A Closer Look at Affinity Ligands

Affinity ligands play an exciting role in the ever-growing sphere of downstream processing. In speaking with BioPharm International®, Phillip Elliott, PhD, associate director of Process Development, BioCina; Jan Bekker, PhD, director, Business Development, Commercial and Technical Operations, BioCina; and Parviz Shamlou, PhD, senior vice-president of Science & Technology, Abzena illuminate the newest innovations with affinity ligands and provide a deeper understanding of the role affinity ligands play in the future of downstream processing.

Breaking down affinity ligands

BioPharm: What are affinity ligands and how do they contribute to the downstream purification process?

Bekker (BioCina): Affinity ligands are molecules that can bind with high affinity to specific targets. Affinity ligands can be used for various purposes, such as detection, purification, or modulation of biological activities. Affinity chromatography is a separation method based on the specific binding interaction between an immobilized affinity ligand and its binding partner. The most common example in the realm of bioprocessing is Protein A, which has an extremely high affinity for some immunoglobulin molecules. Other examples include antibody/antigen, enzyme/substrate, and enzyme/inhibitor interactions. The degree of purification achieved by affinity purification can be quite high depending on the specificity of the interaction. Consequently, it is generally the first step in a purification strategy.

Release of the protein from the ligand is generally achieved by altering parameters such as pH, and sodium chloride concentration or by the addition of a competitor for ligand binding.

Shamlou (Abzena): In nature, there is a vast array of biomolecule interactions where binding of one molecule to another through receptor–ligand interactions is critical for biological function. This concept is applied to the drug development sector and revolutionized manufacture of large complex medicines where use of ligands that have a specific affinity for the drug are used for purification—with antibodies being the most notable success story.

Large-scale manufacturing of a therapeutic monoclonal antibody (mAb) starts with the expression of the antibody in mammalian cells, typically CHO [Chinese hamster ovary] cells, followed by a series of steps to purify the antibody from all the impurities in the cell culture media. Following the removal of cells and cell debris from the media, the first chromatography purification step is based on the specificity (affinity) properties of the antibody. Bioprocess scientists and engineers realized the importance of affinity chromatography in purification of mAbs in the early 1980s and identified a small, 42 kD protein (i.e., Protein A) that has exceptionally high affinity for mAbs in the Fc region.This is an important property that allows for most antibodies to be purified irrespective of their specificity.We know a lot about Protein A ligand since it was first discovered in 1958. Today, Protein A ligand is manufactured and purified on a large scale using recombinant DNA technology. Purfied Protein A ligand is covalently coupled (conjugated) to a solid, porous support to form a matrix that can uniquely and specifically bind to mAbs under the correct biophysical conditions. The matrix is typically in the form of small 50–150 micron beads that are highly porous to provide a large surface area, maximizing antibody binding potential for higher yields from fixed-volume columns.

BioPharm: Why are affinity ligands preferred for biologics purification over other downstream purification techniques?

Elliot (BioCina): Affinity ligands can produce a very high purification factor in a single unit operation and capture product from a dilute process stream. This makes affinity purification very attractive in biopharmaceutical manufacturing operations as it can help to reduce time and cost. Most other ligands used in chromatography operations rely on more general properties of analytes (proteins and other biological molecules), such as charge and hydrophobicity. This can result in many other molecules besides the target molecule binding to and eluting from the stationary phase and hence being carried forward in the purification scheme. The same drawbacks apply to purification processes that do not rely on ligands, such as the size separation performed by size-exclusion chromatography or filtration.

In addition, affinity separations are generally simple to operate, not requiring gradient or other complex operations, and are rapid in execution.

Shamlou (Abzena): Affinity chromatography is based on highly specific attractions between two biomolecules. Importantly, the interactions between the two biomolecules are reversible making them an ideal choice for purification. This is achieved by attaching one of the biomolecules, known as the affinity ligand to a solid matrix which acts as the stationary phase while the other biomolecule, referred to as the target molecule, is in the mobile phase. While there are many ways affinity ligands may be exploited for purification, Protein A affinity ligand for purification of antibodies from is one of the best examples and has proven a powerful mode of chromatography.

Protein A ligand affinity chromatography is simple to operate, robust, rapid, and highly selective with very good resolution. Importantly, and unlike other modes of preparative chromatography, binding of antibody to Protein A affinity ligand is not sensitive to either cell free culture media pH or its osmolality so there is no need for conditioning the feed. The high specificity and ease of operation of Protein A ligand affinity chromatography has made it the technology of choice and the “darling” of the mAb purification platform
process in industry.

Cutting-edge innovations

BioPharm: What are the most cutting-edge innovations in the manufacturing process for affinity ligands, and what drove those innovations?

Shamlou (Abzena): One of the challenges of traditional protein A affinity chromatography is the use of acidic, low pH, elution. The low pH can lead to instability of certain antibodies potentially causing aggregation. A recent innovation is the development of a new generation of Affinity ligands that are like traditional Protein A in all respects except that elution can be performed at around pH 5.

Another challenge with traditional Protein A affinity ligands is the leaching of protein A from the solid support. This reduces the resin lifetime and introduces a process related impurity into the product mainstream. Leached protein A is considered a CQA (critical quality attribute) to measure for with a release specification that must be met before the product can be released.Much progress has been made in recent years to address this issue and the new generations of protein A are more robust and stable.

