A Novel Downstream Monoclonal Antibody Purification Template to Address the BPOG Biomanufacturing Technology Roadmap

• MilliporeSigma

The release of the BioPhorum Operations Group (BPOG) Biomanufacturing Technology Roadmap¹ in July 2017 presented the biopharmaceutical industry with a challenge: to innovate, collaborate, and deliver groundbreaking solutions that will allow manufacturers to dramatically increase production flexibility, speed, and quality while decreasing overall product cost for monoclonal antibodies (mAbs). The roadmap proposes multiple scenarios for drug substance production, for which significant advances in upstream and downstream consumables, systems, and controls are required to enable successful implementation.

Analyses of three of the roadmap scenarios were performed for the commercial production of mAb drug substance, focusing on downstream purification. A novel process template is proposed to meet the targeted volume and mass throughput, process and changeover times, and operational modes — across the breadth of batch, connected, and continuous operations.

The Biomanufacturing Technology Roadmap Scenarios

The BPOG Biomanufacturing Technology Roadmap presents 5- and 10-year visions for facilities and processes that achieve step changes in productivity, capital and operating cost requirements, quality, and reliability. As examples, ultimate goals included a 90% reduction in capital expenditures for new facility construction, a 70% reduction in construction timelines, a 90% reduction in changeover time between products in a facility, and a 10-fold improvement in process robustness as measured by quality outcomes.

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Globally, regulatory agencies are simultaneously promoting new process development efforts and have set up committees and working groups to meet in anticipation of new technology implementation to discuss validation strategies for continuous processing of biological products. Several continuous processes for small molecule therapies have already been approved.

To map out the technologies and capabilities required to meet its vision, BPOG proposed five manufacturing scenarios for production of mAb drug substance. While this article will deal primarily with the requirements for the manufacturing process, a transformation across multiple operational disciplines is required to meet the desired goals. Relevant domains include single-use systems, automation and controls, process analytical technologies (PATs), and software and data management systems. Figure 1 illustrates the required evolution for these disciplines to transform a batch process into a fully continuous process.

Matching Roadmap Scenarios With Optimal Downstream Processes

Three of the roadmap scenarios were intended for intermediate-to-large scale production of 10 to >1,000 kg mAbs per year, typically suitable for clinical and commercial mAb supply. For these three scenarios, a detailed analysis was performed to determine the downstream purification processes that would best match the upstream bioreactor processes and the manufacturing goals set out for each scenario. Where ranges for bioreactor volume, product titer, and process scheduling were given, the most challenging state was considered in order to fully challenge the recommended process. A summary of the three scenarios is given below.

1. Scenario 1 considers an existing facility with 12.5 kL stainless steel bioreactors operated in fed-batch mode. Due to intensification of the seed train process, the production bioreactor produces 10 g/L mAb with 25e6 cell/mL at harvest. The facility has a single downstream processing (DSP) train with one of six bioreactors harvested every 3.5 days. The facility operates 24 hours per day and 7 days per week. This scenario provides greater than 1 metric ton of a single mAb product per year.
2. Scenario 2 is a 2 kL perfusion, single-use bioreactor operated with a cell retention device that allows continuous harvest of the mAb product at a titer of 4 g/L, operating at a perfusion rate of 1.5 vessel volumes per day. The downstream process must also be continuous for the 60-day duration of the bioreactor run. While the facility is open 7 days per week, the process must be “lights out,” allowing for automated operation at night without operator oversight. The scenario provides approximately 100 kg/yr of mAb with the option of running several products in the facility.
3. Scenario 3 considers a 2 kL fed-batch, single-use bioreactor operated with an ultrafiltration cell retention device that retains both the cells and the mAb product. At harvest, the product titer is 20 g/L at a final cell density of 100e6 cells/mL. As in scenario 1, a bioreactor is harvested every 3.5 days using a single DSP train, and the facility operates 24 hours per day and 7 days per week. The scenario provides approximately 10 kg/yr of multiple mAb products, likely for clinical supply.

A Unified DSP Template for Next Generation Process Scenarios

While these three process scenarios differ significantly upstream, the design of corresponding downstream processes that enable connected and continuous processing can realize significant conservation of core technologies and operating methods. Consideration is given to each set of operations below.

