Positively charged polymeric flocculant polydiallyldimethylammonium chloride (pDADMAC)
During processing of large-scale, high-density cell-culture harvest material, depth filtration can be costly. It often requires substantial filter area and thus represents a significant bottleneck. Alternative methods of enhancing clarification efficiency are needed. Clarifying agents such as charged flocculants and precipitants could help alleviate such constraints.
Traditional methods rely on depth filtration, often combined with centrifugation, but these approaches struggle with high-density cultures due to increased biomass and soluble impurities like host-cell proteins (HCPs) and DNA.
To address these challenges, alternative clarification strategies involving flocculation and acid precipitation were explored in this study. Positively charged polymeric flocculants such as polydiallyldimethylammonium chloride (pDADMAC) and citric acid were tested for their ability to aggregate cells, debris, and impurities, thereby improving filtration efficiency. The study compared traditional depth filtration with and without centrifugation to a modified process incorporating flocculants and Clarisolve filters.
Important findings:
- Flocculation and acid precipitation significantly improved filter throughput, with up to a sevenfold increase compared to untreated harvests.
- The combination of pDADMAC or acid pretreatment with Clarisolve filters reduces harvest filtration to a single stage, thereby reducing the complexity of the typically two-stage filter train.
- DNA and HCP removal were enhanced, with pDADMAC showing superior impurity clearance to citric acid precipitation in most cases.
- High–molecular-weight levels, monitored by HPLC size-exclusion chromatography, remained unchanged by either treatment, suggesting that flocculation and acidification are not effective pretreatments for removing antibody aggregates.
In conclusion, pDADMAC flocculation and citric-acid precipitation alone (for centrifuge processes) or in combination with Clarisolve filters (for centrifuge-free processes) can provide an alternative to traditional harvest methods for high-density cell-culture challenges with higher filter throughput and improved HCP and DNA clearance.
Further studies on cytotoxicity clearance of pDADMAC are required. Also further research is needed to optimize large-scale implementation and ensure regulatory compliance.