Mass spectrometry-based multi-attribute method in protein therapeutics product quality monitoring and quality control.

The multi-attribute method (MAM), a liquid chromatography-mass spectrometry (LC-MS)-based peptide mapping method, has gained increased interest and applications in the biopharmaceutical industry. MAM can, in one method, provide targeted quantitation of multiple site-specific product quality attributes, as well as new peak detection. In this review, we focus on the scientific and regulatory considerations of using MAM in product quality attribute monitoring and quality control (QC) of therapeutic proteins. We highlight MAM implementation challenges and solutions with several case studies, and provide our perspective on the opportunities to use MS in QC for applications other than standard peptide mapping-based MAM.

High throughput FAMS - A fatty acid mass spectrometry method for monitoring polysorbate hydrolysis in QC.

Surfactant degradation in biopharmaceuticals has recently gained significant attention in the pharmaceutical industry. Specifically, hydrolytic degradation of polysorbates, leading to the release of free fatty acids potentially forming visible particles, is a key theme in technical development. To address this emerging topic, we present the development of a fully automated liquid-chromatography single quad mass detector method for the quantification of free fatty acids in biopharmaceuticals. For the first time, we have quantified the longer chain fatty acid degradation products of polysorbate, palmitic and stearic acid, allowing reliable detection and early critical insights for process improvements. This high-throughput method was validated underlining its robust performance in an interlaboratory trial as well as high flexibility allowing different robotic platforms and preparation techniques. The combination of automated sample preparation, separation by liquid chromatography and single quad mass detection makes the validated fatty acid mass spectrometry assay ready for routine use in a regulated environment.

mD-UPLC-MS/MS: Next Generation of mAb Characterization by Multidimensional Ultraperformance Liquid Chromatography-Mass Spectrometry and Parallel On-Column LysC and Trypsin Digestion.

For the past few years, multidimensional liquid chromatography-mass spectrometry (LC-MS) systems have been commonly used to characterize post-translational modifications (PTMs) of therapeutic antibodies (mAbs). In most cases, this is performed by fractionation of charge variants by ion-exchange chromatography and subsequent online LC-MS peptide mapping analysis. In this study, we developed a multidimensional ultra-performance-liquid-chromatography-mass spectrometry system (mD-UPLC-MS/MS) for PTM characterization and quantification, allowing both rapid analysis and decreased risk of artificial modifications during sample preparation. We implemented UPLC columns for peptide mapping analysis, facilitating the linkage between mD-LC and routine LC-MS workflows. Furthermore, the introduced system incorporates a novel in-parallel trypsin and LysC on-column digestion setup, followed by a combined peptide mapping analysis. This parallel digestion with different enzymes enhances characterization by generating two distinct peptides. Using this approach, a low retentive ethylene oxide adduct of a bispecific antibody was successfully characterized within this study. In summary, our approach allows versatile and rapid analysis of PTMs, enabling efficient characterization of therapeutic molecules.

Fast analysis of antibody-derived therapeutics by automated multidimensional liquid chromatography - Mass spectrometry.

Characterization of post-translational modifications (PTMs) of therapeutic antibodies is commonly performed by bottom-up approaches, involving sample preparation and peptide analysis by liquid chromatography-mass spectrometry (LC-MS). Conventional sample preparation requires extensive hands-on time and can increase the risk of inducing artificial modifications as many off-line steps - denaturation, disulfide-reduction, alkylation and tryptic digestion - are performed. In this study, we developed an on-line multidimensional (mD)-LC-MS bottom-up approach for fast sample preparation and analysis of (formulated) monoclonal antibodies and antibody-derived therapeutics. This approach allows on-column reduction, tryptic digestion and subsequent peptide analysis by RP-MS. Optimization of the 1D -and 2D flow and temperature improved the trapping of small polar peptides during on-line peptide mapping analysis. These adaptations increased the sequence coverage (95-98% versus 86-94% for off-line approaches) and allowed identification of various PTMs (i.e. deamidation of asparagine, methionine oxidation and lysine glycation) within a single analysis. This workflow enables a fast (<2 h) characterization of antibody heterogeneities within a single run and a low amount of protein (10 µg). Importantly, the new mD-LC-MS bottom-up method was able to detect the polar, fast-eluting peptides: Fc oxidation at Hc-Met-252 and the Fc N-glycosylation at Hc-Asn-297, which can be challenging using mD-LC-MS. Moreover, the method showed good comparability across the different measurements (RSD of retention time in the range of 0.2-1.8% for polar peptides). The LC system was controlled by only a standard commercial software package which makes implementation for fast characterization of quality attributes relatively easy.

Inter-laboratory study to evaluate the performance of automated online characterization of antibody charge variants by multi-dimensional LC-MS/MS.

