Special considerations for conducting genotoxicity tests with protein materials

Standard genotoxicity tests are often inappropriate for testing new biological entities, in particular for recombinant proteins which are nature-identical. Arguments that these may contain mutagenic impurities are not substantiated; however, we have produced evidence that such impurities would be detected amidst a vast excess of protein. Concerns that human patients receiving therapy may be at risk from higher-than-physiological levels of proteins are also somewhat theoretical. However, it is apparent that genotoxicity testing will be required for these products for the time being, even if pragmatic approaches reduce the battery of in vitro tests to Ames and chromosomal aberrations only, and reduce the top dose in vivo to 1000x the human therapeutic dose. There is a number of physical and chemical properties of proteins that demand special approaches to methodology if the tests are to produce accurate results. The potential for adsorption to certain forms of glass and plastic means special care must be taken in dissolving and diluting test solutions; adherence to filters means special low protein binding, non-pyrogenic filters should be used for sterilisation of test solutions, where this is necessary; freeze-dried powders aliquotted in multiple vials should be dissolved in minimal solvent and cascaded from vial to vial rather than trying to empty the solid contents for bulk weighing. As proteins are often supplied in solution, in order to achieve sufficiently high test concentrations, it may be necessary to resuspend test bacteria/cells in the test solutions for short periods of time before centrifuging and resuspending in selective or growth media.(ABSTRACT TRUNCATED AT 250 WORDS)