The Elution Dilemma in Protein A Chromatography

Protein A chromatography is the industry standard capture step for antibody purification, providing high selectivity and scalability. However, the acidic conditions required for antibody elution create an inherent challenge, as low pH can destabilize antibodies and promote aggregation.

In this study, the impact of elution pH and buffer chemistry on antibody aggregation and elution behavior was investigated during purification of an aggregation-prone monoclonal antibody (Antibody X). Different elution conditions were evaluated using the protein A resin WorkBeads™ affimAb Edge, and aggregation levels were analyzed by size exclusion chromatography.

The results demonstrated that aggregation increased at lower pH, while the choice of buffer system significantly influenced the elution profile. Citrate buffers enabled a more uniform pH transition and produced narrower elution peaks compared with glycine-HCl. Among the tested conditions, citrate at pH 3.5 provided the best balance between efficient elution, peak shape, and controlled aggregation. Under these optimized conditions, WorkBeads™ affimAb Edge delivered higher yield and lower aggregate levels compared with widely used commercial protein A resins, demonstrating its suitability for efficient purification of aggregation-prone monoclonal antibodies.

Challenges in Antibody Purifications

Monoclonal antibodies (mAbs), including bispecific formats, and Fc-containing antibody derivatives are important components in many diverse therapies due to their ability to specifically target selected antigens. This specificity also makes mAbs, and derivatives thereof, vital tools in the development of diagnostic kits as well as biomolecular assays for analyte detection. However, to achieve the required selectivity, therapeutic mAbs are subjected to stringent purity requirements, increasing the demand for efficient and economical purification processes. In such a purification regime, protein A resin is considered the gold standard for efficient capture. It is usually the single largest contributor to cost of goods while also being especially receptive to fouling and capacity decay. This makes it a key target for process optimization.

Recent advancements in upstream processing have improved the efficiency of mAb production, which further drives the need for more efficient downstream purification, both in terms of resin capacity and impurity clearance. The main critical impurity challenges include host cell nucleic acids, such as host cell DNA and RNA, host cell proteins, viruses, and mAb aggregates, all of which can cause severe allergenic reactions in treated patients and therefore must be removed. Among these, mAb aggregates are of particular concern, as increased production yields typically lead to higher target concentrations and a greater risk of nucleation. This issue is further accentuated by the increased binding capacities of modern protein A resins, which result in even higher localized concentrations at the binding surface. When combined with the acidic conditions required for elution, which are known to enhance aggregation, the issue is of clear concern.

In addition to the high purity demands, more stringent sustainability requirements force process optimizations to decrease both buffer and resin consumption at the same time as processes need to be both faster and more cost-efficient.

Taken together, these factors mean that modern purification processes require high-performing protein A resins that are robust and flexible, enabling operation at high flow rates and high loading capacities without compromising product quality.

WorkBeads™ affimAb Edge

WorkBeads™ affimAb Edge is a next-generation resin in the affimAb series, engineered for efficient purification of monoclonal antibodies and any Fc-containing fragment or fusion product from laboratory to process scale. The resins are designed to provide optimal purification performance with a dynamic binding capacity (DBC) at >60 mg/mL at 4 min and >70 mg/mL at 6 min residence time.

The improved resin is constructed from a novel agarose-based matrix resulting in porous beads with a tight pore size distribution and very high mechanical stability. In combination with the optimized density of the immobilized alkali-stable protein A ligand, WorkBeads affimAb Edge allows high DBC together with superior pressure flow characteristics. Together this results in faster processing, reduced elution volumes, and robust performance across multiple purification cycles.

With its cost-effective design WorkBeads affimAb Edge provides high loading rates, target recovery and purity resulting in greater process efficiency and improved overall economics for antibody purification workflows.

The Elution Dilemma

Aggregate formation is an inherent challenge in protein A chromatography, since elution requires a shift to low pH, typically pH 2.7–3.5. Under these low-pH conditions protein A undergoes conformational changes that weaken its interaction with the Fc region of the antibody, thereby enabling elution. However, this creates an “elution dilemma”: while sufficiently acidic conditions are required to efficiently release the antibody from the resin, exposure to low pH may compromise antibody stability and promote aggregation.

When pH drops below 4.0, key stabilizing interactions – including hydrogen bonds and salt bridges that maintain the structural integrity of the antibody – are disrupted. As a result, intramolecular and interdomain interactions are weakened, leading to increased conformational flexibility and partial unfolding. This destabilization exposes hydrophobic regions and promotes non-native intermolecular interactions that may initiate nucleation and subsequently lead to aggregation.

Based on these results, citrate at pH 3.5 was selected as the preferred elution buffer for Antibody X as it results in a narrow elution peak, high yield, and moderate HMWS levels that can be removed in later stages of the purification process.

Performance Comparison of Protein A Resins

The preferred elution condition, citrate at pH 3.5 was further evaluated to assess the performance across different protein A resins. Antibody X was purified using WorkBeads affimAb Edge and two widely used commercial protein A resins, MabSelect™ PrismA and SuRe 70.

This comparative evaluation showed that elution peak shapes were comparable among the three tested resins. The capture eluates were further analyzed for aggregate content, yield and purity. Among the evaluated resins, WorkBeads affimAb Edge produced the lowest levels of high molecular weight species (HMWS), demonstrating improved control of aggregation during the capture step. All evaluated resins also demonstrated high yield (>90%) and high purity. Notably, WorkBeads affimAb Edge achieved a yield above 95% and produced the purest eluate among the tested resins.

Conclusions

The choice of elution buffer and pH strongly affects performance in protein A chromatography, as both parameters play a critical role in controlling aggregation and peak shape. A central challenge is the “elution dilemma”, where the low pH required to release the antibody from the protein A ligand simultaneously promotes antibody aggregation. In this study, the effects of elution pH and buffer chemistry were evaluated during the purification of an aggregation-prone monoclonal antibody, Antibody X.

The results confirmed that aggregation is more pronounced at low pH. They also showed that the chemistry of the elution buffer influences how the pH transition propagates through the column, which directly affects the elution profile. Citrate buffer produced a uniform pH transition and narrow elution peaks, whereas glycine-HCl caused a delayed pH transition and broader elution peaks.

Under these optimized buffer conditions, WorkBeads™ affimAb Edge delivered higher yield and lower HMWS levels compared with MabSelect™ PrismA and MabSelect SuRe 70. Together, these results highlight the ability of WorkBeads affimAb Edge to provide superior control of aggregation while maintaining high recovery, supporting increased process productivity and improved cost efficiency in monoclonal antibody purification workflows.

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