Mention industrial cell culture and most people think of mega-processes of tens of thousands of liters. In fact rising protein titers are causing new processes to shrink somewhat. In addition, emerging applications in the culture of stem cells, primary cells, and specialized cells for basic research are helping to create opportunities at the lower end of the scale.
Supply chain reliability, with a view of risk mitigation, is an important trend in bioproduction based on large-scale cell culture, according to Nicolas Barthelemy, svp at Invitrogen (www.invitrogen.com). For cell culture-based firms, this means having redundant supply sources.
Redundancy can mean multiple suppliers, or one supplier running two or more production facilities for critical components such as cells, media, consumables, and supplements. In the latter case, production plants need to be harmonized and covered by a consistent, robust quality system. “Otherwise, redundancy becomes an optical illusion,” says Barthelemy.
Other notable emerging trends in cell culture include the pursuit of greater productivity and higher protein titers, the evolution of piecemeal workflows into integrated workflows (particularly through single-use disposable systems), and the ongoing evolution of media from serum-free to animal-derived component-free to chemically defined.
Not too long ago, biomanufacturers were comfortable with black-box media. “Now, as part of risk-mitigation and quality strategies, companies desire a deeper understanding of the various components of their cell culture media. If they know what’s inside, they can optimize it.” Taking the universe of cell culture users as a whole, Barthelemy estimates that between 35% and 40% of cell culture users employ media that is free of animal-derived components; while as many as 25% use chemically defined media.
The reasons everyone has not switched to these inherently safer media are twofold. First, legacy processes are difficult to change—if it works, why mess with it? Second, looking too closely at black-box bioprocess components opens up a regulatory Pandora’s box. Biomanufacturers who examine processes too closely might find something, and if they do they will probably have to explain it to regulators.
Barthelemy notes several other emerging cell culture trends:
• Biosimilars or follow-on biologics are on the horizon and will ultimately have an enormous impact on demand for cell culture products and capacity. Biosimilars are probably still several years away, and microbial (vs. mammalian) systems will probably be the first ones approved.
• Cell therapy using stem cells, immune system cells, and even somatic cells is at a tipping point—approximately where biotech was in the early 1980s. “There’s a lot of investment and a lot of great science going on here, with no leaders or standards emerging thus far.” Barthelemy predicts numerous applications in conventional and regenerative medicine within 10 years.
• On the research side, interest in primary (as opposed to transformed) cells is growing for direct therapeutic applications, drug testing, ADME studies, and toxicology. Primary cells are typically precursors to animal work because they provide a higher level of predictability in cell assays and generate proteins, as one would expect, from cells in vivo.
Cosmetics companies are particularly interested due to ethical issues related to animal testing. The downside: a limited number of cell divisions, which puts a high premium on feeding and maintaining cells in a healthy state.
Chicken and Egg Question
The emergence of cell culture for vaccine production is one trend that is pushing production-worthy cell culture downward in scale.
Novartis (www.novartis.com) recently broke ground on construction of a $600 million cell culture vaccine plant in Holly Springs, NC. The plant will produce vaccines for seasonal influenza, and vaccines for pandemic flu should an outbreak occur. When the facility is geared up for production in 2011, it will produce about 50 million doses of seasonal flu vaccine and the ability to generate 150 million doses of pandemic flu vaccine within six months after receiving the order.
Virus-based influenza vaccines produced through cell culture offer two major benefits compared with chicken-egg culture. Production is no longer at the mercy of chicken and egg supplies, and manufacturing is more robust, scalable, and predictable. “Our eggs are MDCK cells,” says Ulrich Valley, Ph.D., site head of operations at Novartis. Seed cells are stored in a deep freeze until use and expanded in bioreactors. MDCK cells exhibit robust growth characteristics similar to those of CHO cells.
Vaccine production in cultured cells differs in several respects from cell culture for protein products. Because vaccine doses tend to contain minute quantities of active antigen or live or live-attenuated virus, the cultures are much smaller than for, say, monoclonal antibody production. Cells are chosen specifically for their infectivity with the virus or viruses of interest.
Novartis’ flu vaccines employ two influenza antigens, neuraminidase and hemagglutinin, rather than the whole virus. The antigens are generated by the addition of strong detergent, which also facilitates clearance of undesirable viruses. Additional viral clearance is achieved chemically and through column chromatography.
For its EU approval-pending vaccine, Novartis also tested for about 25 adventitious contaminating viruses, and demonstrated clearance for five major virus families. After production, titers are checked with a single-radiant immunodiffusion assay. The company is also planning to file a BLA in the U.S.
A huge difference between conventional cell culture and that used for influenza virus production is that the latter change every year, and each vaccine is a new product. Culture conditions and yield can and do change. Flu vaccine production is also constricted by time. Generally, batches must be ready by late September or they will not reach the marketplace in time for flu season.
Bioprocessors and cell culture product vendors continue to show that with cell culture a process does not need to be huge to have a huge impact. Nowhere is this more evident than with disposable cell culture systems.
Corning (www.corning.com) produces cell culture products in both glass and disposable plastic formats. Among its offerings is CellCube® disposable cell culture modules with surface areas ranging from 8,500 to 85,000 cm2. The systems come with either plain polystyrene surfaces or Corning’s enhanced CellBIND® surface for improved cell attachment.
