Protein potency, determination

The quality control of immunoglobuhns includes potency tests and conventional tests of safety and sterihty. The potency tests consist of neutrahzation tests that parallel those used for the potency assay of immunosera, except that in the cases of some immimoglobulins the assays are made in vitro, fit addition to the safety and sterihty tests, total protein is determined by nitrogen estimations, the protein composition by... 

The introduction of "fast HPLC" has proven to be particularly valuable in protein analysis. As stated earlier, assay time in RP-HPLC analysis of proteins is typically long compared to that for smaller organic molecules. We have evaluated the use of 0.6-cm ID x 4-cm columns packed with 3-um particles in the analysis of insulin by RP-HPLC for potency determination, related substances, and in peptide mapping (7). The use of the "fast column" allows considerable savings (40-60%) in analysis time, compared to the regular (0.46-cm ID x 25-cm) columns, without loss in resolving power. 

Product Potency Tests. Two assays were developed that measure the potency of the FGF-4 protein. In the first case, a one-step growth promotion assay is conducted on normal, human retinal pigment epithelial cells (ARPE-19). The assay measures metabolic activity (Alamar blue dye metabolism) after infection of ARPE-19 cells with a serial dilution of the drug substance (Ad5FGF-4). The increase in metabolic activity correlated with FGF-4 protein production (determined by an FGF-4 ELISA), increased de novo DNA synthesis (measured by BrdU incorporation), and an increase in cell number. 

Table 4 presents different stages during a cell activation cascade leading to a specific cell function and lists the assay formats commonly used to detect these events. The methods listed in Table 4 are separated into two categories to distinguish more reproducible quantitative assays that are used in industry as quality control assays for a lot release/stability potency determination and the assays that are less suitable for routine use but can be used for characterization of the therapeutic protein and comparability assessment. In some cases the same bioactivity method can satisfy the requirement of both categories, to be representative of MoA and suitable for routine use (quantitative and reproducible). 

Standardization and Testing". RequHemeats are geaerally specified within Hceases Hi the United States, and include a variety of Hi-process tests to assess purity, safety, and potency of the iadividual components and potency and safety of the final product. Potency is standardized by determining the size of the conjugate and the quantitative amount of saccharide that is bound to the carrier protein. General safety and immunogenicity is assessed Hi animals. 

Standardization and Testing". The final vaccine is tested for safety, potency, and residual chemicals. Safety includes testing for endotoxin and stetihty. Potency is evaluated by quantitative determination of the amount of hemagglutinin in the vaccine. Antibody to this glycoprotein is associated with protection. The single radial immunodiffusion (SKID) technique is used to standardi2e the mass of this protein in comparison to a reference preparation. 

Some inhibitors interact very slowly with the enzyme protein, and onset of inhibition thus exhibits time-dependence. These inhibitors are generally referred to as slow-binding inhibitors, and as slow tight-binding inhihitors if the potency of inhibition is extremely high. Analysis of these inhibitory mechanisms is complex because binding and dissociation rate constants may be determined in addition to values. Indeed, a complete analysis may require extensive use of specialized computer software, and the complexities of such analyses preclude their discussion in this chapter. However, the reader is directed to several publications from Morrison s laboratory if a slow-binding mechanism is suspected for an inhibitor of interest (Morrison, 1982 Morrison and Stone, 1985 Sculley and Morrison, 1986 Morrison and Walsh, 1988). 

In 2006, Greis and co-workers reported on the application of MALDI-TOF MS as a tool for rapid inhibitor screening [10]. Different kinases (protein kinase C-a, cAMP-dependent protein kinase) in combination with their substrates were assayed, and the inhibitory potencies of staurosporine and three novel compounds were determined. For all four compounds, IC50 values could be determined, and... 

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