Recombinant DNA technology has now made it possible to produce proteins for pharmaceutical applications Consequently, proteins produced via biotechnology now comprise a significant portion of the drugs currently under development Isolation, purification, formulation, and delivery of proteins represent significant challenges to pharmaceutical scientists, as proteins possess unique chemical and physical properties These properties pose difficult stability problems A summary of both chemical and physical decomposition pathways for proteins is given Chemical instability can include proteolysis, deamidation, oxidation, racemization, and β-elimination Physical instability refers to processes such as aggregation, precipitation, denaturation, and adsorption to surfaces Current methodology to stabilize proteins is presented, including additives, excipients, chemical modification, and the use of site-directed mutagenesis to produce a more stable protein species

Stability of Protein Pharmaceuticals

Rapid extraction of oxygenated metabolites of arachidonic acid from biological samples using octadecylsilyl silica.

The opiomelanotropinergic neuronal and endocrine systems.

The following fractions can be obtained after extraction of biological samples with ODS silica. 1. 1. Arachidonic acid or other nonpolar fatty acids. Apply sample to ODS silica in acidified 30% ethanol. Wash with 30% ethanol (20 ml), water (20 ml), and petroleum ether (10 ml). Elute 20:4 with petroleum ether-chloroform (1:1, 20 ml). 2. 2. Monohydroxyeicosenoic acids. Apply sample in acidified 15–25% ethanol, depending on whether it is desired to obtain a subsequent fraction containing PGs. Wash ODS silica with the same concentration of ethanol (20 ml), water (20 ml), and petroleum ether (20 ml). Elute monohydroxyeicosenoic acids with petroleum ether-chloroform (65:35, 20 ml), chloroform (10 ml) or methyl formate (10 ml), depending on whether or not it is necessary to obtain a subsequent PG fraction. 3. 3. Prostaglandins and thromboxanes. Apply sample to ODS silica in acidified 15% aqueous ethanol. Wash with 15% ethanol (20 ml) followed by petroleum ether (20 ml) or, if it is desired to extract monohydroxyeicosenoic acids, water (20 ml), and petroleum ether-chloroform (65:35, 20 ml). If nonpolar PGs such as 13, 14-dihydro-15-oxo-PGE 2 are to be extracted, petroleum ether should be used. Elute PGs and Txs with methyl formate (10 ml). 4. 4. Monohydroxyeicosenoic acids, PGs, and Txs. Apply sample in acidified 15% aqueous ethanol. Wash ODS silica with 15% aqueous ethanol (20 ml), water (20 ml) and petroleum ether (20 ml). Elute with methyl formate (10 ml). 5. 5. Polar PG metabolites. Apply sample in acidified 0–10% aqueous ethanol (for ω-hydroxy-PGs 10% ethanol can be used). Wash ODS silica with the same concentration of ethanol (20 ml) followed by petroleum ether-chloroform (65:35). Elute PG metabolites with methyl formate (10 ml). 6. 6. PGs extracted from neutral or basic media. Apply sample to the ODS silica in 10–15% aqueous ethanol at the desired pH. Wash with the same concentration of ethanol (10 ml) (for greater speed, it should be possible to eliminate this step), followed by petroleum ether (20 ml). Elute with methyl formate (10 ml). For nonpolar products, better results might be obtained by washing the ODS silica with water prior to petroleum ether.

Rapid extraction of arachidonic acid metabolites from biological samples using octadecylsilyl silica.

The rat calcitonin gene has recently been shown to encode a novel peptide (rat calcitonin gene-related peptide, rCGRP) thought to be produced in nervous tissue after tissue-specific RNA processing1,2. This peptide has so far been identified only in rat tissue, by immunocytochemistry and immunoassay. We now report the isolation of a related (89% homology) peptide from human tissue (hCGRP) which we have sequenced using a novel mass spectrometric approach, fast atom bombardment (FAB) mapping3–5. The human peptide differs significantly from the predicted rCGRP structure in four positions in the amino acid sequence (three effecting charge changes), and the presence of a disulphide bridge and an amide, surmised in the rat work, is proven in the hCGRP molecule. hCGRP was present in plasma from 10 patients with medullary thyroid carcinoma (MTC) and in 6 MTC tumours removed at surgery, suggesting the tissue distribution may differ from that in the rat where the peptide is reported to be absent from thyroid tissue2. hCGRP is shown to have biological activity and it is possible that its presence in MTC plasma may be responsible for some of the symptoms in this disease.

Isolation and characterization of human calcitonin gene-related peptide

Peptides can be adsorbed on octadecasilyl-silica from large volumes of aqueous solution and eluted with aqueous solvent mixtures containing methanol or acetonitrile. These properties may be used for the extraction and purification of peptide fragments in plasma samples collected from rats. After intravenous injection of Synacthen [corticotropin-(1-24)-tetracosapeptide], it was shown that within 2 min the main circulating products were intact peptide and its sulphoxide. In addition, a number of fragments indicative of cleavage at the N- and C-termini were present. Most of the products formed from Synacthen have low biological activity. Somatostatin was rapidly cleaved in vivo and in vitro to a single product, which probably retains biological activity. The absence of other circulating products suggests that somatostatin is only inactivated once it leaves the circulation.

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Use of octadecasilyl-silica for the extraction and purification of peptides in biological samples. Application to the identification of circulating metabolites of corticotropin-(1-24)-tetracosapeptide and somatostatin in vivo.

