Recombinant Proteins - FiberCell Systems

SIMPLIFY YOUR PRODUCTION OF RECOMBINANT AND 'DIFFICULT-TO-EXPRESS' PROTEINS USING HOLLOW FIBER BIOREACTOR

The challenge of producing recombinant proteins

Many laboratories forgo using mammalian expression systems to produce recombinant proteins because it can be a cumbersome process. You’ll need a large number of plates, flasks, or roller bottles. In some cases, you’ll even require the use of a stirred tank vessel. For these methods, the cell culture conditions are inherently non-physiologic.

At FiberCell, we aim to provide you with the best conditions for the most optimal cell culture results through Hollow FIber bioreactors. Click here to read more.

Difficult-to-express proteins

Difficult-to-express proteins are generally considered to be recombinant proteins that are expressed at low titers in mammalian systems. However, this can also include highly complex, highly glycosylated, large and unstable proteins.

Mammalian cells bring out superior solubility, immunogenicity, and bioactivity of a protein as compared to other hosts (such as bacteria, insect cells and yeast).

Cultured mammalian cells are the preferred platform for the production of recombinant proteins for clinical applications due to their capacity for proper protein folding, assembly and post-translational modifications. This superiority of protein quality is also important in the research laboratory to ensure proper biological activity and especially for potentially therapeutic proteins.

In a 3-D hollow fiber bioreactor the continuous process, lack of passaging and the maintenance of biologically homeostatic culture conditions over time result in improved folding and complete and uniform post-translational modifications. The more in-vivo-like growth conditions and lack of shear within an HFBR also results in significantly reduced cell lysis. Host cell proteins, lysozyme and DNA are not released into the culture medium resulting in a product that is cleaner and easier to purify.

Why are Hollow Fiber bioreacters (HFBRs) the preferred method to produce recombinant proteins?

Typical protein harvests from FiberCell Systems’ cartridges contain 100 µg/mL/day or higher. Daily harvests are 2 mg to 10 mg a day.

Hollow Fiber bioreactors (HFBRs) allows your cells to grow in an in-vivo-like, 3D cell culture environment that closely replicates the mammalian system. HFBRs provide:

Easy cell culture scale-up with improved protein fidelity at 100X greater concentrations as compared to flask, roller or spinner culture.
Large amount of surface area for cell attachment and the high cell density permits the use of our CDM-HD serum replacement.
Improved protein folding and more uniform post-translation modifications over extended culture periods.
Protein concentrations in the range of 100 ug to 300 ug/mL/day and the production of 10’s of mg on up to gram quantities of proteins using only a single cartridge.

When paired with our unique protein-free CDM-HD serum, the protein of interest becomes a significant component of the harvested supernatant and the small volumes facilitate purifications.

Interested to find out more about in-vivo-like environments? Click to read more.

Achieve 100X more concentrated secreted proteins than conventional tissue culture supernatants

The simpler way to produce recombinant proteins:

No splitting of cells required; maintenance is just 15 minutes a day.
Cultures can be maintained for several months of continuous production.
Improved protein assembly and folding.
Small harvest volume for easy handling and more efficient down-stream processing.
Reduced cell lysis, less host cell protein and DNA contamination.
Eliminates requirement for adapting cells to suspension culture or medium optimization.
Use of protein-free CDM-HD simplifies purification.

Cartridge Selection

The molecular weight cut off is defined at the 50% retention level as an industry convention. There is also “useful retention” defined as the size of protein that will be retained by 90% or more. This will depend upon the size, shape, post-translational modifications and Stokes radius of the protein in question, not just the molecular weight. For the 5 kd MWCO fiber we define the useful retention as 20 kd. It should be used for proteins in the range of 20 kd on up to about 100 kd. For larger proteins, the 20 kd MWCO fiber should be used. Since the gross filtration rate of the 5kd MWCO fiber is so high there is no penalty (such as the requirement to feed the ECS) with using the 5 kd MWCO fiber.

Large cartridge for Receipt Protein page

Examples of difficult-to-express proteins

Difficult-to-Express Proteins

Highly Glycosylated
High level of post-translational modifications
Low levels of secretion
- IL15 receptor complex
- BITE: Bi-specific T-Cell Engagers
- TRIKE: Tri-Specific Killer Engagers

Structures not found in nature

Recombinant IL12 Expressed in 293T Cells

Stable Native Conformation
25% of total protein in cell culture supernatant
Critical inter-subunit disulfide bond is intact
Produced using CDM-HD protein free medium

IL15 RC is a Difficult to Express Protein

Stable Cytokine Production in HFBR Over 5 Months

The new generation of bio-therapeutics under development are increasing in size and complexity, along with higher specific activities. As new therapeutic strategies are developed, bioengineering moves beyond copying that which is expressed naturally and begins to use novel and unique structures not found in nature, but created in the mind of the bioengineer.

