Early developability screen of therapeutic antibody candidates using Taylor dispersion analysis and UV area imaging detection

Abstract

Therapeutic antibodies represent one of the fastest growing segments in the pharmaceutical market. They are used in a broad range of disease fields, such as autoimmune diseases, cancer, inflammation and infectious diseases. The growth of the segment has necessitated development of new analytical platforms for faster and better antibody selection and characterization. Early quality control and risk assessment of biophysical parameters help prevent failure in later stages of antibody development, and thus can reduce costs and save time. Critical parameters such as aggregation, conformational stability, colloidal stability and hydrophilicity, are measured during the early phase of antibody generation and guide the selection process of the best lead candidates in terms of technical developability. We report on the use of a novel instrument (ActiPix/Viscosizer) for measuring both the hydrodynamic radius and the absolute viscosity of antibodies based on Taylor dispersion analysis and UV area imaging. The looped microcapillary-based method combines low sample consumption, fast throughput and high precision compared to other conventional methods. From a random panel of 130 antibodies in the early selection process, we identified some with large hydrodynamic radius outside the normal distribution and others with non-Gaussian Taylor dispersion profiles. The antibodies with such abnormal properties were confirmed later in the selection process to show poor developability profiles. Moreover, combining these results with those of the viscosity measurements at high antibody concentrations allows screening, with limited amounts of materials, candidates with potential issues in pre-formulation development.

Keywords: APS; Dulbecco phosphate buffered saline; hIgG; Taylor dispersion; Taylor dispersion analysis; CE; UV array imaging detection; active pixel sensor; TDA; capillary electrophoresis; DLS; coefficient of variation; developability assessment; dynamic light scattering; dPBS; fragment antigen-binding; ID; human immunoglobulin G; mIgG; hydrodynamic radius; hydrodynamic radius; S-DAS; internal diameter; OD; millipascal seconds; Rh; monoclonal antibodies; monoclonal antibody; Fab; mouse immunoglobulin G; mAb; outside diameter; mPa.s; scaling factor; STD; selection developability assessment; SEC-LC; size-exclusion liquid chromatography; SF; standard deviation; CV; viscosity.

Figure 1.

Comparison between the Rh measured by TDA and by DLS of a panel of 27 mAbs.

Figure 2.

Linear correlation between the Rh measured by TDA and the molecular mass measured by mass spectrometry (r² = 0.99).

Figure 3.

Rh distribution of a random panel of 110 mAbs. (A) Scatter plot representation of the Rh vs. mAb. The black line corresponds to the average Rh of 5.77 nm. (B) Frequency histogram of the Rh measured at 1.5 mg/ml in PBS. The black line represents the normality fitting.

Figure 4.

Viscosity distribution of a random panel of 100 mAbs. (A) Frequency histogram of the absolute viscosity (${\displaystyle {\eta }_c)}$ measured at 1.5 mg/ml in PBS at 25°C. Shaded bars represent mAbs with a viscosity above the average +2 STD. (B) Scatter plot representation of the viscosity versus Rh. MAbs outside the black box have values 2 STD beyond the average viscosity (0.93 mPa.s) or average Rh (5.77 nm).

Figure 5.

The viscosity profiles of 5 mAb solutions were determined at different protein concentrations. The black line represents the 50 mPa.s empirical threshold for subcutaneous injection.

Figure 6.

Rh determination of proteins by the ActiPix/Viscosizer system. The protein sample is injected into a capillary loop at a constant pressure. UV absorbance at 214 nm is recorded during the first and second passages through the APS detector, respectively. The analysis of peak broadening, time (t) and diffusion (τ), due to Taylor dispersion allows the calculation of the Rh.

Figure 7.

Protein viscosity measured by the ActiPix/Viscosizer system. Example of a UV chromatogram during viscosity measurement. A dilute solution of the tyrosine marker is continuously injected into the pre equilibrated capillary for 2 min. The Δtm (tm2-tm1) is determined by passage through the dual window of the APS detector. The capillary is then re-equilibrated and the Δts (ts2 –ts1) is measured by injection of the protein sample into the capillary.