Peptides can be recovered from tissues by homogenization in a carefully selected extraction medium followed by adsorption from the supernatant on a small bed of octadecasilyl-silica. Recoveries of corticotropins and somatostatin added to a variety of tissues were quantitative, and the peptides undamaged as determined by high-pressure liquid chromatography.

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A rapid method, using octadecasilyl-silica, for the extraction of certain peptides from tissues.

An adrenocorticotrophic hormone (ACTH) was isolated from extracts of the pars distalis of the pituitary of the dogfish Squalus acanthias by gel filtration and ion-exchange chromatography. It had 15% of the potency of human ACTH in promoting cortico-steroidogenesis in isolated rat adrenal cells. Sequence analysis revealed it to be a nonatria-contapeptide with the following primary structure: Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Met-Gly-Arg-Lys-Arg-Arg-Pro-Ile-Lys-Val-Tyr-Pro-Asn-Ser-Phe-Glu-Asp-Glu-Ser-Val-Glu-Asn-Met-Gly-Pro-Glu-Leu. The N-terminal tridecapeptide sequence was identical with the proposed structure of dogfish α-melanocyte-stimulating hormone (α-MSH). On comparison with human ACTH eleven amino acid differences were seen, nine of which are in the 20–39 region of the molecule which is not essential for the steroidogenic activity of ACTH. A peptide identical with the 18–39 portion of this new ACTH was similarly isolated from the neurointermediate lobe of the pituitary where considerable amounts of dogfish α-MSH were found. This supported our view that ACTH as well as having a distinct biological role of its own is also the precursor of α-MSH.

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The isolation and amino acid sequence of an adrenocorticotrophin from the pars distalis and a corticotrophin-like intermediate-lobe peptide from the neurointermediate lobe of the pituitary of the dogfish Squalus acanthias.

A melanocyte-stimulating hormone (MSH) has been isolated from extracts of the neurointermediate lobe of the pituitary of the dogfish Squalus acanthias by gel-filtration and ion-exchange chromatography. It had approximately 1% of the potency of mammalian α-MSH on bioassays in vitro on frog skin and dogfish skin. Sequence analysis revealed it to be a hexadecapeptide with the following primary structure: Asp-Gly-Asp-Asp-Tyr-Lys-Phe-Gly-His-Phe-Arg-Trp-Ser-Val-Pro-Leu. It appears to be related to the β-MSH species of mammalian species but has only the sequence -His-Phe-Arg-Trp- in common with the heptapeptide core -Met-Glu-His-Phe-Arg-Trp-Gly- which is characteristic not only of the MSH peptides but also of the adrenocorticotrophins and lipotrophins studied so far. An α-MSH was also isolated, 50% of which was amidated at the C-terminus group. Sequence data from this study taken in conjunction with those from a previous study (Lowry & Chadwick, 1970b) revealed it to be a tridecapeptide which is identical with the N-terminal sequence of dogfish adrenocorticotrophin.

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Structural studies of α-melanocyte-stimulating hormone and a novel β-melanocyte-stimulating hormone from the neurointermediate lobe of the pituitary of the dogfish Squalus acanthias

1. The fate of corticotrophins in a trypsin-dispersed rat adrenal-cell assay system was investigated with a view to establishing whether differences in the rate of inactivation might contribute to potency differences observed between analogues. 2. Corticotrophin-(1–24)-tetracosapeptide and to a lesser extent synthetic 1–39 corticotrophins were found to be inactivated during incubation with cell suspension. 3. Peptide fragments were isolated by using [[3H2]Tyr23]corticotrophin-(1–24)- tetracosapeptide as a marker. The fragments indicate a peptidase with a predominantly tryptic specificity. 4. The peptidase is present in the extracellular fluid and is released from cells when they are damaged. 5. Cells were fractionated on an albumin gradient. Cells from the zona fasciculata and the zona intermedia or reticularis were present in fractions which produced fluorogenic steroids in response to corticotrophin. 6. Purification of the cells by centrifugation through albumin decreased degradation by peptidases, so that if the assay is carried out with a dilute suspension of purified cells peptide breakdown should not affect the observed potencies of adrenocorticotrophin analogues. 7. No binding of [[3H2]Tyr23]corticotrophin-(1–24)- tetracosapeptide to cells could be detected at low concentrations of the peptide. This indicated that less than 120 receptors/cell are occupied during stimulation by a dose that would elicit approx. 80% of the maximal response.

H. P. J. Bennett

Papers

Use of octadecasilyl-silica for the extraction and purification of peptides in biological samples. Application to the identification of circulating metabolites of corticotropin-(1-24)-tetracosapeptide and somatostatin in vivo.

A rapid method, using octadecasilyl-silica, for the extraction of certain peptides from tissues.

The isolation and amino acid sequence of an adrenocorticotrophin from the pars distalis and a corticotrophin-like intermediate-lobe peptide from the neurointermediate lobe of the pituitary of the dogfish Squalus acanthias.

Structural studies of α-melanocyte-stimulating hormone and a novel β-melanocyte-stimulating hormone from the neurointermediate lobe of the pituitary of the dogfish Squalus acanthias

Fate of corticotrophins in an isolated adrenal-cell bioassay and decrease of peptide breakdown by cell purification.