As technology matures, inefficiencies and process limitations in downstream process chromatography are improved.
By Feliza Mirasol
Selecting an appropriate separation technology for biologic manufacturing is a crucial task in maximizing productivity in downstream bioprocessing. Biomanufacturers consider several key factors when selecting a best-fit process chromatography technology for their products, including the stability of the biologic, compatibility of the buffers used, and the ease with which scale-up can be achieved. Recent developments in process chromatography have addressed challenges in downstream separation and purification and led to improved bioprocessing.
Current challenges
The biopharmaceutical industry continues to face technical and logistical challenges in process chromatography, particularly during scale-up. Although significant work has been done in upstream bioprocessing to remove bottlenecks through the implementation of single-use technologies, better characterization of raw materials, and the use of more precisely controlled bioreactors, improvements in downstream processing have not kept pace, says Nandu Deorkar, vice-president, research & development—Biopharma Production, Avantor.
“In downstream processing, the ultimate goal for biopharma manufacturers is to improve recovery and reduce the cost per gram of protein produced. Currently, over 60% of the cost to produce a new biologic still relates back to downstream steps,” Deorkar says. “It doesn’t help that there’s approximately 30% more effort, such as resources, materials, and equipment, as harvest material goes through downstream purification due to multiple unit operations. Any percentage of improvement in downstream recovery can contribute to improving the ultimate process yield for drug product of the target biologic,” he adds.
Deorkar also notes that there is a lack of diversity in the supply chain of Protein A resin, which is used in the first downstream chromatography step in monoclonal antibody (mAb) manufacturing. “That dearth is seen especially through the limited dynamic binding capacity needed to process the high-titer cell culture harvest,” he says.
The performance reproducibility in column packing, which is essential for ensuring consistent scale-up, can accelerate technology implementation in manufacturing, says Hemanth Kaligotla, chromatography task force manager at Sartorius. “For chromatography offerings based on membranes, both the availability of devices at multiple scales and the performance across the device sizes need to be consistent. Flow distribution in the devices remain critical,” Kaligotla emphasizes.
Slow processing times are another big issue, especially because the biopharma industry is looking to shorten supply chains and make drugs “at the bedside”, says Ian Scanlon, staff researcher, and Zachary Sexton, research engineer, both at Cytiva. Reducing the costs for cleaning and cleaning validation are also challenges that many big companies and contract manufacturing organizations (CMO)/contract development and manufacturing organizations (CDMOs) discuss. “Those two steps often lead to huge amounts of ‘non-productive’ time, even with mitigation strategies. Single-use solutions can eliminate much of that downtime with a simple plug-and-play format,” says Scanlon.
“Two goals of the biopharmaceutical industry are scalability and flexibility,” notes David Beattie, head of Bioprocessing R&D, MilliporeSigma. “A downstream facility should give reliable product qualities from first clinical batches to full-scale manufacturing. It is of great importance to rely on materials, such as resins, buffers and equipment, which are designed for production purposes and available in the amount they are needed,” he states.
“The other goal, flexibility, is of particular importance for novel therapeutics. Bispecific antibodies, antibody-drug conjugates, engineered glycovariants, fusion proteins—all these molecules need to be purified with a templated approach using resins with a predictable behavior,” Beattie continues.
Past challenges
Fortunately, past challenges with downstream process chromatography have been overcome with innovations in technology, equipment, or methodology.
Chromatography has been a workhorse in the purification of biological products for decades, and there is a constant drive to optimize productivity and minimize the cost of goods, Kaligotla states.
“We see an increase in the use of mixed-mode resins due to the additional selectivity they offer for challenging molecules, as well as for the minimal feed stream manipulation needed to develop a robust process. Specific affinity resins are also being developed that enable an effective and straightforward capture step that results in high purity. We can see this trend in new biological targets (e.g., adeno-associated virus [AAV]) where no platform processes have been established yet. Convective flow membrane chromatography and monoliths addressed the previous purification challenges with diffusive matrices for large molecules, such as viruses (> 100 nm), plasmids, or even large protein complexes,” Kaligotla notes.
Process simplification has become a prominent solution, with processes optimized to eliminate non-value-added steps and remove disconnections between unit operations, Kaligotla adds. Process intensification is another viable approach through which equipment footprint could be reduced and manufacturing could be made more flexible.We also see work being done in the space of real-time product quality monitoring for real-time/automated event control to achieve real-time release and continuous processing,” Kaligotla says.
Meanwhile, the amount of effort needed to perform screening experiments poses another major challenge. Customers can benefit from working with vendors that support them with high throughput screening tools with pre-packed consumables, high throughput screening systems, and well-designed studies using robust design of experiment software tools, according to Kaligotla.
