Understanding and Modulating Opalescence and Viscosity in a Monoclonal Antibody Formulation

Abstract

Opalescence and high viscosities can pose challenges for high concentration formulation of antibodies. Both phenomena result from protein-protein intermolecular interactions that can be modulated with solution ionic strength. We studied a therapeutic monoclonal antibody that exhibits high viscosity in solutions at low ionic strength (~20 centipoise (cP) at 90 mg/mL and 23°C) and significant opalescence at isotonic ionic strength (approximately 100 nephelometric turbidity units at 90 mg/mL and 23°C). The intermolecular interactions responsible for these effects were characterized using membrane osmometry, static light scattering and zeta potential measurements. The net protein-protein interactions were repulsive at low ionic strength (~4 mM) and attractive at isotonic ionic strengths. The high viscosities are attributed to electroviscous forces at low ionic strength and the significant opalescence at isotonic ionic strength is correlated with attractive antibody interactions. Furthermore there appears to be a connection to critical phenomena and it is suggested that the extent of opalescence is dependent on the proximity to the critical point. We demonstrate that by balancing the repulsive and attractive forces via intermediate ionic strengths and by increasing the mAb concentration above the apparent critical concentration both opalescence and viscosity can be simultaneously minimized.

Resolving self-association of a therapeutic antibody by formulation optimization and molecular approaches

“…Solution conditions can often be manipulated to minimize the protein self-interactions. 1,[7][8][9][10][11]13 The data reported here show that formulation pH and ionic strength had a large effect on gelation. No gelation was seen at pH below 5.5 and all samples formulated at pH greater than or equal to 6.0 gelled.…”

“…Aggregation, precipitation, crystallization, liquid-liquid phase separation, opalescence and high viscosity are all manifestations of self-interactions that can be promoted by high antibody concentration. 1,[5][6][7][8][9][10][11] The different outcomes are due to the nature of the forces dominating the interactions (electrostatic, hydrophobic, short range attraction) and specific solution conditions. 4 These phenomena differ fundamentally from rapid heat-or chemically-induced aggregation or precipitation in that the intermolecular interactions can be reversed by changing the solution conditions such as pH, ionic strength, temperature or excipients, suggesting that a native or near-native protein conformation is maintained.…”

High-throughput screening for developability during early-stage antibody discovery using self-interaction nanoparticle spectroscopy

“…This is problematic because weak antibody self-and cross-interactions are often responsible for aggregation and polyreactivity, respectively. 6,7,12,[14][15][16][17][18][19] Nevertheless, numerous assays such as self-interaction chromatography (SIC) [20][21][22][23][24][25] and cross-interaction chromatography (CIC) [26][27][28] have been designed to identify these possibly troublesome antibodies early in the discovery program to avoid downstream issues. In these chromatography assays, increased retention of mAbs passing through a column conjugated with identical mAbs or a pool of polyclonal serum antibodies is indicative of attractive self-or cross-interactions, respectively.…”

Computational tool for the early screening of monoclonal antibodies for their viscosities

“…For example, antibodies have high viscosity near their isoelectric point (pI) values at low ionic strength conditions. 2,10 Furthermore, negative charge-based descriptors 1,3,7,9,11 have shown good correlation with the viscosity of highly concentrated solutions.…”