N‐1 semi‐continuous transient perfusion in shake flask for ultra‐high density seeding of CHO cell cultures in benchtop bioreactors

One strategy to enhance the production of biological therapeutics is using transient perfusion in the preculture (N-1 stage) to seed the production culture (N stage) at ultra-high cell densities (>10 x 10⁶ viable cells/mL). This very high seeding density improves cell culture performance by short...

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Vydáno v:	Biotechnology progress Ročník 41; číslo 5; s. e70029 - n/a
Hlavní autoři:	Lemire, Lucas, Reyes, Sebastian‐Juan, Durocher, Yves, Voyer, Robert, Henry, Olivier, Pham, Phuong Lan
Médium:	Journal Article
Jazyk:	angličtina
Vydáno:	Hoboken, USA Wiley 01.09.2025 John Wiley & Sons, Inc Wiley Subscription Services, Inc
Témata:	Agitation Animals Bioreactors Cell Count Cell culture Cell Culture Techniques - instrumentation Cell Culture Techniques - methods Cell death Cell density Cell Survival Centrifugation CHO cell culture CHO Cells Cricetinae Cricetulus Culture Media cumate inducible system Density Exchanging Filtration Flasks Oxygen - metabolism Oxygen transfer Perfusion Perfusion - methods preculture perfusion pseudo-perfusion semi-continuous perfusion Sulfite ultra-high seeding density (UHSD) pseudo‐perfusion preculture perfusion ultra‐high seeding density (UHSD) semi‐continuous perfusion oxygen transfer CHO cell culture cumate inducible system
ISSN:	8756-7938, 1520-6033, 1520-6033
On-line přístup:	Získat plný text
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Shrnutí:	One strategy to enhance the production of biological therapeutics is using transient perfusion in the preculture (N-1 stage) to seed the production culture (N stage) at ultra-high cell densities (>10 x 10⁶ viable cells/mL). This very high seeding density improves cell culture performance by shortening the timeline and/or achieving higher final product concentrations. Typically, an N-1 seed train employs bioreactors with alternating tangential flow filtration (ATF) or tangential flow filtration (TFF) perfusion systems or Wave cell bag bioreactor with integrated filtration membrane, which have costs and technical complexity. Here, we propose an alternative method using semi-continuous transient perfusion through media exchange in shake flasks, which is suitable for benchtop-scale intensification process development. Daily media exchange was necessary to prevent nutrient limitations. The observed limitation of maximum viable cell densities (VCD) in various flask sizes was demonstrated to be due to oxygen limitations through the measurements of maximum oxygen transfer rates (OTR) using the sulfite system. By increasing agitation frequency from 200 to 300 RPM, maximum OTR in 500-mL shake flasks was increased by 62.3%, allowing an increase in maximum VCD of 29.6%. However, in 1000-mL shake flasks, an increase in agitation rate resulted in early cell death. After demonstrating that media exchange in shake flasks by centrifugation had no significant impact on cell growth rates, metabolism, and productivity, a benchtop bioreactor was seeded from semi-continuous transient perfusion cell expansion. The ultra-high cell density seeding resulted in a 49.3% increase in space–time-yield (STY) when compared to a standard low seeding density culture. NRC publication: Yes
Bibliografie:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 14 content type line 23
ISSN:	8756-7938 1520-6033 1520-6033
DOI:	10.1002/btpr.70029