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.
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
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).
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.