The production of viruses, whether for use as viral vaccines, viral vectors for gene therapy, or oncolytic applications, requires complex processes that can translate into high costs, as well as slow development timelines and time to market. In this article, we will present several case studies highlighting the advantages of process intensification using Natrix® single-use membrane chromatography to increase your productivity and reduce your capital and operational manufacturing costs.

The high binding and short residence times of Natrix single-use membrane chromatography, (used in all of the following case studies), are enabled by the combination of a non-woven reinforcing mesh skeleton coupled with a porous hydrogel containing functional groups. The skeleton provides the mechanical strength and durability, while the hydrogel creates a large 3D surface area containing a high density of functional groups with interconnected pores allowing for convective flow channels to achieve high flow rates. 

Transition to a Cost-Effective, Scalable Manufacturing Template

The first case study is based on our collaboration with the Jenner Institute at the University of Oxford. This project was undertaken to transition a labor and time-intensive process for lab-scale production of an adenovirus-based vaccine to a more cost-effective and scalable manufacturing platform suitable for GMP production. The lab-scale production process made use of shaker flasks, centrifugation, and ultracentrifugation and suffered from low throughput and overall process yield, scalability challenges, and high capital expenditure when transitioned to large scale production. 

The ultracentrifugation step was replaced with a NatriFlo® HD-Q strong ion exchanger. The tangential flow filtration (TFF) retentate of a vaccine candidate from the adenovirus vector platform was used for the evaluation. The anion exchanger was loaded to 6 × 1013 viral particles per mL membrane. In the gradient elution, the majority of the virus came off at approximately 22 µSiemens/cm, corresponding to a recovery of 76%, with host cell protein reduced by 85% (data not shown).

While the replacement of the ultracentrifugation with membrane chromatography was a one-for-one exchange, the overall productivity of the process was significantly improved, resulting in reduced capital investments at scale. Moreover, it became a fully single-use process that drastically reduced the downtime and expenditures associated with unit cleaning, storage, upkeep, and any associated validation requirement. 

Improve Productivity and Scalability without Compromising Purity

Compared to vaccines, oncolytic virotherapies require high quality and high purity for patient treatment via intravenous administration. In this project, we developed a high-productivity, scalable process for use with Newcastle Disease Virus (NDV) as an oncolytic virotherapy without compromising the purity. This work was done in collaboration with the University of Guelph. 

High-titer NDV was produced from pathogen-free embryonated chicken eggs. The traditional process included depth filtration, TFF, and sucrose-gradient ultracentrifugation with a process duration of nine hours plus two-day dialysis and PEG concentration to remove concentrated sucrose, which can be toxic when delivered intravenously. The lengthy process has low productivity, with a processing unit that is difficult and expensive to scale up (Figure 1).

Figure 1. Comparison of traditional and next-generation processes using membrane chromatography for the production of an oncolytic virus.

TFF and ultracentrifugation steps were replaced with NatriFlo HD-Q anion-exchange membrane. The entire purification process was completed in 30 minutes with greater than 90% recovery, compared to 9 hours and 65-70% recovery previously. This increase in recovery improved downstream throughput and process economics while enabling the downsizing of an upstream production — in this case, the number of eggs required — by sevenfold. NDV was concentrated 13x compared to the starting feed, while 99.9% HCP clearance (3.2 LRV) enabled single-step purification.

This process intensification strategy can help drive down costs by streamlining the production process and reducing the capital and operational costs required for a scaled-up manufacturing process. In the case of viruses produced in cell culture, downsizing of upstream processes, as a result of increased downstream recovery, could potentially enable the switch from a stainless-steel bioreactor to a single-use solution, resulting in significant capital expenditure and operational savings. 

Reducing the Manufacturing Footprint 

Process intensification can drive down the costs of traditional inactivated viral vaccines by reducing the overall manufacturing footprint. This project represents a collaboration with Univercells and Batavia Biosciences and was funded by the Bill & Melinda Gates Foundation in support of the World Health Organization’s (WHO) Global Polio Eradication Initiative. The objective was to deliver an intensified manufacturing platform, with a significantly reduced cost of goods (COGS), and ultimately improved the affordability of inactivated poliovirus vaccines for use in low- and middle-income countries.

