Liquid-liquid phase separation of a monoclonal antibody at low ionic strength: Influence of anion charge and concentration

- Antibody solution undergoes liquid-liquid phase separation (LLPS) at the isoelectric point.
- Citrate and oligovalent anions induce LLPS at pH below the isoelectric point.
- Citrate is assumed to bind to the antibody resulting in charge neutralization.
- Citrate-antibody binding was complementarily proven by using buffer equilibration.

Liquid-liquid phase separation (LLPS) of a monoclonal antibody solution was investigated at low ionic strength in the presence of oligovalent anions, such as citrate, trimellitate, pyromellitate and mellitate. Phase separation was observed at the isoelectric point of the antibody at pH 8.7 as well as in more acidic pH regions in the presence of the tested oligovalent ions. This can be attributed to charge neutralization via binding of the oligovalent anions to the positively charged antibody. The influence of the anion concentration on liquid-liquid phase separation with respect to the net charge of the antibody was examined. Similarities to the formation of a complex coacervate were shown to apply. These findings enable us to understand the usage of excipients to rationally induce or avoid liquid-liquid phase separation at low ionic strength. Furthermore we present a method to directly examine the competition of different ions for the solvation shell, called buffer equilibration.