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