Continuous background correction of refractive index signal to improve monoclonal antibody concentration monitoring during UF/DF and SPTFF operations.

Inline refractive index (RI) has the potential for monitoring protein concentration during final bulk concentration. While useful for monitoring and controlling product concentration, RI is sensitive to the respective background buffer being used for processing. This raises concerns around variations in buffer preparations, and during diafiltration where the buffer background is a mixture of different buffers during exchange. This study evaluated whether the use of a RI probe in the permeate line could facilitate continuous background subtraction (dual RI) and improve concentration monitoring during ultrafiltration/diafiltration and single pass TFF concentration for IgG1 and IgG4 antibodies. The proposed dual RI strategy yielded reductions in % error compared to the use of a single refractive index estimate from the retentate line (6.18% vs 8.63% for IgG4 and 2.65% vs 8.85% for IgG1) during traditional ultrafiltration/diafiltration. The improvement in IgG estimates were best during diafiltration where the continuous background subtraction of the permeate RI-enabled continuous monitoring of antibody material without knowledge of what the background buffer was compared to the use of a single RI estimate (6.47% vs 10.79% for IgG4 and 3.29% vs 19.59% for IgG1). In contrast minimal improvement to accuracy was obtained when using SPTFF as a concentration step. The ability to monitor product concentration changes via the proposed dual RI approach removes the need for complex calibrations, minimal worry about changing buffer backgrounds during diafiltration, and could enable better process control during product concentration in the cGMP manufacture of biologics.