Small-scale bioreactor supports high density HEK293 cell perfusion culture for the production of recombinant Erythropoietin

Schwarz, Hubert; Zhang, Ye; Zhan, Caijuan; Malm, Magdalena; Field, Raymond; Turner, Richard; Sellick, Christopher A.; Varley, Paul; Rockberg, Johan; Chotteau, Véronique

doi:10.1016/j.jbiotec.2019.12.017

Cited by 45 publications

(47 citation statements)

References 38 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…HEK293 cell lines have been reported to have a pseudotriploid genome with the adenoviral DNA inserted on chromosome 19 19,22,23 . The organization of the HEK293 genome is continuously evolving through the events of chromosomal translocations and copy number alterations, suggesting that long-term cultivation and subcloning of cells result in karyotypic drift 22,24 . Such abnormalities and genomic instability is, however, characteristic for immortalized cells and have also been reported for CHO cells [25][26][27][28] .…”

mentioning

confidence: 99%

“…Two examples are 293T 29 and 293E 30,31 cell lines, constitutively expressing the temperature sensitive allele of the large T antigen of Simian virus 40 29 , or the Epstein-Barr virus nuclear antigen EBNA1, respectively 30,31 . In addition, several HEK293 cell lines have been adapted to high-density suspension growth in serum-free medium [32][33][34] , enabling large-scale cultivation and bioproduction in bioreactors 24 . Two industrially relevant suspension cell lines are 293-F and 293-H (Gibco, Thermo Fisher Scientific), which both enable fast growth and high transfectivity in serum-free medium.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Evolution from adherent to suspension: systems biology of HEK293 cell line development

Malm

Saghaleyni

Lundqvist

et al. 2020

Sci Rep

Self Cite

View full text Add to dashboard Cite

The need for new safe and efficacious therapies has led to an increased focus on biologics produced in mammalian cells. The human cell line HEK293 has bio-synthetic potential for human-like production attributes and is currently used for manufacturing of several therapeutic proteins and viral vectors. Despite the increased popularity of this strain we still have limited knowledge on the genetic composition of its derivatives. Here we present a genomic, transcriptomic and metabolic gene analysis of six of the most widely used HEK293 cell lines. Changes in gene copy and expression between industrial progeny cell lines and the original HEK293 were associated with cellular component organization, cell motility and cell adhesion. Changes in gene expression between adherent and suspension derivatives highlighted switching in cholesterol biosynthesis and expression of five key genes (RARG, ID1, ZIC1, LOX and DHRS3), a pattern validated in 63 human adherent or suspension cell lines of other origin.

show abstract

mentioning

confidence: 99%

mentioning

confidence: 99%

Evolution from adherent to suspension: systems biology of HEK293 cell line development

Malm

Saghaleyni

Lundqvist

et al. 2020

Sci Rep

Self Cite

View full text Add to dashboard Cite

show abstract

“…Based on the knowledge of the established fed‐batch process for the production of this enzyme, the values of

q_{a a}

and

{AA}_{B R}

for the key amino acids at mid‐phase of the FB were assumed to represent the cell need in the EFB and the HCDP and taken as targets

q_{aa}^{target}

and

{AA}_{BR}^{target}

, respectively. The set points for the perfusion rate

D

, cell density

C_{v}

and thus CSPR

= \frac{D}{C_{v}}

were constant and selected according to our previous experience of high cell density perfusion with CHO and HEK293 cells (Gomis‐Fons et al, 2020; Schwarz et al, 2020; Zhang et al, 2020). Equation (2) could be rewritten as follows:

{AA}_{m e d i u m} = \frac{q_{aa}^{target} \times ​ C_{v}}{D} + {AA}_{BR}^{target} italic.

