The biomanufacturing industry is under increasing pressure to boost productivity and reduce the cost of biologics. Process intensification is an important strategy that offers many benefits toward meeting these goals. It provides the technical benefits of raising viable cell density, increasing product yield, and/or reducing batch length for the same productivity. From an operational perspective, process intensification offers a way to increase capacity while utilizing existing footprint and equipment. With these productivity improvements, process intensification also provides a path for reducing product cost and decreasing time to market, providing a business benefit as well.

In this Ask the Expert session, we spoke with Earl Pineda, Repligen about the benefits of upstream process intensification and how the XCell^® ATF System can ease implementation and speed success.

Our questions and answers are below.

What are the benefits of process intensification?

Process intensification has been around since the 1980s.  It was originally intended for the continuous harvest of unstable proteins, but it is now being used more broadly in different modalities for both productivity and throughput improvements.

Process intensification is primarily used for three technical benefits in standard steady-state perfusion or short-term perfusion:

• To increase cell culture density
• To increase titer of the molecule or product of interest.
• Steady state N-perfusion can be long and much more productive per run, or intensified N-1 perfusion coupled with high-density seeding of a fed-batch can achieve the same productivity over a shorter run enabling more batches in a campaign.

Process intensification provides the operational benefit of increasing capacity in an existing facility footprint by increasing productivity using current unit operations and equipment.

Lastly on the business side, by moving from a batch to an intensified perfusion process, you can reduce the cost per gram per month for an antibody or other product of interest. Depending on the phase of development, you might be able to decrease the time to market. For example, if you are running a clinical batch with process intensification, you could produce enough product in a single run as opposed to multiple fed batches that may suffer from batch-to-batch variation.

There can also be environmental benefits of process intensification, including reduced energy use, reduced waste, reduced reagent use, and a smaller facility footprint.

When implementing process intensification in upstream processes are there any hurdles that companies should be aware of?

Implementing process intensification can feel challenging for some who have not done it before. If someone has worked in fed-batch for a long time, perfusion requires a bit of a mind shift. Perfusion can appear to be more complex than fed-batch because you must manage liquid handling of fresh media and spent media and level control of the bioreactor working volume.  But once you have everything in place, it’s like cruise control.

Repligen has done a great job of simplifying the change from batch operation to perfusion mode by providing equipment that is plug-and-play, especially in commercial scale manufacturing. XCell ATF System is a great example of this, as it provides a simple process intensification solution that is easy to implement, scale, and operate.

Are there any ways to simplify process intensification for upstream processes?

As I mentioned in the last question, the XCell ATF System really does ease the transition to process intensification. I have been working on the XCell ATF System over the last 20 years and since then it has been adopted as the gold standard for cell retention in the biopharma industry. A key reason for this is the efficiency and simplification of the perfusion operation that comes with this technology.

Regardless of what the end user selects for intensification, there are a few considerations that are worth looking at when implementing process intensification.

Understanding your facility constraints to target the appropriate cell densities and productivity targets.

It is important to know your facility so that you can target where you want to be in terms of the degree of intensification, more specifically the cell densities that you want to achieve. You may be able to reach 100-200 million cells per milliliter for example, but you need to be sure that your facility can handle large volumetric changes to the operation and that your bioreactor is configured to support such high cell densities.

Ensure proper development at bench-scale and linear scale-up to process.

Another area to consider is proper development at bench scale so that the process is scaled up accordingly and you eventually achieve the same results at your manufacturing scale. A good supplier partner can really help here by working with you to make sure that the process scales correctly.

Understanding that the process control is different between batch and continuous modes of operation.

Finally, process control and automation are slightly different between batch and continuous operations, so it is important to be sure that you are aware of these upfront. Again, a good supplier partner can work with you so that you are fully aware of any differences.

How does Alternating Tangential Flow work and why is it better?

Most people are familiar with tangential flow filtration, if you look at alternating tangential flow (ATF), it is attained by the action of a diaphragm pump moving upward (pressure stroke) and downward (exhaust stroke). This bi-directional flow results in a back flush action that keeps the filter clean and allows for longer filter life, making the ATF a very simple and effective technology to boost productivity.

When the filter is continuously cleaned, it enables the continuous operation to run reliably for runs as short as 5 days with N-1 intensification to significantly longer N-bioreactor perfusion runs, in some cases over 60 days with a single or no filter changeout.

In addition, some perfusion technologies can take up an entire room at production scale, but the ATF footprint is much smaller, imagine less than that of a refrigerator. So, it is a small addition to a company’s cell culture operation and is a technology that has been known and tested as reliable for over a decade.

What applications are best suited for the XCell ATF System?

