IMAC Purification of CHO-Expressed Recombinant Proteins

Researchers from WuXi Biologics (Shanghai, China) expanded upon immobilized metal affinity chromatography (IMAC)’s applicability using larger cell proteins. Their findings were published in the Journal of Chromatography A (1).

Immobilized metal affinity chromatography (IMAC) is a type of affinity chromatography where proteins or peptides are separated according to their affinity for metal ions that have been immobilized by chelation to an insoluble matrix (2). This process is widely used for purifying His-tagged proteins, which are DNA sequences that specify strings of six to nine histidine residues, across different expression systems (3). IMAC relies on coordinative interactions between transition metal ions chelated on the resin and the electron-donating amino acid side chains, primarily histidine, on a protein’s surface. To enhance specificity, polyhistidine tags are often added to the N- or C-terminus of the target protein, enabling selective binding with IMAC resins. Prior to purification, the target protein binds selectively to the resin, while impurities, such as host cell proteins (HCPs) and viruses are removed through tailored wash steps.

IMAC has been used for a variety of applications including for small-scale purification of Escherichia coli lysates, large-scale industrial use of the process for proteins expressed in mammalian cells remains limited, due to specific processing and biosafety concerns. Key process-related limitations include:

• The need for pre-diafiltration of harvested cell culture fluid to ensure compatibility with IMAC media, which contain reducing agents and chelating substances that would typically cause significant metal stripping and yield loss.
• Requirements for metal stripping and recharging of the resin, which can increase process time and buffer consumption.
• Lower binding capacity compared to other capture resins, raising resin costs.
• Elevated levels of impurity post-IMAC, which increases the burden on subsequent polishing steps.

In this study, the scientists purified a Chinese hamster ovary (CHO)-expressed and secreted recombinant protein with His-tag using a nickel (Ni) Sepharose excel resin, which proved resistant to ethylenediaminetetraacetic acid (EDTA) and reducing agents (4,5). With this arrangement, they hoped to optimize loading, washing, and elution conditions to maximize protein recovery and HCP clearance.

The resin demonstrated a maximum load capacity of 10 ng/mL. Further, it was discovered that incorporating detergents (Triton X-100, PS80), a solvent (TnBP), or 2-propanol in washing buffers significantly improves HCP removal, all while maintaining low salt concentrations in the elution buffer, enhancing both yield and product quality. Resin lifetime studies conducted under optimal conditions showed acceptable yields, stable product quality attributes (PQAs), and effective cleaning over 54 purification cycles.

To align with ICH Q5A (R2) and European Medicines Agency (EMA) guidelines on viral safety, future studies will aim to clarify whether viral clearance occurs through removal or inactivation when detergent and solvent washes are involved in IMAC. If validation of the IMAC’s steps’ inherent viral removal capability is preferred, appropriate detection methods are essential to avoid overestimating log reduction values (LRV) caused by chemical-mediated virus inactivation.

By comprehensively optimizing critical IMAC parameters coupled with resin lifetime studies, the researchers hope to bridge current knowledge gaps in industrial application, allowing accelerated and broader adoption of IMAC technology for biologics manufacturing.

References

(1) Jing, X.; Qin, R.; Ma, T.; Xu, K.; Lv, F. Optimization of Immobilized Metal Affinity Chromatography for a Recombinant Protein Expressed in CHO Cells. J. Chromatogr. Open 2025, 8, 100230. DOI: 10.1016/j.jcoa.2025.100230

(2) PROTEIN | Determination and Characterization. ScienceDirect 2003. https://www.sciencedirect.com/topics/nursing-and-health-professions/immobilized-metal-affinity-chromatography (accessed 2025-7-7)

(3) His-Tagged Proteins – Production and Purification. Thermo Fisher Scientific 2025. https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/his-tagged-proteins-production-purification.html (accessed 2025-7-7)

(4) Chinese Hamster Ovary Cell. ScienceDirect 2021. https://www.sciencedirect.com/topics/medicine-and-dentistry/chinese-hamster-ovary-cell (accessed 2025-7-7)

(5) George, T.; Brady, M. F. Ethylenediaminetetraacetic Acid (EDTA). StatPearls 2023. https://www.ncbi.nlm.nih.gov/books/NBK565883/ (accessed 2025-7-7)