MiMode PuraBead® HL4: 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 MiMode PuraBead® HL4, a mixed-mode chromatography resin developed by Astrea Bioseparations, for polishing monoclonal antibody (mAb) feedstreams. Unlike other mixed-mode resins on the market, MiMode PuraBead® HL4 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.
MiMode PuraBead® HL4 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, MiMode PuraBead® HL4 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 MiMode PuraBead® HL4 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 MiMode PuraBead® HL4 and demonstrates its efficiency as a flow-through polisher in CHO-derived IgG purification workflows.
Starting materials & primary capture
To evaluate the performance of MiMode PuraBead® HL4 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 MiMode PuraBead® HL4’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 MiMode PuraBead® HL4 as a single-step polish to remove HCP and HCDNA
MiMode PuraBead® HL4 allows for increased separation and selectivity by utilizing both hydrophobic and hydrogen bonding interactions simultaneously. This puts MiMode PuraBead® HL4 at an advantage over traditional ion-exchangers because it retains functionality in a broad range of conductivity load conditions compared to ion-exchange 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 MiMode PuraBead® HL4 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 MiMode PuraBead® HL4’s effectiveness as a singlestep polishing solution.
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 MiMode PuraBead® HL4 column, the final concentration of aggregates was 2.9%, which is a nearly 60% reduction in the overall aggregate concentration.
Table 2: Concentrations of HCP and HCDNA before and after MiMode PuraBead® HL4.
MiMode PuraBead® HL4 single-step polish analysis
Figure 1: Analysis of MiMode PuraBead® HL4 elution pool, showing 79% reduction in HCP and 93% reduction in HCDNA.
MiMode PuraBead® HL4 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 MiMode PuraBead® HL4 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 MiMode PuraBead® HL4 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 MiMode PuraBead® HL4 column in flow-through mode.
Table 3: Reduction of high molecular weight aggregates using MiMode PuraBead® HL4.
MiMode PuraBead® HL4 as part of a multistep polishing process
While MiMode PuraBead® HL4 is highly effective as a singlestep 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.
MiMode PuraBead® HL4 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 bind-and-elute mode, MiMode PuraBead® HL4 can reduce impurities such as HCP to below the limit of quantitation (see Table 4).
Its versatility makes MiMode PuraBead® HL4 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.
MiMode PuraBead® HL4, 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, MiMode PuraBead® HL4 offers a distinct binding profile, making it especially effective for capturing impurities that are challenging to remove with conventional approaches.
MiMode PuraBead® HL4 also complements traditional ion exchange resins in multistep polishing strategies, offering orthogonal selectivity and enhanced impurity clearance.
Whether used alone or in combination, MiMode PuraBead® HL4 supports robust, scalable, and regulatory-compliant purification of mAbs, helping manufacturers deliver safe, highpurity 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.