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N-1 Perfusion for Fed-Batch

N-1 perfusion refers to the use of a cell retention device to achieve high cell density in the seed train step before the production bioreactor (N), achieving a more rapid and robust process while maintaining an existing Fed-Batch based production scheme.

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N-1 perfusion

A form of seed train intensification, N-1 perfusion refers to the intensification of cell growth in the step prior to the production bioreactor (N). This process intensification is done by attaching a cell retention device, such as the XCell ATF® Device, to the N-1 bioreactor to attain high cell density and viability. This seeds the production bioreactor at a higher starting cell density and shortens the production bioreactor run time. This can dramatically increase the facility output without direct change to the core production process.  A robust cell retention device is required to attain a high cell density inoculum for the production bioreactor.

Start intensification with N-1 

The combination of implementation ease and productivity increase make N-1 perfusion the most frequent starting point for upstream intensification. Once in place, further intensify the seed train, add HPH to the N bioreactor or transition to continuous. 

More on Seed Train intensification

Boost throughput, save time 

N-1 perfusion intensifies the Fed-Batch process, increasing facility throughput. Seeded at a higher initial density, cells in the N bioreactor grow faster, reach a higher max VCD and produce more product than traditional Fed-Batch. Harvest sooner and increase the number of batches per year as compared to a standard fed batch process.  Alternatively, maintain the N-bioreactor run time to increase overall yield. 

Maintain a Fed-Batch process 

N-1 perfusion intensification increases N bioreactor productivity and throughput while maintaining a fed batch process.  No perfusion or media exchange of the N bioreactor is required, allowing the N bioreactor to maintain a single and discreet fed batch harvest point.  

Minimize validation impact 

Intensify with minimal process modifications and regulatory edits. No additional equipment is connected to the N bioreactor, no media exchange occurs during the production run and cell viability is higher, typically leading to lower impurity values.  

N-1 Performance Data

N Bioreactor reaches 350 x 106 VCD 

N-1 perfused Fed-Batch reaches 350 million cells/mL VCD in just 6 days.  In comparison, standard fed-batch achieves 30 million cells/mL viable cell density (VCD) over a course of 14 days (data not shown).  Productivity of the N bioreactor after with an N-1 perfusion increased 20-fold (data not shown)  while maintain viability above 90%. 

Higher cell density and viability  

  • 10X VCD over Fed-Batch  
  • VCD as high as 350 x 106 cells/mL
  • 50% reduction in production time 
  • 20-fold increase in productivity 
  • Lower COGs 

N-1 Perfusion increases VCD 10X and reduces vaccine production time 50% 

The perfused N-1 bioreactor cell density rapidly increases from 8 to 80 million cells/mL (10X VCD) in 4-5 days. Inoculation of the N bioreactor with the N-1 perfused culture establishes a 10X higher initial viable cell density over fed-batch (A). After 3 days, the N-1 inoculated bioreactor achieves 35 million VCD. In comparison, the control requires 6 days to achieve 25 million VCD (B). Leverage a higher VCD to either increase throughput or increase yield. Harvest of the N-1 Fed- Batch bioreactor at day 8 is equivalent to harvesting the fed-batch control at day 14, saving 6 days of N bioreactor suite time (C). Alternatively, harvest the N-1 fed-batch process at day 14 for a 25% overall yield increase. 

  • N-1 bioreactor reaches 10X VCD in 4-5 days
  • N-1 perfused Fed-Batch bioreactor reached max VCD of 35 million compared to 25 million with traditional Fed-Batch
  • Max VCD is achieved 3 days earlier
  • Harvest 6 days earlier for same yield OR
  • Achieve 25% yield increase by day 14
N-1 Bioreactor vcd
Production bioreactor VCD
Production Bioreactor

n-1 perfusion technologies

Alternating Tangential Flow (ATF) Filtration

An award-winning technology, ATF allows the removal of spent media while keeping cells in culture. Applied using an XCell ATF® device attached to a bioreactor, ATF minimizes cell shear and keeps cells in constant equilibrium with bioreactor contents. This results in faster cell growth at higher densities with higher productivity. Today, the XCell ATF® device, in single-use or reusable format, is the leading perfusion device for mAb and rProtein production.

Xcell ATF® Technology

XCEll ATF® Devices

XCell™ Controllers

Potent cell culture supplements

Repligen developed and manufactures cell culture supplements that provide the benefits of serum supplementation while maintaining an animal-free process.

  • LONG®R3 IGF-I (LONG R3) is 200 times more potent and two times more stable in cell culture than insulin
  • LONG® EGF is a like-for-like replacement for native EGF or recombinant human EGF (hEGF)

CELL CULTURE SUPPLEMENTS

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