Evaluation of Heavy-Chain C-Terminal Deletion on Product Quality and Pharmacokinetics of Monoclonal Antibodies.

Due to their potential influence on stability, pharmacokinetics, and product consistency, antibody charge variants have attracted considerable attention in the biotechnology industry. Subtle to significant differences in the level of charge variants and new charge variants under various cell culture conditions are often observed during routine manufacturing or process changes and pose a challenge when demonstrating product comparability. To explore potential solutions to control charge heterogeneity, monoclonal antibodies (mAbs) with native, wild-type C-termini, and mutants with C-terminal deletions of either lysine or lysine and glycine were constructed, expressed, purified, and characterized in vitro and in vivo. Analytical and physiological characterization demonstrated that the mAb mutants had greatly reduced levels of basic variants without decreasing antibody biologic activity, structural stability, pharmacokinetics, or subcutaneous bioavailability in rats. This study provides a possible solution to mitigate mAb heterogeneity in C-terminal processing, improve batch-to-batch consistency, and facilitate the comparability study during process changes.

Kinetic Modeling of Methionine Oxidation in Monoclonal Antibodies from Hydrogen Peroxide Spiking Studies.

UNLABELLED: When isolator technology is applied to biotechnology drug product fill-finish process, hydrogen peroxide (H2O2) spiking studies for the determination of the sensitivity of protein to residual peroxide in the isolator can be useful for assessing a maximum vapor phase hydrogen peroxide (VPHP) level. When monoclonal antibody (mAb) drug products were spiked with H2O2, an increase in methionine (Met 252 and Met 428) oxidation in the Fc region of the mAbs with a decrease in H2O2 concentration was observed for various levels of spiked-in peroxide. The reaction between Fc-Met and H2O2 was stoichiometric (i.e., 1:1 molar ratio), and the reaction rate was dependent on the concentrations of mAb and H2O2. The consumption of H2O2 by Fc-Met oxidation in the mAb followed pseudo first-order kinetics, and the rate was proportional to mAb concentration. The extent of Met 428 oxidation was half of that of Met 252, supporting that Met 252 is twice as reactive as Met 428. Similar results were observed for free L-methionine when spiked with H2O2. However, mAb formulation excipients may affect the rate of H2O2 consumption. mAb formulations containing trehalose or sucrose had faster H2O2 consumption rates than formulations without the sugars, which could be the result of impurities (e.g., metal ions) present in the excipients that may act as catalysts. Based on the H2O2 spiking study results, we can predict the amount Fc-Met oxidation for a given protein concentration and H2O2 level. Our kinetic modeling of the reaction between Fc-Met oxidation and H2O2 provides an outline to design a H2O2 spiking study to support the use of VPHP isolator for antibody drug product manufacture. LAY ABSTRACT: Isolator technology is increasing used in drug product manufacturing of biotherapeutics. In order to understand the impact of residual vapor phase hydrogen peroxide (VPHP) levels on protein product quality, hydrogen peroxide (H2O2) spiking studies may be performed to determine the sensitivity of monoclonal antibody (mAb) drug products to residual peroxide in the isolator. In this study, mAbs were spiked with H2O2; an increase in methionine (Met) oxidation of the mAbs with a decrease in H2O2 concentration was observed for various levels of spiked-in peroxide. The reaction between Met and H2O2 was 1:1, and its rate was dependent on mAb and H2O2 concentrations. Consumption of H2O2 by Met followed pseudo first-order kinetics; the rate was proportional to mAb concentration. Formulations containing trehalose or sucrose had faster consumption rates than formulations without the sugars, which could be due to excipient impurities. Based on H2O2 spiking study results, we can predict the amount of Met oxidation for a given mAb concentration and H2O2 level. Our modeling of the reaction between Fc-Met oxidation and H2O2 provides an outline to design a H2O2 spiking study that supports using VPHP isolators during manufacture of mAb products.

Site-specific tryptophan oxidation induced by autocatalytic reaction of polysorbate 20 in protein formulation.

PURPOSE: Tryptophan (Trp) oxidation leading to atypical degradation of a protein (Fab) formulated with polysorbate 20 (PS20) was investigated. Such atypical Trp oxidation was discussed in relation to a kinetic model that involves initiation of oxidizing free radical through an autocatalytic reaction. METHODS: Ion-exchange chromatography and peptide mapping were used to determine Trp oxidation. Peroxides in PS20 and free radicals in Fab samples were detected by fluorometric assay and electron paramagnetic resonance (EPR), respectively. RESULTS: PS20 with increased peroxides level led to degradation of Fab stored at 30°C. Degradation was characterized as Trp50 oxidation, which was not observed in a Fab variant where His31 was replaced. EPR peaks related to known spin adducts of 5,5 dimethylpyrroline N-oxide were detected in Fab exhibiting Trp oxidation, indicating free radicals were present. Trp oxidation of Fab observed in several drug product lots with different degradation rates fits an autocatalytic reaction model that involves free radicals. EDTA, catalase, and free tryptophan prevented oxidation. CONCLUSIONS: A metal-binding amino acid, His31, was responsible for Trp50 oxidation of Fab induced by peroxides in PS20 present in the protein formulation. Oxidation was induced by autocatalytic degradation of PS20 and could be inhibited by antioxidants.

In vitro stability and compatibility of tenecteplase in central venous access devices.

Central venous access devices (CVADs) aid in the delivery of nutritional support, infusion therapy, and hemodialysis. Maintaining continuous flow through these devices is challenging, because they are susceptible to complications such as thrombi occlusion. Therefore, CVADs may require treatment with anticoagulant or thrombolytic agents. Using these agents as locking solutions has been widely investigated; however, few publications have described the compatibility of the therapeutic with the CVAD itself. The objective of this investigation was to evaluate the in vitro stability and compatibility of a thrombolytic biologic agent, tenecteplase, with various CVAD materials. Tenecteplase was reconstituted to 1 mg/mL with either sterile water for injection or bacteriostatic water for injection (0.9% benzyl alcohol) then incubated in glass vials, polysulfone/silicone vascular access ports, and polyurethane or silicone catheters for up to 96 hours. Biochemical assays including protein monomer, protein one-chain, and in vitro bioactivity were used to assess tenecteplase's compatibility with the investigated diluents and materials every 24 hours. Antimicrobial testing was also performed for up to 28 days on bacteriostatic water for injection-reconstituted samples only. Our results showed tenecteplase to be compatible with both types of diluents (in glass vials) and catheters for up to 72 hours. Furthermore, tenecteplase was compatible with the polysulfone/silicone vascular access ports for up to 24 hours. Finally, bacteriostatic water for injection-reconstituted tenecteplase effectively met USP criteria for the inhibition of growth of micro-organisms. This study serves as an example of a best practice to evaluate the in vitro stability and compatibility of a biologic agent with CVAD materials.