Pharmaceutical Perspective on Opalescence and Liquid-Liquid Phase Separation in Protein Solutions

Mol Pharm. 2016 May 2;13(5):1431-44. doi: 10.1021/acs.molpharmaceut.5b00937. Epub 2016 Apr 5.


Opalescence in protein solutions reduces aesthetic appeal of a formulation and can be an indicator of the presence of aggregates or precursor to phase separation in solution signifying reduced product stability. Liquid-liquid phase separation of a protein solution into a protein-rich and a protein-poor phase has been well-documented for globular proteins and recently observed for monoclonal antibody solutions, resulting in physical instability of the formulation. The present review discusses opalescence and liquid-liquid phase separation (LLPS) for therapeutic protein formulations. A brief discussion on theoretical concepts based on thermodynamics, kinetics, and light scattering is presented. This review also discusses theoretical concepts behind intense light scattering in the vicinity of the critical point termed as "critical opalescence". Both opalescence and LLPS are affected by the formulation factors including pH, ionic strength, protein concentration, temperature, and excipients. Literature reports for the effect of these formulation factors on attractive protein-protein interactions in solution as assessed by the second virial coefficient (B2) and the cloud-point temperature (Tcloud) measurements are also presented. The review also highlights pharmaceutical implications of LLPS in protein solutions.

Keywords: B2; Tcloud; aggregation; crystallization; formulation; light scattering; liquid−liquid phase separation; opalescence; phase diagram; protein−protein interactions; stability; thermodynamics.

Publication types

  • Review

MeSH terms

  • Antibodies, Monoclonal / chemistry
  • Chemistry, Pharmaceutical / methods
  • Humans
  • Hydrogen-Ion Concentration
  • Iridescence / drug effects*
  • Osmolar Concentration
  • Pharmaceutical Solutions / chemistry*
  • Pharmaceutical Solutions / pharmacology*
  • Proteins / chemistry*
  • Solutions / chemistry*
  • Solutions / pharmacology*
  • Temperature
  • Thermodynamics


  • Antibodies, Monoclonal
  • Pharmaceutical Solutions
  • Proteins
  • Solutions