Trends in Pharmaceutical Analysis: A Technology Forum - - Chromatography Online
Trends in Pharmaceutical Analysis: A Technology Forum

The Column
Volume 10, Issue 9, pp. 2 to 6

Q: How are methods used for quality control and characterization of biopharmaceuticals different from those used with small-molecule pharmaceuticals?

AVS: Biopharmaceuticals tend to be more complex in primary and secondary structure. In the past decades we have seen the arrival of various forms of biopharmaceuticals, all with their own specificity. Following the first set of compounds made through genetic engineering, we have seen the coming of monoclonal antibodies and the conjugated forms of biopharmaceuticals, made in order to enhance their pharmacokinetic performance. All of these biopharmaceuticals require proper characterization such as a study of glycosylation patterns and checking for the presence of deamidated products. Thinking about nucleic acid-based materials as oligonucleotides, the determination of their sequence can be done using MS coupled to a separation technique. Purity testing can gain from the combination of different orthogonal techniques, such as ion exchange liquid chromatography and sieving capillary electrophoresis.

For proteins, sequencing techniques and tryptic maps can also perform structure confirmation. But the biopharmaceutical field is in need of techniques that allow quality assessment of intact proteins. The latter are indeed the compounds that will be administered to the patient, and their activity and quality are determined by the structure of the intact protein.

TvW: Biopharmaceuticals cover a wide range of compound classes and, when compared to small molecules, the classification of purity and impurity is not that well defined. Historically, many people working in biopharmaceuticals have a background in small molecules and the ICH Q3A/B guidance may be followed as a way of ensuring quality, however, this may not always be feasible or required. Often "fingerprints" are used for characterization purposes. For biopharmaceuticals, higher reporting and identification levels of impurities are acceptable because of the larger process variation anticipated.

For both small-molecule pharmaceuticals and biopharmaceuticals, high-end technology is available and is more often applied as supportive data for product characterization in regulatory filings. For routine quality control analysis, however, the classic methods, such as ELISA and SDS-page for antibodies, are still in place.

HN: Typically, the "purity" of biopharmaceuticals extends beyond the level of identifying or quantifying components that are not the intended active ingredient. Biopharmaceuticals may consist of mixtures of iso-forms and slightly (differently) modified proteins that can all represent (some) activity. Therefore, profiling the composition of these mixtures is an important part of biopharmaceutical analysis in characterization and quality control. Parameters evaluated often include: Folding and association using spectroscopic techniques (circular dichroism, fluorescence); oxidation, deamidation, and N- and C- terminal heterogeneity using typtic peptide mapping; charge heterogeneity using cation-exchange chromatography (CEX) or capillary isoelectric focusing (CIEF); and glycosylation using digestion or deglycosylation with reversed-phase LC, anion exchange chromatography (AEC), or matrix-assisted laser desorption–ionization time-of-flight (MALDI-TOF), and receptor assays.

Following on from the fact that drug activity results from the combined effect of many individual contributions, at least one (overall) activity assay (often cell-based) is always included.

Furthermore, the diversity of product- and process-related impurities is generally much wider for biopharmaceuticals than for small-molecule pharmaceuticals. As a result, the number of methods needed to cover all of these is generally much wider too. These include: Product-related impurities: Soluble aggregation is tested using size-exclusion chromatography (SEC); and cleavage, decomposition, or proteolysis is tested using SEC, SDS-page, or CE. Process-related impurities: Host cell proteins are tested using immunological techniques; DNA impurities are analyzed using real-time polymerase chain reaction (qPCR); and individual generally xenobiotic process additives are analyzed using immunological, chromatographic, or spectroscopic techniques. Other: Bioburden or virus-related testing is carried out using compendial techniques; and general parameters are also tested using compendial techniques. Please note that my focus here has primarily been on antibody biopharmaceuticals.


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