With the widespread adoption of precision medicine, an increasing number of therapeutic biologics such as bispecific antibodies, antibody-drug conjugates (ADCs), and CAR-T cell therapies have been approved for marketing. Meanwhile, pharmaceutical enterprises are actively expanding their biopharmaceutical pipelines, leading to intensified R&D competition in therapeutic biologics. Therefore, adopting appropriate process development strategies at the early R&D stage helps shorten the research cycle and reduce R&D costs, delivering strong competitive advantages.
This article reviews the screening strategies for resins commonly used in downstream purification of biologics, including affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography, size-exclusion chromatography, and multimodal chromatography, aiming to facilitate rapid downstream purification process development.
Prior to designing the downstream purification workflow for biologics, it is essential to clarify the objectives of the purification process, such as balancing yield and purity and controlling residual impurity levels. In addition, a comprehensive evaluation of the properties of the target product should be conducted, including molecular weight, isoelectric point, structural characteristics, and affinity properties. Finally, the stability of the target product must be fully investigated, covering pH tolerance, thermal stability, and solubility, so as to preliminarily determine the temperature conditions and buffer system for purification. After fully characterizing the target product, appropriate chromatographic modes can be selected for resin screening and process optimization.
1 Affinity Chromatography
Affinity chromatography is a highly selective purification method based on specific biological interactions between two molecules, enabling capture of target molecules from complex matrices. When applying affinity chromatography for purification, it is necessary to fully evaluate the structure and affinity binding sites of the target product in advance. For instance, recombinant proteins fused with histidine tags during vector construction can be purified using nickel-based affinity resins; those with GST tags are suitable for glutathione affinity chromatography.
For the capture of therapeutic antibodies (monoclonal antibodies, bispecific antibodies and multispecific antibodies), resin selection is generally based on antibody structure. Antibodies with an intact Fc region are routinely captured using Protein A and Protein G affinity chromatography.
After determining the type of affinity resin, 3 to 4 commercial resins of the same type can be selected for screening based on matrix compatibility, cost accessibility and supply lead time. For affinity chromatography relying on specific ligand-analyte interactions, the peak symmetry factor only needs to be maintained at 0.8–1.8 during column packing.
Moreover, platform affinity chromatography protocols can be adopted for resin screening. Typically, 250 mM imidazole is used for elution in nickel affinity chromatography; for antibody purification with Protein A resins, elution is performed at pH 3.0–4.0. With standardized platform methods, the yield and purity of the target product can be determined. In general, the primary screening of affinity resins can achieve a yield of over 85% and SEC purity above 80%.
Further resin optimization can be carried out according to predefined process targets. Once the optimal resin is selected, Design of Experiments (DOE) can be applied to optimize critical process parameters, including loading pH, conductivity, dynamic binding capacity, washing for impurity removal, and elution conditions.
Ion Exchange Chromatography
Ion exchange chromatography separates target products from impurities based on charge differences and is mainly applied in the polishing stage of purification. It is categorized into anion exchange chromatography and cation exchange chromatography according to ion exchange types. Two operational modes are available based on purification purposes:
Flow-through mode: The target product does not bind to the resin while impurities are retained on the chromatographic medium.
Bind-and-elute mode: The target product binds to the resin and is subsequently eluted with a graded salt solution.
The selection of suitable resins and operational modes requires confirmation of purification objectives and the isoelectric point (pI) of the target product. The pI value can be calculated theoretically from the amino acid sequence or determined experimentally via capillary isoelectric focusing (CIEF).
For impurities with a lower pI than the target product, anion exchange chromatography in flow-through mode is the preferred option. The buffer pH is adjusted between the pI values of impurities and the target product — higher than the impurity pI and lower than the product pI. Under this condition, the target product carries a positive charge and is collected in the flow-through fraction, while negatively charged impurities bind to the anion resin and are removed during regeneration. This mode also effectively eliminates negatively charged viral particles.
