Utilizing N-1 Perfusion to Increase Productivity and Intensify Processes

Perfusion technology is a tool to increase productivity that can be applied at different stages during upstream cell culture for biotherapeutics products. Perfusion allows removal of waste by-products and replenishment of fresh nutrients so that the cells are able to grow and reach higher cell densities and higher productivity. N-1 perfusion, the application of the perfusion technology in the step immediately before the production bioreactor, is a good option for processes that exhibit low productivity. Join Claudia Berdugo, Ph.D., Director of Process Development at Catalent Biologics, where she discusses N-1 perfusion and how Catalent Biologics works toward process improvements for each client to enable them to move their pharmaceutical products forward with confidence.

Question 1

What is perfusion and what is N-1 Perfusion?

Perfusion is a technology that allows removal of waste by-products and replenish with fresh nutrients so the cells always have a nice environment to grow and can reach higher cell densities and higher productivity. N-1 refers to the step previous to the production bioreactor, N-1 perfusion is the application of the perfusion technology in the step immediately before the production bioreactor, since higher cell density will be reached in the N-1 stage the production reactor or N stage can be inoculated at a higher seed density than with a traditional seed train.

Question 2

What other applications can perfusion be used for besides N-1?

Perfusion technology is a tool to increase productivity and can be applied at different stages in the upstream cell culture, for example :

Cell banks: cells can be grown in creating cell banks since higher cell density can be obtained large number of vials can be generated or bags at a very high cell density that can be used later to start the seed train or even the production bioreactor

Seed train intensification: Higher cell density reached as we pass cells previous to the reactor and can inoculate reactor with higher cell density, in addition bioreactor steps can be skipped

Production bioreactor: Perfusion can be applied in the production reactor at different production modes

  • Concentrated fed batch: where no  product is removed
  • Steady State: constant removal of product and cells reach steady state concentration
  • Dynamic Perfusion: product can be removed and cells reach high peak VCD

Question 3

What are the benefits of N-1 perfusion? Why do some companies want use N-1 perfusion as part of their process?

As the bioreactor is inoculated with higher cell density the duration of the bioreactor can be reduced achieving the same titer as in fed batch, alternatively higher titer can be reached with a longer duration in the feed batch when inoculated at higher cell density from a perfusion N-1

Question 4

Can you speak to the media consumption and if the additional media cost is offset by other factors?

Media consumption in perfusion and N-1 perfusion are key points to consider and need to be optimized to avoid impact on CoG. Media manufacturers have been working on potential solutions to provide media suitable for the high VCDs and consequently high metabolism expected in this mode of culture. Specifically, with N-1 perfusion, the impact on CoG is less severe than in production perfusion because the perfusion mode is shorter (usually 5 to 7 days), and the volume is lower than a production reactor. Traditional media used for Fed Batch can still be used for N-1 perfusion, and the cost of media consumption can be offset by reducing the duration of the production bioreactor and for the higher productivity expected from the production bioreactor inoculated at higher cell density from N-1 perfusion.

Question 5

What types of programs are the best fit for N-1 perfusion?

N-1 perfusion Is a good option for processes with really low productivity. We work with our customers to evaluate their needs to determine which approach best fits their molecule and program goals.

Question 6

What will be the next steps for the future of N-1 perfusion technology?

N-1 perfusion can be incorporated into a production process in a variety of ways.  It can be incorporated into process intensification as part of a fully continuous process.  Or it can be performed in a hybrid batch/continuous process in which the upstream and/or downstream processes remain in batch mode. The main goal is to increase productivity and facility utilization.

Question 7

For seed train expansion, do you find that you need a more complex media with perfusion? Have you found any supplements that are very useful to support high densities?

