A proposed in vitro cellular insulin assay to replace the USP bioidentity test

The goal of our investigation was to develop and assess a nonradioactive in vitro assay for insulin that could replace the in vivo USP Bioidentity Test(2). We have developed and tested a cell culture bioassay for insulin that is based on this hormone's primary biological effect, the stimulation of glucose uptake and metabolism, We have compared the precision and accuracy of an in vitro cellular bioactivity assay with the USP Bioidentity Test, total nitrogen measurement, and high-performance liquid chromatography (HPLC) assay of particular components. We propose that this cellular assay can be used to confirm that the hormone complex is in a biologically active conformation that can interact productively with relevant membrane receptors, Therefore, it could usefully replace the USP Bioidentity Test. [NOTE- It might be clearer if the names of these tests be changed, The USP Bioidentity Test might better be entitled the ''Biological Activity Test,'' and the HPLC test be entitled the ''Test for Insulin Content,'' The HPLC procedures furnish the amount of insulin, and related analogs (e.g., proinsulin) in the test material, It is possible to assign a theoretical potency which may be used for labeling, by adding the amounts of the components, adjusted where appropriate with a factor. The biological test would be an assay to confirm overall biological potency. There must be a reasonable relationship of results from the two test, within specified limits, the validity of which would be confirmed by the statistical calculations from the cell culture test data.] This cell culture bioassay uses 3T3-L1 preadipocytes that have been differentiated to adipocytes. Following differentiation of these cells, glucose transport and metabolism become robust and highly sensitive to insulin. The insulin-dependent disappearance of glucose from the media can be measured spectrophotometrically with a 96-well microplate reader, The assay shows a high level of ao curacy, and by virtue of its almost 3-fold lower index of precision and its narrow 95% confidence limits, this assay has also been found to be more precise when compared to the USP Bioidentity Test as currently formulated. Since rabbit blood sugar measurement has evolved over time into a bioidentity test rather than a true assay for determining precise potency, we have exceeded the rigors of required testing by showing the reproducible Precision and accuracy of this cell culture assay. The index of precision for the rabbit test averaged -0.41 +/- 0.56 (mean +/- standard deviation (SD), n=18), while for the cellular assay the index of precision was -0.15 +/- 0.07, (n - 60). The 95% confidence interval for eight rabbit tests averaged +/- 39%, while for the cellular glucose metabolism assays the 95% confidence interval averaged +/- 19%, We have also used human proinsulin; [des-64, 65] human proinsulin; [Asp(B10) des-(B28-30]) human insulin; [Lys(B28), Pro(B29]) human insulin; and human insulin dimers to test the specificity and flexibility of the assay. Potencies relative to human insulin in this cellular assay generally agreed with previously determined in vivo relative potencies for all analogs except the human insulin dimers (87% in this cellular assay versus 15% from previously published in vivo data) and [Lys(B28), Pra(B29]) human insulin (74% in this cellular assay versus 100% in vivo). A real virtue of the biological assay lies in its use of a sigmoidal dose-response relationship, where 6 doses of insulin bracket the calculated insulin concentration at the middle of the dose-response curve (EC(50)) and yield the expected receptor-mediated biological effect. The: USP Bioidentity Test measures a small difference in rabbit blood glucose at two selected doses of insulin, but does not provide the same information as a test that uses a wide range of biological activity.