The first step of downstream operations in monoclonal antibody (mAb) manufacturing is the removal of cells and cellular debris from cell culture via centrifugation and depth filtration, prior to capture chromatography (Han et al., 2003; Kelley et al., 2009; Shukla et al., 2007). The separation of cells and cell debris can be facilitated by the addition of flocculants (Aunins and Wang, 1989; Han et al., 2003; Roush and Lu, 2008; Thommes and Etzel, 2007). Different flocculants and precipitants have been explored as components to improve clarification efficiency, process yield, and clearance of impurities during the primary mAb recovery step from mammalian cell culture (Hove et al., 2010; McNerney et al., 2011; Peram et al., 2010; Romero et al., 2010; Roush and Lu, 2008; Schirmer et al., 2010; Shan et al., 1996; Wang et al., 2009). Notably, positively charged flocculants, in particular polyamines (Peram et al., 2010), divalent cations (Romero et al., 2010; Shpritzer et al., 2006), chitosan (Riske et al., 2007), and polydiallyldimethylammonium chloride (PDADMAC) (McNerney et al., 2011; Zhao et al., 2012) have been shown to be successful in inducing flocculation as a result of interactions between the flocculant and the negatively charged surfaces of cells and cell debris. A key benefit of flocculation is a potential reduction in process related impurities such as HCP, host DNA, and viruses (Akeprathumchai et al., 2004). However, purification performance in this step is culture dependent (Roush and Lu, 2008) and it would be challenging to develop this step as an alternative to downstream polishing chromatography steps. Further, due to their toxicity, flocculants must be removed from the final antibody product (Gagnon, 2012; Schirmer et al., 2010), possibly requiring the inclusion of an additional separation step and in-process monitoring or clearance studies of residual polymers.
In this study, a novel stimulus responsive polymer (Jaber et al., 2011), partially benzylated poly(allylamine), termed smart polymer E (SmP E) in this article, was investigated as a potential flocculant to address the mAb clarification and purification challenges discussed above. SmP E is a salt tolerant, cationic polymer with hydrophobic residues that undergoes a soluble to insoluble transition when exposed to multivalent anions such as phosphate ions and thus can be employed for flocculation and/or precipitation. The polymer response to the stimulus of multivalent anions is likely due to strong interactions between multivalent anions and the closely arranged amine repeat units present on the polymer, resulting in a collapse and aggregation of polymer chains which renders it insoluble. It is this unique property that allows its highly efficient binding to negatively charged impurities such as HCP and host DNA under typical cell culture conditions while enhancing the removal of cells and cell debris and controlling the level of residual polymer. Through design of experiments (DOE) condition screening and optimization of flocculation in hundreds of experimental runs, we developed an improved harvest process by flocculation followed by depth filtration. The novel process demonstrates high step recovery, improved clearance of cells and cell debris, as well as efficient reduction of process and product related impurities such as HCP, host DNA, and high molecular weight species (HMW) for four different mAbs. Improvement in filterability was observed during the depth filtration step using Clarisolve™ 40MS filters. This process achieved levels of residual impurities in the Protein A eluate that meet the requirements of drug substance and thus alleviates the purification burden in subsequent chromatography operations.
In addition, the efficient clearance of residual polymer was demonstrated using fluorescence tagged SmP E. The residual polymer level was reduced to <0.1 ppm in the subsequent Protein A chromatography. Therefore, no additional ion exchange chromatography is likely needed for polymer removal. The mechanism of impurity removal in this new harvest/clarification step was also explored. The developed process can be easily incorporated into current purification platforms, offering a solution in cases where mAbs demonstrate poor purification process performance. Methods described here for developing the SmP E flocculation process could also be applied to process development using other defined flocculants.