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18 Pharmaceutical Technology ® Bio/Pharma Outsourcing Innovation eBook February 2024 PharmTech.com Biologics applied to the drug development sector and revolu- tionized 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 mono- clonal 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 puri- fication step is based on the specificity (affinity) proper- ties 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 exception- ally 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 tech- nology. 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 typi- cally in the form of small 50–150 micron beads that are highly porous to provide a large surface area, maximiz- ing antibody binding potential for higher yields from fixed-volume columns. PharmTech: Why are affinity ligands preferred for biologics purification over other downstream purifi- cation 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 biophar- maceutical manufacturing operations as it can help to reduce time and cost. Most other ligands used in chro- matography 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 bind- ing 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 per- formed by size-exclusion chromatography or filtration. In addition, affinity separations are generally simple to operate, not requiring gradient or other complex op- erations, and are rapid in execution. Shamlou (Abzena): Affinity chromatography is based on highly specific attractions between two bio- molecules. 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 antibod- ies from is one of the best examples and has proven a powerful mode of chromatography. Protein A ligand affinity chromatography is sim- ple to operate, robust, rapid, and highly selective with very good resolution. Importantly, and unlike other modes of preparative chromatography, binding of an- tibody 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 PharmTech: What are the most cutting-edge innova- tions in the manufacturing process for affinity ligands, and what drove those innovations? Shamlou (Abzena): One of the challenges of tra- ditional 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 af- finity ligands is the leaching of protein A from the solid support. This reduces the resin lifetime and in- troduces a process related impurity into the product mainstream. Leached protein A is considered a CQA (Critical Quality Attribute) to measure for with a re- lease specification that must be met before the prod- uct can be released. Much progress has been made in recent years to address this issue and the new genera- tions of protein A are more robust and stable. Elliott (BioCina): A very interesting recent develop- ment from Cytiva is the ProteinSelect system which consists of a tag protein sequence added to the N-ter- minus 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-cleav- age 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 natu- ral affinity ligand as well as more rapid purification of new drug targets. The need to have more rapid puri- fication of a variety of proteins for drug development