The Applications - BioPharm International - Excipients

The Applications - BioPharm International
Mar 10, 2006
BioPharm International
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.
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