Site-Specific Structural Changes in Long-Term-Stressed Monoclonal Antibody Revealed with DEPC Covalent-Labeling and Quantitative Mass Spectrometry.

Studies of structural changes in mAbs under forced stress and storage conditions are essential for the recognition of degradation hotspots, which can be further remodeled to improve the stability of the respective protein. Herein, we used diethyl pyrocarbonate (DEPC)-based covalent labeling mass spectrometry (CL-MS) to assess structural changes in a model mAb (SILuMAb). Structural changes in the heat-stressed mAb samples were confirmed at specific amino acid positions from the DEPC label mass seen in the fragment ion mass spectrum.

Effect of salt modulators on the elution behavior of insulin and the separation of product-related impurities in reversed-phase chromatography.

In this work, the effect of the salt modulators potassium chloride, ammonium chloride, ammonium sulfate, and potassium sulfate on the elution behavior of insulin in reversed-phase chromatography with ethanol as the organic modifier was investigated. Without the addition of salt modulators, insulin shows the formation of multiple peaks under non-linear loading conditions, presumably due to an aggregate formation equilibrium. Flow rate and temperature did not influence the appearance of multiple peaks.

Mechanistic modeling of cation exchange chromatography scale-up considering packing inhomogeneities.

In process development and characterization, the scale-up of chromatographic steps is a crucial part and brings a number of challenges. Usually, scale-down models are used to represent the process step, and constant column properties are assumed. The scaling is then typically based on the concept of linear scale-up.

Anti-Langmuir elution behavior of a bispecific monoclonal antibody in cation exchange chromatography: Mechanistic modeling using a pH-dependent Self-Association Steric Mass Action isotherm.

The objective of this scientific work was to model and simulate the complex anti-Langmuir elution behavior of a bispecific monoclonal antibody (bsAb) under high loading conditions on the strong cation exchange resin POROS™ XS. The bsAb exhibited anti-Langmuirian elution behavior as a consequence of self-association expressed both in uncommon retentions and peak shapes highly atypical for antibodies. The widely applied Steric Mass Action (SMA) model was unsuitable here because it can only describe Langmuirian elution behavior and is not able to describe protein-protein interactions in the form of self-association.

Two peak elution behavior of a monoclonal antibody in cation exchange chromatography as a screening tool for excipients.

Aggregation of proteins is a critical quality attribute and a major concern during the purification of therapeutic proteins, like monoclonal antibodies. In-solution experiments applying different stress scenarios, e.g.