The process integrated a number of advanced technologies, including a novel single-use fixed-bed bioreactor and an innovative cation exchange with HIC modality chromatography membrane (Natrix Sb), which allows single-step purification that increased the throughput by 80-fold. These high-productivity upstream and downstream technologies were integrated into an automated, continuous production platform with a small manufacturing footprint and low production costs. 

The single-step purification of one isotype (sIPV-3) is shown in Figure 2. The clarified sIPV feed was loaded to over 81,000 D-antigen units per mL membrane. At elution, the material was concentrated by 35-fold with very good recovery and a purity profile meeting the WHO’s requirement for HCP and DNA. With this high load and recovery, one cycle from the scale-down device-generated sufficient sIPV-3 material for over 9,000 doses of vaccine. With a 500 mL device for large scale manufacturing, we extrapolated that one cycle can supply 1.2 million doses of sIPV-3 material.

Figure 2. Single-step purification with Natrix Sb chromatography membrane.

The merging of high-productivity technologies in the upstream and downstream process enables the entire production process to be self-contained into a series of six-square-meter isolators that can be placed in a BSL-3 pod. The intensified, integrated process enabled a significant reduction in their manufacturing footprint and enabled the realization of pod-based micro-facilities. With less than $25 million in capital investments in the facility and reduced operational costs, it is now possible to supply 40 million doses of trivalent sIPV vaccine annually, at less than $0.30 per dose, which is a five-fold reduction compared to the current process and output.

Exploring the Power of Single-Use Affinity Chromatography

For many viruses that are expressed in more complex systems, traditional purification process usually involves multiple steps including precipitation, centrifugation, gradient ultracentrifugation, size exclusion, and ion-exchange chromatography to achieve the target purity, but at the expense of low overall process yield, high capital and operational cost, as well as limited process flexibility and robustness. Purification process intensification, in this case, may require a more powerful separation technique to streamline the process without compromising critical quality attributes. Moreover, due to the diversity of virus types and different production methods used in upstream, many times a unique purification process is required for every virus type and sometimes even every strain. The large investment of time and money in the downstream process and facility development recurs when new molecules join the pipeline. To address these challenges, one possibility is to explore affinity-based development and manufacturing platform. Affinity chromatography is one of the most powerful separation techniques, thanks to the highly specific interactions between target molecules and immobilized ligand. It potentially enables a platformable purification strategy where each molecule can be purified with a different affinity media followed by one or two polishing steps, but the overall downstream scheme and facility only require minor or no modification. When coupled with high productivity single-use technology, the cost savings and flexibility can be improved even further.   

As an outlook for potential future improvement, in collaboration with the University of Guelph, we conducted a proof of concept study for purifying viruses with affinity membranes. A commercially available ligand was coupled to the activated membrane. Influenza virus produced in cell culture was loaded to the membrane at 2 × 106 HA unit per mL membrane; at elution, 95% recovery was achieved, with over 99% HCP removed and 30-fold virus concentration (Figure 3). 

Figure 3. Purification of egg-based influenza virus using affinity membrane chromatography delivered a 30-fold concentration and 99.9% HCP clearance (3LRV).

In extrapolation, allantoic fluid harvested from one million eggs could be purified in one eight-hour shift with a 500 mL affinity membrane device. Compared to affinity resin using the same commercially available ligand, the productivity can be significantly improved because the cycle time is shortened due to the reduced residence time. Comparing current manufacturing approaches for active influenza virus, the multi-step purification process can be simplified into a clarification step followed by an affinity chromatography step, with the final material meeting the purity requirements and sent to inactivation for further processing.

Conclusion

As demonstrated in these case studies, process intensification using membrane chromatography can deliver significant advantages for a variety of virus production needs.

To learn more about these case studies and NatriFlo HD-Q membrane chromatography, please visit: www.emdmillipore.com/natrix

 

Ranjeet Patil is a Segment Head for Vaccines and Gene Therapies and Mochao Zhao is a Global Product Manager for Single-Use Membrane Chromatography, both with MilliporeSigma.

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