Determination of Protein–Protein Interactions in a Mixture of Two Monoclonal Antibodies

Priyanka Singh, Aisling Roche, Christopher F. van der Walle, Shahid Uddin, Jiali Du, Jim Warwicker, Alain Pluen, Robin Curtis

Research output: Contribution to journalArticlepeer-review


The co-formulation of monoclonal antibody (mAb) mixtures provides an attractive route to achieving therapeutic efficacy where the targeting of multiple epitopes is necessary. Controlling and predicting the behaviour of such mixtures requires elucidating the molecular basis for the self and cross protein-protein interactions, and how they depend on solution variables. While self interactions are now beginning to be well understood, there do not exist any systematic studies of cross interactions between mAbs in solution. Here, we have used
static light scattering to measure the set of self and cross osmotic second virial coefficients in a solution containing a mixture of two mAbs, mAbA and mAbB, as a function of ionic strength and pH. mAbB exhibits strong association at low ionic strength, which is attributed to an electrostatic attraction that is enhanced by the presence of a strong short-ranged attraction of non-electrostatic origin. Under all solution conditions, the measured cross interactions are intermediate the self interactions and follow similar patterns of behaviour.
There is a strong electrostatic attraction at higher pH values, reflecting the behaviour of mAbB. Protein-protein interactions become more attractive with increasing pH due to reducing the overall protein net charges, an effect which is attenuated with increasing ionic strength due to screening of electrostatic interactions. Under moderate ionic strength conditions, the reduced cross virial coefficient, which reflects only the energetic contribution to protein-protein interactions, is given by a geometric average of the corresponding self coefficients. We show the relationship can be rationalized using a patchy sphere model, where the interaction energy between sites i and j is given by the arithmetic mean of the i-i and j-j interactions. The geometric mean does not necessarily apply to all mAb mixtures and is expected to break down at lower ionic strength due to the non-additivity of electrostatic interactions.
Original languageEnglish
Article number10.1021/acs.jpcc.9b06648
Pages (from-to)4775-4786
JournalMolecular Pharmaceutics
Issue number12
Early online date15 Oct 2019
Publication statusPublished - 2 Dec 2019


  • monoclonal antibody
  • protein-protein interactions
  • electrostatics
  • osmotic second virial coefficients
  • biopharmaceutical formulations


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