Characterization of highly concentrated antibody solution - A toolbox for the description of protein long-term solution stability

Abstract

High protein titers are gaining importance in biopharmaceutical industry. A major challenge in the development of highly concentrated mAb solutions is their long-term stability and often incalculable viscosity. The complexity of the molecule itself, as well as the various molecular interactions, make it difficult to describe their solution behavior. To study the formulation stability, long- and short-range interactions and the formation of complex network structures have to be taken into account. For a better understanding of highly concentrated solutions, we combined established and novel analytical tools to characterize the effect of solution properties on the stability of highly concentrated mAb formulations. In this study, monoclonal antibody solutions in a concentration range of 50-200 mg/ml at pH 5-9 with and without glycine, PEG4000, and Na₂SO₄ were analyzed. To determine the monomer content, analytical size-exclusion chromatography runs were performed. ζ-potential measurements were conducted to analyze the electrophoretic properties in different solutions. The melting and aggregation temperatures were determined with the help of fluorescence and static light scattering measurements. Additionally, rheological measurements were conducted to study the solution viscosity and viscoelastic behavior of the mAb solutions. The so-determined analytical parameters were scored and merged in an analytical toolbox. The resulting scoring was then successfully correlated with long-term storage (40 d of incubation) experiments. Our results indicate that the sensitivity of complex rheological measurements, in combination with the applied techniques, allows reliable statements to be made with respect to the effect of solution properties, such as protein concentration, ionic strength, and pH shift, on the strength of protein-protein interaction and solution colloidal stability.

Keywords: Conformational and colloidal stability; monoclonal antibodies; phase diagram; thermal stability; viscoelasticity; viscosity; zeta-potential.

Figure 1.

Monomer, HMW, and LMW contents of the tested antibody determined with the help of size-exclusion chromatography. Plotted are conditions after buffer exchange with original mAb concentrations of 120 and 180 mg/ml, at pH 5, pH 7, and pH 9 without additive (a) and with 150 mM glycine (b), as well as samples at pH 7 with 160 mM Na₂SO₄ and samples at pH 9 containing 1.2 (m/V)% PEG4000 (c).

Figure 2.

ζ-potential values of mAb at 10 mg/ml, pH 5–11, with and without 150 mM glycine, 2 (m/V)% PEG4000, and 160 mM Na₂SO₄, respectively, in solution determined by laser Doppler microelectrophoresis.

Figure 3.

Comparison of the viscosity values of samples with mAb concentrations of 120 and 180 mg/ml at pH 5, pH 7, and pH 9 with and without additive in solution.

Figure 4.

T_m1, T_m2, and T_agg values of mAb samples in a concentration range from 1–180 mg/ml at pH 7 without additive in solution determined over a temperature ramp ranging from 20–90°C.

Figure 5.

Comparison of T_m1 (a) and T_agg (b) values of samples with mAb concentrations of 120 and 180 mg/ml at pH 5, pH 7, and pH 9 with and without selected additives.

Figure 6.

ω_CO values of mAb samples without additive in solution as a function of protein concentration and pH (a). Impact of pH with an additive in solution, namely, 150 mM glycine (b), 160 mM Na₂SO₄ (c), and 2 (m/V)% PEG4000 (d), on the viscoelastic response. The gray area symbolizes the critical ω_CO region. Above this region, the viscoelastic response indicates stable mAb solutions. ω_CO values below this region indicate samples that might undergo phase transition.

Figure 7.

Scoring of the phase behavior of mAb in a pH range from 5 to 9, a protein concentration range from 120 to 225 mg/ml, and (a) 0–250 mM glycine, (b) 0–2 (m/V)% PEG4000, and (c) 0–160 mM Na₂SO₄ visualized as 3D plots. The plates were stored at a constant temperature of 20°C. The scoring was done after 40 d of incubation based on the visual evaluation of the pictures taken by the Rock Imager. The blue round symbols stand for samples containing soluble mAb molecules, the small light red squares symbolize light precipitation in the middle of the well, and the dark red squares symbolize heavy precipitation.

Figure 8.

Scoring of analytical results is shown in relation to the long-term colloidal stability (a). The subplots from top to bottom show the predicted impact at pH 5, pH 7, and pH 9 with mAb concentrations of 120 and 180 mg/ml without additive in solution, with the addition of 150 mM glycine, 1.2 (m/V)% PEG4000, and 160 mM Na₂SO₄, respectively. The scoring of the phase behavior at t₄₀ of similar samples is depicted on the right hand side for easier comparison (b).