Opalescence Measurements: Improvements in Fundamental Knowledge, Identifying Sources of Analytical Biases, and Advanced Applications for the Development of Therapeutic Proteins.

Abstract

Opalescence of biopharmaceutical solutions can indicate suboptimal colloidal stability and is therefore a generally undesirable attribute that requires investigation and potentially remediation. While there are numerous instrumentation options available for measuring opalescence, cross-instrument comparisons and detailed knowledge of analytical biases have been limited. Here, we highlight key findings from a multi-instrument investigation where differences in reported opalescence values are explained with particular emphasis on how the optical configuration and detector properties of each instrument affect the response of the sample and the primary formazin standards required for instrument calibration. In doing so, the particle size distribution, angular-dependent light scattering properties and refractive index of the primary formazin standard material are characterized and presented. Finally, the advanced application of a 90° angle light scattering instrument is presented as a suitable approach for making low volume, temperature controlled, nephelometric measurements of opalescence. Moreover, we demonstrate how this approach enables the simultaneous evaluation of key physical properties, such as hydrodynamic size, that are pertinent to investigations of opalescent biopharmaceuticals but have historically required the use of separate instrumentation. The findings reported here address key knowledge gaps and provide opportunities for improving the efficiency and inter-laboratory comparability of opalescence measurements for biopharmaceuticals.