Cell-culture and fermentation are high-yielding processes that effectively produce complex biologic molecules with desirable bioactivities. Even so, there are many impurities present in the harvest cell-culture fluid obtained after removal of the cells or yeast and media. Host-cell proteins, host-cell DNA, other process-related contaminants, and product-related impurities (e.g., aggregates, incorrectly modified or unfolded proteins, etc.) must be removed. A combination of chromatography steps using different resin chemistries is used to purify the biologic drug substance to an acceptable level. The number and types of chromatography steps depend on the type of molecule and the nature of the contaminants (similar vs. dissimilar). Drug manufacturers are always seeking ways to improve the efficiency and productivity of chromatographic separations. Suppliers of chromatography resins are developing new resin chemistries and binding mechanisms to meet this need.
Several interaction modes
There are four major chromatographic techniques employed to purify biologic drug substances: affinity, ion-exchange (IEX), mixed- or multi-mode, and hydrophobic-interaction chromatography (HIC). Metal chelate, size-exclusion, and hydroxyapatite resins are also used on a limited basis, according to Andrew Bulpin, head of process solutions strategic marketing and innovation with MilliporeSigma.
Monoclonal antibodies (mAbs), which today account for approximately 50% of the total revenues of all biopharmaceuticals according to David Westman, global product marketing manager for Cytiva, are typically purified using a platform approach in which protein A chromatography resins are used in the first capture step. Polishing is then achieved using some combination of anion exchange (AEX), cation exchange (CEX), mixed mode, and HIC resins.
“Protein-A based affinity chromatography is very popular and more widely used because it typically affords purities above 95% in one step,” observes Nandu Deorkar, vice-president of research and development for Avantor. IEX chromatography is widely used due to its versatility, while HIC is an orthogonal technique to IEX and, therefore, the two are often used in series, according to Westman. Generally for mAbs, both strong CEX and AEX resins are preferred, according to Shelly Parra, global field applications senior manager with Thermo Fisher Scientific.
Mixed-mode and multi-mode resins are considered second-generation IEX chemistries. “Traditionally with IEX chromatography, one type of ionic interaction occurs. In multi-mode and mixed-mode chromatography, two interactions take place simultaneously, or one right after the other. This approach is becoming more popular because it allows for better separations of very closely related impurities, with the opportunity to avoid a step in the process,” Deorkar explains.
Many separation mechanisms
The different interaction modes separate biologic drug substances from impurities through different binding mechanisms. “In all cases, resins are selected because of their ability to separate the target molecule from process- and product-related impurities,” Westman notes.
IEX resins, according to Bulpin, have ionic groups on their surfaces comprising weak or strong acids (cation exchangers) or bases (anion exchangers). These groups discriminate between different macromolecules (e.g., proteins, antibodies, etc.) due to the net charges of the target compounds. Salt, or a pH shift, is then used to disrupt the interactions and, if used for capture, to free the biologic drug substance from the resin. IEX resins are noted for their versatility and robustness and thus are applicable for most biologic separations.
IEX resins can be used for capture of biomolecules or polish, according to Parra, with these different chemistries used depending on the application. For example, she notes that strong CEX resins like sulfopropyl are suitable for mAb aggregate removal and charge variant separation, while strong AEX resins are suitable for mAb polish due to their ability to bind low pH impurities such as DNA, endotoxin, leached protein A, and viruses. “Different ion exchange chemistries can offer unique selectivity depending on the purification needs, so a variety of chemistries are tested based on the application,” Parra says.
Affinity resins such as protein A, on the other hand, separate biomolecules based on highly specific biological interactions, according to Westman. “More specifically, protein A resins have affinity ligands on their surfaces that have the appropriate shape and chemical substituents to allow for a three-dimensional steric fit with the Fc portion of antibodies,” says Bulpin. Within the main site of interaction, according to Deorkar, ionic charges exist for other interacting sites. “Similar reverse charges are present on the ligand as well, so they fit together like two pieces of a puzzle,” he explains.
The result, according to Westman, is high-purity recovery under generic conditions in just one separation step and enables a plug-and-play approach that leads to reduced process development times. In fact, according to Parra, highly selective affinity resins with antibody-like specificity can significantly improve a purification process making it simpler and more economical for a variety of molecules from mAbs, to recombinant proteins, to viruses.
