Analytical considerations in the development of protein purification processes.
Successful and rapid process development requires the availability of suitable analytical methods at all stages of the development cycle (see Tables 2 and 3). The methods should be selected first to ensure the identity, potency, and purity of the product and second to facilitate the process development. Some assays are particularly valuable early in the recovery process (and early in the development cycle) because they are typically robust and can be used with crude samples that contain many components. Popular examples include SDS-PAGE, IEF, and immunoassay, which allow the process scientist simply to monitor the yield and removal of impurities on a broad scale. Each step should be monitored with a biological assay, so that the specific activity of the target protein can be followed and steps that cause a loss of activity can be quickly detected. At the end of the recovery process (and development cycle) more sophisticated analytical methods, for example RP-HPLC and mass spectrometry, are used to detect and characterize variants of the desired product. Highly process-specific assays, such as host cell impurity ELISAs, are developed at this point, and the key methods for process monitoring and final product characterization are selected. While each process development project is distinct, in all cases the use of well-validated complementary analytical methods will provide greater assurance of a timely and successful project.