An international study was conducted to evaluate the performance and reliability of an online multi-dimensional (mD)-LC-MS/MS approach for the characterization of antibody charge variants. The characterization of antibody charge variants is traditionally performed by time-consuming, offline isolation of charge variant fractions by ion exchange chromatography (IEC) that are subsequently subjected individually to LC-MS/MS peptide mapping. This newly developed mD-LC-MS/MS approach enables automated and rapid characterization of charge variants using much lower sample requirements. This online workflow includes sample reduction, digestion, peptide mapping, and subsequent mass spectrometric analysis within a single, fully-automated procedure. The benefits of using online mD-LC-MS/MS for variant characterization include fewer handling steps, a more than 10-fold reduction in required sample amount, reduced sample hold time as well as a shortening of the overall turnaround time from weeks to few days compared to standard offline procedures. In this site-to-site comparison study, we evaluated the online peptide mapping data collected from charge variants of trastuzumab (Herceptin®) across three international laboratories. The purpose of this study was to compare the overall performance of the online mD-LC-MS/MS approach for antibody charge variant characterization, with all participating sites employing different mD-LC-MS/MS setups (e.g., instrument vendors, modules, columns, CDS software). The high sequence coverage (95%-97%) obtained in each laboratory, enabled a reproducible generation of tryptic peptides and the comparison of values of the charge variants. Results obtained at all three participating sites were in good agreement, highlighting the reliability and performance of this approach, and correspond with data gained by the standard offline procedure. Overall, our results underscore of the benefit mD-LC-MS/MS technology for therapeutic antibody characterization, confirming its potential to become an important tool in the toolbox of protein characterization scientists.

Monitoring modifications in biopharmaceuticals: Toolbox for a generic and robust high-throughput quantification method.

Monoclonal antibodies (mAbs) constitute one of the most important and fastest growing sector within the pharmaceutical industry. The variety of different formats and the large molecule sizes of the mAbs result in an inherent complexity. In addition, the posttranslational modifications (PTMs) that can occur during production, formulation and storage pose a major analytical challenge for their characterization. These PTMs may affect the safety, efficacy and/or pharmacokinetic profile of the medicinal product. Therefore, strict quality and stability monitoring, in particular of (potentially) critical quality attributes (CQAs), is mandatory. Focusing on these needs, a toolbox for different approaches of peptide mapping in the routine quality control (QC) environment was developed. This toolbox includes an automated sample preparation with an optimized buffer system in combination with a Single Quad LC-MS system, which provides the flexibility to handle a high-throughput workflow. Our results demonstrate that a Single Quad LC-MS system is suitable for a routine work environment to monitor modifications by using a user-friendly system.

Monitoring polysorbate hydrolysis in biopharmaceuticals using a QC-ready free fatty acid quantification method.

Hydrolysis of the non-ionic surfactant polysorbate upon long-term storage poses significant challenges to development of biopharmaceutical liquid formulations. Low concentrations of intact surfactant may compromise its protective properties and thus affect protein stability. In addition, accumulation of polysorbate hydrolysis products is increasingly put into context with the formation of visible and subvisible particulates based on the low solubility of the main degradation products. Despite of this potential negative impact on product quality, quantification of the released free fatty acids is performed commonly in an indirect and consequently insensitive manner by determining the remaining PS content or by cumbersome methods, which are unsuitable for routine testing in quality control laboratories. For this purpose, this study describes the development and qualification of a label-free, reliable liquid-chromatography single quad mass detector (LC-QDa)-based method capable of resolving slight changes in the free fatty acid profile which can be readily integrated into quality control facilities. The practical utility of the herein described method is outlined by a case study on the real-time storage stability of a formulated monoclonal antibody.

Fast and Automated Characterization of Antibody Variants with 4D HPLC/MS.

Characterization of unknown monoclonal antibody (mAb) variants is important in order to identify their potential impact on safety, potency, and stability. Ion exchange chromatography (IEC) coupled with UV detection is frequently used to separate and quantify mAb variants in routine quality control (QC). However, characterization of the chromatographic peaks resulting from an IEC separation is an extremely time-consuming process, involving many cumbersome steps. Presented here is an online four-dimensional high performance liquid chromatography-mass spectrometry (4D HPLC/MS) approach, developed to circumvent these limitations. To achieve this, a 2D HPLC system was extended through the introduction of additional modules, hence enabling fully automated bioseparation of mAbs, fractionation of peaks, reduction, tryptic digestion, and reversed-phase (RP) separation of resulting peptides followed by MS detection. The entire separation and analytical process for an unknown peak is performed in less than 1.5 h, leading to a significant time savings, with comparable sequence coverage. To show the comparability with the traditional offline process, a proof of concept study with a previously characterized mAb1 is presented in this paper.