Hydrophobic interaction resins separate molecules based on differences in hydrophobicity and are primarily used in high-salt conditions and for challenging resolutions, according to Westman. “HIC can be an orthogonal and powerful chromatography tool used in both bind/elute and flow-through modes of operation,” asserts Orjana Terova, product manager for purification with Thermo Fisher Scientific. In cases where the molecule is highly hydrophobic or contains a very hydrophobic conjugate, it can be difficult to elute even in low salt conditions. “Recently developed HIC resins from Thermo Fisher Scientific offer a differentiating range of selectivities by altering the hydrophobicity of the resin surface. These resins are appropriate for a wide variety of hydrophobic molecules while also addressing mAb aggregate removal in flow-through mode in low salt conditions,” she comments.
With mixed-mode resins, two different interactions occur. They can both be ionic interactions, or they may be ionic and hydrophobic.
Optimizing resin chemistries
“Resin development is basically driven by what is required at the end of a process--whether it is the ability to bind more protein, more separation in less time, high selectivity, or low pressure,” says Deorkar. For high selectivity, the ligand interaction is changed, which is where engineered mixed-mode chemistry comes in. Increased binding capacity can be achieved by changing the linker chemistry on the surface of the polymer so the ligands can be spaced equally in an optimized way, allowing more molecules to be attached. The resin particles can also be optimized with respect to shape, size, and mechanical properties (i.e., compressible, non-compressible, can be crushed under pressure, etc.) to, for instance, achieve particles with high surface areas but that do not afford high pressures, according to Deorkar.
Process development scientists must also balance multiple factors when developing new processes to address current challenges and industry demands. “Capacity, resolution, and the speed at which the process can be run must all be simultaneously optimized,” asserts Parra. Process development scientists also need to consider the ability to achieve good capacity and separation under higher salt concentrations for added flexibility and process simplicity, according to Parra. “Using tangential flow filtration (TFF) between steps reduces product yield and lengthens process times. A salt-tolerant resin can be very beneficial to a purification process by decreasing the need for dilution or eliminating TFF steps all together,” she notes.
“Next-generation IEX resins that offer high capacity, high resolution, and salt tolerance at faster flow rates have significantly improved process flexibility and allow for easy adaptation to integrated processing,” Parra continues. “By increasing the throughput, process flexibility, and process performance, the costs associated with purification can be decreased and more productive processes can be realized,” she states.
Ion exchange resins that offer high capacities at high linear flow rates help to scale-up processes for the economic production of large-volume mAb drugs, says Bulpin. “The base beads of these materials exhibit good mechanical strength while maintaining high porosity,” he says. In addition, Bulpin notes that the three-dimensional presentation of the ligands in the open pore spaces of ion exchange ligands is important. “This effect can be achieved through the use of so-called tentacles, grafted linear chains presenting the ion exchange functions in a more flexible way that allows multi-point interactions with the proteins of interest,” he observes.
The past decade has, in fact, seen a steady increase in usage of multimodal chromatography resins, according to Westman. Because these resins separate molecules based on more than one mode of action, they are being used when other chromatography techniques do not give the desired selectivity. He adds that they are also commonly used in polishing steps during mAb purification.
Multimodal resins most often separate molecules based on ligand properties, but there are examples of multimodal resins for which the base matrix design contributes to the separation. Cytiva has, for instance, developed a resin with a dual-layer design that combines size-exclusion separation via the inactive shell with adsorption chromatography via the ligand activated core. “This resin chemistry is beneficial for the separation of large molecules, such as viruses, vaccines, and immunoglobulin M,” says Westman.
Biosimilars and other challenges
The increased trend toward the development of two-step chromatography processes and higher protein doses is requiring more performance from chromatography products, according to Parra. “Process development scientists need new tools that will clear even more impurities in a single step and achieve even cleaner biologic drug substance,” she says.
Biosimilars also present chromatographic challenges. “For biosimilars, it is necessary to produce a comparable pattern of protein variants. That requires removal of closely related byproducts in proteins, such as charge or glycosylation variants,” Bulpin says. He adds that the drive away from bind/elute chromatography steps to more efficient flow-through steps is impacting resin development. Single-use chromatography is another challenging area. “To date it has been difficult to provide cost-effective devices that can also be scaled up,” notes Bulpin.
