The journey from gene to clinic for a protein therapeutic is long and sometimes torturous, requiring a wide range of knowledge, expertise and capabilities. An industry-wide imperative to accelerate the journey with the same or fewer resources simply adds more pressure. 

Below, we describe a novel approach for the “pragmatic” implementation of single-use technologies in templated downstream processes for monoclonal antibody (MAb) production. We describe how this approach reduces process development time, reduces the time and effort required for equipment specification and procurement, and most importantly, enables these activities to be conducted in parallel thus shortening overall project timelines.

Template Processes + Single-Use

Developing a downstream process for MAb purification is considered to be a “solved problem”. The use of template processes is common among large manufacturers based on experience with multiple molecules. The advantages of applying a template approach to the downstream processing of MAbs are well known and include standardization of unit operations, buffers, certain operating parameters, and equipment. Standardization allows streamlining and minimizing process development, rapid scale-up and facilitation of technology transfer.

In parallel with a template approach to downstream processing, we are seeing increased adoption of single-use technology, especially in the area of small-scale or clinical-scale production. The drivers for this adoption are numerous:

• Reduce/eliminate cleaning and cleaning validation costs

• Eliminate concerns of carryover

• Reduce turnaround time between batches/campaigns

• Facilitate duplication of suites in multiple locations

• Enable use of same equipment with various MAbs resulting in flexibility, rapid scale-up and reduced operator training

While the evidence for the benefit of single-use systems continues to grow, examples for the entire downstream process train have been limited.

The Need for Speed

In the development of clinical supply, speed is more important than creation of a fully optimized process. Clearly the process must deliver material of acceptable quality, yield, and purity but optimization for a commercial process can be left until the molecule shows promise in the clinic. As such, a pragmatic approach to minimize time and effort makes sense.

Such an approach can be facilitated through a combination of single-use technologies and a template approach to downstream processing in which parameters are already pre-selected to reduce process development efforts. This approach is expected to be of particular value for smaller, emerging companies who are unlikely to have the experience needed to establish a downstream processing template. Further, use of pre-packaged devices, systems, and ancillaries can reduce the time and effort required for specification, procurement, and installation.

Process Development Benefits

We can divide process development into two elements – effort and risk. “Effort” reflects the number of trials or experiments that need to be performed to screen multiple products and device alternatives from various vendors and the number of operating parameters that need to be determined. Taking one unit operation as an example, we may have two to three different vendor products to screen, a couple of different types from each vendor and multiple operating parameters to determine. For example, with clarification filters, flux and capacity need to be determined, while with chromatography resins, buffer composition, residence time and capacity all need to be assessed. “Risk” encompasses what protocol is to be followed, what data to collect, what analyses to perform and what scale-up rules to follow. These factors will apply to each unit operation and the effort and risk will multiply by the number of unit operations.

In the approach we describe below, we have pre-selected from our range of purification and filtration devices, based on experience and pre-selected certain operating parameters. In doing so, we have effectively reduced the number of devices and parameters to screen.  At the same time pre-configured standard systems reduce the specification, delivery, and implementation time and effort. The risk element has also been considerably reduced through the provision of protocols, data collection, analysis, and scale-up tools.

Proof of Principle

To demonstrate proof of principle, we undertook process development for two different monoclonal antibodies from two different cell lines. We then scaled one of the processes to the 100L level using the sizing and device selection tools we have developed. These allowed us, based on process development data, to determine not only the membrane area or resin volume required for a particular bioreactor volume and titer, but also the specific device and system size, and associated bag and mixer sizes from our Mobius single-use range.

 

Figure 1. Impurity clearance for two different bench scale
monoclonal antibody production runs.
Specification limits are indicated by horizontal red lines

Figure 1 shows that the process delivered effective clearance of impurities for both of the monoclonal antibody feedstreams at bench scales; impurity levels were below targeted levels at the end of the process. We used bulk drug substance specifications based on typical industry values of <10 ppm for host cell protein and leached protein A and <10 ppb for DNA.

Figure 2.  Impurity clearance for scaled up process.

We then scaled up the process in a 100L bioreactor for one of the antibodies. Figure 2 compares bench and pilot scale data. Similar impurity clearance profiles indicate the process readily and robustly scales. Figure 3 compares yield from bench and process scale. Overall yield at both scales was approximately 85%, which is very satisfactory for a clinical scale process. Product quality attributes were also assessed. As shown in Figure 4, there were no differences in the change variant distribution pattern between scales or between unit operations. Distribution of change variants was very similar in final pool from both scales.

Figure 3. Comparison of yields from bench and process scale.

The overall process required five months from start to finish. Process development time required three months during which all assays and a confirmatory run of the final process at bench scale was completed. The preparation time for pilot scale required about one month and included a water run which was found to be exceedingly helpful to confirm the workflow and ensure everything fit properly together. The two pilot scale runs, including assays, required another month.

Figure 4.  Distribution of change variants was unaffected by unit operations
at both scales. Carboxypeptidase B digestion confirmed that basic peaks are HC C terminal lysine variations.

Table 1 summarizes the hardware and disposables used in the pilot scale runs. Note that only one chromatography system was required. Although the process had three chromatography steps in sequence, only one was needed due to the disposable flow path.

Table 1.  Summary of hardware and disposables used in pilot scale runs.
Note that this does not include chromatography resins or TFF cassettes which
are typically used for a number of batches.

This project demonstrates the value of combining a process template with a pre-configured, single-use process train. Benefits included reduced time and effort for process development as well as equipment specification and procurement, which translated into sizable time and resource savings.

Lessons Learned/Conclusion

The need to develop a clinical scale process for a new monoclonal antibody provided a further case study to explore the benefits of the Mobius ClinicReady for MAbs approach.

Here the challenge was to deliver a clinical GMP process, within 12 months, involving process development as well as facility design and construction based on implementation of single-use systems.

Minimal fitting was required for the downstream process development to the Mobius ClinicReady Template, enabling completion of process development and start of pre-clinical production within a six-month time frame.

Meanwhile, the facility construction was underway. Key was the fact that in parallel with these activities, the selection, specification and ordering of the clinical scale devices, systems and ancillaries, including mixer and bag sizes, was able to be completed, using the sizing and selection tools developed as part of the Mobius ClinicReady for MAbs package. In this case the downstream production train was matched to the requirements of a CellReady 200L single-use bioreactor.

This meant that as soon as the clean rooms were completed and qualified we were able to install the single-use equipment, validate it and be ready to begin clinical scale manufacturing. The ability to perform the key phases in parallel resulted in achieving our aggressive 12 month target.

In comparison to a traditional stainless steel facility, we estimate time savings of at least 6 months and a cost savings of approximately EU 3M on an overall project cost of EU 8-10M. The major savings come from reduced utility requirements and no CIP requirement.

In summary, this pragmatic approach to process development and clinical scale specification and implementation can deliver benefits in reducing effort and shortening timelines to produce clincial material. Much of the complexity around getting a molecule to clinic is removed, and clients are able to accomplish their goals while allowing them to focus on their product and less on process specifics.

Acknowledgements

The authors wish to acknowledge the work of Elizabeth Goodrich, Sue Walker and Venkatesh Natarajan from the EMD Millipore, Biomanufacturing Sciences Network, Applications Engineering Group, for performing the proof of concept studies.