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.

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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.