Guest Column | June 20, 2025

Ask The Pros — The Latest In Downstream HCP Mitigation

A conversation with Aylin Mohammadzadehmarandi, Penn State, and Younghoon Oh, Johnson & Johnson Innovative Medicine

Scientist working in laboratory-GettyImages-2215842283

Host cell proteins (HCPs) are one of the most persistent, frustrating process contaminants in biomanufacturing, and the science for mitigating them grows, like most science, slowly and incrementally. 

Process development expert Gopinath Annathur has spent his decades-long career studying and developing biomanufacturing processes, finding solutions to prevent and remove HCPs. He’s worked in senior process development and manufacturing technical operations roles at AGC Biologics, Biocon, Pfizer, and most recently as director of biologics process development at Chinook Therapeutics.

Lately, a few nagging questions about HCP mitigation have emerged from the literature.

Together with Bioprocess Online and Life Science Connect, Annathur assembled some of the most pressing ones, and we queried other Bioprocess Online contributors. They’re experts in the areas of downstream, upstream, and analytical process development who have deep experience working with HCPs.

We hear from Younghoon Oh, Ph.D., a Johnson & Johnson scientist whose Ph.D. research focused on HCP persistence, and from Aylin Mohammadzadehmarandi, a Ph.D. candidate at Penn State whose work explores ultrafiltration and protein retention.

This is the first in a collection of articles looking at HCP issues. Here, we explore some of the last places where they may be addressed — downstream processes.

Chromatin presents a recognized challenge in biological processes because it does not adhere to the separation rules expected for pure DNA. What practical tools, novel technologies, and strategies might one utilize as part of a robust process design to mitigate the influence of chromatin on HCP clearance?

Chromatin is a complex of DNA and histone proteins. While histones are basic and positively charged, the overall net charge of chromatin under physiological conditions is negative due to the DNA phosphate backbone. This net negative charge allows chromatin to interact strongly with positively charged surfaces, a property that can be leveraged for its removal during bioprocessing.

A highly effective tool for chromatin removal is the 3M Harvest RC Chromatographic Clarifier. This device employs a synthetic fibrous anion exchange (AEX) media, which is positively charged and thus ideally suited to bind and remove negatively charged chromatin and DNA from cell culture harvests. The Harvest RC clarifier can process high-density CHO cell cultures (>40 million cells/mL) in a single step, achieving high product recovery and efficient separation of both soluble and insoluble contaminants. Downstream of the AEX media, a 0.2 µm PES membrane distributes flow and protects subsequent sterilizing-grade filters, while also enabling simple process endpoint monitoring via pressure readings. By removing chromatin and DNA upstream, the clarifier significantly reduces the burden of these foulants and associated HCPs on downstream purification steps, improving both product purity and process robustness.

Building on this foundation, high-performance countercurrent membrane purification (HPCMP) represents a particularly promising novel approach. HPCMP exploits highly selective diffusion transport to remove impurities, including HCPs and chromatin-associated contaminants, in a truly continuous process. As demonstrated in our recent study (Mohammadzadehmarandi, Zydney 2023), when cell culture feeds were first prefiltered through the 3M Harvest RC clarifier, HPCMP provided more than a 100-fold reduction in HCP levels while maintaining greater than 95% mAb yield during 48 hours of stable, continuous operation. Integrating these advanced clarification and membrane-based steps not only mitigates chromatin’s impact on HCP clearance but also enhances process robustness and scalability, paving the way for fully continuous manufacturing of mAbs.

— Aylin Mohammadzadehmarandi, Department of Chemical Engineering, The Pennsylvania State University

Histones are notorious contaminant proteins from host cells that act as strong anion exchangers in solution. They bind to product proteins, aggregates, or purification media, resulting in HCPs sneaking through along with the product. What approaches would one take to effectively remove them from product pools and ensure resins are cleaned properly?

Histones, due to their strong positive charge, can bind to product proteins and purification resins, making them difficult to remove and leading to persistent HCP contamination. In traditional chromatography-based processes, these interactions often require rigorous cleaning and washing protocols to restore resin performance and prevent product carryover. Cleaning-in-place (CIP) with sodium hydroxide or chaotropic agents is commonly used to remove tightly bound histones and other foulants from reusable chromatography columns.In contrast, the HPCMP technique offers a distinct advantage: it is a single-use, single-pass approach. The hollow fiber membrane modules used in HPCMP are not reused, eliminating the need for complex cleaning or regeneration steps that are necessary for chromatography resins. This not only simplifies the process but also reduces the risk of cross-contamination and resin degradation over time. By integrating upstream clarification (such as with the 3M Harvest RC clarifier) to remove DNA and chromatin and employing single-use HPCMP modules for continuous HCP and histone removal, the overall process becomes more robust and less reliant on extensive cleaning protocols.