Media optimization is highly recommended for perfusion processes. Customized media can be designed with the right balance of components appropriate for high cell density cultures. Every process may require a different balance of nutrients for production, but for N-1 perfusion, it is likely that a base media with specific supplementation works well to achieve the goal of increasing cell density while avoiding accumulation of toxic metabolites for the cells. For production perfusion, the optimization may need to include additional supplementation to maintain growth and achieve the desired product quality.

Question 8

How much automation are you able to incorporate into your perfusion processes for production? Can it be a fully automated or close to fully automated process?

The industry is working toward solutions that enable fully automated processes that will allow better control and improve robustness of the process. Automation includes various components. Some of them are well established and routinely used in perfusion, such as capacitance probes. Other components that are part of perfusion systems require additional work to be integrated with the bioreactor systems. For example, automated cell bleeding can be achieved by integrating a continuous pump with an on-line cell density probe. For production perfusion, advanced automation tools are being developed to monitor product quality as well. A collaborative approach is needed between vendors and facility owners to achieve full automation of N-1 and perfusion processes.


Question 9

We are having difficulty in maintaining a constant working volume in our production bioreactors. We have been trying to balance the harvest and feed pumps at a consistent perfusion rate, but we keep seeing that this is not maintaining the same working volume.

Different factors can contribute to challenges to maintain constant volume, including pump accuracy. When pumps are operating for long periods of time, calibration may be compromised and may need a flow sensor. Controlling working volume weight is a good alternative that can be achieved by having a weight cell in the bioreactor.

Question 10

Have you had any issues with regulatory validation of N-1 perfusion processes?

Perfusion processes have specific aspects to be considered for process validation that are different from traditional fed batch processes such as: extended cell generations and associated genetic stability during production, consistent process operation, and product characterization consistency at all stages of the production process. Other process validation guidance should be considered and properly applied to N-1 perfusion and production perfusion processes.

Question 11

What production scale is best suited for perfusion in manufacturing?

The production scale should be defined based on the needs of the program. Production perfusion has been demonstrated by different companies in scales that include, 200L, 500L, 1,000L and 2,000L for clinical and commercial production.

Question 12

How do you recommend determining the starting perfusion rate for an antibody production process?

Perfusion rate depends on cell line and culture media. It should be estimated for optimal growth rate and productivity. A good source that advises steps to determine perfusion rate is here: https://www.repligen.com/technologies/xcell-atf/FAQ-perfusion

Question 13

We are having an issue with foaming. Could you provide some suggestions? I’d like to avoid using antifoam because of clogging issues.

Bioreactor spargers and conditions may impact foaming. The amount of antifoam can be minimized by optimizing mass transfer conditions including P/V and gassing strategy and sparger type and/or size of sparger pores. Max challenge studies can be performed to understand the effect of antifoam in the culture and clogging. The filter can be regularly changed when fouling is detected through changes in permeate flow pressure.

Question 14

How much time can be saved by using perfusion for seed train?

During the seed train, passage number may be reduced and vessels can be skipped. Time savings may not be significant in seed train, but the production time may be reduced due to the high cell density inoculation and potential to reach peak productivity earlier in the culture as compared with fed batch.

Question 15

What kind of process optimization do you recommend for using perfusion in manufacturing? I have seen data that you can get good results with less development time. Do you agree?

The development and optimization required to implement a perfusion system in manufacturing depends on different factors including the molecule and cell line. Depending on those factors, cell culture process prior knowledge and, if available, platform knowledge can be leveraged to reduce or improve efficiency of development and scale up of the perfusion process.

Question 16

How do you handle sampling during runs in your N-1 set up?

Sampling in the bioreactor is performed using sample ports, as in traditional fed batch cultures. Additional sample ports can be installed on permeate and cell bleed lines if needed.

Question 17

How do you calculate cell specific perfusion rate?

Perfusion rate depends on cell line and culture media. It should be estimated for optimal growth rate and productivity. A good source that advises steps to determine perfusion rate are here: https://www.repligen.com/technologies/xcell-atf/FAQ-perfusion

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