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 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

High-performance liquid chromatography of proteins

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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G E Purdon

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.