By: Feliza Mirasol
Recent innovations in chromatography resins offer promising advantages in downstream bioprocessing.
With ongoing efforts to increase monoclonal antibody (mAb) productivity in large-scale biomanufacturing, biomanufacturers continuously seek advancements in downstream processing technologies, with downstream operations as a focal point because it frequently represents a process bottleneck. Recent advances in chromatography resins can effectively impact the purification unit operation with positive consequences that reverberate both upstream to the bioreactor and downstream to filtration steps.
Ongoing downstream challenges
Constraints still remain in downstream processing of mAbs, which continue to be the most successful segment of biological molecules in the market in terms of required volumes, says Henrik Ihre, director of Global Strategic Technologies at Cytiva. Ihre also notes that there continues to be strong demand for new mAbs to address new therapeutic areas and rising requests from customers of contract development and manufacturing organizations (CDMOs) for process intensification, which can improve process economy and shorten time-to-market.
A generic capture step for mAbs and Fc fusion proteins has been robustly established by classical Protein A affinity resins, says Oliver Schub, director of Business Development at Navigo Proteins, a scaffold protein engineering and custom affinity resin development company. In contrast, all therapeutics proteins/non-Fc biologics are left without a generic affinity capture step. Development of an individual, multi-step chromatography downstream processing (DSP) for every recombinant protein anew increases overall development costs and timelines, and hence prolongs time-to-market, Schub notes. “The problem is that, with every DSP step, product yields decrease, while bioprocessing time and equipment occupancy goes up, along with DSP manufacturing risks,” Schub says.
He also emphasizes that the challenge is amplified by non-mAb recombinant proteins, which often express at lower levels. Additionally, without an initial affinity step, larger process volumes need to be managed over longer times. He concluded that conventional multi-step DSP development bears the increased risk of commercial viability, where yield, impurity levels (such as host cell proteins or product-related impurities) or overall costs simply do not need meet manufacturing requirements.
Simplifying purification
Protein-specific affinity resins effectively reduce the number of iterations and the development time of purification processes, Schub states. “Coupling a protein-specific affinity ligand to a suitable chromatography bead creates the corresponding affinity resin. The underlying affinity ligand molecule plays a critical role in the performance required for clinical and commercial GMP [good manufacturing practice] manufacturing,” he adds.
Schub notes that extending the performance of conventional Protein A ligands/resins to bind to non-Fc molecules, thereby creating novel affinity resins, combines the best of both worlds. “The result would be a generic, efficient mAb-like DSP with the same equipment/facility, yet for recombinant proteins,” he says. He explains that this can be achieved by simply replacing a conventional Protein A column with one that has the specificity for the respective recombinant protein. The resulting general DSP performance characteristics would remain largely unchanged.
To achieve this goal, Navigo created a unique technology (Precision Capturing) designed to generate target protein-specific affinity ligands. The engineered ligand is based on an artificial Protein A scaffold. “Navigo created large and diverse libraries of artificial Protein A molecules and, from those [libraries], selects the ligands that bind the target of interest. We then couple the selected ligand to chromatography beads and deliver small-scale samples to the client to confirm custom affinity resin performance in-house,” says Schub. “Subsequent resin scale-up is then taken on by a preferred Navigo manufacturing partner, such as Repligen, who provides large-scale custom affinity resins for clinical/commercial GMP purification.”
A recent project focused on SARS-CoV-2 demonstrated the ability to deliver a resin with high affinity, high specificity, and caustic stability on an expedited timeline. In partnership, Navigo and Repligen completed ligand discovery, ligand selection, small-scale production, and scaled-up manufacturing in 10 months. In February of 2021, Repligen launched a new resin (NGL COVID-19 Spike Protein Affinity Resin) for the purification of COVID-19 vaccines. This resin provides vaccine manufacturers the ability to quickly implement a high-purity capture step while decreasing processing time and significantly improving overall yield, notes Vikas Gupta, vice-president of Downstream Bioprocessing at Repligen.
Affinity-based chromatography enables high purity product, says Ihre. From the early steps of downstream processing, affinity chromatography is already working to push through high purity product, which puts less stress on subsequent purification steps, he says. However, the technique comes with its own challenges. On the one hand, affinity chromatography allows for the development of downstream purification platforms; on the other hand, protein-based affinity resins are often more expensive and more sensitive to standard cleaning protocols, Ihre cautions.
“In general, I would say that if the target molecule is either difficult to purify and/or is seen as a high-value target, then affinity chromatography is an appealing alternative from a cost and time-to-market perspective, especially if there are similar molecules from the manufacturer that could be purified with the same affinity resin,” Ihre states.
Why the resins?
Much focus in the improvement of downstream separation and purification is centered on the innovation of resins and/or resin technology. “Resins based on porous beads have indeed been the foundation for which manufacturers have been able to develop and bring new bio-molecules to the market, and resins will keep on being a cornerstone in this industry for decades to come,” says Ihre.
Meanwhile, manufacturers working on new targets, where existing beads may not be ideal from a bead and pore-size perspective, will require novel resins with performances that are further refined around key parameters, such as resolution, selectivity, pressure/flow properties, and capacity to ensure competitive downstream solutions into the future, Ihre says. “Such improvements, combined with new tools, such as in-silico modeling of downstream processes and high-throughput screening of optimal process conditions at small scale, will enable a faster route to more efficient downstream protocols in the future,” he states.
The strong attention around custom affinity chromatography resins for non-Fc biologics is based on the fact that the biopharmaceutical industry would like to further rely on its experience with using Protein A-like resins, their performance and track-record, as well as their regulatory acceptance and the industry’s familiarity with the equipment needed to use them, says Schub.
Meanwhile, an innovation such as a fiber-based protein A chromatography resin offers advantages over traditional resins. “Until recently, chromatography based on different porous bead formats has been the best available tool to design new resins, but there have always been limitations with the traditional bead format as well,” explains Ihre. “For instance, when it comes to the purification of truly large molecules, such as large proteins, viral vectors, or plasmids, these molecules may not be able to enter the pores of the beads, resulting in overall low binding capacities,” he points out. However, a new Fibro format (1) (from Cytiva) has shown to be an effective complement for such large target molecules, owing to the more open and accessible pore structure combined with the ability for rapid cycling and low backpressures, Ihre emphasizes.
Reference
1. Cytiva, “Cytiva Further Advancing Fiber Chromatography Technology with Next Release of Fibro Technology,” Press Release, Jan. 27, 2021.