For flow-through resin screening, high-throughput screening combining DOE with 96-well plates is commonly adopted. Key evaluation factors include resin matrix (rigid resins feature high pressure resistance; soft resins offer superior hydrophilicity and non-specific adsorption resistance), particle size (smaller particles provide higher resolution but higher backpressure, while larger particles deliver lower resolution with better pressure tolerance), and dynamic binding capacity (generally over 50 g/L for flow-through mode). Screening results are evaluated comprehensively based on yield and quality attributes.
For impurities with a higher pI than the target product, cation exchange chromatography in bind-and-elute mode is typically adopted. Salt gradient elution is used for initial resin screening, impurity washing exploration and elution salt concentration scouting. Based on gradient elution results, DOE is applied to convert linear gradient elution into step elution, followed by verification of product quality attributes.
Column chromatography is prioritized for resin screening in bind-and-elute mode, as 96-well plate screening requires 12–24 hours for sufficient binding and cannot achieve high throughput. Attention should also be paid to resin particle size, salt concentration for washing and elution — high salt conditions may compromise the stability of biologics and interfere with subsequent analytical detection — with yield and quality attributes as core evaluation indicators.
Hydrophobic Interaction Chromatography
Hydrophobic interaction chromatography (HIC) separates substances based on differences in hydrophobicity between target products and impurities, and is primarily used for removing high-molecular-weight aggregates. Since the hydrophobicity of target products cannot be directly quantified, preliminary trials with resins carrying ligands of varying hydrophobicity are required for process design. The hydrophobicity of common ligands follows the order: Butyl < Octyl < Phenyl. Binding and adsorption tests with different HIC resins enable preliminary prediction of product hydrophobicity.
After characterizing product hydrophobicity, sample pretreatment is required. Target molecules bind to HIC resins under high-salt conditions and elute at low salt concentrations. Therefore, optimizing the salt concentration (ammonium sulfate is commonly used) for sample loading is critical. After confirming the ligand type and loading salt concentration, linear gradient elution from high to low salt is applied for resin screening and process optimization, which can be further converted to step elution via DOE.
Size-Exclusion Chromatography
Size-exclusion chromatography (SEC), also known as molecular sieve chromatography, achieves separation based on molecular weight differences. At the process development and resin screening stage, the molecular weight of the target product and impurities should be clarified in advance via SDS-PAGE and CE-SDS.
The core consideration for SEC resin screening is the fractionation range of pore size. Larger pore sizes correspond to wider separation ranges. For example, 75 Å resins feature a small pore size with a separation range of 3–70 kDa, while 200 Å resins have larger pores covering 10–600 kDa. Resins should be selected according to purification requirements. Additionally, non-specific adsorption between the resin and target product should be minimized during selection.
Multimodal Chromatography
In recent years, multiple resin suppliers have developed multimodal chromatography resins, with mainstream types including cation-hydrophobic and anion-hydrophobic combinations. Multimodal chromatography integrates the high resolution of ion exchange chromatography and the high salt tolerance of hydrophobic interaction chromatography, allowing sample loading under high-salt conditions and offering greater operational flexibility for the purification of complex samples.
In multimodal resin screening, product hydrophobicity is a key factor. Highly hydrophobic products tend to bind tightly to resins via combined ionic and hydrophobic interactions, leading to difficult elution and low yield under high-salt conditions. For such products, resins with low ligand density are recommended, or arginine can be added to the buffer to weaken hydrophobic interactions. For products with moderate hydrophobicity, resin screening and process optimization can be conducted through multiple gradient trials, including single salt gradient, single pH gradient, pH gradient at fixed salt concentration, salt gradient at fixed pH, and dual salt-pH gradient elution.
Conclusion
This article summarizes the application scope of affinity, ion exchange, hydrophobic interaction, size-exclusion and multimodal chromatography in downstream purification of biologics, and elaborates resin screening strategies for each chromatographic mode. It is expected to provide process development references for downstream R&D teams and support efficient downstream purification workflow development of biopharmaceutical products.
