Better expression of recombinant proteins from CHO and HEK293

Quickly produce fully bioactive, complex recombinant proteins

If your project requires significant amounts of fully bioactive, complex recombinant protein then choosing your culture method will be an important decision, especially if the protein is particularly difficult to express. A compact FiberCell hollow fibre bioreactor can quickly generate hundreds of milligrams of highly valuable, well-folded and -glycosylated protein from stably transfected cells growing under gentle “3D” ultra-high density conditions. It will enable you to keep total control of the production process in a standard cell culture lab requiring just an incubator shelf and laminar flow cabinet. The single-use cartridge modules can be used over weeks, or even months, and will require just 15 minutes maintenance per day (>10e9 cells per 20 ml cartridge module with scale up to 70 ml cartridges possible.)

  • Obtain 100’s of milligrams to gram quantities of well-folded and uniformly glycosylated rec. protein
  • Achieve higher yields of complex multi-subunit proteins compared to other culture methods
  • Produce at a concentration 10 to 100 X higher than flask or rollers
  • Avoid laborious cell passaging
  • Harvest continuously over weeks, or months if needed, with low maintenance
  • Easily switch to simple, low-cost protein-free media […find out how]

Small footprint system

Continuously culture >1 billion cells in a 20ml FiberCell cartridge. A FiberCell bioreactor fitted with one or two cartridges fits on one shelf of a standard lab incubator. 70 ml cartridges can contain up to 50 billion cells.

Eliminate the hassle of flasks or roller bottles

The idea of setting up your own mammalian cell culture process may be daunting. However, the hassle of passaging cells in large numbers of flasks or roller bottles can easily be avoided. Continuous high-cell-density culture using a compact FiberCell hollow fibre bioreactor will enable you to keep control of the production process within your own lab without the expense of outsourcing. Different types of disposable cartridge modules are available to match the type and quantity of any protein, antibody or other secreted product you may wish to generate in future.

High yield production of complex, bioactive recombinant proteins

Obtain well-folded and glycosylated recombinant protein

The high density, zero-shear stress, constant pH culture environment of a FiberCell bioreactor is ideal for generating significant amounts of highly pure, correctly folded and fully glycosylated recombinant protein. Several relevant customer publications can be found at the foot of his page.

Comparison – FLASK vs. HFBR

A complex hexamerized IgG consisting of six IgG1 subunits held together with three IgA tails was produced in flask and FiberCell culture.

In flasks approximately 40% of the protein was secreted as an unfolded monomeric subunit whereas >90% was correctly folded in FiberCell culture.

476 mg of protein was produced in two months in a harvest volume of less than 5 L using FiberCell cartridge C2018. (Data courtesy of Dr. Jim Arthos, Bethesda, MD.)

Recombinant IgG production example

Production of a Recombinant IgG in 293 Cells Using FiberCell Systems CDM-HD (Serum-Free Medium)

Cartridge C2011 was used to produce 276 mg of protein in a total volume of 900 mL over a 2 month period using serum-free medium. The bioreactor was run over weekends without media changes; most cell types demonstrate high resistance to lactate under hollow fibre culture conditions.

Immediate adaptation to serum-free medium possible

Adaptation from DMEM-FBS to serum-free medium

SDS-Page gel of successive harvests of a secreted recombinant protein from a 293 cell line in the FiberCell HF Bioreactor. Starting media is DMEM with 10% FBS (Monday, left). Shows a one-step switch to serum free medium (Hyclone) while maintaining high levels of the secreted target protein.

Switch from FBS to CDM-HD serum replacement (293T]

C2011 cartridge was used to produce rec. IgG with 293t cells. Note the reduction in contaminating serum proteins after switching to CDM-HD. By harvesting every day elimination of serum proteins would be even faster than shown. Note how the IgG becomes the dominant protein in the unpurified supernatant.

Switch to CDM-HD serum replacement (BHK]

BHK cells expressing PEDF using DMEM 10% FBS followed by a switch to DMEM 10% CDM-HD on day 17 with no adaptation.

Data demonstrates stable glucose uptake rate and no loss of PEDF production when using CDM-HD.

Conclusion:
BHK cells can be cultured in hollow fibre bioreactors using protein free medium with no loss of production.

Cartridge used Cat # C2008
Cells inoculated 3 X 108 cells
Medium Consumed 14 L
Total protein produced 99.8 mg
Total volume of harvest 164 mL
Average concentration 0.6 mg/mL

Produce 2 mg to 10 mg per day

Hollow fibre cartridge module options (BHK]

5 Kd or 20 Kd MWCO fibre cartridges are available in 20 ml (medium) or 70 ml (large) sizes. These cartridges have high flux polysulfone fibres to provide highest possible cell density. You should be able to count on producing 100µg/ml/day of recombinant protein. See : cartridge modules

Customer Publications :

Humanization of JAA-F11, a Highly Specific Anti-Thomsen-Friedenreich Pancarcinoma Antibody and In Vitro Efficacy Analysis: Tati, S. et al.; Neoplasia,2017 19:716-733 [Open Access]

Antigenicity and Immunogenicity of a Trimeric Envelope Protein from an Indian Clade C HIV-1 Isolate: Priya, R.S. The Journal of Biological Chemistry 2015 290: 9195-9208 [Open Access]

A Humanized Anti-CD22-Onconase Antibody-Drug Conjugate Mediates Highly Potent Destruction of Targeted Tumor Cells: Weber, T. et al; Journal of Immunology Research Volume 2015 (2015), Article ID 561814. [Open Access]