Elliott (BioCina): A very interesting recent development from Cytiva is the ProteinSelect system which consists of a tag protein sequence added to the N-terminus of a target protein and an affinity ligand for that tag. Once the protein of interest binds to the affinity ligand on the resin via the tag, refolding and self-cleavage of the tag-ligand complex occurs, such that the protein of interest is eluted without any trace of the tag. This will allow more widespread use of this affinity ligand with proteins that do not have a current natural affinity ligand as well as more rapid purification of new drug targets. The need to have more rapid purification of a variety of proteins for drug development drove this innovation, as well as the availability of these self-cleaving sequences.

The future of affinity ligands

BioPharm: In what direction is affinity ligand development headed moving forward?

Shamlou (Abzena): Another popular affinity ligand for purification of antibodies is Protein G, which has a molecular mass of 21.9 kDa and has a different binding affinity to antibodies compared to Protein A.

Affinity ligand is a powerful technology and, beyond Protein A and G affinity ligands, in recent years, affinity chromatography matrices have been developed with high selectivity for purification of, for example, recombinant viral vectors for gene therapy applications.In one example, camelid single domain antibodies that recognize adeno-associated virus (AAV) viral vector capsids are proving very effective. Another affinity matrix also provides high affinity for recombinant human growth hormone (hHGH) by using an immobilized 14 kD single variable domain of heavy chain (VHH) antibody that is recombinantly produced in yeast. The single-domain heavy chain antibody fragments from Camelidae have also been utilized successfully as affinity ligands for purification coagulation factors to help patients with hemophilia type A and B.

Elliott (BioCina): Several factors must be considered to obtain a successful separation in affinity chromatography. For instance, it is necessary to immobilize the ligand onto or within a chromatographic support. The immobilization process should ideally create a stable affinity ligand without significantly altering the binding properties of the ligand. If the affinity column is to be reused for multiple cycles, care must also be taken to select application and elution conditions that allow both effective dissociation of retained targets and good column regeneration without permanently damaging the ligand. As an example, much engineering has recently gone into modifying the Protein A ligand to ensure it can withstand the stringent cleaning and sanitization procedures required for use in a pharmaceutical setting without becoming inactive and no longer being able to bind its target. Similarly, a ligand should not bind its corresponding analyte too strongly, such that the target analyte cannot be eluted without using conditions that damage the analyte. These issues represent areas of ongoing research in the development of affinity ligands. Continued growth is expected in the future for affinity chromatography as further advances are made in the ligands, supports, immobilization schemes, and potential applications.

Recent work has explored the use of molecularly imprinted polymer surfaces (MIPS) for the purification of proteins. MIPS are polymer stationary phases that contain pockets or cavities specific to the target analyte. Due to the lower cost of these surfaces compared with biological affinity ligands, developments in this area are expected to continue and be highly beneficial.

BioPharm: What role do you see affinity ligands playing in the future of downstream purification, especially with emerging biologic therapeutic modalities?

Bekker (BioCina): Due to their specificity and high purification factor, affinity ligands will continue to play a major role in the downstream purification of biological molecules. Cell affinity chromatography is another important application involving target purification by affinity-based separations. Examples of affinity ligands that have been used for this purpose are lectins, which can interact with glycoproteins on cell membranes, and antibodies, which can bind to specific surface proteins. With the expected increase in cell-based therapies, the development of ligands for these purposes should see them continue to find a large role in the future.

Elliott (BioCina): Recently, oligo-deoxythymidine acid (oligo-dT) ligands have been developed and immobilized to chromatography stationary phases to provide a highly specific ligand for the polyadenylated tail of mRNA [messenger RNA] molecules. The emergence of this class of therapeutic molecules (mRNAs) drove the development of the corresponding affinity ligand that could simplify their purification. As with cell therapies, this new ligand will play an important role in mRNA’s future development.

Shamlou (Abzena): Gene therapy is an emerging therapeutic modality that needs significant process development including the application of novel affinity ligands during purification. A key step in the production of gene therapy products is the use of high-quality viral vectors with [AAV] vector and lentiviral vector (LV) being the most popular platforms. Robust manufacture of high-quality vectors has proven to be a major bottleneck in the growth of gene therapy. Titers are relatively low during production and robust purification of the viral particles from impurities has proven to be a challenge. For example, viral particles are physically much larger than proteins and antibodies. Using traditional resins with small pore size leads to low binding capacity because of very low diffusion rates into the pores of the resin. LV particles are also exceptionally sensitive to pH, salt concentration, shear, and temperature making their purification more challenging. These challenges currently mean there is no ‘platform’ downstream purification for viral particles.

A new generation of vector serotype specific and non-specific affinity ligand chromatography resins are proving successful in capturing LV and AAV particles from cell-free media and promise the potential of a platform approach to purification of AAV and LV particles. AAV and LV affinity ligands are, however, exceptionally expensive making commercial scale an issue for many drug manufacturers.

About the author

Daria Husni is assistant editor to BioPharm International®.

Article details

BioPharm International®

Vol. 37, No. 4

April 2024

Pages: 21-24

Citation

When referring to this article, please cite it as Husni, D. A Closer Look at Affinity Ligands. BioPharm International® 2024 37 (4).