This new class of proteins includes bi-specific T-cell engager (BITE) antibodies and tri-specific killer engagers (TRIKE) which utilize unique, newly created structures for their novel functionality. A HFBR system from FiberCell Systems allows any laboratory to take advantage of the superior folding, glycosylation and complete post-translational modifications that only expression in mammalian cells can provide. HFBRs are an effective method for producing milligram to gram quantities of recombinant proteins. The harvested product is concentrated and contaminating proteins, DNA, RNA, and proteases are reduced significantly.

The use of CDM-HD renders the medium economical and chemically defined. Cultures can be maintained for long periods of time, meaning that scalability of the system is determined by length of culture, not new equipment. Protein-free medium and high concentration product simplifies downstream processing, and can result in increased yields by reducing the steps required for purification. A HFBR from FiberCell Systems is the ideal method for the production of 50 mg on up to gram quantities of recombinant proteins from mammalian cells, and is particularly useful for the production of difficult-to-express proteins.

WE HAVE A VARIETY OF CARTRIDGES TO SUIT YOUR APPLICATION

Cartridge/Flowpath Specifications

Catalog No.	Size	Surface Area	Fiber Type	Packing Density	ECS Vol	MWCO 50%	Max. Cell#	Oxygenator
C2025D	Small	450 cm²	High flux PS	50%	3.2 mL	20 kD	10⁸	.65 m
C2008	Medium	4000 cm²	Low flux PS	50%	20 mL	5 kD	10⁹	4 m
C2011	Medium	4000 cm²	High flux PS	50%	20 mL	20 kD	10⁹	4 m
C5011	Medium	4000 cm²	High flux PS	50%	20 mL	20 kD	2 x 10⁹	6.1 m
C2003	Large	1.2 m²	Low flux PS	50%	70 mL	5 kD	5 x 10¹⁰	6.1 m
C2018	Large	1.2 m²	high flux PS	50%	70 mL	20 kD	5 x 10¹⁰	6.1 m

Suggested Ordering for Recombinant Protein Cell Culture

Bioreactor
P3202	The FiberCell Systems Duet more info
Cartridges
Catalog No.	Size	Surface Area	Fiber Type	Packing Density	ECS Vol	MWCO 50%	Max. Cell#
C2003	Large	1.2 m²	low flux PS	50%	70 mL	5 kD	5 x 10¹⁰	more info
C2008	Medium	4000 cm²	low flux PS	50%	15 mL	5 kD	10⁹	more info
C2011	Medium	4000 cm²	high flux PS	50%	15 mL	20 kD	10⁹	more info
C2018	Large	1.2 m²	high flux PS	50%	70 mL	20 kD	5 x 10¹⁰	more info
Reservoir Caps
A1005	33 mm Reservoir Cap more info
A1006	38 mm Reservoir Cap more info
A1008	45 mm Reservoir Cap more info
Serum Replacement
CDM-HD more info

Click here for our US list pricing for 2023. We offer tiered discounted pricing for quantity purchases of our products, 10-25 units, 25+ units, and 50+ units. Please enquire for details. We recommend the most appropriate products for your applications so please feel free to email or phone us to discuss your application and provide you with a quote at info@fibercellsystems.com, 301-471-1269, 240-440-2662. We can accept credit cards and purchase orders from accredited institutions.

1. Production of a Hexeramized Recombinant IgG from CHO Cells in the FiberCell Systems C2018 Cartridge

Production of a hexeramized IgG consisting of 6 IgG1 subunits held together by 3 IgA tails was expressed in a CHO cell line and produced in a 1.2 m2, 20 kD MWCO hollow fiber bioreactor. This is a classic example of a difficult-to-express protein; a very large, highly glycosylated, complex, engineered structure not seen in nature. See results here.

2. HEK 293 culture expressing heterodimeric IL-15 maintained in a HFBR for over 4 months of continuous production

Efficient production of this noncovalently linked but stable heterodimer in HEK293 cells is demonstrated in a 5 kD MWCO HFBR. Using the HFBR demonstrated consistent, continuous product of the IL over 150 days of culture with no change in glycosylation or conformation. See results here.

3. Recombinant IgG production in HEK 293 cells

Recombinant immunoglobulin produced by 293t cells in our C2011 cartridge demonstrated the reduction in contaminating serum proteins after switching to CDM-HD. See results here.

Interested to find out more? Read on for more case studies on recombinant proteins.

WE ARE HERE TO HELP

For detailed information on producing recombinant proteins using HFBRs in your laboratory, contact us or request for a quote.