— Aylin Mohammadzadehmarandi, Department of Chemical Engineering, The Pennsylvania State University

What depth filtration step parameters and filter design aspects benefit HCP reduction?

Recent work from Andrew Zydney’s group has revealed essential insights into how depth filter design and operating parameters influence HCP removal during bioprocess clarification. In particular, Chu et al. (2023) systematically compared fully synthetic and diatomaceous earth (DE)-containing depth filters to elucidate the mechanisms governing HCP binding.

Their study demonstrated that filter media composition is a primary determinant of HCP removal efficiency. The fully synthetic X0SP filter (polyacrylic fibers with synthetic silica) exhibited strong electrostatic binding, especially for positively charged proteins, while the DE-based X0HC filter (cellulose fibers with DE) favored removal of hydrophobic proteins, with electrostatic interactions also contributing. Notably, certain proteins (e.g., ovotransferrin) showed high binding to X0HC, likely due to interactions with metals present in DE.

Filter structure also plays a critical role. The X0SP filter had a greater pore area and smaller average pore diameter, increasing its internal surface for protein adsorption. Multilayer designs, as in X0SP, allow for better utilization of adsorptive capacity, particularly in the second layer.

Operating parameters such as buffer conductivity and flow rate further impact HCP reduction. Lower buffer conductivity enhances electrostatic interactions, improving the binding of charged proteins. Optimizing flow rate and filter loading prevents premature fouling and maximizes HCP capture.

In summary, optimal HCP reduction in depth filtration is achieved by selecting filter media tailored to the charge and hydrophobicity of target HCPs, leveraging multilayer synthetic designs for greater capacity and tuning buffer conditions and flow rates to favor adsorption. 

— Aylin Mohammadzadehmarandi, Department of Chemical Engineering, The Pennsylvania State University

What additives and wash step buffer conditions are commonly employed to disrupt HCP binding to the product during protein A chromatography?

I believe that addressing this question is quite complex, as the specific wash buffers and additives employed can differ significantly among various companies, and many of these details may not be publicly available. While arginine often comes up in discussions on this topic, its application might be limited due to intellectual property considerations. Other wash additives and buffers that are commonly referenced in the public domain include sodium chloride, sodium caprylate, guanidine hydrochloride, histidine hydrochloride, Triton, propylene glycol, a combination of urea and isopropanol, and high pH buffers. 1,2,3,4,5,6,7,8,9

Furthermore, it may be beneficial to explore wash additives that have been assessed for their ability to disrupt interactions between HCPs and mAbs using immobilized mAb resin.10,11,12

Additionally, it appears that the effectiveness of any wash strategy can be influenced by the specific sequence and structure of the mAb being purified.2,3,8

— Younghoon Oh, Ph.D., Johnson & Johnson Innovative Medicine

How effective are spiking studies using purified mAbs into cell culture supernatant for characterizing HCP co-elution in downstream chromatography columns? What are the key challenges and potential pitfalls of this approach?

In this approach, a cell culture supernatant containing no antibody is spiked with a purified mAb before applying to a chromatography column. In my view, the use of mAb spiking has contributed to a better understanding of HCP retention mechanisms, especially in the context of the debates surrounding HCP-mAb binding versus HCP-resin binding.

Based on studies to date, I believe that this approach has been helpful in illustrating that HCP-mAb binding could be a primary mechanism for HCP persistence during protein A chromatography (although it is not the only mechanism contributing to persistence).

However, when considering polishing chromatography, I think that the challenge stems more from the representativeness of the HCP solution used rather than the mAb spiking method itself. This aspect can complicate both the execution and interpretation of such studies. Typically, once the product has reached the polishing stages of the antibody purification process, HCP levels might already be sufficiently low, which could hinder effective analytical assessment. Therefore, the challenge lies in selecting appropriate HCPs for these studies; while I believe the mAb spiking method has value, introducing a higher concentration of non-representative HCPs into the column might compromise the study  representativeness. Conversely, using lower levels of HCPs can present analytical challenges. Additionally, if the focus is on specific problematic HCPs and their behavior during polishing steps, ensuring a truly representative population of those HCPs could introduce further complexities.

