FAMS-A Targeted Fatty Acid Mass Spectrometry Method for Monitoring Free Fatty Acids from Polysorbate Hydrolysis.

Polysorbates are the predominant surfactants used to stabilize protein formulations. Unfortunately, polysorbates can undergo hydrolytic degradation, which releases fatty acids that can accumulate to form visible particles. The detection and quantitation of these fatty acid degradation products are critical for assessing the extent of polysorbate degradation and the associated risks of particle formation.

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.

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.

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.

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.

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.

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.

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.

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.