The low bioavailability of orally delivered peptides, which ranges from 1% to 10%, should not be problematic from a therapeutic standpoint since most peptides are highly potent. However, it carries special significance for manufacturing cost and scale, particularly for peptide drugs with high dosing requirements (e.g., Fuzeon and insulin).
Although great strides have been made in reducing costs and improving scalability for peptide production through chemical synthesis, recombinant technology has become the method of choice for the large-scale manufacture of larger peptides (25 amino acids or more).
Recombinant expression of foreign proteins in microorganisms and cells provides the best combination of cost-effectiveness, scalability, and environmental safety.
The manufacture of peptides in recombinant organisms began in the early 1980s. Since then, many different host cells and organism types have been used to produce peptides.
Microbial fermentation, particularly in E. coli, has several significant advantages over mammalian cell culture. E. coli fermentations are rapid, predictable, free of downstream contaminants associated with cells, and less costly than cell culture.
Conventional bacterial fermentation systems are not without their limitations, however. For example, the relatively small size and lack of tertiary structure of most peptides makes them susceptible to rapid degradation in the cytoplasm of expressing bacteria and yeast.
This drawback may be mitigated by expressing the product with a much larger protein fusion partner, which generally protects the peptide from proteolysis. Liberation of the product from the fusion partner, however, requires chemical or enzymatic cleavage, which adds at least two processing steps (cleavage and purification) and results in significantly reduced peptide yield.
Also, lysing the bacterial cell to release the peptide product causes release of all the bacterial proteins as well as DNA and bacterial endotoxins. These process-related contaminants then need to be purified away from the peptide of interest, which further increases the number of purification steps required.
An ideal expression system, therefore, would be one that allowed for the production of peptides without a fusion partner and secreted the expressed peptide from the cell into the growth medium, thus leaving the bacterial cells intact.
Unfortunately, obtaining excreted products from E. coli is difficult because the organisms do not normally excrete peptide or protein products and they produce proteases that break down foreign proteins intracellularly.
A further complication in producing peptide hormones in bacteria or yeast is the frequent requirement that these products be amidated at the C-terminus of the hormone for full biological activity. Prokaryotes lack peptidylglycine a-amidating monooxygenase (PAM), the enzyme that carries out this post-translational amidation, therefore, peptides produced in E. coli are not C-terminally amidated.
To address these issues, Unigene has developed a manufacturing platform that efficiently produces amidated peptide hormones through the use of two recombinant cell lines. The glycine-extended precursor of the desired peptide is first produced in recombinant E. coli using a direct expression technology.
The expression construct incorporates an upstream signal sequence that causes the peptide to translocate from the cytoplasm to the periplasm, at which point the signal sequence is cleaved. Due to further innovations in the growth conditions and the components of the growth medium, the peptide is then excreted into the growth medium. The E. coli host cell is a protease-minus cell that allows for the accumulation of the peptide in the growth medium without significant degradation.
Since E. coli does not excrete appreciable quantities of endogenous proteins, the peptide product in the conditioned medium provides a relatively enriched starting material for purification, thus reducing the number of purification steps and increasing the yields from purification.
After purification, the peptide is treated in vitro with PAM, which is separately produced from recombinant CHO cells. PAM quantitatively converts a variety of C-terminally glycine-extended peptides to the corresponding peptide amides at a mass ratio of enzyme to substrate of 1:1000 or greater, depending on the glycine residue’s immediate neighbor. Hence, the quantity of PAM needed is a small fraction of the amount of peptide to be produced, and the higher cost of production of PAM in CHO cells does not add appreciably to the overall cost of the process.
One or more chromatography steps then separates amidated product from precursor and other minor contaminants. After purification and amidation, the peptide is typically >98% pure.
The direct expression process is readily scalable up to 20,000 liters with no loss of productivity. Yields will vary depending on the peptide, but such products as salmon calcitonin, parathyroid hormone analogs, glucose regulatory peptide analogs, secretin, and growth hormone releasing factor have been expressed at up to 1g/liter of intact peptide. In instances where peptide degradation occurred in the growth medium, changes in the nutrient feed significantly reduced it.
There has never been a more exciting time to be involved in peptide pharmaceutical development. Oral delivery methods have changed the paradigm for peptide drugs irrevocably and for the better. The results of human genome research should provide peptide drug candidates for years to come.