Phase behavior of an intact monoclonal antibody

Abstract

Understanding protein phase behavior is important for purification, storage, and stable formulation of protein drugs in the biopharmaceutical industry. Glycoproteins, such as monoclonal antibodies (MAbs) are the most abundant biopharmaceuticals and probably the most difficult to crystallize among water-soluble proteins. This study explores the possibility of correlating osmotic second virial coefficient (B(22)) with the phase behavior of an intact MAb, which has so far proved impossible to crystallize. The phase diagram of the MAb is presented as a function of the concentration of different classes of precipitants, i.e., NaCl, (NH4)2SO4, and polyethylene glycol. All these precipitants show a similar behavior of decreasing solubility with increasing precipitant concentration. B(22) values were also measured as a function of the concentration of the different precipitants by self-interaction chromatography and correlated with the phase diagrams. Correlating phase diagrams with B(22) data provides useful information not only for a fundamental understanding of the phase behavior of MAbs, but also for understanding the reason why certain proteins are extremely difficult to crystallize. The scaling of the phase diagram in B(22) units also supports the existence of a universal phase diagram of a complex glycoprotein when it is recast in a protein interaction parameter.

FIGURE 1

Schematic representation of a generalized protein phase diagram.

FIGURE 2

Effect of the injected protein concentration on the retention of the MAb. The experiment was conducted at pH 4.2 (100 mM Na-acetate) in the MAb-immobilized column.

FIGURE 3

B₂₂ profile of IDEC-152 in NaCl at different pHs.

FIGURE 4

B₂₂ profile of IDEC-152 in (NH₄)₂SO₄ and PEG-400 at pH 7.6.

FIGURE 5

Phase behavior of IDEC-152 MAb in NaCl at 30°C and pH 7.6.

FIGURE 6

Phase behavior of IDEC-152 MAb in (NH₄)₂SO₄ at 30°C and pH 7.6

FIGURE 7

Phase behavior of IDEC-152 MAb in PEG-400 at 30°C and pH 7.6.

FIGURE 8

Phase diagram of IDEC-152 MAb showing liquid-liquid coexistence.

FIGURE 9

Phase diagram of IDEC-152 MAb in the format of a generic protein phase diagram. Calculations were made according to Haas and Drenth (51). Assumptions: = 1.665 × 10⁻¹⁹ cm³; = 0.37; M = 144000 g/mol; = 1.4362 g/cm³; f = 0.3217. Existence of binodal and spinodal within the crystal phase is not realistic. However, it is shown to visualize their approximate location.