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


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

BioPharm International

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.

ISSUES WITH RECOMBINANT PRODUCTION OF PEPTIDES 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 C-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.

Figure 1. Dual Recombinant Process for the Production of Amidated Peptide Hormones
ADVANTAGES OF DIRECT EXPRESSION RECOMBINANT TECHNOLOGY 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.6,7 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 E.coli cell into the growth medium.

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

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