(M0930-08-49) Novel Strategies for Process-Related Impurity Clearance during Biologics Manufacturing

Purpose: Host organisms used to manufacture biologics also produce process-related impurities. Monitoring and reducing the prevalence of these impurities is a vital part of recombinant protein purification because of their potential to impact drug safety, stability, and efficacy. Host cell proteins (HCPs) and DNA are impurities that may co-elute with recombinant proteins during downstream purification due to inadequate resin selectivity. Further, residual HCPs, such as lipases, can degrade polysorbates and other excipients used to improve drug stability. Polishing chromatography can clear these impurities at the cost of product recovery, requiring the development of novel filtration and column purification modalities for HCP and DNA removal. Additionally, efficient HCP and DNA removal can help maximize the resin binding capacity for traditional downstream trains. This work evaluated the DNA, HCP, and lipase clearance capabilities of a novel peptide-based resin (Resin A) and a charged filter (Filter B) for harvest cell culture fluid (HCCF) containing monoclonal antibodies (mAbs).

Methods: HCCF from two mAbs expressed in Chinese Hamster Ovary (CHO) cells with varying titers, conductivity, and impurity levels were evaluated. HCCF flowthrough from Resin A and Filter B was fractionated every 0.25 column volume (CVs) and 50 L/m2 of area, respectively. Filter and column products were further purified using Protein A Chromatography. Products from Resin A, Filter B, and Protein A Chromatography were analyzed for residual HCPs (rHCP), residual DNA (rDNA), and Lipase Activity (LAA).

Results: Figures 1a and 1b demonstrate the impurity clearance capability of Resin A. rHCP levels in earlier fractions are below that of untreated HCCF for mAb1 and rHCP levels remained below that of untreated HCCF for mAb2 regardless of column loading (Figure 1a). A 15-fold reduction of rHCP and a 2.6-fold reduction in LAA was observed between Resin A treated and untreated Protein A Products (PAPs) for mAb1 (Figure 1b). Similarly, a 6.4-fold and 1.8-fold decrease in rHCP and LAA, respectively, was observed for mAb2 (Figure 1b). Figure 2 focuses on the impurity clearance capabilities of Filter B for the program with a higher impurity burden, mAb2. A 14.6-fold and 5.2-fold decrease in rHCP and LAA, respectively, was observed (Figure 2). Additionally, the charged nature of Filter B results in complete DNA clearance.

Conclusion: DNA and HCP removal is a significant challenge in the downstream purification of recombinant proteins. Reducing the HCCF impurity burden can improve overall process robustness and final drug product stability. Our work shows that Resin A offers slightly more HCP clearance, but Filter B doubles the lipase activity removal of Resin A. This work demonstrates the potential of these modalities to change downstream process development and lays the groundwork for adopting them in the future.

Figure 1a: Residual HCP in Resin A Flowthrough for mAb1 and mAb2

Figure 1b: Residual HCP and Lipase Activity in Untreated and Resin A Treated Protein A Chromatography Products (PAPs) for mAb1 and mAb2

Figure 2: Residual HCP and Lipase Activity in Untreated and Filter B Treated Protein A Chromatography Products (PAPs) for mAb2