Aggregate removal can be a challenge in downstream purification processes for many proteins, including monoclonal antibodies (mAbs). During process development, fractions are typically collected during purification with pooling decision made afterwards with offline Size Exclusion Chromatography. This is both laborious and time consuming. In this work, we demonstrate connecting an AKTA Avant purification system to a Wyatt TREOS: an inline multi-angle light scattering (MALS) detector that directly calculates average molecular weight, Mw, using first principles. With only minimal apriori protein characterization, the monomer, dimer, and higher-order oligomer fraction content can then be determined. The TREOS can also send a start/stop fractionation trigger to the Avant based on a preset Mw or percent aggregate criteria. The Mw analysis and fractionation occurs in real-time with only a 15 second delay, eliminating the need for analysis after purification. We applied this unique Process Analytical Technology (PAT) tool for monitoring and control of average Mw in various purification applications. Both Flowthrough and Bind and Elute Hydrophobic Interaction Chromatography (HIC) were performed where only protein within a preset Mw was collected. In the case of Flowthrough HIC, the loading limit can be more aggressive as loading can be set to not stop until a specified Mw (or aggregate level) is observed in the breakthrough. This maximum usage of a resin’s aggregate binding capacity can reduce the cycles and buffers needed for processing batches. Finally, the TREOS was connected to an Avant in a continuous production process to monitor an Anion Exchange Chromatography step for several days. Any deviation from the expected Mw (and thus aggregate content) was controlled by signaling the AKTA Avant to discard the material. The novel Avant-MALS system demonstrated here could be used as a powerful tool for real-time aggregate monitoring during process purification steps.

Downstream process control of aggregates using light scattering and real time molecular weight

255th National Meeting (2018)

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