There is unprecedented pressure on the biopharmaceutical industry to improve the performance of monoclonal antibody (mAb) development and manufacturing processes. There are several factors essential to addressing this challenge, including flexibility, in which product change-over time is reduced; quality, as defined by increased robustness and reliability; speed, in which production and product release are accelerated; and cost reductions in manufacturing and capital expenditure.
Improving mAb Development and Manufacturing Processes
Improvements in processes and productivity can enable smaller and simpler facilities and lower costs, leading to several advantages, including the ability to:
- Improve competitiveness and long-term sustainability,
- Enable business models for new biologics and novel therapies,
- Supply developing countries with affordable biologics, and
- Reach emerging markets profitably.
A key strategy for delivering higher productivity and enabling more flexible manufacturing processes is the adoption of single-use technologies. We have witnessed major improvements in upstream processes with single-use bioreactors and perfusion strategies that can deliver significantly higher titers. In parallel, single-use approaches are being incorporated into downstream processes, including clarification, tangential flow filtration, virus filtration and final fill. Owing to a lack of more productive and efficient alternatives, the use of low-throughput chromatography processes requiring large and expensive units has continued, however, preventing implementation of a fully single-use downstream flow path.
Resin-based columns are often oversized to match new achievements in upstream productivity, and this translates into a large footprint requiring a large amount of maintenance, quality assurance and quality control labor. Such high capital and high operating–expense facilities require amortization to fully utilize resin media’s lifetime to achieve any kind of cost efficiency. While costs can be reduced via economies of scale for large-volume products, this is not the direction in which industry is heading. Moreover, amortization is a luxury that usually is not realized during clinical production; media lifetime is rarely fully utilized due to a limited quantity requirement and the high failure rate in clinical development.
An important initiative that is gaining momentum is the use of membranes for high-productivity chromatographic purification processes that match upstream efficiency without the need for oversizing. Capital expenditures can be reduced due to the possibility of downsizing the chromatography unit operation, which derives from lower costs for media and supporting systems and hardware. Elimination of column packing, unpacking, cleaning and storage between batches, as well as associated labor and validation requirements, further reduces the operational expenses. In parallel, the single-use, plug-and-play nature of membrane chromatography in downstream operations using this approach can increase flexibility, resulting in more rapid changeovers, which can increase production and support creation of a multi-product facility.
Advancements in Membrane Chromatography
In contrast to resin chromatography, where the mass transfer is dominated by diffusion and requires long residence time to achieve decent binding capacity, membrane chromatography relies on a conventional flow that enables high flow–rate operation. However, the typical highly permeable membrane structures that facilitate very short residence time operations exhibit modest binding capacity due to a limitation of binding site density. Advances in membrane science that have focused on resolving this fundamental trade-off have resulted in affinity and ion-exchange membranes with a combination of high dynamic antibody-loading capacities with <10 second residence time, opening the way to an intensified high-productivity, truly single-use purification platform.
The high-binding, short residence times of single-use membrane chromatography presented in this article are enabled by the combination of a non-woven reinforcing mesh skeleton and porous hydrogel containing functional groups (Figure 1). The skeleton provides mechanical strength and durability, while the hydrogel creates a large three-dimensional surface area that contains a high density of functional groups with interconnected pores allowing for convective flow channels to achieve high flow rates. The high density of binding sites, together with a macroporous structure, enables high binding capacity for not only proteins, but also large molecules, such as viruses and DNA, at seconds resident time.
This innovative membrane chromatography design offers a rapid binding mechanism that can be exploited to achieve very short operating residence times — on the order of seconds — without compromising binding capacity, which together enable very high productivity purification processes. Below, we describe the use and benefits of a fully single-use membrane chromatography approach for intensified capture followed by intermediate and polishing purification for host cell protein (HCP), aggregate removal and viral clearance.
Protein A Membrane for Intensified Capture
A novel Protein A affinity chromatography membrane has been developed for rapid multi-cycling bind and elutes the capture of monoclonal antibodies. This platformable tool provides similar or better host cell protein clearance than Protein A resins over a wide range of mAb feeds (Figure 2) with a much shorter residence time requirement (6 seconds versus 2–12 minutes), improving productivity significantly while achieving the required impurity reduction.
Protein A capture is typically bound by a fixed time expected to complete the process (usually 1–2 days) and a fixed process amount coming from the bioreactor. Given these stringent parameters and inherent long residence time requirement, Protein A resin columns are often oversized according to the process time requirement instead of capacity, which requires taking account of lifetime utilization. This approach not only limits the productivity of Protein A resin columns to approximately 10 g/L/h but also increases costs due to the need for additional media and larger hardware.
In contrast, Protein A membranes provide high throughput without the need for oversizing. By operating at residence times of seconds rather than minutes, the cycle time can be reduced from several hours to several minutes, which enables a rapid multi-cycling approach. Operated under the rapid multi-cycling mode, these membranes can significantly increase process productivity, achieving >200 g/L/h. Table 1 highlights the significant advantage offered by Protein A membranes when running in rapid cycling mode in comparison to traditional resin chromatography operation. (Table 1)
Together with consistent performance over 100+ cycles (Figure 3), rapid multi-cycling enables right-sizing of the capture step based on capacity need. Moreover, the rapid multi-cycling approach promotes full utilization of the media life in a single batch without the need for amortization, ultimately improving process economics, especially in the consideration of drugs’ life cycle, including the large expenditure during clinical development. Fully utilizing media lifetime at a per batch/campaign basis is an effective way to mitigate high risk during clinical development, where a large investment in resin media will not be amortized if drug candidates fail the trials.
Cation Exchange (CEX) Membrane for High-Productivity Aggregate and HCP Clearance
The use of CEX membrane in flow-through mode delivers efficient aggregate and HCP removal at a 12.5x higher mAb load capacity compared with beads. This high productivity intermediate purification reduces process time and buffer consumption and allows:
- Right-sized purification media,
- Reduced equipment footprint, and
- True single-use operation.
Anion exchange (AEX) Membrane for Effective Impurity and Viral Clearance at Unmatched Load Capacity
The high ligand density on our advanced AEX membrane provides a more robust impurity removal and viral clearance and a >60x higher mAb load capacity than beads to further reduce media volume requirement (Figure 4). The impurity clearance performance (HCP and viral clearance) is independent of the load up to 20 kg/L, enabling downsizing of the unit operation. Good viral clearance is achieved over a range of buffer and pH conditions, which provide a wide design space for operation. Performance is maintained at high conductivity (10 mS/cm) even in phosphate buffer, which reduces feed dilution or buffer exchange requirement for a simplified process with reduced cost factors.
The combination of cation and anion membrane adsorbers enables high-throughput flow-through aggregate and impurity removal in a convenient, single-use format.
Conclusion
New, innovative membrane chromatography technology enables a new paradigm for mAb manufacturing, offering key benefits to address productivity and cost challenges:
- Critical quality attributes are comparable to reference resin processes
- Improved productivity enables smaller columns and facilities
- Novel Protein A membrane enables fully single-use mAb manufacturing
- Potential affinity membranes for other applications enables process intensification
- Fully single-use processes enable flexible, low-cost facilities and promote better facility utilization
Note: Content originally published in European Biopharmaceutical Review.