Addressing Host Cell Protein Challenges in Monoclonal Antibody Production

Overview

Host cell proteins (HCP) have a serious impact on downstream processes in antibody manufacturing, and their removal presents a significant obstacle in antibody purification. To address this issue, Purolite™ Praesto™ Jetted A50 HipH was designed to elute at higher pH, thereby reducing both aggregation and host cell protein levels as discussed in this paper.

Host Cell Proteins and Their Impact

Host cell proteins (HCP) introduce many challenges in monoclonal antibody downstream manufacturing. HCP are expressed during the production of biotherapeutics by the host organism, typically by Chinese Hamster Ovary (CHO) cells. This standard mammalian host cell produces over 6000 proteins, of which HCPs are directly secreted into the cell culture fluid or released from lysed host cells during culture and harvest¹. The presence and levels of HCP depend on various factors including host cell type, clone type, the product being expressed, and upstream conditions. HCP levels in CHO Clarified cell culture fluids (CCFs) of 30 monoclonal antibodies range from 5 to 6 logs ppm, showcasing the complexity of HCP in the process feed stream and creating considerable challenges for downstream processes².

HCP Characterization and Regulation

Various methods are employed to measure and characterize HCP, such as immunoassays (ELISA) and mass spectrometry. HCP levels are considered a critical quality attribute — while regulatory agencies do not specify allowed levels of HCP in the final drug product, conventional practice aims to limit the levels of HCP to a maximum of 100ppm due to potential adverse immune responses in patients. Proteolytic HCP may also impact product quality of the final drug by cleaving the product during storage, highlighting the importance of reducing HCP to the lowest reasonable level. Previous studies have found that marketed monoclonal antibodies and derivatives exhibit an average HCP level of 20 ppm, indicating an ongoing effort to manage HCP levels³.

Approaches to Mitigate HCP Challenges

Industry and regulatory standards utilize prior knowledge and a quality by design (QBD) approach to ensure product quality and manage risks, as guided by the ICH Q6B guideline. Unit operations upstream and downstream are designed to decrease HCP presence or remove it from the feed stream⁴. For instance, post-harvest clarification utilizing depth filters can effectively remove a subset of HCP through size exclusion or charge interaction. 

Protein A Affinity Chromatography and HCP Removal

Protein A affinity chromatography, commonly used in monoclonal antibody manufacturing, offers selective capture of target molecules while allowing other impurities to flow through. Modified pH washes and additives have been utilized to increase HCP removal during the protein A step, but residual HCP quantities, including “difficult to remove” HCP, have been reported to persist, potentially due to non-specific binding to chromatography media or co-elution with antibodies⁵.

Furthermore, the acidic elution (pH 3.5–4) commonly employed to release antibodies from Protein A resins increases the propensity for protein aggregation, particularly in Bi-Specific Antibodies (BsAb), Fc-fusions, and other mAb derivatives^6,7.  Aggregation has been identified as a key factor in the persistence of HCP, complicating efforts to clear aggregates with current methodologies^4,8.

Decreasing the levels of HCP, aggregates and other impurities in the protein A eluate pool alleviates the challenge in purification and enables a lower chromatography matrix (resin, membrane) volume for clearing HCP impurities in subsequent steps. In a conventional three-step chromatography process aggregate removal is typically restricted to a single polishing step, thus reducing their level also enhances process robustness^9,10.

Thus, mitigating protein aggregation during the protein A capture step can increase product yield, enhance product quality, and improve process economy.

Conclusion

Host cell proteins are process-related impurities that pose challenges for producing monoclonal antibodies and derivatives. Removing these impurities is crucial for product quality, and introducing sufficient log reduction in HCP levels during a whole chromatographic workflow is paramount to developing a robust and efficient process. Puroilte Praesto Jetted A50 HipH resin has been developed to allow elution of bound monoclonal antibodies at a higher pH that that typically seen with Protein A affinity resins. This leads to a lower level of aggregation, particularly with complex antibody architectures like bispecific antibodies. Reduced aggregation effectively facilitates robust and superior clearance of HCP compared to other commercial protein A resins, significantly enhancing product purity and quality. Combining Purolite Praesto Jetted A50 HipH with polishing resins that give good HCP clearance can lead to unprecedented levels of host cell protein removal.

References

1. Baycin Hizal, D. et al. “Proteomic Analysis of Chinese Hamster Ovary Cells.” Journal of Proteome Research vol. 11 (2012): 5265–5276.
2. Luo, H. et al. “ Formation of transient highly-charged mAb clusters strengthens interactions with host cell proteins and results in poor clearance of host cell proteins by protein A chromatography.” Journal of Chromatography A vol. 1679 (2022): 463385.
3. Molden, R. et al. “Host cell protein profiling of commercial therapeutic protein drugs as a benchmark for monoclonal antibody-based therapeutic protein development.” vol. 13 no 1 (2021): e1955811.
4. Ito, T. Et al. “Host cell proteins in monoclonal antibody processing: Control, detection, and removal.” Biotechnology Progress vol. (2024); e3448.
5. Shukla, AA. et al. “Host cell protein clearance during protein A chromatography: Development of an improved column wash step.” Biotechnology progress vol. 24, 5 (2008): 1115-21.
6. Shukla AA, et al. “Protein aggregation kinetics during Protein A chromatography Case study for an Fc fusion protein.” Journal of Chromatography A vol. 1171 (2007): 22–28
7. Wang, FAS. et al. “Evaluation of mild pH elution protein A resins for antibodies and Fc-fusion proteins.” Journal of Chromatography A vol. 1713 (2024): 464523
8. Herman, CE. et al. “Behavior of host-cell-protein-rich aggregates in antibody capture and polishing chromatography.” Journal of Chromatography A vol. 1702 (2023): 464081
9. Andrade, C. et al. “An Integrated Approach to Aggregate Control for Therapeutic Bispecific Antibodies Using an Improved Three Column Mab Platform-Like Purification Process.” Biotechnology Progress vol. 35, 1 (2019): e2720
10. Guo, G. et al. “A potential downstream platform approach for WuXiBody-based IgG-like bispecific antibodies.” Protein Expression and Purification vol. 173 (2020): 105647