The Applications

As we've shown, biopharmaceutical companies have many options when they choose an expression system: bacteria, yeasts, insect or mammalian cells, and transgenics. Each system has its advantages and disadvantages. Company decision makers must ask themselves several important questions: How much product must be made? How complex is the molecule? Does it require posttranslational modifications to be biologically active? The expression system determines what kind of contaminants will be present and in what quantities. It also determines economic factors: the time scales involved, expression levels obtained, and various regulatory issues. Each potential expression system must be evaluated for its ability to produce economically the maximum amount of biologically active product. Purification methods may be different for products that come from the different host systems. And regulators are more familiar with some systems than with others.

Bacteria

Advantages: Established regulatory track record; well-understood genetics; cheap and easy to grow; inexpensive media; high expression levels quickly — sometimes within five days; fast-growing (growth time measured in minutes); easy characterization (with few adventitious agents).

Disadvantages: Proteins are not usually secreted (so cell disruption step complicates harvesting); contain endotoxins; are microheterogeneous; no posttranslational modifications; possibility of incorrect protein folding; harvesting can damage proteins.

Products made by bacteria. Insulin produced in E. coli at Eli Lilly and Company was the first FDA-licensed drug produced through recombinant DNA technology. Genentech, Inc., uses E. coli to produce recombinant human growth hormone. Other companies use bacteria to produce the interleukin-2 lymphocyte growth factor and interferon, a cytokine.

Because the plasmid cloning vector used with bacteria can accommodate up to 10,000 base pairs of DNA, it limits the size of proteins that can be produced by bacterial fermentation. Because each amino acid in a protein is coded by three base pairs of DNA, there is a limit to the length of code that a bacterial plasmid vector will handle. Insulin is a small chain of about 51 amino acids with a molecular weight of about 6,000 daltons. Interferon's 165–166 amino acids give it a molecular weight of about 20,000 daltons. Human interleukin-2 is about 15,000 daltons. Other therapeutic proteins can be much larger, but all of these molecules are huge in comparison to more traditional drugs like acetaminophen (151 daltons), glucosamine (179 daltons), ibuprofen (206 daltons), or prednisolone (360 daltons).

Yeasts

Advantages: "Generally recognized as safe" by regulators; long history of use; genetics well understood; no endotoxins; high expression levels fairly quickly (two to eight weeks); protein is secreted for easy harvesting; fast growth (hours); inexpensive media; proteins usually properly folded; posttranslational modifications.

Disadvantages: Overglycosylation can ruin protein bioactivity, safety, activity, potency, or clearance; can contain immunogens or antigens; nonnative proteins are not always properly folded.

Products made by yeasts. S. cerevisiae and S. pombe have both been used in large-scale fermentation processes for beer and other products for hundreds of years. Yeasts produce several recombinant vaccines. Biotechnology can produce recombinant proteins that induce a patient's immune system to create antibodies against the organisms that normally express those proteins. The first such vaccine was a hepatitis B virus vaccine produced by recombinant yeast. Yeasts are currently being used to produce human insulin. Aventis is using yeast as an excipient to make human serum albumin. The cost of recombinant protein production in yeasts usually runs between $50 and $100 per gram of final product.

The yeast artificial chromosome (YAC) constructed by molecular biologists can accommodate recombinant DNA up to 100,000 base pairs long. Because of this, yeasts can produce larger recombinant proteins than bacteria can. Examples include streptokinase, human serum albumin, and tissue necrosis factor. P. pastoris has been shown to produce all of those in addition to smaller proteins like hirudin, aprotinin, and gamma interferon.

Insect Cells

Advantages: Posttranslational modifications; properly folded protein; product is secreted; fairly high expression levels; expresses within about four weeks; baculoviruses are harmless to humans.

Disadvantages: Minimal regulatory track record and short history of use; slow growth, expensive media; baculovirus infection is an extra step in the process; contain immunogenic host cell proteins; some incorrect glycosylation; mammalian viruses can infect the cells in warm culture.

Products made by insect cells. BEVS insect cell expression is the least frequently used expression system for fermentation or cell culture in the biopharmaceutical industry. It is a relatively new technology that some companies are beginning to put into use. Those include Pfizer, Wyeth-Ayerst, Bayer, and Human Genome Sciences, which is working to remove certain genes from the 100 or so that make up the baculovirus genome in hopes of easing purification of insect-cell sourced products.

Novavax is using insect cell culture to produce virus-like particles (the protein shells of viruses without the genetic material inside) for use as vaccines, therapeutics, and in diagnostics, viral structure crystallography, and viral drug-delivery systems.

Mammalian Cells

Advantages: Usually fold proteins correctly; usually make correct posttranslational modifications; can secrete protein; good regulatory track record; only choice (except for transgenics) for the largest, most complicated proteins.