The two most common applications are N-1 perfusion and long-term perfusion. With N-1 perfusion we are typically taking an existing facility and looking at how we can increase the seeding density of the final production bioreactor. It is a short 5-day process and by using ATF you can increase the viable cell density in the seed train so that you are starting at a much higher VCD in the bioreactor. This approach leads to immediate process gains. N-1 perfusion boosts throughput and therefore the number of clinical molecules while having the least amount of impact on process/facility/equipment change. Intensified seed trains can reduce whole unit operations and time to production bioreactor, in some cases by up to 15-30 days

The second application, long-term perfusion, is run at a desired fixed viable cell density in steady-state production and generates significantly more product as a function of time as compared to traditional fed-batch cell culture. With long-term perfusion, you can mimic your fed-batch process by running continuously for two weeks or up to 60 days or longer depending on how much product you want to generate from the process. There is also a modified fed-batch application called high productivity harvest (HPH). In that application, you are modifying an existing fed-batch process and introducing continuous operations toward the end of the run to prevent the fed-batch culture from crashing and get an extra boost in productivity.

There are also other applications including media exchange for gene therapy prior to the infection/transfection step or cell expansion for cell therapy where the cells are the product.

Is the XCell ATF System scalable?

The ATF technology has been designed to be linearly scalable, which differentiates it from other cell retention technologies in the bioprocess market.  We have identified key scale-up parameters that when equalized from bench scale to manufacturing scale, produce the same results at all scales.  Furthermore, Repligen filters are designed with certain specifications such as path length and ID that facilitate this linear scalability.

And from a controller perspective, the team at Repligen has worked diligently over the last few years to modernize the controllers and develop the XCell^® Lab Controller and XCell^® Large Scale Controller, which is a next-generation platform. The algorithms that drive the ATF device operation are identical between bench scale and manufacturing scale. This ensures equivalency in ATF control at all scales and enables a smooth tech transfer when the time comes.

The XCell Lab Controller is compatible with XCell ATF devices ATF1, ATF2, and ATF4 which can accommodate bioreactors from 0.5L to 20L, while the XCell LS Controller is compatible with ATF4, ATF6, and ATF10 devices which can accommodate bioreactors 50L up to 5000L, which the largest commercial implementation of ATF that we are aware of.

What kind of molecule types has the XCell ATF System been used with?

The XCell ATF System is now being used in over 500 sites globally, so it works with a wide range of molecules. The most common molecules are monoclonal antibodies, where it is in use in over 40 commercial upstream processes. Other popular applications are recombinant proteins, viral vectors, vaccines, and cell therapies.

If you look at cell lines, the XCell ATF System is in use with over 25 different cell lines including CHO, HEK-293, SF9, Per.C6, iPSC, and hPSC. It is even being employed in the cultivated food market to reduce the cost per pound of cultivated meats.

For customers who are considering the XCell ATF System for process intensification, is training and support available?

Yes, Repligen has field application scientists who are highly trained and experienced in intensified cell culture and provide customer end-to-end support.  They come in and look at the customer’s process and can advise on the best options for process intensification, based on the product of interest, yield targets, the physical setup and facility, and business needs. The FAS work with the end user from start to finish to ensure that the end user is comfortable with the process that has been developed and that it is ready to scale.

Repligen also has a very strong technical sales support team that is ready to assist in moving the process forward with project engineering support to ensure the process is scaled appropriately. For instance, if the end user is going to a GMP or clinical facility, the team makes sure that the customer has selected the right equipment, the automation, and the hardware to run the process as designed.

After this, post-sales support is available through Repligen’s field service and applications teams. With broad and deep experience across different functions from applications to engineering, Repligen offers complete support. Many customers have never heard of ATF, yet we can get them moving on their project very quickly by employing the full range of support and expertise that we offer.

About the Expert:

Earl Pineda is based in France and is currently managing the Field Applications team for Europe and Asia at Repligen.  Earl has been in the biotech industry for about 20 years, and since working at Refine Technologies and now at Repligen, he has helped hundreds of companies successfully implement the XCell® ATF technology all over Europe and Asia over the past 13 years.  He has previously worked at major biotech companies like Amgen, Novartis, and Biomarin, within different research functions in process development, MSAT, and tech transfer teams.  An expert in perfusion process, Earl has vast experience with cell retention technologies, including ATF, TFF, gravitational cell settlers, acoustic cell retention, and microcarrier-based systems.  At Biomarin, Earl helped develop several now commercial perfusion processes for Aldurazyme and Naglazyme.  At Refine and Repligen, Earl has helped develop the Single-use ATF and has also designed the user interfaces for several ATF controllers, including C24 and the new XCell Lab and XCell Large Scale.