Clarification

The optimal methods for harvest and clarification of the cell culture fluid are likely the most disparate among the three scenarios. At the 12.5 kL fed-batch bioreactor scale (Scenario 1), centrifugation remains the most suitable technology for solid/liquid separation. Depth filtration will be required for secondary clarification. Flocculation should be considered to increase the efficiency of the centrifugation process and of colloid removal by the depth filters.²

For the 2 kL perfusion bioreactor (Scenario 2), the cell retention device provides a clarified harvest stream on a continuous basis. Secondary clarification via depth filtration is required in some cases to reduce colloids or act as a guard against column fouling in the capture step. For the 2 kL fed-batch bioreactor (Scenario 3), flocculation followed by 2-stage depth filtration offers robust clarification at reasonable filter capacities, even at an elevated cell titer of 100e6 cell/mL.³ Sterile filtration ahead of the capture step acts as a bioburden reduction and column guard step across all three scenarios.

Capture

Protein A remains the robust workhorse for mAb capture processes, with two options for intensification. Protein A multicolumn chromatography (MCC) enables continuous loading of clarified cell culture harvest with a reduction in resin and buffer volumes of approximately 20%. Process times can also be reduced through the implementation of higher loading flowrates in the case of rigid resins.⁴ MCC does introduce additional process complexity with the chromatography system, the control scheme, and the need to pack and qualify multiple columns for a single operation. However, for the 12.5 kL bioreactor Scenario 1 dedicated to the production of single product, the protein A lifetime can be fully utilized over the course of many batches, making this a cost-effective operation.

For Scenarios 2 and 3, where smaller batches of multiple products need to be processed through the facility, a single-use capture option would be cost-advantageous. Several suppliers are currently developing protein A membranes with very short residence times for product capture.⁵ These devices can be rapidly cycled on a semi-continuous basis to utilize the full lifetime of the protein A in a single batch. The typical result is 10- to 30-fold increases in productivity as measured in grams of mAb purified per liter of media per hour.⁶ This type of single-use device could be easily integrated into disposable assemblies designed for processing of a single lot with rapid changeover for the next lot or product. This option has the added advantage of eliminating the need for, and cost of, cleaning and storage of a protein A resin column.

Continuous Inline Virus Inactivation

As a continuous inline process, replacement of traditional low pH virus inactivation, performed batch-wise using two to three tanks for product titration and hold, was found to be advantageous for all scenarios.

Continuous inline virus inactivation allows for inline titration of the protein A eluate, incubation in a coiled tube chamber for ~10 min, and inline neutralization under constant flow conditions.⁷ Rapid virus inactivation kinetics have been demonstrated with greater than 5 logs of xenotropic murine leukemia virus-related virus (XMuLV) and pseudorabies virus (PRV) reduction in ≤5 minutes at pH ≤3.7. Product quality can be enhanced by reducing the product’s residence time under unstable conditions. The flowrate and incubation chamber volume are matched to the process volume and validated low pH hold time. This design is easily accommodated for single-use operation, reduces facility footprint, and reduces process time while achieving robust virus inactivation.

Flow-Through Polishing

For all three scenarios, elimination of intermediate hold steps in the polishing sequence offers significant opportunities to connect multiple operations into a constant flowrate, continuous process. To achieve this, bind-and-elute chromatography - which effectively introduces a hold step during the binding and column wash steps - and intermediate product hold tanks must be eliminated. Polishing operations must focus on the specific binding or filtration of trace product- and process-related impurities.

These may include mAb aggregates, host cell DNA and protein, leached protein A, and/or viruses. A novel flow-through polishing operation has been proposed using activated carbon filtration, anion and cation flowthrough chromatography, and virus filtration. This combination achieves product quality requirements with significantly reduced process times, buffer volumes, and facility footprint.⁸ Early testing of this method indicates effective scalability to the clinical scale with existing technology that could be implemented for the proposed scenarios.⁹

Continuous Ultrafiltration and Diafiltration

The final step of the mAb purification process is the concentration and diafiltration of the product into its formulation buffer. Continuous concentration via single-pass tangential flow filtration (SPTFF) has proven to be a robust process for implementation throughout the purification process; it can be used for initial concentration and final over-concentration steps on either side of the diafiltration step.¹⁰ Methods for continuous diafiltration have been proposed, including multistage membrane devices and a tank cycling technique.¹¹

The combination of single-pass TFF and continuous diafiltration methods allows for continuous production of formulated drug substance. Selection of these technologies should depend on the goals of the process impacted by the manufacturer’s definition of a batch or lot of mAb product. For instance, if the batch is defined by mass, then a fixed volume of product may be collected before performing a batch ultrafiltration/diafiltration (UF/DF) process. Such processes may leverage single-pass TFF for overconcentration of the formulated drug substance. If the batch is defined by process time, on the other hand, continuous production of formulated drug substance using single-pass TFF and continuous diafiltration would be advantageous.

