Biopharmaceuticals and in particular therapeutic proteins produced using recombinant DNA technologies are generally complex, heterogeneous, and subject to a variety of enzymatic or chemical modifications during expression, purification, and long-term storage. Hence a full detailed structural characterization, quantitation, purity assay and evaluation of the biological activity are strictly required for the quality assessment.
The aim of the course is to point out the main applications of the most updated techniques for assessing the chemical and biological properties of biopharmaceuticals. In particular the course is based on the following topics:
Structural Characterization and Confirmation: molecular weight by mass spectrometry, sequences by LC-MS/MS sequencing of peptide fragments from digests, peptide map, isoforms pattern,
disulphide bridges (S-S bridge) maps, glycosylation maps, post translational modification studies (PTM), positions of conjugation e.g. protein-toxin conjugates using LC-MS and LC-MS/MS to determine loading and binding sites, extinction coefficient determination, spectroscopic profiles by UV-VIS, conformational integrity, higher-order structure, heterogeneity of the sample by omics techniques;
Impurities: product related impurity analysis, determination of variants such as truncated forms, modified forms and aggregates (soluble and insoluble);
Contaminants: in biopharmaceuticals there are potential risks associated with host cell contaminants derived from bacteria, yeast, insect, plants, and mammalian cells. For products derived from insect, plant, and mammalian cells, or transgenic plants and animals, there may be an additional risk of viral infections. The presence of cellular host contaminants can result in allergic reactions and other immunopathological effects. The adverse effects associated with nucleic acid contaminants are theoretical but include potential integration into the host genome. Different methods to assess contamination of biopharmaceuticals will be illustrated, including methods to assess immunogenicity and immunotoxicity;
Quantitation: intact protein analysis (spectroscopic and MS methods), peptide signature based methods;
Pharmacodynamic characterization of biopharmaceuticals: Potency determination refers to the quantitative measurement of the biological activity of a given product. Biological activity is a critical quality attribute; therefore, potency testing is an essential component of quality control. During the course, various procedures for potency testing will be shown, including ligand and receptor binding assays, cell culture-based assays, biochemical assays (such as enzymatic assays) and animal-based assays. In more detail, regarding animal models, in recent years there has been much progress in the development of models that are thought to be similar to the human disease. These animal models include induced and spontaneous models of disease, gene knockout(s), and transgenic animals. These models may provide further insight, not only in determining the pharmacological action of the product, pharmacokinetics, and dosimetry, but may also be useful in the determination of safety (e.g., evaluation of undesirable promotion of disease progression). In certain cases, studies performed in animal models of disease may be used as an acceptable alternative to toxicity studies in normal animals. Some of the main animal models used will be shown.