One of the major causes of delay in the pharmaceutical development process is inadequate forward planning on the use of analytical methods. Nowhere is this more acute than in the implementation of cell-based assays. It is never too early to develop these methods as the need to identify, quantify, and measure the potency of the drug substance is vital in producing meaningful and interpretable data.
Before going into more detail regarding the development of an analytical package, there are a number of considerations to bear in mind. First, we have found at Covance that it often takes longer than expected to develop, evaluate, validate, and transfer the majority of assays. This is even more of an issue for bioassays that can take six months or longer to transfer and validate.
Second, acceptance criteria should not be fixed too early. During development, the assay may have been carried out over many months by one or two dedicated individuals who gained significant experience and familiarity. This may not be representative of the actual release studies which, due to volume and scale-up, may need to be performed by a greater number of analysts in different locations.
Finally, it is often the detail that is not written into the SOP that can turn out to be the critical parameter that increases variability (e.g., such apparently minor details as angle of pipetting, ambient temperature, and the number of times an incubator door is opened and closed).
In this article, we look at the assays themselves and provide an overview of the key areas to manage to avoid unnecessary delays in the establishment of a robust and validated analytical package for large molecules (Figure).
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.