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Perfusion Cell Culture

Perfusion, or upstream continuous bioprocessing, has been practiced since the 1980s towards maximizing facility utilization, expanding process flexibility and minimizing costs.  Innovative technologies, sophisticated control logic systems, cell culture supplements and single-use assemblies have tremendously simplified implementation, establishing perfusion as the cornerstone of intensification and continuous processing.

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long-term perfusion

Perfusion cell culture utilizes a cell retention device and continuous media exchange to achieve and maintain high cell densities and viabilities over extended periods of time, typically weeks. The cell retention device retains cells inside the bioreactor, while fresh media is added, and product of interest, waste products and spent (or depleted) media are continuously removed. Fresh media is provided at the same rate that product and spent media are removed from the bioreactor. Hollow fiber-based membrane filters acts as the most reliable and commonly used membrane type.  Long term perfusion is just one application of perfusion and contrasts with shorter time based perfusion applications, such as High Productivity Harvest (HPH) that focus on intensifying fed patch processes over the course of days. Perfusion may also be used to intensify the seed train with reduced steps and faster overall time to production. 

Early perfusion technologies, developed during the 1980s, frequently negatively impacted cell health and entailed complicated engineering scale-up. Despite promising advantages, adoption was not widespread.  In addition, advances in cell line engineering, media composition, and bioreactor design generated multiple-fold increases in product titer from batch and fed-batch cell cultures, temporarily reducing the need for, and the impact of, perfusion cell culture approaches. 

The recent pursuit of alternative manufacturing strategies that can boost facility capacity, throughput, and productivity with reduced costs and scale of operation has led to a renewed interest in perfusion technology and its benefits. Technology innovations, such as XCell ATF®, using novel alternating tangential flow filtration, have been instrumental in solving historical challenges and in shifting the paradigm towards perfusion as a key platform for modern upstream processing. 

Perfusion Enables 10X VCD and 10X Product than Fed-Batch

Perfusion achieves 100-130 million VCD compared to 13 million for Fed-Batch. Perfusion culture viability is maintained above 90% over a period of 17 days. Fed-Batch viability begins to drop after day 9 to approximately 70% at days 14-15. Higher VCD and viability of perfusion cultures translates to an increase in total product yield. Perfusion process yields approximately 30-35 g. Fed-Batch process achieves 3 g in approximately 15 days. 

Higher cell density, higher viability 

  • Unoptimized perfusion process achieves up to 10X VCD over optimized Fed-Batch  
  • Perfusion enables higher cell growth rates and healthier cultures 

Higher productivity 

  • Higher cell density and viability of perfusion culture enables 10X yield increase over Fed-Batch  

Technologies for long-term perfusion

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 is the leading perfusion device for mAb and rProtein production.

Resources

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Continuous Biorprocessing: Industry Best Practices volume 2 

Continuous Biorprocessing: Industry Best Practices volume 1 

Special Report on Continuous Bioprocessing: Upstream, Downstream, Ready for Prime Time?
by Angelo DePalma
BioProcess International, May 2016

Continuous Manufacturing: A Changing Processing Paradigm
By Randi Hernandez
BioPharm International, Apr 01, 2015

A Novel Seed-Train Process: Using High-Density Cell Banking, a Disposable Bioreactor, and Perfusion Technologies
by Benjamin Wright, Mike Bruninghaus, Mike Vrabel, Jason Walther, Neha Shah, Seul-A Bae, Timothy Johnson, Jin Yin, Weichang Zhou and Konstantin Konstantinov
BioProcess International, March 2015

Developing an Integrated Continuous Bioprocessing Platform: Interview with Konstantin Konstantinov 
by Maribel Rios
BioProcess International, December 2012

A Brief History of Perfusion Biomanufacturing: How High-Concentration Cultures Will Characterize the Factory of the Future
by John Bonham-Carter and Jerry Shevitz
BioProcess International, October 2011