It should be further noted that single-pass TFF has seen implementation throughout the purification process. In front of the protein A capture, concentration of the clarified harvest reduces loading times, increasing productivity by 50 to 100 percent. Likewise, concentration of the feed to the anion exchange chromatography step using single-pass TFF increases the capacity of the media for trace impurities while decreasing load times.¹²

Integration of an Intensified Downstream Purification Process

The technologies and methods for an intensified downstream purification process have come into focus, but the challenges of integrating these operations into an optimized process remain. From the analysis performed, the following key decision and gaps were identified as critical.

Decision: Intensified, Connected or Continuous?

The technologies and methods discussed above compose a toolbox from which a process can be constructed to meet the requirements for the molecule (stability, quality profile, etc.), facility, production capacity, and cost. Continuous processing is not a goal, in itself, but may be a solution for a given case. The ultimate decision with respect to intensified, connected, or continuous processing is often a business decision based on specific constraints of a biomanufacturer’s available infrastructure, process experience, digital capabilities, and capacity requirements.

For an existing facility, a combination of flow-through polishing chromatography and single-pass TFF can significantly reduce the need for intermediate hold tanks and process pool volumes. For a facility with a perfusion bioreactor for mAb production, implementing a continuous capture and virus inactivation step may offer the flexibility required without the need for continuous polishing or UF/DF operations. Where maximizing capacity is critical, a fully continuous downstream process may be the best option. Using the toolbox approach incrementally to produce a hybrid process of intensified or continuous steps allows one to reduce the development costs and risks of implementing new process technologies.

Technology Gaps to Be Addressed

Single-use technologies

Single-use, pre-sterilized devices and flowpaths are key enablers for continuous processing. For long-duration processes - 60 days or more with perfusion bioreactors - bioburden control is critical, and sterile devices and flowpaths eliminate a significant risk to sterility. The ability to exchange single-use devices when capacity is exhausted offers an advantage over multi-use technologies. At this time, gamma-stable chromatography, virus filtration, and depth filter devices that can be incorporated into single-use assemblies are needed but are not widely available in the industry.

Membrane chromatography

With the advent of protein A membrane chromatography and the need for single-use devices in continuous processing, the time for implementation of membrane chromatography throughout the downstream process has come. These devices will need to be high-throughput, capable to deal with elevated product and impurity loading from cell culture fluid, to address the extended batch times of the intensified upstream processes. Gammastability will also be required to allow for sterile installation and operation.

Automation and controls

Development and commercialization of systems, automation, and software platforms that exploit the benefits of new separation technologies and methods are the final step toward integrating these individual operations into connected and continuous processes. Automation and control schemes must be enabled with both feed-forward and feed-back process control. Minimizing process disruptions and preserving process parameters within the validated operating space will assure product quality and reproducibility.

While this is not an exhaustive list, other notable gaps include: suitable process analytical technologies, regulatory guidance and experience, and validated scale-down models for process development and representative applications studies.

Summary

A new template for downstream mAb purification processes has been proposed which can be tailored to operate across a wide range of process volumes for batch, connected, or fully continuous processing. This template consists of intensified harvest operations with flocculation for clarification; multicolumn or rapid cycling membrane protein A chromatography for capture; continuous inline virus inactivation; flow-through polishing; and continuous UF/DF.

Intensification of each of these operations offers benefits in cost and risk reduction, flexibility, and process time savings. The technologies and methods can then be assembled into connected and continuous processes as required to meet operational, facility, and business needs. Implementation of this new DSP template will require single-use, pre-sterilized devices and flow paths to enable continuous processing, high-throughput, membrane-based separation technologies. It will also necessitate the development and commercialization of systems, automation, and software platforms that exploit the benefits of new technologies while maintaining stringent process control.

Author Biography

Mike Felo has 20 years of biopharmaceutical industry experience. With an undergraduate degree in chemical engineering and a master’s in biotechnology from the University of Pennsylvania, Felo began his career in clinical, GMP manufacturing before tackling late-stage process development and technology transfer. Previously the head of single-use solutions at MilliporeSigma, he is now the director of downstream process integration, assembling the portfolio of technologies and products enabling intensified, connected and continuous purification of monoclonal antibodies.

References

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