Oral Delivery and Recombinant Production of Peptide Hormones, Part II: Recombinant Production of Therapeutic Peptides

July 1, 2004
Nozer M. Mehta

BioPharm International, BioPharm International-07-01-2004, Volume 17, Issue 7

This technology includes an efficient and scalable in vitro enzymatic amidation step for peptide hormones.

Last month, we described oral delivery technology for delivery of therapeutic peptides.1 We now describe a recombinant technology for the efficient and cost-effective production of these peptides. The two technologies are complementary to each other for developing a useful therapy. We will describe a direct expression process that is scaleable from 1 to 1,000 L without significant loss of fermentation productivity or yield following downstream purification. This platform technology for recombinant production enables the development of orally delivered peptide hormone drugs for chronic administration in a variety of therapeutic areas such as osteoporosis and diabetes.


For peptide hormones that are 25 amino acids or greater, recombinant production in bacteria or yeast offers the potential to be more cost-effective and environmentally acceptable than chemical synthesis, particulary at production scales of hundreds of kilograms per year. However, the relatively small size and lack of tertiary structure of most peptides make them susceptible to rapid degradation in the cytoplasm. Moreover, greater than 50% of the known peptide hormones and neurotransmitters are post-translationally modified by the addition of an amide group to the


-terminus of the peptide. Under normal physiologic conditions, amidated peptides are expressed as glycine-extended hormone precursors, and subsequently processed to yield the mature amidated hormone.

Amidation of the peptide is often required for full biological activity of the hormone.2 The enzyme that performs this post-translational modification is peptidylglycine α-amidating monooxygenase, or PAM.3 Since PAM is not present in prokaryotes, peptide hormones that are produced in E. coli are not C-terminally amidated.

The degradation problem can be circumvented by expression of these peptides with a larger protein as a fusion partner, which allows the resulting fusion protein to accumulate relatively undegraded in a soluble or insoluble form in the cytoplasm of the host cell.4,5 Although large amounts of fusion protein can be made in this manner to give yields on the order of grams/liter, there are several disadvantages. Liberation of the fusion partner from the peptide hormone by chemical or enzymatic cleavage can be difficult and expensive, and the resulting yield of the peptide is greatly diminished, since the peptide represents only a fraction of the entire fusion protein. Also, purification from the large number of cytoplasmic proteins that are released by cell lysis, as well as from the fusion partner itself, may require several steps, which can increase the cost of production and reduce the overall yield of the peptide hormone.


We developed a direct expression process for the efficient production of amidated peptide hormones that involves two recombinant cell lines (Figure 1). In the main path, a glycine-extended precursor of the peptide is produced in recombinant

E. coli

cells using a proprietary direct expression technology.


The glycine-extended peptide is produced with an upstream signal sequence that translocates the peptide from the cytoplasm to the periplasm. The signal sequence is cleaved in the periplasm, and the peptide is secreted from the


cell into the growth medium.

Figure 1. Dual Recombinant Process for the Production of Amidated Peptide Hormones

High levels of the peptide are obtained due to several desirable features of the system. These features include the use of a unique, high-expression plasmid vector, a protease-deficient host cell, and a fermentation protocol that allows for high-density cell mass and secretion of the peptide across the outer cell membrane into the growth medium. Since E. coli secretes very few endogenous proteins, the glycine-extended peptide hormone can be recovered from the conditioned medium in a relatively enriched form, which reduces the number of downstream purification steps.

Figure 2 shows a high-density SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) separation of crude conditioned medium from 1 L fed-batch fermentations producing four different peptide hormones: glycine-extended forms of salmon calcitonin, two analogs of a glucose regulatory peptide, and a parathyroid hormone analog. In each lane, the major band corresponds to the secreted recombinant peptide, which represents the major protein species in the crude conditioned medium. The identities of the four peptides have been confirmed by Western blots with the corresponding specific antibodies, and the intactness of each peptide has been confirmed by separation by reversed phase high-performance liquid chromatography (RP-HPLC), followed by mass spectroscopy of the peptide peak (data not shown). This SDS-PAGE experiment confirms that the secreted peptides are intact and that there are few contaminating proteins that need to be removed during purification.