CellCube systems are sold with accessory products such as a controller, oxygenator, circulation pump, and oxygen probe. Applications include production of cells, viruses, and proteins. According to applications manager Todd Upton, Ph.D., CellCube is used almost entirely in manufacturing. “A large number of pharmas and biotechs use these for protein and virus production as well as for vaccines at up to millions of doses per year.”
From Dr. Upton’s perspective, end users are looking for more efficient use of incubator or growth capacity space for experimental and production cell lines. “Our customers want products that enable them to grow adherent cells at higher density.” Another focus these days is on primary cells—hepatocytes, neurons, islet cells, bone cells, and others—as well as stem cells. “These all require specialized surface for attachment.”
For these cells, Corning has developed the 3-D nanofiber Ultra-Web™ synthetic surface, which, according to the company, provides a growth environment that closely resembles the biological niches of stem cells and other specialized cells. “It’s more in vivo-like than polystyrene,” says Dr. Upton. In April the company introduced four new products based on Ultra-Web—two 96-well microtiter plates and two tissue culture dishes.
Stem cell culture is another emerging small culture technique with a potentially large impact. According to COO Hugh Ilyine of Stem Cell Sciences (SCS; www.stemcellsciences.com), the supply of stem cells is the critical component of this business.
Established stem cell techniques harvest cells from animal or cadaver tissues. More exciting are recent developments that collect stem cells from a variety of human tissues for example bone marrow and embryo-related tissue. These sources will eventually provide stem cell researchers with a wide variety of cells in a reliable, predictable format, which is expected to lead to commercialization of therapeutic stem cells.
SCS focuses on the production side of stem cells, which means fully defined media products to grow and control stem cell biomass, and to induce differentiation. The company is currently scaling up its technologies for growing cells, including media, at its new facility in Cambridge, U.K.
Within SCS’ SC Proven line of media products are the PassAID™ medium supplement and the HEScGRO™ medium for human embryonic stem cell culture. PassAID is a media supplement containing a Rho-associated kinase inhibitor for the enzymatic passaging of human embryonic stem cells. HEScGRO medium is claimed to be the first human embryonic stem cell growth medium that is complete, serum-free, animal-component free, and ready to use.
Also offered are ESGRO Complete™ clonal-grade medium that enables mouse stem cells to grow in the absence of fetal bovine serum or primary mouse fibroblast feeder cells and NDiff™ Neuro medium supplements for in vivo differentiation of murine embryonic stem cells. All of the SCS’ media products are available through Millipore (www.millipore.com).
In addition to media and nutrients, SCS licenses its stem cell production, selection, and genetic engineering technologies to research and commercial organizations. Among its licensees are Merck, to which SCS also sells media for growing and differentiating cells.
BrainStorm Cell Therapeutics’ (BCT; www.brainstorm-cell.com) principal business is to treat neurodegenerative diseases through autologous reimplantation of adult stem cells. Because the cells are harvested from the eventual patient, BrainStorm sidesteps not only the ethical issues related to embryonic stem cells, but the potential for tumorigenicity from heterologous transplants.
Using a procedure developed at Tel Aviv University by Eldad Melamed, M.D., Brainstorm scientists harvest mesenchymal stem cells from the bone marrow cells from patients, propagate them to create a large mass of cells, then differentiate them into the desired neurological cell types. Mesenchymal cells are notable for their dual ability to create identical daughter cells or turn into somatic cells. For neurodegenerative disease treatment that means specialized neuron-like cells that release dopamine and glial-derived neurothrophic factor.
The company is targeting Parkinson’s disease, amyotrophic lateral sclerosis, and multiple sclerosis. It has developed an animal model of Parkinson’s in which implanted nerve cells can cure or delay the onset of disease. BCT is now looking to turn the process into a robust, reproducible protocol suitable for human testing.
Embryonic stem cells grow easily, as do those taken from fetal tissues. Adult stem cells, currently a hot area of research, are more difficult to propagate. BCT puts a lot of effort into growing adult stem cells in large quantities, according to Avinoam Kadouri, Ph.D., chief technology adviser. “The problem,” says Dr. Kadouri, “is the low native abundance of highly desirable mesenchymal and progenitor cells.”
The breakthrough for BCT was a protein-containing media that is animal-derived component-free. Dr. Kadouri describes the media as the significant factor that allows propagation of stem cells without differentiation. Once the cell mass is large enough, the cells are allowed to differentiate into dopamine-secreting astrocytes (for neurodegenerative diseases), or bone, fat, or other cells. The mixture of added growth factors and cytokines dictates what direction the cells take.
Getting really small with cell culture is useful for research, particularly for ADME, toxicology, drug discovery, cell line engineering, media optimization, as well as for monitoring the health of a large culture. Nikon Instruments (www.nikoninstruments.com) recently introduced a microscope product, the BioStation CT, that allows users with minimal microscopy experience to conduct imaging of live cells locally or by remote operation over a computer network. BioStation CT allows investigators to manage, observe, and record vital cell characteristics (growth, morphology, protein expression) from multiple cultures under environmentally controlled conditions.
The microscope can also measure multiple parameters within cells through user-selected settings for magnification, fluorescence wavelengths, viewing axes and dimensions, and multiple fluorescence modes.