Sexton notes that ligand development has helped enhance the robustness of processes in development. “A ligand that has a higher affinity for its substrate or that is more resistant to harsher cleaning procedures gets preference because it saves the customers money and time,” Sexton states. Hardware innovations, such as simulated moving bed (SMB), periodic counter-current (PCC), or other systems, which allow users to make more efficient use of a resin are still emerging, he adds.
“We are also seeing that, as regulatory limits impose restrictions for ensuring customer safety, like toxicology and cleaning validation for GMP [good manufacturing practices] environments, off-the-shelf technology becomes more appealing,” Sexton states.
The development of pressure stable chromatography resins, which allows seamless scale-up with high linear velocities, was another innovation that helped the industry address past chromatography challenges, Beattie says. Another innovation addressing the need for increased flexibility is the adoption of single-use membrane-based chromatography technologies, he adds.
“Traditional, resin-based chromatography columns are often oversized due to throughput limitations and are ill-suited to flexible manufacturing. These factors impose challenges on the design of purification schemes for manufacturing of biotherapeutics,” Beattie states. MilliporeSigma, for example, developed a membrane adsorber that addresses these challenges by combining high binding capacity and high flow rates.
Membrane adsorbers deliver enhanced process flexibility and robustness and reduce cost as well as risk. Benefits include faster processing, which increases productivity; eliminating the need for column packing, which saves labor and time; eliminating the need for cleaning between batches, which allows for rapid switch over; eliminating the risk of cross-contamination between batches; and allowing for a smaller facility footprint, Beattie enumerates.
Deorkar adds that finding efficiencies and economies of scale in downstream processing involves more complex analysis and optimization. Improvements may be reached after investigating aspects of current purification steps and technologies. Such improvements include expanding the use of mixed-mode and multimode chromatography resins, particularly since using resins to target ligands for increased selectivity can help to process targeted molecules more efficiently; exploring ways to make chromatographic buffers more effective, which can be done by using new kinds of additives and prepackaged single-use buffer materials to streamline buffer exchange steps.; and making wider use of data analysis tools. Data can help quality and process optimization engineers gain deeper insight into complex material interactions in downstream process steps, particularly related to raw material characterization, Deorkar says. He adds that variability in raw materials can present serious issues that impact downstream efficiency and can lead to long investigations and potential delays in making drugs available for patients. Advanced characterization of materials, therefore, is a best practice adopted from semiconductor manufacturing that can provide biopharma manufacturers with further insights into the variability of materials within the integrated supply chain.
“New choices for Protein A, such as, for example, a new recombinant Protein A resin developed by Avantor, [can]addresses challenges of limited supply,” Deorkar continues. “The new resin is built on a proprietary ligand using an alternative supply chain. Additionally, current Protein A product formulations require flammable chemicals for storage and transportation, which require specific safety precautions and incur additional shipping and handling costs. Avantor addresses this problem by packaging its new resin in a non-flammable storage buffer that does not require restrictions or special handling in storage and transportation related to flammability,” Deorkar explains.
Continuous chromatography
As continuous bioprocessing gains more attention, there is the need to evaluate whether current process chromatography technologies pose any limits or risks to implementing continuous processing. According to Scanlon, there are definite risks in moving toward continuous bioprocesses, both on the technology/implementation side and in the regulatory side of things. Among the issues discussed frequently are how one defines where a batch starts and ends and how one can minimize the impact of contamination when something inevitably goes wrong.
“It’s relatively easy with batch processes because of its definite containment. When everything blends and your monitoring is in-line, you become aware of the problem very quickly, but making that separation of what is affected or unaffected is crucial. You almost need second-order indicators—indicators of when something is moving towards that out-of-spec range to make you aware before a large continuous separation process becomes non optimal, resulting in an expensive loss of material and a potentially long and involved problem-solving investigation,” Scanlon says.
“We’ve seen vast improvements in adsorber characteristics, be it resins or membranes. These larger pore structures allow for better pressure characteristics over a longer lifetime. Perfusion titers will increase over time like batch processes have previously. This will create a need for downstream process improvements that are being developed today,” Scanlon adds.
Continuous processing also makes things more complex, from flow path complexity to connections and programming to keep processes in sync, Scanlon continues. Added to the complexity is the fact that CMOs/CDMOs use equipment from different suppliers. “Getting interoperability in a continuous process could be challenging if the equipment isn’t designed to do so and could potentially require some sort of in-between hardware that may need to be custom-made,” Scanlon observes.
“The industry is moving towards intensified, connected, and continuous bioprocessing, and we expect about 30% of new molecules moving into clinical manufacturing will be based on intensified processing in upstream or downstream operations,” observes Beattie.
