Effect of Annealing on Visible-Bubble Formation and Stability Profiles of Freeze-Dried High Concentration Omalizumab Formulations.

In the clinical application of freeze-dried highly concentrated omalizumab formulations, extensive visible bubbles (VBs) can be generated and remain for a long period of time in the reconstitution process, which greatly reduces the clinical use efficiency. It is necessary to understand the forming and breaking mechanism of VBs in the reconstitution process, which is a key factor for efficient and safe administration of biopharmaceutical injection. The effects of different thermal treatments on the volume of VBs and stability of omalizumab, mAb-1, and mAb-2 were investigated. The internal microvoids of the cake were characterized by scanning electron microscopy and mercury intrusion porosimetry. Electron paramagnetic resonance was applied to obtain the molecular mobility of the protein during annealing. A large number of VBs were generated in the reconstitution process of unannealed omalizumab and remained for a long period of time. When annealing steps were added, the volume of VBs was dramatically reduced. When annealed at an aggressive temperature (i.e., -6 °C), although the volume of VBs decreased, the aggregation and acidic species increased significantly. Thus, our observations highlight the importance of setting an additional annealing step with a suitable temperature, which contributes to reducing the VBs while maintaining the stability of the high concentration freeze-dried protein formulation.

Monitoring of low-molecular-weight protein aggregation by CE-SDS as a complementary method to SE-HPLC.

Capillary electrophoresis with sodium dodecyl sulfate (CE-SDS) has long been proven to have excellent performance in the analysis and characterization of therapeutic proteins. However, it is rarely used for the detection of low-molecular-weight proteins or peptides. Our research has proved the ability of CE-SDS to characterize the purity of low-molecular-weight proteins (i.e., <10 kDa) and even polypeptides. In this article, insulin glargine was used as a model protein, and CE-SDS was used to analyze the samples damaged by heating and light exposure. The monomers, dimers, and trimers of insulin glargine were effectively separated, and the results of the mass spectrometry also confirmed the existence of two kinds of insulin aggregates. For comparison, the size-exclusion high-performance liquid chromatography (SE-HPLC) only showed a single aggregate peak. In addition, the denaturation conditions caused only the covalent aggregates to appear in the CE-SDS analysis. These advantages also make CE-SDS an excellent supplementary technology to the traditional SE-HPLC, providing biopharmaceutical analysts with more information.

Characterization and exploration of an artifact in the reducing capillary electrophoresis-sodium dodecyl sulfate analysis of the 'me-too' drug zuberitamab related to rituximab.

For monoclonal antibody (mAb) drugs, the 'me-too' drug is a pharmacologically active compound that is structurally similar to the first-in-class drugs, acting on the same target and is used for the same therapeutic purposes, but it may differ in drug-drug interactions and adverse drug reactions. Capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) has been widely used for quality evaluation of mAb drugs. The properties of the detected substances can interfere with the credibility and accuracy of the method. In the routine comparison analysis for both innovator rituximab and 'me-too' drug zuberitamab samples, an uncommon artifact related to the heavy chain (HC) of zuberitamab was observed in reducing CE-SDS and interfered with our identification of the purity of samples. In this work, the overall hydrophobicity of the HCs of rituximab, zuberitamab, and several other common mAbs was characterized and determined by reversed-phase high-performance liquid chromatography. Additionally, the local hydrophobicity and surface charge were compared using Expasy ProtScale and PyMOL software simulations. We concluded that noncovalent protein aggregation can be related to strong hydrophobicity and low electrostatic repulsion of local amino acid regions, which complicates drug quality control. These findings shed light on the relationship between protein aggregation and the local hydrophobicity region, and broaden the way to analyze the detection 'artifacts' in reducing CE-SDS studies of therapeutic proteins.

Elimination of light chain tailing in reducing capillary electrophoresis with sodium dodecyl sulfate analysis of a monoclonal antibody.

Capillary electrophoresis with sodium dodecyl sulfate (CE-SDS) is a common analytical technique for investigating the purity and molecular size heterogeneity of monoclonal antibody (mAb) drugs. In reducing CE-SDS analysis of mAb-A, the light chain (LC) peak exhibited severe tailing, seriously affecting the purity analysis. The purposes of this investigation are to clarify the source of tailing and develop a more appropriate CE-SDS method to eliminate LC tailing. The degree of LC tailing was closely related to the mAb concentration, SDS concentration, and injection amount, and more hydrophobic detergents, such as sodium hexadecyl sulfate (SHS) and sodium tetradecyl sulfate (STS), could be used instead of SDS to obtain better peak shapes. The results also indicated that the tailing was caused by the binding problem associated with SDS, and SHS/STS could provide a more stable and uniform complexation for the LC. In summary, the method we developed successfully eliminated the LC tailing and provided a robust characterization of mAb-A in reducing CE-SDS analysis.

Investigation of an Uncommon Artifact during Reducing Capillary Electrophoresis-Sodium Dodecyl Sulfate Analysis of a Monoclonal Antibody with Dynamic Light Scattering and Reversed Phase High-Performance Liquid Chromatography.

PURPOSES: In reducing capillary electrophoresis sodium dodecyl sulfate (CE-SDS) analysis of a monoclonal antibody (mAb-1), the peak area ratio of heavy chain (HC) to light chain (LC) was out of balance, while multiple artifact peaks were observed following the migration of HC. The main purposes of this study were to describe the techniques utilized to eliminate this artifact and clarify the root cause for this interesting phenomenon. METHODS: We optimized the CE-SDS analysis of mAb-1 by a vairety of techniques including changing the concentration of protein or replacing SDS with a more hydrophobic surfactant (i.e., sodium hexadecyl sulfate (SHS) or sodium tetradecyl sulfate (STS) instead of SDS) in sample and/or the sieving gel buffer. Dynamic light scattering (DLS) and reversed phase high-performance liquid chromatography (RP-HPLC) were used to study the protein-surfactant complex. RESULTS: The artifact could be partially mitigated by reducing the protein concentration and replacing SDS with SHS or STS in the sample and/or the sieving gel buffer solutions. Due to replacing a more hydrophobic surfactant, the HC-surfactant complex formed was more resistant to dissociation, preventing additional hydrophobic HC-HC interaction and aggregation, thus eliminating the artifact problem. CONCLUSIONS: DLS and RP-HPLC are powerful supplementary techniques in characterizing the protein-surfactant complex, and hydrophobic surfactants such as SHS and STS could afford more normal electropherograms during the analysis of mAbs.