BACKGROUNDThe biopharmaceutical industry is increasingly interested in the analysis of trace metals due to their significant impact on product quality and drug safety. Certain metals can potentially accelerate the formation of degradants or aggregates in biotherapeutic proteins, leading to drug product quality concerns. A better understanding of metal-mAb interactions would aid in the development of purification processes and formulations, thereby ensuring better drug quality and safety. An efficient analytical method to analyze metal-mAb interactions and identify metal binding molecules is needed.RESULTSThis article presents a novel approach combining native size-exclusion chromatography (nSEC)-UV detection with inductively coupled plasma mass spectrometry (ICPMS) and nano-electrospray ionization (nano-ESI) high resolution mass spectrometry (HRMS) for simultaneous detection of metals and identification of metal binding molecules, including biotherapeutics and small molecules in drug substances and products. With this method, several metals, including chromium, manganese, iron, cobalt, nickel, copper and zinc, were found to bind the bispecific antibody mAb1. In addition, iron and copper were found to bind small molecules, such as histidine, citrate or sucrose present in drug substances. The developed platform was further used to study an IgG1 mAb (mAb2). A high iron binding capacity was observed for mAb2. Through integrated enzyme assisted subunit analysis, we further identified that iron binds predominantly the mAb Fab domain.SIGNIFICANCEThis LC-UV-ICPMS-HRMS method enables deeper understanding of metal-mAb interactions and offers information on both metal and metal-binding molecules in a single experiment, thus saving time and minimizing sample consumption. Consequently, this method can effectively and directly facilitate exploration of metal-mAb interactions during drug development.

01 Aug 2024·Journal of Chromatography A

An inert tracer: The binding site of a fluorescent dye on the antibody and its effects on Protein A chromatography

Article

Author: Eppink, Michel ; Lali, Narges ; Dirksen, Eef ; Kokke, Bas ; Tsiatsiani, Liana ; Jungbauer, Alois ; Satzer, Peter ; Elffrink, Walter

Fluorescently labeled antibodies are widely used to visualize the adsorption process in protein chromatography using confocal laser scanning microscopy (CLSM), but also as a tracer for determination of residence time distribution (RTD) in continuous chromatography. It is assumed that the labeled protein is inert and representative of the unlabeled antibody, ignoring the fact that labeling with a fluorescent dye can change the characteristics of the original molecule. It became evident that the fluorescently labeled antibody has a higher affinity toward protein A resins such as MabSelect Sure. This can be due to slight differences in hydrophobicity and net charge, which are caused by the addition of the fluorescent dye. However, this difference is eliminated when using high salt concentrations in the adsorption studies. In this work, the site occupancy of two labeled antibodies, MAb1 (IgG1 subclass) and MAb2 (IgG2 subclass) conjugated with the fluorescent dye Alexa Fluor™ 488 was elucidated by intact mass spectrometry (MS) and peptide mapping LC-MS/MS, employing a sequential cleavage with Endoproteinase Lys-C and trypsin and in parallel with chymotrypsin alone. It was shown that the main binding site for the dye was a specific lysine in the heavy chains of the MAb1 and MAb2 molecules, in positions 188 and 189 respectively. Other lysine residues distributed throughout the protein sequence were labeled to a lot lesser extent. The labeled antibody had a slightly different affinity to MabSelect Sure although its primary binding site (to Protein A) was not affected by labeling, despite the secondary region responsible for binding to the protein A was partly labeled. Overall, the fluorescent-labeled antibodies are a good compromise as an inert tracer in residence time distribution and chromatography studies because they are much cheaper than isotope-labeled antibodies; However, the differences between the labeled and unlabeled antibodies should be considered.

01 Apr 2024·Molecular Pharmaceutics

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

Article

Author: Gao, Han ; Ge, Xin-Zhe ; Xu, Jie ; Fang, Wei-Jie ; Liu, Jia-Wei ; Wang, Si-Tao

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.

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