One area of bottlenecking in continuous mAb polishing chromatography is the cation exchange step, which is traditionally operated in bind and elute mode to provide clearance of product-related aggregate, Beattie points out. “While bind and elute chromatography can be operated in a continuous manner using multi-column systems, buffer consumption is relatively high and product elution often occurs under high salt conditions, which drives the need for dilution or buffer exchange to maintain compatibility with unit operations further downstream. A more efficient purification strategy is to operate the cation exchange step in a product flow-through mode, rather than bind and elute mode,” explains Beattie. “From a continuous processing perspective, a flow-through cation exchange step is more easily integrated with other product flow-through purification steps, such as anion exchange polishing or virus filtration, without requiring conductivity adjustment or dilution.”
While some bioprocessing is operating on a semi-continuous basis, major limitations for complete continuous chromatography exist, such as the linking of different unit chromatography and filtration unit operations. Multiple chromatography steps use different types of buffer systems, according to Deorkar.
“As part of these process flows, buffer exchange is required and is typically difficult to link. If you think of a train, it is very easy to jump from cart to cart with a bridge. But without this connector, the train will separate and lose part of its cargo. What is needed is a seamless connector to bring chromatography and buffer exchange work together. Once this is accomplished, the supply of various buffers would need to be assured. This could be achieved by using hydrated concentrated buffers or ready-to-hydrate pre-weighed direct dispense delivery of solids to enhance flexibility and efficiency of buffer prep operations,” Deorkar explains.
Kaligotla supports the idea that the integration of multiple unit operations while maintaining robust process control and minimizing risk is an essential focus for continuous bioprocessing. “The current process technologies, such as functionally closed single-use manifolds for [chromatography] skids, pre-packed columns, and robust sensor components, enable long-term processing in a bioburden-reduced environment. The technologies are slowly maturing, and there are continued efforts on real-time in-line analytics to optimize continuous processing,” Kaligotla says.
Risks associated with these maturing technologies include the high cost of consumables, the integrity of the flow paths over extended use, limited leachable and extractable data, and bioburden concerns, Kaligotla points out.
The application of more recently developed technologies, such as single-pass ultrafiltration/diafiltration, and as well as continuous virus inactivation step post-Protein A, allows for steps to run in a truly continuous mode,” Kaligotla says
Downstream response to upstream scale-up
What is the downstream response to upstream scale up as the industry sees increasing titers in upstream processing resulting from increased sizes of single-use bioreactors and optimized cell culture conditions? The response in downstream processing will likely be realized in different ways for different facilities, Beattie believes.
In multi-product facilities, higher titer upstream processes can reduce the number of batches needed to produce a target product mass. This provides an opportunity to increase the number of molecules per facility, Beattie observes. “This goal cannot be achieved without highly productive single-use purification solutions which feature high throughput, quick change-over, and low cross-contamination risk,” Beattie states.
Another strategy involves increasing the total output of a single product in an existing facility, Beattie continues. “Downstream, this results in not only increased column size, but also a corresponding increase in buffer volumes that can be overcome with the use of buffer concentrates.”
Increasing the size of downstream equipment is also a response to increased upstream output, Deorkar chimes in. In addition, multi-cycling may be possible if unit operations allow faster flow. “This is like continuous processing within a unit operation. In this case, pressure is placed on the buffer train, so it is necessary to ensure that the buffer prep operation is efficient and scalable,” Deorkar states.
However, increasing the size of downstream equipment also means that costs are increased, says Sexton. He notes, though, that the industry will innovate and solve this problem creatively. “Some of that will include increasing size in the downstream sphere. However, it may include a smaller footprint to make sure existing equipment is operating as efficiently and as effectively as possible. The easy solution is to make everything larger, but the better and more effective (and more exciting) solution is to make current processes more efficient,” Sexton states.
“There is also the argument that, as we trend toward highly efficacious, personalized medicine and closing supply chain gaps, the paradigm will shift more toward smaller batches of material made in region,” Sexton adds.
The higher titers in upstream processing can impact the number of downstream operating cycles and the total process times, adds Kaligotla. The higher titers also further create challenges with process scheduling; the higher upstream productivity shifts the bottleneck from upstream to downstream. “The higher the titer, one can expect to see a corresponding increase in the level of impurities, increasing the need for depth filter area downstream. A practical way to address this productivity increase is by utilizing a multicolumn approach. This approach has recently gained traction due to the availability of functionally closed single-use manifolds that consist of robust sensor components that enable long-term processing in a bioburden-reduced environment at clinical or commercial-stage manufacturing.” Kaligotla also says that the material cost-savings and enhanced productivity benefits make this unit operation more attractive.