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Self-interaction of macromolecules has been shown to play an important role in a number of physical processes, including crystallization, solubility, viscosity, and aggregation. Peptide self-interaction is not as well studied as for larger proteins, but should play an equally important role. The osmotic second virial coefficient, B, can be used to quantify peptide and protein self-interaction. B values are typically measured using static light scattering (SLS). Peptides, however, do not scatter enough light to allow such measurements. This study describes the first use of self-interaction chromatography (SIC) for the measurement of peptide B values because SIC does not have the molecular size limitations of SLS. In the present work, SIC was used to measure B for enfuvirtide, a 36-amino acid therapeutic peptide, as a function of salt concentration, salt type, and pH. B was found to correlate strongly with solubility and apparent molecular weight. In general, the solubility of enfuvirtide increases with pH from 6 to 10 and decreases as the salt concentration increases from 0 to 0.5M for three different salts. The effect of peptide concentration on B was also investigated and shown to have a significant effect, but only at high concentrations (>80 mg/mL).

A method is described to enhance the resolution and detection sensitivity of proteins, peptides, and amino acids in capillary electrophoretic analysis of solution mixtures. The method consists of derivatizing the analytes with fluorescamine, which is normally used as a fluorogenic reagent for compounds containing a reactive primary amine functional group, and then using the derivative as an ultraviolet chromophore to enhance detection sensitivity (measured at 280 nm) in capillary electrophoresis. The results demonstrated a significant improvement in the separation and detection sensitivity of the derivatized analytes as compared to their underivatized counterparts. The use of chromophores, such as fluorescamine, in capillary electrophoresis facilitates the analysis of components of solution mixtures, such as pharmaceutical formulations, that could not be resolved and/or detected by conventional capillary electrophoresis procedures.

Several methods are available for determining the components present in a typical pharmaceutical dosage form. Although some of these methods are simple and have received wide pharmaceutical industry acceptance over many years, several limitations still need to be overcome. Capillary electrophoresis was examined as an alternative method for the determination of a recombinant cytokine in a pharmaceutical dosage form. The results show that capillary electrophoresis can separate the recombinant cytokine from its dosage form matrix adequately, rapidly and with minimum sample volume requirements. Owing to its high efficiency, high resolution and simplicity, capillary electrophoresis appears to be very applicable to pharmaceutical samples. For reliable quantification of the resulting data, however, it is essential to optimize the experimental conditions, such as separation buffer and capillary column temperature. The advantages and potential of capillary electrophoresis for the separation of active ingredients and excipients in pharmaceutical formulations are discussed.

Capillary electrophoresis has proved to be a versatile method for the determination of proteins, peptides and amino acids in pharmaceutical formulations. For quantification of the capillary electrophoresis data, however, significant errors may result if the analysis is performed using improper separation conditions. The peak area response for protein analytes, which is generally low in conventional UV detection, may also vary dramatically depending on the nature of the buffer used in the separation. This paper describes the effects of various buffer constituents and analytical conditions on the capillary electrophoretic separation and quantification of a humanized monoclonal antibody in bulk form and in a typical therapeutic formulation. For optimum peak area response and reproducibility, protein derivatization with an appropriate chromophore (e.g., fluorescamine) and separation in the presence of a moderate ionic strength buffer containing lithium chloride, tetramethylammonium chloride or trimethylammonium propylsulfonate is recommended. General guidelines for the determination of proteins by capillary electrophoresis and a rationale for the use of internal standards to improve the quantification of data are also discussed.

A purified glycoprotein, designated RSL-major, was isolated from the rat sublingual gland by means of the procedure of Tettamanti and Pigman. It was found to be homogeneous by analytical ultracentrifugation, to have a mol. wt. of 2-2 times 10-6, and to contain 81 percent (W/W) of carbohydrate, which consists mainly of sialic acid, 2-acetamido-2-deocy-D-glucose, 2-acetamido-2-deocy-D-galactose, and D-galactose in the molar ratio of 1.4:1.4:1.0:1.5; small amounts of fucose and mannose [1.2 and 2.8 percent (W/W), respectively] were also present. The sialic acid residues were resistant to the action of V. cholerae neuraminidase. This resistance was completely abolished by removal of the O-acetyl groups contained in the sialic acid. The sialic acid in RSL-major appeared to be a mixture of N-acetyl-4-O-acetyl- and N-acetyl-4, 7(8)-di-O-acetylneuraminic acids. The carbohydrate to protein attachment of RSL-major was shown, by alkaline beta-elimination reaction, to consist of an O-glycosyl linkage between 2-acetamido-2-deoxy-D-galactosyl residues in the oligosaccharide chains and seryl and threonyl residues in the protein core. The average oligosaccharide, contained in RSL-major, was postulated to be a heptasaccharide. A second material, designated RSL-minor, and also isolated from the ratsublingual gland, was obtained as a mixture of glycoprotein(s) and hydroxylapatite gel, and was not purified further.

The visco-elastic properties of salivary secretions are due to high molecular-weight glycoproteins, known as mucins. Mucins are composed of numerous oligosaccharide side-chains O-glycosidically linked through 2-acetamido-2-deoxy-alpha-D-galactose to the hydroxyl groups of seryl and threonyl residues of the protein core; on the average, every fourth amino acid residue is involved in such a bond. This work conveys their isolation and purification, compiles the compositional analysis of several mammalian submaxillary and sublingual mucins; defines the conditions of the alkaline beta-elimination reaction, its mechanism, and importance in structural studies of glycoprotens, and briefly discusses the influence of stimuli on mucous secretions, as well as biosynthesis, structural diversity, and physiological role of salivary mucous glycoproteins.