Figure 2. High-Density SDS-PAGE of Crude Conditioned Medium from Four Different Direct Expression Fermentations

The other path produces 75 kDa bi-functional PAM in recombinant Chinese hamster ovary (CHO) cells (Figure 1). A readily scalable batch-culture bioreactor-production protocol has been developed. The recombinant CHO cells are grown in a medium that is free of proteins and animal-sourced components, and soluble PAM is secreted into the growth medium. PAM is separately purified from the conditioned culture medium by two column-chromatography steps to yield a preparation that is free of contaminating proteases and shows a single major 75 kDa band on SDS-PAGE.


Glycine-extended peptides are purified from the conditioned growth medium by a three-column purification procedure. Conversion of the peptide-glycine substrate to the peptide-amide by purified PAM is carried out in an

in vitro

reaction with the appropriate buffer and co-factors.

PAM is able to quantitatively convert a variety of glycine-extended peptides to the amide form at a mass-ratio of enzyme to substrate as low as 1:1,000 or greater, depending on the penultimate amino acid of the peptide. Hence, the amount of PAM needed is a very small fraction relative to the amount of peptide to be produced, and the high cost of producing PAM in CHO cells does not add appreciably to the overall cost of the process.

Following amidation, the bioactive peptide is separated from any remaining glycine-extended precursor and other minor contaminants by one or more chromatography steps. The purified peptide is lyophilized and stored at -80°C until formulated into the final product.


The fermentation protocol used in the direct expression process has been shown to be readily scalable from 1 to 1,000 L for several recombinant cell lines with no significant loss of productivity. The peptide yield from any fermentation varies depending on the peptide. Examples of peptides that have been successfully expressed with this direct expression technology are salmon calcitonin, analogs of parathyroid hormone, glucose regulatory peptide analogs, secretin, and growth hormone releasing factor. For some peptides, yields of approximately 1gram/liter of intact peptide have been achieved.

The direct expression technology also offers advantages in purification. No cell lysis is required to recover the peptide product, and, hence, the peptide does not need to be purified away from the large number of cytoplasmic and periplasmic proteins, nor from the high-molecular-weight DNA that would be liberated from lysed cells. E. coli secretes few endogenous proteins, so there are relatively few contaminating proteins in the crude conditioned medium. Since no fusion partner is used, no chemical or enzymatic cleavage is required to liberate the peptide from the fusion partner. The potential for contamination of the final product by DNA and endotoxin is greatly reduced. The yield of peptide following purification and amidation is generally at least 50%, and the purity of the amidated peptide is typically over 98%.


The direct expression fermentation technology, as well as the purification steps that utilize low-pressure chromatography, is readily scalable. Several proprietary peptides have been expressed and purified in 1,000 L batches at Unigene's manufacturing plant. Considering very large scale, for a peptide that yields 500 mg/L after purification, a single 20,000 L fermenter with the appropriate downstream purification suite, at one batch per week, would be large enough to supply about 500 kg per year. It should be pointed out that the direct expression technology can also be applied to peptides that are not


-terminally amidated. For such non-amidated peptides, CHO cell fermentation is unnecessary, and the peptide yield after purification may be higher since the amidation and downstream purification steps are eliminated.


Vicki Ray and Christopher Meenan have been largely responsible for development of the direct expression cloning and fermentation technology. Angelo P. Consalvo developed the downstream purification and amidation steps. Duncan Miller contributed to the development of the PAM enzyme clone-and-fermentation protocol. Process scale-up and cGMP manufacture of peptides at the 1,000 L scale are conducted under the direction of Paul Shields.


1. Mehta NM. Oral delivery and recombinant production of peptide hormones. Part I: making oral delivery possible.

BioPharm International

2004 June; 17(6): 38-43.

2. Eipper BA, Stoffers DA, Mains RE. The biosynthesis of neuropeptides: peptide α-amidation. Annu. Rev. Neurosci. 1992 15: 57-85.

3. Mehta NM, et al. Purification of a peptidylglycine α-amidating enzyme from transplantable rat medullary thyroid carcinomas. Arch Biochem Biophys. 1988, 261(1): 44-54.

4. Ray MVL, et al. Production of recombinant salmon calcitonin by in vitro amidation of an Escherichia coli produced precursor peptide. Bio/Technology. 1993 11: 64-70.

5. Shuman HA and Silhavy TJ. The art and design of genetic screens: Escherichia coli. Nat Rev Genet 2003. 4(6): 419-431.

6. Ray MVL, et al. Production of salmon calcitonin by direct expression of a glycine-extended precursor in Escherichia coli. Protein Expression and Purification 2002 26: 249-259.

7. Mehta NM, et al, inventors. Direct expression of peptides into culture media, US Patent 6,103,495, 2000 August 15.

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