Ask The Pros — The Latest In Upstream HCP Mitigation

A conversation with upstream processing and HCP experts from across industry

Fewer process-related impurities in the harvest ease the load on downstream operations, yet host cell protein (HCP) mitigation is still largely viewed as a downstream challenge.

Advances in LC-MS-based HCP profiling and targeted cell line engineering are beginning to change that, enabling upstream strategies that reduce impurity burden before purification even begins.

Recognizing the potential impact — lower costs, improved stability, and safer biologics — Life Science Connect partnered with CMC technical expert Gopinath Annathur to explore emerging approaches. We asked leading experts how upstream interventions can reshape HCP control. Here’s what they shared. Answers have been edited for clarity.

What new cell line developments improve viability and incorporate knockouts of high-risk HCPs for industry use?

With advances in HCP quantification methods, such as LC-MS HCP analysis, manufacturers can now pinpoint exactly which HCPs are present as impurities. When products are faced with consistent contamination with problematic HCP(s), upstream cell line modifications can be performed to remove that protein from the host cell’s genome; however, not all HCPs can be removed without impacting cell viability or other product attributes.

— Anthony Blaszczyk, United States Pharmacopeia

What upstream preventive actions for cell culture and harvest steps would mitigate chromatin interference in downstream purification steps concerning HCP clearance?

Several upstream strategies can be employed. These include engineering CHO cells to reduce problematic HCP expression and enhance viability, optimizing media and culture conditions to limit cell lysis and DNA release, and carefully timing the harvest to avoid late-stage degradation.

Gentle harvest techniques, such as low-shear pumping and appropriate centrifugation, further reduce chromatin release. Chemical flocculants (e.g., PEI, pDADMAC) and acid precipitation can aggregate and remove DNA–HCP complexes before filtration, while nucleases like benzonase enzymatically digest DNA to reduce viscosity and disrupt HCP binding.

Finally, advanced clarification technologies, such as AEX hybrid filters, can capture soluble DNA and HCPs directly during harvest, improving Protein A column performance and downstream purity. Integrating these approaches reduces the impurity burden early on, enhancing the overall robustness of biologics purification.

— Derrick Zhang, United States Pharmacopeia

What HCP risk factors has the industry learned for upstream culture conditions and downstream purification steps that will give us the ability to predict HCP risk now so that PAT-based control can be implemented in the future? 

HCP analysis by LC-MS has allowed for the monitoring of specific HCPs during production. By doing this, manufacturers can not only monitor total HCP clearance at each production step but also monitor the clearance of every detected HCP at every step. This advanced control allows optimization that can target clearance of problematic HCPs. Notably, the HCP profiles of common expression systems like CHO cells have been extensively characterized, and many problematic HCPs, such as lipases, proteases, and other process-disrupting or immunogenic proteins are now well documented. This knowledge base enables risk-based monitoring strategies, where manufacturers can prioritize tracking of known high-risk HCPs while also using the broader LC-MS profile to identify process deviations, such as increased cell lysis, that may introduce additional HCP burden.

— Anthony Blaszczyk, United States Pharmacopeia

The only one that I can think of is managing cell viability prior to harvest. Cell debris (and HCP) increases with decreasing cell viability. Every process that I've worked on has a harvest criterion of >70% viability, whether it’s for mAb or viral vector production.  

— Peter Alexander, industry consultant

What cell culture media design advances, including additives, will help reduce HCP levels while maintaining other CQAs and product titer?

Media additives like Pluronic F68, a non-ionic surfactant, help protect cells from shear stress during agitation and gas sparging by stabilizing bubbles and membranes, reducing lysis and subsequent DNA/HCP release.

In high-density cultures, higher concentrations or additional shear protectants are often used. Anti-foam agents are also selected to minimize foam-related shear. Optimizing these parameters helps preserve cell integrity, reducing chromatin and HCP levels in the harvest and easing the burden on downstream purification.

— Derrick Zhang, United States Pharmacopeia

From my experience, the small molecules on the market now for boosting AAV production that maintain cell viability, but halt cell doubling, are very helpful in reducing the HCP load in products. Harvesting three times fewer cells in the bioreactor helps to reduce the starting point of HCP and therefore reduces HCP in drug substance.

– Seth Levy, Modalis

What cell culture conditions before, during, and after harvest influence the level and population distribution of HCP seen in downstream steps? 

In addition to media optimization, harvest-stage strategies can significantly reduce chromatin release. Harvesting at high viability (>80%) minimizes late-stage cell lysis and prevents spikes in DNA and HCP levels that can overwhelm downstream purification. Gentle handling during harvest using low-shear pumps, optimized centrifugation, and smooth piping further limits mechanical cell breakage. Some processes incorporate cooling steps or chelating agents (e.g., EDTA) to stabilize cells and reduce lysis. These approaches aim to preserve cell integrity during harvest, reducing chromatin and HCP release and improving downstream clearance efficiency.

— Derrick Zhang, United States Pharmacopeia

Two-stage depth filtration followed by a 0.2 µm sterilizing-grade filter is generally able to reduce HCP load going into the first chromatography step.

— Peter Alexander, industry consultant

Does continuous cell culture improve HCP clearance downstream and resolve high-risk HCPs? 

By itself, continuous manufacturing will not optimize HCP clearance downstream. However, process analytical technology can result in better control of every production step. This control and continued monitoring does allow for real-time adjustments to process parameters, which, if properly controlled, can be adjusted to improve HCP clearance. In order for this to be successful, a thorough understanding of your process is required, along with detailed knowledge of the effects various process parameters have on CQAs, including HCP production.

— Anthony Blaszczyk, United States Pharmacopeia

I don't have any direct experience with continuous cell culture but would expect that a steady accumulation of dead cells occurs — even with a high steady-state cell viability. The perfusion filter may allow clearance of some soluble HCPs, but I'm not sure to what extent that occurs.

— Peter Alexander, industry consultant