Induction and Activation of Latent Transforming Growth Factor-β1 Are Carried out by Two Distinct Domains of Pregnancy-specific Glycoprotein 1 (PSG1): Ballesteros, A. et al.; The Journal of Biological Chemistry 2015 290: 4422-4431 [Open Access]

Better and faster: improvements and optimization for mammalian recombinant protein production: Almo,S., Love, J.; Curr Opin Struct Biol 2014 ; 26: 39–4 [Open Access]

Efficient production and purification of recombinant human interleukin-12 (IL-12) overexpressed in mammalian cells without affinity tag: Jayanthi S. et al.; Protein Expr Purif. 2014 Oct; 102: 76–84. [Open Access]

Free IL-12p40 Monomer Is a Polyfunctional Adaptor for Generating Novel IL-12–like Heterodimers Extracellularly: Abdi, K. et al.; The Journal of Immunology 2014 192 (12): 6028-6036 [Open Access]

A novel tetrameric gp350(1-470) as a potential Epstein-Barr virus vaccine:Cui,X. et al.; Vaccine 2013 31(30): 3039-3045 [Open Access]

Overcoming drug-resistant herpes simplex virus (HSV) infection by a humanized antibody: Krawczyk, A. et al.; 2013 100 (17): 6760-6765 [open access] [materials and methods]

Cloning, Expression, and Functional Characterization of TL1A-Ig:
Khan, S. et al.; The Journal of Immunology 2013 190 (4): 1540-1550 [Open Access]

Comparison of intradermal and intramuscular delivery followed by in vivo electroporation of SIV Env DNA in macaques: Kulkarni, V. et al.; Human Vaccines & Immunotherapeutics Vol 9, 2013 (10). [Open Access]

Characterization and Favorable in Vivo Properties of Heterodimeric Soluble IL-15•IL-15Rα Cytokine Compared to IL-15 Monomer: Chertova, E. et al.; The Journal of Biological Chemistry 2013 288: 18093-18103. [Open Access]

Circulating IL-15 exists as heterodimeric complex with soluble IL-15Rα in human and mouse serum: Bergamaschi, C.. et al.; Blood 2012 120 (1) [Open Access]

Antiviral Breadth and Combination Potential of Peptide Triazole HIV-1 Entry Inhibitors: McFadden, K. et al.; Antimicrob. Agents Chemother. 2012 56 (2): 1073-1080 [Open Access]

T Cell Costimulation by TNFR Superfamily (TNFRSF)4 and TNFRSF25 in the Context of Vaccination: Schreiber, T. et al.; The Journal of Immunology 2012 189 (7): 3311-3318 [Open Access]

Purification and characterization of recombinant human renin for X-ray crystallization studies: Wu, Z. et al.; BMC Biochemistry 2008 9:19 [Open Access]

Murine Pregnancy-Specific Glycoprotein 23 Induces the Proangiogenic Factors Transforming-Growth Factor Beta 1 and Vascular Endothelial Growth Factor A in Cell Types Involved in Vascular Remodeling in Pregnancy: Wu JA et al., Biol Reprod. 2008 Dec;79(6):1054-1061 [Open Access]

Expression of SEAP (secreted alkaline phosphatase) by baculovirus mediated transduction of HEK 293: Jardin BA et al., J Biotechnol. 2008 Jun 30;135(3):272-80. [Abstract]

Purification of soluble CD14 fusion proteins and use in an electrochemiluminescent assay for lipopolysaccharide binding: Burkhardt, M. et al., Protein Expr Purif. 2007 Jan;51(1):96-101 [Abstract]

Recombinant-protein production in insect cells utilizing a hollow-fibre bioreactor: Baxter SC et al., Biotechnol Appl Biochem. 2006 Nov;45(Pt 3):167-72. [Abstract]

Biochemical and biological characterization of a dodecameric CD4-Ig fusion protein. Implications for therapeutic and vaccine strategies: Arthos, J.; Cicala, C et al., JBC 2002;277(13):11, 456-464 [Open Access]

Other references :

(1) Effect of Shear Stress on Intrinsic CHO Culture State and Glycosylation of Recombinant Tissue-Type Plasminogen Activator Protein: Senger, R.S., Nazmul Karim, M., Biotechnology Progress, Volume 19, Issue 4,2003,Pages 1199–1209 [Open Access]

Starter kit for rec. protein production MW > 100 kd

Cat.# Description Qty
P3202 FiberCell® Systems Duet 1
C2011 High Flux PS Cartridge 20 kd (3000 cm2 – 20 ml)* 1
A – – – 33mm, 38mm or 45mm Reservoir Cap 1
CDM-HD Serum replacement 1

* C2018 High Flux PS Cartridge (20 kd – 1.2 m2 – 70 ml) is also an option. This large size cartridge offers greater productivity but consumes more media. Recommended for advanced users. Please enquire.

Starter kit for rec. protein production MW < 100 kd

Cat.# Description Qty
P3202 FiberCell® Systems Duet 1
C2008 Low Flux PS Cartridge 5 kd (3000 cm2 – 20 ml)* 1
A – – – 33mm, 38mm or 45mm Reservoir Cap 1
CDM-HD Serum replacement 1

* Option: C2003 Low Flux PS Cartridge (5 kd – 1.2 m2 – 70 ml) providing 4 X more fibre surface area.

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