study the impact of PS-20 concentration on protein photostability. RESULTS: We identified and characterized light-induced His-adducts of mAb-1 by LC-MS/MS. We showed that the levels of light-induced His-adducts generally correlate with the solvent accessibility of His residues in the protein. In addition, the presence of PS-20 at concentrations commonly used in protein drug formulations can significantly increase the levels of light-induced His-adducts. CONCLUSIONS: Since His residues are present in a conserved region in the Fc domain, and may be present in the complementarity-determining region (CDR), the impact on the biological functions of the His-adducts observed here should be further studied to evaluate the risk of their presence.

Light-Induced Covalent Buffer Adducts to Histidine in a Model Protein.

PURPOSE: Light is known to induce histidine (His) oxidation and His-His crosslinking in proteins. The crosslinking is resulted from the nucleophilic attack of a His to a photooxidized His from another protein. The goal of this work is to understand if covalent buffer adducts on His residues can be generated by light through similar mechanisms in nucleophilic buffers such as Tris and His. METHODS: A model protein (DNase) was buffer exchanged into nucleophilic buffers before light exposure. Photogenerated products were characterized by tryptic peptide mapping with mass spectrometry (MS) analysis. Several buffer adductions on His residues were identified after light exposure. To understand the influencing factors of such reactions, the levels of adducts were measured for six nucleophilic buffers on all His residues in DNase. RESULTS: The levels of adducts were found to correlate with the solvent accessibility of the His residue. The levels of adducts also correlate with the structure of the nucleophile, especially the steric restrictions of the nucleophile. The levels of adducts can be higher than that of other His photoreaction products, including photooxidation and crosslinking. CONCLUSIONS: In nucleophilic buffers, light can induce covalently-linked adducts to His residues.

Structure-Based Correlation of Light-Induced Histidine Reactivity in A Model Protein.

Light is known to induce covalently linked aggregates in proteins. These aggregates can be immunogenic and are of concern for drug product development in the biotechnology industry. Histidine (His) is proposed to be a key residue in cross-link generation ( Pattison , D. I. Photochem. Photobiol. Sci. 2012 , 11 , 38 - 53 ). However, the factors that influence the reactivity of His in proteins, especially the intrinsic factors are little known. Here, we used rhDNase, which only forms His-His covalent dimers after light treatment to determine the factors that influence the light-induced reactivity of His. This system allowed us to fully characterize the light-induced covalent dimer and rank the reactivities of the His residues in this protein. The reactivities of these His residues were correlated with solvent accessibility-related parameters both by crystal structure-based calculations of solvent-accessible surface area and by hydrogen-deuterium exchange (HDX) experiments. Through this correlation, we demonstrate that the photoreactivity of His is determined by both solvent accessibility and structural flexibility. This new insight can explain the highly complex chemistry of light-induced aggregation and help predict the aggregation propensity of protein under light treatment.

Glycation of antibodies: Modification, methods and potential effects on biological functions.

Glycation is an important protein modification that could potentially affect bioactivity and molecular stability, and glycation of therapeutic proteins such as monoclonal antibodies should be well characterized. Glycated protein could undergo further degradation into advance glycation end (AGE) products. Here, we review the root cause of glycation during the manufacturing, storage and in vivo circulation of therapeutic antibodies, and the current analytical methods used to detect and characterize glycation and AGEs, including boronate affinity chromatography, charge-based methods, liquid chromatography-mass spectrometry and colorimetric assay. The biological effects of therapeutic protein glycation and AGEs, which ranged from no affect to loss of activity, are also discussed.

Charge variants in IgG1: Isolation, characterization, in vitro binding properties and pharmacokinetics in rats.

Antibody charge variants have gained considerable attention in the biotechnology industry due to their potential influence on stability and biological activity. Subtle differences in the relative proportions of charge variants are often observed during routine biomanufacture or process changes and pose a challenge to demonstrating product comparability. To gain further insights into the impact on biological activity and pharmacokinetics (PK) of monoclonal antibody (mAb) charge heterogeneity, we isolated the major charge forms of a recombinant humanized IgG1 and compared their in vitro properties and in vivo PK. The mAb starting material had a pI range of 8.7-9.1 and was composed of about 20% acidic variants, 12% basic variants, and 68% main peak. Cation exchange displacement chromatography was used to isolate the acidic, basic, and main peak fractions for animal studies. Detailed analyses were performed on the isolated fractions to identify specific chemical modification contributing to the charge differences, and were also characterized for purity and in vitro potency prior to being administered either subcutaneously (SC) or intravenously (IV) in rats. All isolated materials had similar potency and rat FcRn binding relative to the starting material. Following IV or SC administration (10 mg/kg) in rats, no difference in serum PK was observed, indicating that physiochemical modifications and pI differences among charge variants were not sufficient to result in PK changes. Thus, these results provided meaningful information for the comparative evaluation of charge-related heterogeneity of mAbs, and suggested that charge variants of IgGs do not affect the in vitro potency, FcRn binding affinity, or the PK properties in rats.

The effect of low intensity ultraviolet-C light on monoclonal antibodies.

As part of an investigation to identify potential new viral reduction strategies, ultraviolet-C (UV-C) light was examined. Although this technology has been known for decades to possess excellent virus inactivation capabilities, UV-C light can also introduce significant unwanted damage to proteins. To study the effect on monoclonal antibodies, three different antibodies were subjected to varying levels of UV-C light using a novel dosing device from Bayer Technology Services GmbH. The range of fluencies (or doses) covered was between 0 and 300 J/m(2) at a wavelength of 254 nm. Product quality data generated from the processed pools showed only minimal damage done to the antibodies. Aggregate formation was low for two of the three antibodies tested. Acidic and basic variants increased for all three antibodies, with the basic species increasing more than the acidic species. Peptide maps made for the three sets of pools showed no damage to two of the three antibody backbones, whereas the third antibody had very low levels of methionine oxidation evident. Samples held at 2-8 degrees C for 33 days showed no increase in aggregates or charge variants, indicating that the proteins did not degrade and were not damaged further by reactive or catalytic species that may have been created on exposure to UV-C light. Overall, UV-C light was shown to induce very little damage to monoclonal antibodies at lower fluencies and appears to be a viable option for viral inactivation in biotechnology applications.

A study in glycation of a therapeutic recombinant humanized monoclonal antibody: where it is, how it got there, and how it affects charge-based behavior.

The glycated form of a basic recombinant humanized monoclonal antibody (rhuMAb) was separated and quantitated by boronate affinity chromatography using optimized shielding reagents. Characterization on the isolated glycated material by peptide mapping analysis, using liquid chromatography-mass spectrometry (LC-MS) and tandem mass spectrometry (MS/MS) sequencing techniques, identified eight reactive lysine primary amine sites. The glycation reaction extent was similar among the various reactive sites, ranging from approximately 1 to 12%, and a single histidine residue separated the most and least reactive sites. Boronate chromatography run in a linear gradient mode separated monoglycated rhuMAb from higher order glycated species and indicated that the majority ( approximately 90%) of glycated rhuMAb is monoglycated. Low-level glycation on a heavy chain lysine located within a complementarity-determining region (CDR) did not significantly affect binding activity in potency measurements. The glycated forms also behaved as slightly more acidic than the nonglycated antibody in charge-based separation techniques, observable by capillary isoelectric focusing (cIEF) and ion exchange chromatography (IEC). The boronate column has significantly increased retention of aggregated rhuMAb material under separation conditions optimized for the monomer form. Recombinant protein glycation initially occurred during production in mammalian cell culture, where feed sugar and protein concentrations contribute to the total overall glycation on this antibody product.