Glucagon is a critical drug for the treatment of diabetes. The potency assay for glucagon is a challenging ex vivo procedure using a primary culture of rat hepatocyte cells. PBL is perhaps the only independent laboratory in the world offering this assay.
PBL has performed insulin bioassays including biopotency and bioidentity tests according to USP <121> for over fifteen years.
The Bioidentity Test is a qualitative test used as a batch release test.
- We can give you a verbal result within 3 weeks and a report within four weeks. The timeline can be shortened if we have commitment and advanced notice so we can order and acclimate animals.
- The assay uses 8 rabbits assigned in four groups of two animals.
- The point of the bioidentity test is to determine the potency of a test article (solution or powder).
- To pass the USP criteria for the bioidentity test, the potency of the insulin contained in the test article must be greater than 15 mg/dL. (e.g. if the potency of a test formulation containing 1.0% insulin (weight/volume) is 0.2 Units/mL, then the insulin potency is 0.2 Units/0.01 = 20 Units/mg insulin and the solution passes the test).
The Biopotency Test is a quantitative test used to attain an accurate measurement of the potency with a statistically significant 95% confidence limit.
It takes about four weeks, four assays to complete a set and attain a confidence interval. Although it usually takes four assays to attain a result within USP compliance, we cannot guarantee that that will happen. There is the possibility of attaining a value with fewer assays. We only charge for the assays performed.
- The report takes about 7-10 working days after the completion of the last replicate.
- The assay uses 24 animals, four groups of six each.
- The dosing and blood collection procedures are the same as for the 8 rabbit bioidentity test.
- The endpoint of the assay is a potency value with a 95% confidence limit.
- USP <121> requires the confidence limit to be not greater than approximately +/- 10 percent of the potency.
- The level of accuracy is rarely attained with one 24-rabbit assay and normally requires the combined results of several.
- In our experience it normally takes four 24-rabbit assays to get a potency value and confidence limits that pass the USP <121> criteria.
In both assays animals receive subcutaneous injections of solutions of a known insulin standard or an equivalent solution of the test article on two different days, 3-7 days apart. The solutions are set at 1Unit and 2 Units/mL. If the animals in a group receive the high dose of the standard on one day, they receive the low dose of the test article on the 2nd day and vice versa. On both days, blood is collected 1 hour and 2.5 hours after dose administration. The potency is calculated by comparing the plasma glucose response to the test article versus the standard.
For raw powder, 50-100 mg of sample is enough to do all testing, whether for bioidentity or biopotency. This is because a 40 Unit/ml stock solution is prepared from the powder in USP insulin diluent and is good for six months at 2-8C and all the dosing solutions can be prepared from it.
For formulations, we normally make the dosing solutions directly from the formulations each time they are needed. About 2 mL per assay is needed (1mL per dosing day x 2 dosing days), thus 2 mL for a bioidentity test or 8 mL for a four-assay quantitative test.
Somatropin is the recombinant version of the human growth hormone produced by the pituitary gland. USP <126> outlines an in vivo bioidentity test which should be conducted on each batch of drug product or bulk drug substance. Both in vivo and in vitro methods are outlined in USP <126>. PBL performs only the in vivo method which is desribed below.
In vivo somatropin bioassay summary:
- Place male hypophysectomized Sprague Dawley rats on a fixed diet. Hypophysectomized rats do not contain a pituitary gland.
- After 72 hours weigh the rats.
- Reweigh the rats seven days later and use only rats that are in good health and have not gained or lost 10% of their body weight.
- Place approximately 10 rats into control, standard and test groups.
- Each day for 10 days subcutaneously inject 0.1 mL of the control, standard, or sample solution into the appropriate rats.
- Record the weight of each animal at the start of the test and at approximately 18 hours after the 10th injection.
- Determine the change in body weight and compute the potency of the sample relative to the standard. The calculated potency must not be less than 2 USP SomatropinUnits/mg.