Taylor dispersion analysis compared to dynamic light scattering for the size analysis of therapeutic peptides and proteins and their aggregates.

Purpose: To evaluate Taylor dispersion analysis (TDA) as a novel method for determination of hydrodynamic radius of therapeutic peptides and proteins in non-stressed and stressed formulations and to compare it with dynamic light scattering (DLS).

Methods: The hydrodynamic radius of oxytocin, bovine serum albumin, various monoclonal antibodies (type IgG) and etanercept at concentrations between 0.05 and 50 mg/ml was determined by TDA and DLS.

Probing bis-ANS binding sites of different affinity on aggregated IgG by steady-state fluorescence, time-resolved fluorescence and isothermal titration calorimetry.

Fluorescent dyes, for example, 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid dipotassium salt (bis-ANS) are extensively used to detect nonnative protein structures in therapeutic protein products, for example, during formulation development by monitoring the greatly enhanced dye fluorescence upon binding to nonnative species. Our aim was to characterize the level of heterogeneity of bis-ANS binding sites in a thermally stressed monoclonal antibody (IgG) formulation by steady-state fluorescence, time-resolved fluorescence and isothermal titration calorimetry (ITC), and to obtain apparent dissociation constants (Kd ) by data fitting. Because the methods differ in their underlying measurement principles, they provide different information on binding properties of bis-ANS to thermally stressed IgG.

Misconceptions over Förster resonance energy transfer between proteins and ANS/bis-ANS: Direct excitation dominates dye fluorescence.

Our aim was to disprove the widespread misconception that Förster resonance energy transfer (FRET) is the only explanation for observing fluorescence from ANS (8-anilino-1-naphthalenesulfonic acid) and bis-ANS (4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid, dipotassium salt) following excitation at 280nm in the presence of protein. From ultraviolet (UV) absorption spectra and fluorescence emission spectra of bis-ANS and ANS in buffer and ethanol, direct excitation at 280nm was found to be the dominant mechanism for the resulting dye fluorescence. Furthermore, Tyr/Trp quenching studies were performed for solutions of N-acetyl-l-tryptophanamide, heat-stressed immunoglobulin G (IgG), and bovine serum albumin (BSA) by monitoring changes in steady state fluorescence spectra and time-resolved fluorescence decays as a function of dye concentration.

Fluorescent molecular rotors as dyes to characterize polysorbate-containing IgG formulations.

Purpose: The aim was to evaluate fluorescent molecular rotors (DCVJ and CCVJ), which are mainly sensitive to viscosity, for the characterization of polysorbate-containing IgG formulations and compare them to the polarity-sensitive dyes ANS, Bis-ANS and Nile Red.

Methods: IgG formulations with polysorbate 20 or 80 were stressed below the aggregation temperature and analyzed by steady-state and time-resolved fluorescence and by HP-SEC with UV and fluorescent dye detection (Bis-ANS and CCVJ). Furthermore, commercial protein preparations of therapeutic proteins (Enbrel 50 mg, Humira 40 mg and MabThera 100 mg) were aggregated accordingly and analyzed with CCVJ fluorescence and HP-SEC.

Structural properties of monoclonal antibody aggregates induced by freeze-thawing and thermal stress.

Aggregation of monoclonal antibodies can be induced by freeze-thawing and elevated temperature, typical stress factors during development, production and storage. Our aim was to characterize structural properties of aggregates formed after freeze-thawing and thermal stressing of humanized monoclonal IgG(1) antibody (IgG). Formulations with 1.

Online fluorescent dye detection method for the characterization of immunoglobulin G aggregation by size exclusion chromatography and asymmetrical flow field flow fractionation.

The aim of this study was to develop an online fluorescent dye detection method suitable for high-pressure size exclusion chromatography (HP-SEC) and asymmetrical flow field flow fractionation (AF4). The noncovalent extrinsic fluorescent dye 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (Bis-ANS) was added to the mobile phase or the sample, and the fluorescence emission at 488nm was recorded on excitation at 385nm. By combining HP-SEC and AF4 with online dye detection, it was possible to simultaneously detect heat-induced aggregation and structural changes of monomeric and aggregated immunoglobulin G (IgG); an increase in Bis-ANS fluorescence was observed in both the aggregate and monomer fractions.