Protein Chemistry Techniques
Protein chemistry techniques are used throughout the drug-development pathway to provide characterization, batch release, and stability-indicating assays together with pharmacopoeial methods.
These assays confirm the size (SDS-PAGE, SE-HPLC), charge distribution (IEX-HPLC, IEF), concentration (UVA280), physical characteristics (pH, appearance, osmolality, Karl Fisher, sub visible particulates), glycoproteins (O-and N-glycans, sialic acid), and impurities (deamidation, methionine oxidation, host-cell proteins) of the protein therapeutic and also any excipients.
At early stages of development, characterization of a protein therapeutic is essential for determining the methods that will be required to support toxicology, clinical, and stability studies as well as batch-to-batch consistency and comparability. In our experience, the main focus needs to be on developing appropriate assays that may require adaptation and on defining appropriate system suitability criteria.
Complete protein characterization provides essential information that can ease this process and pinpoint those assays that may need further development to support impurity analysis.
Problems can occur during the transition to Phase III, which requires validated assays and the need for impurity and excipient assays, which may not have been anticipated. Again, this process is eased by assessing assay robustness as early as possible and prior to validation. At this stage, the development of host-cell protein assays also becomes a priority as the manufacturing process is locked down and the reference host-cell protein can be identified. Both these processes require additional time and ideally should be planned up to a year before they are required.
There is always a balance between the ideal development plan and cost considerations. However, good characterization at an early stage, robustness testing, and planning provide useful information and experience to draw upon.
The Table gives an indication of the methodologies frequently employed during the drug-development life cycle within protein chemistry. While not exhaustive, the techniques reviewed in this article reflect those typically used to support characterization, batch release, and stability. Where appropriate, assays have been identified that can be used in a later phase to give additional information in support of batch release and/or stability testing.