— Younghoon Oh, Ph.D., Johnson & Johnson Innovative Medicine

References:

  1. Chollangi, S., Parker, R., Singh, N., Li, Y., Borys, M., & Li, Z. (2015). Development of robust antibody purification by optimizing protein-A chromatography in combination with precipitation methodologies. Biotechnology and bioengineering, 112(11), 2292–2304. https://doi.org/10.1002/bit.25639
  2. Cui, T., Chi, B., Heidbrink Thompson, J., Kasali, T., Sellick, C., & Turner, R. (2019). Cathepsin D: Removal strategy on protein A chromatography, near real time monitoring and characterisation during monoclonal antibody production. Journal of biotechnology, 305, 51–60. https://doi.org/10.1016/j.jbiotec.2019.08.013
  3. Han, J., Yang, J., Wang, Y., & Li, Y. (2019). The adequate amount of sodium chloride in Protein A wash buffer for effective host cell protein clearance. Protein expression and purification, 158, 59–64. https://doi.org/10.1016/j.pep.2019.02.016
  4. Herman, C. E., Min, L., Choe, L. H., Maurer, R. W., Xu, X., Ghose, S., Lee, K. H., & Lenhoff, A. M. (2023). Behavior of host-cell-protein-rich aggregates in antibody capture and polishing chromatography. Journal of chromatography. A, 1702, 464081. https://doi.org/10.1016/j.chroma.2023.464081
  5. Holstein, M., Cotoni, K., Bian, N. (2015). Protein A intermediate wash strategies. BioProcess International, 13(2), 52-58.
  6. Hu, L., Tang, J., Zhang, X., & Li, Y. (2021). Sodium caprylate wash during Protein A chromatography as an effective means for removing protease(s) responsible for target antibody fragmentation. Protein expression and purification, 186, 105907. https://doi.org/10.1016/j.pep.2021.105907
  7. Luo, H., Du, Q., Qian, C., Mlynarczyk, M., Pabst, T. M., Damschroder, M., Hunter, A. K., & Wang, W. K. (2022). 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, 1679, 463385. https://doi.org/10.1016/j.chroma.2022.463385
  8. Shukla, A. A., & Hinckley, P. (2008). Host cell protein clearance during protein A chromatography: development of an improved column wash step. Biotechnology progress, 24(5), 1115–1121. https://doi.org/10.1002/btpr.50
  9. Sisodiya, V., Lequieu, J., Rodriguez, Mc., McDonald, P., & Lazzareschi, K. P. (2012). Studying host cell protein interactions with monoclonal antibodies using high throughput protein A chromatography. Biotechnology journal, 7, 1233-1241.
  10. Aboulaich, N., Chung, W. K., Thompson, J. H., Larkin, C., Robbins, D., & Zhu, M. (2014). A novel approach to monitor clearance of host cell proteins associated with monoclonal antibodies. Biotechnology progress, 30(5), 1114–1124. https://doi.org/10.1002/btpr.1948
  11. Levy, N. E., Valente, K. N., Choe, L. H., Lee, K. H., & Lenhoff, A. M. (2014). Identification and characterization of host cell protein product-associated impurities in monoclonal antibody bioprocessing. Biotechnology and bioengineering, 111(5), 904–912. https://doi.org/10.1002/bit.25158
  12. Liu, X., Chen, Y., Zhao, Y., Liu-Compton, V., Chen, W., Payne, G., & Lazar, A. C. (2019). Identification and characterization of co-purifying CHO host cell proteins in monoclonal antibody purification process. Journal of pharmaceutical and biomedical analysis, 174, 500–508. https://doi.org/10.1016/j.jpba.2019.06.021

About The Experts:

Aylin Mohammadzadehmarandi is a Ph.D. candidate in chemical engineering at Penn State University, specializing in high-performance countercurrent membrane purification (HPCMP) as a novel approach for continuous platform for therapeutic purification. Additionally, part of her research focuses on the effect of buffer excipients on protein sieving loss. She is the recent recipient of the Women in Chemical Engineering Award from AIChE, the Best Presentation Award from the MAST (Membrane Applications Science and Technology) Center, and the Leadership Scholarship from Penn State University. Her industrial experience includes three years of research and development in downstream and formulation development at CinnaGen Biopharma (Iran), where she developed advanced protein characterization and purification methods for monoclonal antibodies.

Younghoon Oh, Ph.D., is a Senior Scientist at Johnson & Johnson Innovative Medicine. Prior to his involvement in the current subject, Younghoon had worked in the fields of microbial metabolic engineering and fermentation, contributing to the publishing of multiple scientific papers and inventions. He earned his Ph.D. in Chemical and Biomolecular Engineering from the University of Delaware, investigating HCP persistence under the guidance of Prof. Lenhoff. Currently, he serves as a DSP lead or scientific integrator for multiple antibody-drug conjugate programs. Currently, he acts as a DSP lead and scientific integrator for multiple antibody-drug conjugate programs. Additionally, he leads a team dedicated to downstream technology development to address specific HCP challenges and is instrumental in cross-functional and multi-company initiatives aimed at reducing HCP-related risks.