Purification processes for mAbs have improved significantly over the past 20 years, but Westman notes that shortcomings remain to be addressed. In particular, the binding capacity of Protein A resins is still lagging behind other chromatography techniques, such as IEX chromatography. “An increase in binding capacity will be beneficial for process economy reasons when purifying steadily increasing upstream titers,” he states. Current protein A resins are also exposed to feeds with high concentrations of cell-culture nutrients yet are more sensitive to the high concentrations of sodium hydroxide needed for cleaning and sanitization, which increases the risk for bioburden issues, according to Westman. “We believe that both binding capacities and chemical resistance can be further improved for future protein A resins,” he observes.
The increased diversity of pipeline therapeutic molecules is a major challenge for chromatographic separation. “The development of novel scaffolds for therapeutic molecules (e.g., bispecific antibodies, antibody fragments, smaller protein drugs, and cell-based therapies) will have an impact on the development of new resins. There is a need for more economic downstream processes to ensure that modern therapeutic regimes can be offered to more patients worldwide,” observes Bulpin.
Adds Terova: “The landscape in molecules is changing with more difficult and unique modalities and therapies leading to new challenges for purification. As more and more challenging molecules are introduced into the industry pipeline, new purification tools are needed with unique functionality.”
The platform approach, which has been so successful for the development and manufacture of mAbs, cannot easily be applied for such diverse drug substances, according to Westman. As a result, each separate biomolecule may need extensive process development, which will impact both time and cost. “When a targeted, specific affinity purification solution does not exist for a biomolecule, the protein purification scheme can be very complex. As the number of required purification unit operations increases, the product yield decreases. Yield drives cost of goods (COG), so not having an affinity purification solution can greatly impact the COG for biotherapeutic manufacturing,” Terova explains.
With the increase in more challenging molecules and the desire to make processes more efficient, she adds that there has been a significant increase in interest for custom resins on high-performing beads. Most chromatography vendors have developed new affinity resins designed for use with the next-generation biologic products being developed today, including antibody fragments, bispecific antibodies, and antibody drug conjugates (ADC), as well as cell and gene therapies and vaccines.
To address the need for different selectivities, new modalities have to be developed that offer more than one mode of interaction, according to Bulpin. He adds that these interaction principles will not only be attached to chromatographic beads, but to a variety of different base matrices, such as adsorptive membranes, monolith, or fiber-based materials. The main driver for new matrices is the wish for pre-sterilized single-use materials that offer both process economy and performance.
As suppliers expand their portfolios of resins to enable efficient separations of newer biomolecules, they must also consider the potential to increase the column capacity, binding/ separation capacity, resin lifecycle, and/or column cleaning efficiency, according to Deorkar. “In addition to these opportunities to optimize performance, it is also important to look at improving the overall ecosystem around the chromatography process, by considering the buffers, cleaning agents, etc., and how they all work together in tandem,” he comments. “Chromatography doesn’t work in isolation.”
Continuous manufacturing is an important topic for cost-efficient and fast production of protein drugs. “Continuous chromatography enables shorter bed heights with lower pressure flow constraints, enabling the use of smaller beads with higher binding capacities and better resolution,” notes Westman. By going to continuous chromatography, resin utilization and capacity and separation efficiency are increased by using smaller particle size resins and operating at slightly higher pressures in smaller columns, according to Deorkar. The higher particle surface areas result in greater efficiency.
The ultimate goal for continuous manufacturing, according to Bulpin, would be to purify the target protein in an all flow-through mode, binding only the impurities and eliminating the need to store the target protein in an intermediate buffer tank. “Given the interest in continuous processing, planning for use in a continuous chromatography process must be taken into account when developing purification tools,” Parra states. She does add, however, that many in industry feel there are enough good tools in the industry now to make continuous processing a reality.
Switching chromatography processes from batch to continuous isn’t a simple exercise, however. Deorkar believes that the best way to make improvements in chromatography processes is to first reduce the number of steps. A process with three or four chromatography steps will not be easy to move to a continuous process. “Multi-mode, mixed-mode resin chemistries that offer the potential to reduce the number of steps will, therefore, play an important role in facilitating the adoption of continuous chromatography,” he concludes.