Practical Pipetting Optimization Strategies for Accurate Concentration Measurement of High-Concentration Protein Formulations

Abstract

High-concentration protein formulations for subcutaneous administration have developed rapidly, while viscosity-related pipetting errors remain a key bottleneck for accurate protein concentration quantification, with commercial detection instruments showing significant limitations in direct measurement of these samples. Here, we developed two pipetting-optimized quantification strategies using two high-concentration monoclonal antibody formulations with distinct viscosities as models: a motorized continuous pipette-based direct measurement method and a weight-corrected method. We validated the high accuracy and repeatability of the motorized continuous pipette for high-viscosity liquid handling, and systematically compared the performance of our optimized methods with the traditional manual pipetting method and three mainstream commercial protein analyzers. Direct measurements using the three evaluated analyzers showed formulation-dependent deviations under the tested conditions. Both optimized workflows improved pipetting precision and provided concentration estimates with improved repeatability for the two high-concentration mAb formulations examined. These findings support motorized pipetting and weight correction as practical options when viscosity compromises volumetric transfer, while their applicability to other protein modalities and formulations with intermediate viscosity requires further validation.

Graphical Abstract