Disadvantages: Expensive media; slow growth; may contain allergens and contaminants from bovine sources; require extensive characterization (many adventitious agents are harmful to humans and mammalian cells alike); complicated purification; expensive, $500–5,000/gram of final product.

Products made by mammalian cell culture. Mammalian cell lines and hybridomas usually can be counted on to fold human proteins properly and to perform the correct posttranslational modifications that make proteins work. As an expression system used by the biopharmaceutical industry, mammalian cell culture is second only to bacterial cell culture in frequency of use. The first drug to be produced commercially by mammalian cell culture was tissue plasminogen activator (tPA), used to dissolve blood clots. Another recombinant protein produced by mammalian cells is a glycoprotein called factor VIII, which induces blood clotting. Hemophiliacs used to be treated with factor VIII purified from human blood, which led to the infection of thousands of people with HIV. Now the protein comes from a much safer source: genetically engineered CHO cells.

The molecular weight of tPA is about 70,000 daltons, and factor VIII is much larger, almost a million! So are some of the monoclonal antibodies (like Genentech's Herceptin and Ortho Biotech's OKT-3) being produced by hybridoma cells in culture. These are all complex proteins that can be produced only by the cells of higher animals. Genentech's CHO facility in Vacaville, CA, can produce over 1,000 kg of MAb per year in fermentors totaling about 150,000 liters of capacity. That's considered one of the most efficient mammalian cell culture facilities in the world.

Transgenic Animals

Advantages: Capable of complex protein processing and of very large proteins; very high expression levels; correctly fold proteins; easy scaleup; low-cost production ($20–50/gram of final product).

Disadvantages: Little regulatory experience; unknown potential for viral contamination; variable expression levels; long time scales; unanswered purification questions; continuous production complicates definition of batches and lots; questions regarding observance of cGMPs on the farm; unresolved public image problems.

Products made by transgenic animals. Several products are currently being developed by the three main companies involved in transgenic animal production of biopharmaceuticals: PPL Therapeutics in Scotland, Pharming in the Netherlands, and Genzyme Transgenics in the US. PPL primarily uses sheep and rabbits to produce alpha-1 antitrypsin, bile salt stimulated lipase, collagen, superoxide dismutase, factors VIII and IX, fibrinogen, human serum albumin, blood-coagulation factor XIV, calcitonin, and some smaller peptides. Pharming focuses on cattle for its production of alpha glucosidase, C-1 esterase inhibitor, collagen, fibrinogen, factors VIII and IX, and lactoferrin. Genzyme is using goats to produce alpha-1 proteinase inhibitor, antithrombin III, beta interferon, calcitonin, human serum albumin and growth hormone, tPA, and a malaria vaccine.

Some recombinant proteins expressed in the milk of transgenic mammals are too big for bacteria or yeasts to handle. For example, the molecular weight of collagen is 130,000 daltons, and fibrinogen is a whopping 400,000 daltons!

Transgenic Plants

Advantages: Shorter development cycles than animals; easy storage of seed banks; easy scale-up; good expression levels (up to 1 kg purified recombinant protein/acre of crop); well-understood genetics; no plant viruses known to infect humans (so viral characterization unnecessary); low-cost production ($10–20/gram of product).

Disadvantages: Potential for new contaminants (soil fungi and bacteria, plant-sourced impurities and metaboliytes, pesticides, herbicides); posttranslational modifications differ from those made by animal cells; contain possible allergens; unresolved public issues.

Products made by transgenic plants. Integrated Protein Technologies is the division of Monsanto that is using genetic engineering technology to produce therapeutic proteins in transgenic corn. In late March 2000, IPT had six recombinant protein products in Phase II clinical trials. Dow AgroSciences is another player in this arena.

CropTech Corporation is using transgenic tobacco to produce human lysosomal enzymes, among other things. Human glucocerebrosidase is currently produced by mammalian cell culture. Patients with Gaucher's disease receive the drug every two weeks for life, costing an average $160,000 a year and making it one of the most expensive drugs on the world market, partly due to manufacturing costs. CropTech researchers have introduced the gene for glucocerebrosidase into tobacco and shown that an active enzyme is produced in the leaves. Leaves from a single tobacco plant could make enough enzyme for one dose. In addition to tobacco, corn, and other crops, duckweed is being investigated as a potential plant source of therapeutic proteins.

ProdiGene already sells recombinant avidin and beta-glucuronidase produced in transgenic corn plants as research and diagnostic reagents through a partnership with Sigma-Aldrich. The company also partnered with Genencor to produce industrial enzymes, and with EPIcyte to produce therapeutic antibodies. ProdiGene is also working to produce vaccines for hepatitis and transmissible gastroenteritis virus in edible form. Edible vaccines are also being produced by the Boyce Thompson Research Center, which genetically modified potatoes and tomatoes to produce recombinant vaccines for cholera, Norwalk virus, and hepatitis B.