Better expression of recombinant proteins from CHO and HEK293

Quickly produce fully biologically active, complex recombinant protein

If your project requires significant amounts of well-folded, assembled and glycosylated 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 with a medium sized 20ml cartridge can quickly generate hundreds of milligrams of highly valuable fully biologically active protein from stably transfected adherent or suspension cells growing under gentle “3D” ultra-high density conditions. Examples of successful projects include: highly valuable multi-subunit cytokines, fusion proteins and humanized mAbs. A FiberCell bioreactor enables you to keep total control of your production process in a standard cell culture lab and requires just the space of an incubator shelf. The single-use cartridge modules are typically operated for several weeks, or can be for several months, requiring 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 of well-folded, uniformly glycosylated rec. protein with 20 ml cartridge
  • Achieve much 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
  • Eliminate your laborious cell passaging procedures
  • No need to wean cells over to serum-free media – CHO and HEK293 don’t require serum in HFBR
  • Uses simple, affordable, GMP-compliant, protein-free media […learn about serum free operation]

Small footprint system

Continuously culture >1 billion cells in a 20ml FiberCell cartridge and routinely harvest concentrated supernatant. 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 and offer a productivity equivalent to daily harvests from 200 roller bottles.

“…the high yields of pure IL-12 obtained using the method described in this study will provide an avenue to embark on structural studies aimed at the development of novel IL-12 based therapeutics for the treatment of cancers and infectious diseases.”

See : https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4164965/

Eliminate the hassle of flasks or roller bottles

The idea of scaling up your 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 yields of complex biologically active 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.)

Do you need 500 mg or more?

Large FiberCell Hollow Fiber Cartridge Module

70 ml cartridge modules for 5 x 10e10 cells

FiberCell C2003 5 Kd and C2018 20 Kd cartridge modules enable you to maintain huge numbers of cells with little maintenance and are recommended for projects to produce >=500 mg of recombinant protein. FiberCell hollow fibre cartridges typically produce around 100µg/ml/day. See cartridge modules page.

Duet pumps maintaining 4 x 10e11 cells

The compact hollow fibre culture set-up on the right is producing around 2 grams per month of a highly valuable cytokine. The large autoclavable carboys minimize hands on time. Result: a highly productive, easily affordable and low maintenance system running free of serum or extraneous media proteins.

Large FiberCell Hollow Fiber Cartridge Module

Recombinant IgG production example

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

A medium sized 20 ml C2011 cartridge module was used to produce 276 mg of protein in a total volume of 900 mL over a 2 month period using serum-free medium. Bioreactor media was not changed at weekends but this had little overall impact on productivity.

Click to Enlarge Image

3D high density culture conditions

Game-changing culture conditions

The high flux polysulfone hollow fibres in FiberCell cartridges enable you to maintain HEK293 or CHO cells, for example, at highest possible cell density (>10e8 cells per ml) without shear stress. Cells proliferate around the fibres in a more physiologic in vivo-like environment compared to flask culture and are not subjected to passaging since cell mass is gently reduced during the routine harvesting procedure.  Culture conditions in this system are ideal for superior protein quality and biological activity. You should be able to count on harvests of 2 mg to 10 mg per day of fully biologically active protein, depending on the cartridge size used, see : cartridge modules.

Cells lose dependence on serum

Under the high density conditions described above cells can be maintained free of serum in the media. In fact, once the culture is established it is generally possible to make a single-step switch to a low-cost, simplified DMEM-based media. Several examples are provided below. FiberCell provides a popular serum-substitute specifically formulated for the bioreactor, see: CDM-HD.

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.

Gel showing purity of target recombinant 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

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]

PSG9 Stimulates Increase in FoxP3+ Regulatory T-Cells through the TGF-β1 Pathway: Jones, K. et al. PLOSone 2016 July [Open Access]

Kinetic and Spectroscopic Study of Peptidylglycine Monooxygenase: Chauhan,S.. 2016 [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]

The HHM Motif at the CuH-site of Peptidylglycine Monooxygenase is a pH-Dependent Conformational Switch: Kline, C.D. et al, Biochemistry 2013 52(15): 2586–2596
[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]

Insights into egg coat assembly and egg-sperm interaction from the X-ray structure of full-length ZP3: Han, L. et al.; Cell 2010 143(3):404-15 [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]

Alteration of Hepatitis A Virus (HAV) Particles by a Soluble Form of HAV Cellular Receptor 1 containing the Immunoglobulin- and Mucin-Like Regions: Silverstein, E., Journal of Virology, 2003 p. 8765-8774 [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.

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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