Highly sensitive glycosylamine labelling of O-glycans using non-reductive ß-elimination.

When developing biopharmaceuticals, glycans are the most important posttranslational protein modifications that must be addressed because they affect the between-protein interactions that maintain homeostasis. The glycan profile may be defined as a critical quality attribute of a biopharmaceutical. Comprehensive analysis of protein glycosylation must overcome challenges such as the release, labelling, separation and detection of O-glycans. In contrast, N-glycans can be readily released non-reductively from peptide backbones using an enzyme such as peptide N-glycosidase F. We developed a highly sensitive protocol using RapiFluor-MS to label glycosylamines for O-glycan analysis combined with a non-enzyme treatment for efficient release of the reduced O-glycans from the glycoproteins. Here we used the cytotoxic T lymphocyte associated protein 4-immunoglobulin G (Ig) fusion protein and fetuin as models for O-glycan analysis and compared the analytical methods glycopeptide mapping, 2-AB labelling and RapiFluor-MS labelling. The structures of major O-glycans and low-abundance O-glycans were successfully identified using the third technique, which detected the O-glycans with high sensitivity.

Pharmaceutical development of a parenteral formulation of conivaptan hydrochloride.

Conivaptan hydrochloride injection (Vaprisol®) was developed for the treatment of hyponatremia. Because the drug is very slightly soluble in water, pH control and cosolvency techniques were used to achieve the optimum concentration required for clinical trial material. Stability studies on retained samples of the clinical trial material for early-phase trials showed white visible particulates mainly in the headspace of the glass ampoule long after completion of the trials. The mechanism for generation of the particulate matter was formation of freebase of conivaptan hydrochloride because of increase in pH. The pH of the formulation for late-phase clinical trials, primary stability studies, and commercial production was fine-tuned to prevent particulate formation. The formulation contains propylene glycol and ethanol. Considering the nature of the cosolvent used in the formulation, the amount of di(2-ethylhexyl)phthalate (DEHP) delivered from an infusion system was evaluated, and we confirmed that the level of DEHP was lesser than that mentioned in the guideline. In the course of the scale-up studies for commercialization, the formulation failed the filter integrity test after the compounding solution was filtered. The dimethylsiloxane extracted from the silicon tubing used for solvent transfer coated the filter surface, which resulted in suppression of the bubble point value. The formulation and manufacturing process enabled conivaptan hydrochloride to be approved and launched in the market as a parenteral formulation. LAY ABSTRACT: Formulation scientists have recognized a trend that promising new chemical entities in the drug discovery phase often do not have ideal physicochemical properties for formulation. In particular, poor solubility is one of the challenges for development of a parenteral dosage form. Here, we describe the case of such a new chemical entity, a very slightly soluble hydrochloric salt, which was handed over from a drug discovery research laboratory to a pharmaceutical laboratory. pH control and cosolvency techniques were used to achieve the optimum concentration required for the product. However, a couple of issues emerged; we performed confirmatory studies in the course of development, and the results of these studies were used to design the formulation and manufacturing process. Most prominent issues were particulate matter formation on the inner surface the headspace of the ampoule and failure of the filter integrity test. In addition, we discuss the root causes and the mechanisms of the above issues and the measures taken to prevent them. A part of them are crucial, and information obtained from this study is important for development of new chemical entities as injectables in the future.

Excipient hydrolysis and ester formation increase pH in a parenteral solution over aging.

Recently, the number of drug substances that are poorly water-soluble has increased dramatically. This makes improving solubility one of the most critical tasks in pharmaceutical development today. In this study, the physicochemical stability of an injectable solution of conivaptan hydrochloride salt was investigated. Because its free form is hydrophobic, the drug substance was solubilized in a co-solvent system, 40% of which was composed of different alcohols. Since the free form is also alkaline, lactic acid was added to the co-solvent system to further improve its solubility. Remarkably, the pH of the solution was found to increase gradually over time. Considering the physicochemical nature of the drug substance, uncontrolled increases in pH would pose a potential threat of reducing solubility and forming precipitates. For this reason, a risk evaluation was performed. The evaluation revealed that the pH increase was caused by the hydrolysis of lactic acid oligomers as well as by the ester formation occurring between lactic acid and the alcohols. High concentrations of lactic acid supplied as an excipient usually contain lactic acid oligomers, which are hydrolyzed into lactic acid monomers upon dilution with water. Commercial software was used to determine the pK(a) values of the lactic acid oligomers, which were found to be lower than that of lactic acid monomers. This indicates that hydrolysis causes the pH to increase. Ester formation consumes the acid, which also causes the pH to increase. However, both hydrolysis and ester formation equilibrated by the 16-month time point when stored at 25 degrees C. This information allowed the upper limit of the pH increase to be determined molecularly, thereby ensuring product quality through the prevention of precipitate formation due to reduced solubility. Increased awareness of the importance of risk evaluation in pharmaceutical development is critical as these kinds of chemical reactions between excipients constitute a potential risk factor, but tend to be overlooked.