Chinese hamster ovary (CHO) cells are widely used to produce recombinant proteins, including monoclonal antibodies (mAbs), through various process modes. Traditionally, fed batch (FB) processes have been the standard. However, the shift towards high-density perfusion processes is driven by increased productivity, flexible facility footprints, and lower costs. Ensuring the clearance of process-related impurities, such as host cell proteins (HCPs), is crucial in biologics manufacturing. While purification processes remove most impurities, integrated strategies are being developed to enhance biologics purity and address high-risk HCPs. Current understanding of HCP expression dynamics in cell culture is limited. This study utilized data-independent acquisition (DIA) proteomics to compare the proteomic profiles of cell culture supernatants from 14 FB clones and 3 perfusion clones, all expressing the same mAb from the same host cell line. Results showed that perfusion processes enhance cell growth and productivity, exhibiting distinct proteomic profiles compared to FB processes. Perfusion processes also maintain a more stable HCP profile across clones, especially for 46 problematic HCPs monitored. Cluster analysis of FB proteomics revealed distinct abundance patterns and correlations with process parameters. Differential abundance analysis identified significant protein differences between the two processes. Compared to FB, the perfusion process may provide a less stressful cellular environment. This is the first extensive study characterizing HCPs expressed by different clones under different process modes. Further research could lead to strategies for preventing or managing problematic HCPs in biologics manufacturing.

Host cell proteins (HCPs) are a significant class of process-related impurities commonly associated with the manufacturing of biopharmaceuticals. However, due to the increased use of crude enzymes as biocatalysts for modern organic synthesis, HCPs can also be introduced as a new class of impurities in chemical drugs. In both cases, residual HCPs need to be adequately removed to ensure product purity, quality, and patient safety. Although a lot of attentions have been focused on defining a universally acceptable limit for such impurities, the risks associated with residual HCPs on product quality, safety, and efficacy often need to be determined on a case-by-case basis taken into consideration of residual HCP profile in the product, the dose, dosage form, and administration route etc. Here we describe the unique challenges for residual HCP control presented by the biocatalytic synthesis of a Merck investigational stimulator of interferon genes protein (STING) agonist, MK-1454, which is a cyclic dinucleotide synthesized using E. coli cell lysate overexpressing cyclic GMP-AMP synthase (cGAS) as a biocatalyst. In this study, a holistic characterization of residual protein impurities using a variety of analytical tools, together with in silico immunogenicity prediction of identified proteins, facilitated risk assessment and guided process development to achieve adequate removal of residual protein impurities in MK-1454 API.