HCPure™: Host cell impurity removal in CHO workflows
Monoclonal antibodies (mAbs), widely produced in CHO cell lines, require rigorous purification to remove host cell proteins (HCP), host cell DNA (HCDNA), and aggregates to meet safety and regulatory standards. This application note evaluates the performance of HCPure™, a mixedmode chromatography resin developed by Astrea Bioseparations, for polishing monoclonal antibody (mAb) feedstreams. Unlike other mixed-mode resins on the market, HCPure™ uses hydrophobic interactions as the primary binding mode, enabling effective impurity removal under a wide range of conditions.
Most therapeutic antibodies are produced in CHO cells to reduce immunogenicity risk. However, downstream purification must effectively remove host cell proteins (HCP), residual host cell DNA (HCDNA), and aggregates to meet regulatory standards. Traditional polishing steps often require multiple chromatography modes, such as ion exchange for HCP and hydrophobic interaction for aggregates, adding time, complexity, and buffer consumption. Many of these steps operate in bind-and-elute mode, which can lead to yield loss and require extensive process optimization. Likewise, many alternative mixed-mode resins on the market rely on ion exchange as the primary binding mechanism and are typically used in bind-and-elute mode, which can increase process complexity and risk of product loss.
HCPure™ offers a streamlined alternative as a mixed-mode chromatography resin designed specifically for flow-through operation. It combines hydrophobic interaction and hydrogen bonding to selectively bind impurities while allowing the target antibody to pass through. This simplifies the purification process by removing HCP, HCDNA, and aggregates in a single polishing step, reducing processing time and buffer use without compromising yield.
Unlike traditional ion-exchange resins, HCPure™ performs effectively across a broader range of conditions, including higher conductivity, making it particularly effective in feedstreams where conventional resins struggle. Most alternative mixed-mode resins depend primarily on ionic interactions, whereas HCPure™ uses hydrophobic interaction
as the primary binding mode. This offers a distinct mechanism of action that enhances clearance of challenging impurities. This application note outlines the unique binding properties of HCPure™ and demonstrates its efficiency as a flow-through polisher in CHO-derived IgG purification workflows.
Starting materials & primary capture
To evaluate the performance of HCPure™ in a representative workflow, CHO cell cultures expressing two forms of IgG were used to generate feedstocks. Protein A affinity chromatography served as the primary capture step, with the eluate subjected to both single and multistep polishing processes. These experiments assessed HCPure™’s ability to remove HCP, HCDNA, and aggregates while maintaining IgG yields. Impurity levels were quantified using ELISA for HCP, Picogreen assay for HCDNA, and size-exclusion chromatography (SEC) for aggregates, as shown in Table 1.
Using HCPure™ as a single-step polish to remove HCP and HCDNA
HCPure™ allows for increased separation and selectivity by utilizing both hydrophobic and hydrogen bonding interactions simultaneously. This puts HCPure™ at an advantage over traditional ion-exchangers because it retains functionality in a broad range of conductivity load conditions compared to ionexchange polishing steps which would require dilution.
Table 1: Concentrations of target mAbs, HCP, aggregates, and HCDNA post-Protein A.
In this study, Protein A eluate from CHO-expressed IgG was loaded onto HCPure™ in flow-through mode at pH 8.0 and 3 mS/cm. The results showed a 79% reduction in host cell proteins and a 93% reduction in host cell DNA, confirming HCPure™’s effectiveness as a single-step polishing solution.
Table 2: Concentrations of HCP and HCDNA before and after HCPure™
HCPure™ single-step polish analysis
pool
Figure 1: Analysis of HCPure™ elution pool, showing 79% reduction in HCP and 93% reduction in HCDNA.
HCPure™ is effective at removing high molecular weight aggregates
Product aggregation remains a crucial issue and results not only in product loss but also affects product safety. During manufacturing of protein therapeutics, the protein is exposed to various stresses, before and after Protein A capture, which can result in the formation of aggregates.
The unique properties of the mixed-mode mechanism of HCPure™ make it effective at removing these aggregates, as high molecular weight aggregates bind more tightly due to the exposed hydrophobic groups in the denatured chains of aggregate.
In order to demonstrate the effectiveness of HCPure™ in removal of aggregates, samples of Protein A eluate were purified through a strong-cation exchanger and then held at low pH and high salt to encourage aggregation to occur, before being passed through a HCPure™ column in flow-through mode.
The data in Table 3 below shows 6.7% aggregation in the load sample, which is higher than most processes would produce. After this highly aggregated sample was passed through the HCPure™ column, the final concentration of aggregates was 2.9%, which is a nearly 60% reduction in the overall aggregate concentration.
Table 3: Reduction of high molecular weight aggregates using HCPure™
HCPure™ as part of a multistep polishing process
While HCPure™ is highly effective as a single-step polishing solution, some users prefer a multistep approach for maximum impurity removal. Following Protein A capture, anion exchange chromatography is often used to remove HCP, but it is less effective against high molecular weight aggregates, which require hydrophobic interaction chromatography.
HCPure™ offers a unique advantage in this context. it complements ion exchange by providing hydrophobic interactions, enabling enhanced clearance of residual HCPs and aggregates. When used after a cation exchanger in bindand-elute mode, HCPure™ can reduce impurities such as HCP to below the limit of quantitation (see Table 4).
Its versatility makes HCPure™ a valuable tool in both single and multistep purification strategies.
Table 4: HCP contamination is reduced below limit of assay quantitation after being combined with cation exchange chromatography.
Conclusion
Due to the critical importance of monoclonal antibodies (mAbs) as therapeutic products, downstream purification must meet stringent safety and regulatory standards. This typically requires multistep workflows involving ion exchange and hydrophobic interaction chromatography to remove host cell proteins (HCP), DNA, and aggregates.
HCPure™, a mixed-mode chromatography resin, simplifies this process by enabling efficient removal of HCP, HCDNA, and high molecular weight aggregates in a single step. Its dual-mode mechanism (primarily hydrophobic interaction) allows for high performance even at elevated conductivity, reducing process complexity and buffer consumption. Unlike other mixed-mode resins on the market, HCPure™ offers a distinct binding profile, making it especially effective for capturing impurities that are challenging to remove with conventional approaches.
HCPure™ also complements traditional ion exchange resins in multistep polishing strategies, offering orthogonal selectivity and enhanced impurity clearance.
Whether used alone or in combination, HCPure™ supports robust, scalable, and regulatory-compliant purification of mAbs, helping manufacturers deliver safe, high-purity biologics more efficiently.
Astrea Bioseparations is a world class provider of chromatography adsorbent and resin services. With over 30 years of chromatography manufacturing expertise, we deliver a unique and trusted service in close partnership with our clients. For more information, please don’t hesitate to reach out at sales@astrea-bio.com or visit astreabioseparations.com.