Development of pDADMAC Flocculation Harvest for Intensified Fed-Batch Process

The separation of cells, cell debris, and host cell related impurities from the target product in biologics manufacturing becomes increasingly challenging as cell density and titer in the upstream bioreactor rise. Traditional filtration methods require large membrane areas or multiple stages, leading to facility fit issues and increased costs. To address these challenges, flocculation with cationic polymers has been explored, but certain key questions regarding its successful application and scale-up remain unanswered.

This study aims to investigate the use of pDADMAC flocculation for consistent flocculate morphology from bench to pilot scale and effective removal of pDADMAC during downstream processing. Cell broth obtained from an intensified upstream process, capable of achieving a peak cell density of 38 million/mL, was used. Real-time imaging with Easyviewer was employed to quickly screen flocculation conditions based on cell density, viability, and pDADMAC concentrations. EasyViewer, both in the bioreactor and flow-cell setup, proved to be a versatile tool for evaluating flocculate particle morphology across different scales.

Successful application of pDADMAC flocculation at a 500L pilot scale achieved a loading of over 130 L/m2 with minimal pressure buildup using only one stage of depth filtration. Additionally, this study provides the first quantitative data, obtained through reverse phase HPLC, for pDADMAC mass balance analysis. The majority of residual pDADMAC after flocculation was found to be removed during the high salt wash in Protein A chromatography. Extensive characterization of flocculated and non-flocculated samples demonstrated that the inclusion of the flocculation step reduces impurity levels at the early stage of downstream purification. Compared to the control group, a 40% reduction in HCP concentrations (measured by ELISA), a 27% reduction in the number of HCP species (measured by mass spectrometry), and complete removal of DNA were observed. The study also addressed potential risks of disulfide bond reduction and particle formation after harvest due to residual pDADMAC.

These results expand the downstream purification toolbox for harvesting cell broth with high cell density and low viabilities. Furthermore, they provide valuable insights into the integration of flocculation-depth filtration with the current platform process for biologics purification.