Meeting Complexity Demands with Innovative Affinity Ligands

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BioPharm International, BioPharm International, October 2023, Volume 36, Issue 10
Pages: 19–20

The innovation of affinity ligands must take stability into consideration for present and future purification applications.

Affinity chromatography, which has been used for nearly 40 years, is the preferred method for separating and purifying large-molecule therapeutics in downstream processing. For the most part, affinity chromatography makes purifying biological molecules simple and straightforward because of the method’s selectivity, scalability, and ease of process development (1).

The advancement of increasingly complex biomolecules in the drug development pipeline, however, drives the continual push to improve downstream processing steps. Innovations in chromatography technology, such as the development and enhancement of affinity ligands, has enabled the improvement and optimization of affinity chromatography.

The preference for affinity chromatography

With decades of use under its belt, affinity chromatography has been shown to be a preferred downstream processing technique. This preference is due to the fact that affinity chromatography offers high selectivity, resolution, and capacity in most protein purification schemes, emphasizes Khaled S. Mriziq, PhD, senior manager, Marketing, Process Chromatography, Protein Purification Business, Bio-Rad Laboratories. Mriziq explains that, compared to other downstream purification methods, affinity chromatography has the advantage of utilizing a protein’s biological structure or function for purification, and, as a result, purifications that would otherwise be time consuming and complicated can often be easily achieved. “Affinity chromatography is increasingly used in platform monoclonal antibody (mAb) processes,” he notes.

Affinity chromatography can reduce a multi-step downstream process to a process where the number of steps is significantly reduced, which minimizes cumulative yield losses, asserts Brittany Crocco, Product Manager—Chromatography Resins, Repligen. This advantage makes affinity chromatography, and the significant process economic improvements it delivers, an obvious technology choice from process simplification and improved process economy perspective, she states.

The role of affinity ligands

Although resins are the workhorse of chromatography methods, ligands were developed to increase the specificity with which the resins can capture target molecules.

“Beyond the high selectivity required, [the ligands] must also be easily conjugated to the base matrix, enable efficient elution in conditions mild enough to ensure the target stability, and be chemically stable to enable robust operation. This is especially important when the resin needs to be cleaned-in-place, for instance using sodium hydroxide at concentrations even as high as 0.5 M,” Crocco explains.

While stability and the ability to easily reuse resins may not be of high importance early in the drug lifecycle, the cost considerations for such maintenance at the commercial level will become significant. Thus, in the further development of innovative ligands for these applications, stability must be a key feature consciously designed from the start, Crocco concludes.

Beyond high specificity, affinity resins for new modalities must have the same basic characteristics as those expected in the more mature recombinant protein (e.g., mAb) field: capacity and stability, stresses Crocco.


Over the past few decades, affinity ligands have been improved, with the main advances seen in immobilization chemistry, ligand engineering, and base matrix design. “IMAC-based ligand resins have seen several improvements in recent years, including the development of smaller base beads for higher resolution and better ligand stability to minimize ionic leachables in chromatography steps,” notes Mriziq, who adds that modifications of the ligand have also improved dynamic binding capacity, allowing for the accommodation of higher titers in the feedstream.

“Chromatographers are wiser now, and they understand the cost pressures to cycle capture resins extensively to improve process economics,” Crocco observes.

Crocco points out that the more a resin is cycled, the more drug each liter of resin produces, and hence the cost contribution of the affinity resin towards each dose of the drug is lower. But to be able to cycle the resin more frequently requires an effective cleaning process. “The industry standard cleaning agent is sodium hydroxide, which is very effective, hence the affinity resin needs to be stable in sodium hydroxide, minimally 0.1 molar, but preferably 0.5 molar. Not all classes of affinity ligands have the required stability, however, and the past 10 years have seen the emergence of new affinity ligand types that are inherently much more stable and able to withstand the rigors of bioprocessing,” Crocco states.

While modern Protein A resins have set the bar high, chromatographers expect and require similar sodium hydroxide stability and long resin lifetime in affinity resins for other modalities as well, Crocco also emphasizes. “The evolution of the newer, more stable ligands means that, now, chromatographers don’t need to compromise and can always seek a resin that can be cleaned with hydroxide,” she concludes.

Future outlook

“Diversity of ligand libraries used for ligand screening significantly increases chances for discovery of unique ligands that, when put on the right solid support, can address very unique purification challenges,” says Crocco. “For example, Repligen starts with more than 50 ligand scaffolds to identify the best ligands with predefined requisite characteristics. This procedure sometimes includes unique selectivity requirements. For instance, where a unique selectivity is required that is not attainable with conventional resins (such as separation of isoforms, truncations, active vs non-active forms of enzymes, etc.), affinity chromatography can be applied as a polishing step with properly selective ligands,” Crocco explains.

Current innovations include ions and ligands that are stable, as described earlier. Low levels of ligand leaching as well as the ability to remain stable against harsh cleaning reagents are critical for operators during manufacturing, Mriziq confirms. “Voice of the customer has driven these innovations,” he states.

Looking toward the future of downstream purification, the complexity of emerging biologics is making affinity purification attractive. “Due to their selectivity, scalability, and ease of process development, affinity ligands enable a simpler platform purification process, resulting in a faster time to market,” says Mriziq.

Meanwhile, adeno-associated virus purification workflows or non-mAb biologics are driving the innovation of newer affinity chromatography approaches, Mriziq adds.


1. Łącki, K.M.; Riske, F.J. Affinity Chromatography: An Enabling Technology for Large-Scale Bioprocessing. Biotechnol J. 2020, 15 (1), e1800397. DOI: 10.1002/biot.201800397

About the author

Feliza Mirasol is the science editor for BioPharm International.

Article Details

BioPharm International
Volume 36, No. 10
October 2023
Pages: 19–20


When referring to this article, please cite it as Mirasol, F. Meeting Complexity Demands with Innovative Affinity Ligands. BioPharm International 2023, 36 (10), 19–20.