…”

Section: Resultsmentioning

confidence: 99%

Process intensification to produce a difficult‐to‐express therapeutic enzyme by high cell density perfusion or enhanced fed‐batch

Särnlund

Jiang

Chotteau

2021

Biotech & Bioengineering

View full text Add to dashboard Cite

Intensified bioprocesses have caught industrial interest in the field of biomanufacturing in recent years. Thanks to new technology, intensified processes can support high cell densities, higher productivities and longer process times, which together can offer lower cost of goods. In this study two different intensified process modes, high cell density perfusion and enhanced fed‐batch, were evaluated and compared with a conventional fed‐batch process for a difficult‐to‐express therapeutic enzyme. The intensified process modes were cultivated with a target cell density of 100 × 106 cells/ml and with alternating tangential flow filtration, ATF, as cell retention device. The processes were designed to resemble an established optimized fed‐batch process using the knowledge of this process without new dedicated optimization for the intensified modes. The design strategy included decision of the ratio of feed concentrate to base medium and glucose supplementation, which were based on target cell‐specific consumption rates of key amino acids and glucose, using a targeted feeding approach (TAFE). A difficult‐to‐express therapeutic enzyme with multiple glycosylation sites was expressed and analyzed in the different production processes. The two new intensified processes both achieved 10 times higher volumetric productivity (mg/L/day) with retained protein quality and minor changes to the glycan profile compared to the fed‐batch process. The study demonstrates the potential of using intensified processes for sensitive complex enzymes. It is shown here that it is possible to transfer a developed fed‐batch process into high cell density processes either in intensified fed‐batch or steady‐state perfusion without new dedicated optimization. The results demonstrated as well that these intensified modes significantly increase the productivity while maintaining the desired product quality, for instance the same amount of product was obtained in 1 day during the perfusion process than in a whole fed‐batch run. Without any prior optimization of the perfusion rate, the high cell density perfusion process resulted in only 1.2 times higher medium cost per gram produced protein.

show abstract

“…Intensified fed‐batch cultures were performed in glass stirred tank DASbox bioreactors (Eppendorf) in 200 ml working volume, according to the method for perfusion operation described in Schwarz et al (2020), except for the hollow fiber cartridges, which were ultra‐filters. Three cultures, cult_01, cult_02, and cult_03, were performed using alternating tangential flow XCell ATF2 (Repligen) cell separation device mounted with a 50 kDa cutoff ultra‐filtration hollow fiber filter (UF HF).…”

Section: Methodsmentioning

confidence: 99%

Antibody capture process based on magnetic beads from very high cell density suspension

Brechmann

Schwarz

Eriksson

et al. 2021

Biotech & Bioengineering

Self Cite

View full text Add to dashboard Cite

Cell clarification represents a major challenge for the intensification through very high cell density in the production of biopharmaceuticals such as monoclonal antibodies (mAbs). The present report proposes a solution to this challenge in a streamlined process where cell clarification and mAb capture are performed in a single step using magnetic beads coupled with protein A. Capture of mAb from non‐clarified CHO cell suspension showed promising results; however, it has not been demonstrated that it can handle the challenge of very high cell density as observed in intensified fed‐batch cultures. The performances of magnetic bead‐based mAb capture on non‐clarified cell suspension from intensified fed‐batch culture were studied. Capture from a culture at density larger than 100 × 106 cells/ml provided an adsorption efficiency of 99% and an overall yield of 93% with a logarithmic host cell protein (HCP) clearance of ≈2–3 and a resulting HCP concentration ≤≈5 ppm. These results show that direct capture from very high cell density cell suspension is possible without prior processing. This technology, which brings significant benefits in terms of operational cost reduction and performance improvements such as low HCP, can be a powerful tool alleviating the challenge of process intensification.

show abstract

Small-scale bioreactor supports high density HEK293 cell perfusion culture for the production of recombinant Erythropoietin

Cited by 45 publications

References 38 publications

Evolution from adherent to suspension: systems biology of HEK293 cell line development

Evolution from adherent to suspension: systems biology of HEK293 cell line development

Process intensification to produce a difficult‐to‐express therapeutic enzyme by high cell density perfusion or enhanced fed‐batch

Antibody capture process based on magnetic beads from very high cell density suspension

Contact Info

Product

Resources

About