April 15, 2018 (Vol. 38, No. 8)
John J.S. Cadwell President and CEO FiberCell Systems
Mammalian Systems Excel in 3D Conditions Provided by Hollow Fiber Bioreactors
Every difficult-to-express protein is difficult in its own way. It may be highly complex. It may be highly glycosylated. It may be large and unstable. Or it may be a recombinant protein that is expressed at low titers in a mammalian system. This last possibility happens to concern many researchers and biomanufacturers. Yet a protein’s solubility, immunogenicity, and bioactivity can be superior when the protein is expressed in mammalian cells versus 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 modification. This superiority of protein quality is also important in the research laboratory to ensure proper biological activity and especially for potentially therapeutic proteins.
The expression of recombinant proteins in mammalian cells in the typical research laboratory can be such a cumbersome process that the use of mammalian expression systems is avoided. Large numbers of plates, flasks, or roller bottles are required, and large-volume spinner flasks or an expensive stirred-tank bioreactor may be needed for scaleup. Processes requiring low-density suspension cultures, or 2D-flask-based processes, along with the use of serum are inherently nonphysiologic.
Classical batch-style 2D cultures are well understood, robust, and convenient, but they are not biologically relevant systems. Proteins are expressed at low concentrations, post-translational modifications may be incomplete, and the presence of serum or other proteins in the medium complicates purification.
Hollow fiber bioreactors (HFBRs) provide a more physiologic, in vivo-like 3D environment than do other cell culture methods, and can also result in improved protein folding and more uniform post-translation modifications over time in a continuous, perfusion-based process. The Table lists the advantages of hollow fiber bioreactors.
Three fundamental characteristics differentiate hollow fiber cell culture from any other method:
- Cells are perfused and bound to a porous matrix, not to a plastic dish, microcarrier bead, or other impermeable support. Consequently, the cells experience an in vivo-like environment (Figure 1).
- The molecular weight cut-off (MWCO) of the fiber can be controlled.
- The ratio of surface area to volume is extremely high, 150–200 cm2/mL, resulting in high cell densities.
Cells bound to a perfused porous support do not require splitting, and passage number becomes irrelevant. Cells in an HFBR maintain viability in a post-confluent manner for extended periods of time—months or longer. The lack of passaging and the maintenance of biologically homeostatic culture conditions 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 result 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.
HFBRs are available with a 5-kD and 20-kD MWCO. Secreted proteins are retained and concentrated within the extracapillary space (ECS) of the cartridge. Here, concentrations are 50–100× higher than in flasks, and the effects of cytokines can also be controlled. The small diameter of the fibers (200 µm O.D.) creates a surface-area-to-volume ratio of 150–200 cm2/mL.
When coupled with the high gross filtration rate of the polysulfone fibers, the exchange of nutrients and waste products across the fibers is very rapid. Cell densities of 1–2 × 108 cells per mL are achieved. These are close to in vivo-like densities.
CDM-HD is a chemically defined, protein-free, animal-component-free, cGMP-compliant serum replacement optimized for the HFBR. The specific high-density cell culture conditions inside an HFBR are different enough from standard culture conditions that a cell culture medium can be simplified and optimized to take advantage of these conditions. CDM-HD can support cell cultures in an HFBR but not in flasks or other low-density culture systems. It is a direct manifestation of the different cell culture conditions found in an HFBR
The use of CDM-HD in an HFBR eliminates contaminants found in serum such as lipids, endotoxin, proteins, viruses, and other adventitious agents. The use of protein-free media results in much cleaner harvests of products and simplified purification. Yields can be improved by reducing the number of purification steps required. Chemically defined CDM-HD also simplifies regulatory compliance.
Production of Recombinant IgG
Production of a hexeramized IgG consisting of six IgG1 subunits held together by three IgA tails was expressed in a CHO cell line and produced in a 1.2-m2, 20-kD MWCO HFBR. This is a classic example of a difficult-to-express protein—a large, highly glycosylated, complex, engineered structure not seen in nature. When grown in flask culture (Figure 2, top trace) about 40% of the protein was expressed as an incompletely folded monomeric subunit.
When the same cells were cultured in the HFBR module (Figure 2, bottom trace), more than 90% of the protein was expressed as a properly folded hexamer because of the superior cell culture conditions inside the hollow fiber cartridge. In this study, 478 mg of purified protein was produced in eight weeks with a harvested volume of less than 5 L.
Production of IL-15 Receptor Complex
The interleukin-15 receptor complex (IL-15-RC) is one of the ultimate difficult-to-express proteins. It consists of two subunits held together by hydrophobic interaction, and it is 45% glycosylated. The IL-15-RC heterodimer demonstrates superior pharmacokinetics and in vivo bioactivity compared to the single chain IL-15 expressed in Escherichia coli. Expression in standard cell culture systems is problematic.
Efficient production of this noncovalently linked but stable heterodimer in HEK293 cells is demonstrated in a 5-kD MWCO HFBR. Cell supernatants (20 mL) were harvested daily for up to five months and assayed for IL levels by ELISA (Figure 3).
This is an important cytokine with potential clinical applications as a lymphocyte growth and activation factor. Although monomeric E. coli-produced IL-15 is in the initial stages of clinical testing, this form of the molecule poses multiple challenges for clinical use due to its instability and rapid plasma clearance.
HEK293 cells produce correctly folded and glycosylated human IL-15-RC heterodimeric cytokine that shows greater stability and longer half-life. The superior bioactivity of IL-15-RC in the heterodimeric form is the result of the presence of the IL-15 receptor contributing to increased stability of the protein in vivo. These properties offer the potential to allow lower and less frequent dosing and simpler delivery methods, with increased convenience for both patients and caregivers.
Protein research at the laboratory scale is the basis for the development of therapeutic products. It is critical that these be produced in a form that retains all of the characteristics of the final product so that results seen the research laboratory can be extended to the clinic.
Expression of proteins and especially difficult-to-express proteins in mammalian systems is efficient and cost-effective in an HFBR. There are many advantages to working with a protein that is correctly folded with tertiary structure intact:
1. Solubility is maintained.
2. Proper bioactivity is sustained.
3. Antigenicity and immunogenicity characteristics are retained.
4. Half-life and pharmacokinetics are improved.
New biotherapuetics under development are increasing in size and complexity, 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. These structures are not found in nature; rather, they emerge from the mind of the bioengineer.2 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.
An HFBR system allows any laboratory to take advantage of the superior folding, glycosylation, and complete post-translational modifications that only expression in mammalian cells can provide. In vivo-like cell densities, constant provision of nutrients and removal of waste products, and total lack of shear result in complete and uniform post-translation modifications over continuous production cultures.
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 (Figure 4), meaning that scalability of the system is determined by length of culture, not new equipment.
1. Chertova E, Bergamaschi C, Chertov O, et. al. Characterization and Favorable in Vivo Properties of Heterodimeric Soluble IL-15-IL15Rα Cytokine Compared to IL-15 Monomer. June 21, 2013. J. Biol. Chem. 288(25): 18093–18103.
2. Cui X, Cao Z, Chen Q, et al. Rabbits Immunized with Epstein-Barr virus gH/gL or gB recombinant proteins elicit higher serum virus neutralizing activity than gp350. July 25, 2016. Vaccine 34(34): 4050–4055.