With the prevalence of diabetes increasing worldwide at unprecedented rates in recent decades, the development of relevant drug therapies have expanded as well. Medications for regulating blood glucose levels are available in numerous permutations, and innovations in drug dosage and delivery methods appear frequently on the market.
Chief among the pharmaceuticals/injectables used in diabetes management is insulin, which lowers glucose levels. Glucagon, on the other hand, increases glucose levels and is often used for the treatment of hypoglycemia.
Both insulin and glucagon are pancreatic hormones secreted in response to changes in blood glucose. Recombinant insulin and glucagon can be mass-produced as a protein therapeutic, which can be modified to create analogs of the original proteins with certain desirable activity profiles.
The determination of biological potency plays a key role in the development and control of biological and biotechnology-derived products. Chapters <121> and <123> of USP outline procedures for testing the bioidentity and biopotency of synthetically produced insulin and glucagon.
BIOIDENTITY AND BIOPOTENCY TESTING
Before synthetic insulin and glucagon products are used in clinical settings, they undergo a number of tests to verify their activity, concentration, and efficacy. These tests, called bioassays, vary in complexity and scope and may assess either quantitative or qualitative characteristics of the substance
· Biopotency tests quantitatively measure a product’s biologic activity
· Bioidentity tests qualitatively determine the identity of a compound by examining its physiological effects.
Standard bioassay procedures for the pharmaceutical industry are outlined in the U.S. Pharmacopeia and National Formulary (USP-NF).
Procedures for insulin assays are usually performed in conformance to USP <121>. Both the bioidentity and biopotency tests described in this chapter involve the rabbit blood sugar method, where four test groups of rabbits receive an injection of either a standard insulin solution or one of several sample solutions diluted to different potencies, and their blood glucose levels are measured periodically for several hours afterwards. A day to a week after the first injection, the rabbits receive a dose from another one of the insulin solutions, and their blood glucose is measured again. The rabbits’ response to the injections is extrapolated from these measurements, and this data can be used to calculate the potency of the sample solutions (not less than 15 USP units/mg to meet the bioidentity test requirements). These computed potencies are further analyzed in the biopotency test to establish a relative value with a 95% confidence interval; in order to meet the criteria of the test, this confidence interval must fall within +/- 10% of the computed potency value.
The USP requires a minimum of two replicates for this assay, though it generally takes 4-6 replicates to meet the specified confidence interval.
The bioidentity test for glucagon outlined in USP <123> is a challenging ex vivo procedure in which the drug’s effects are assessed on a primary culture of rat liver cells. As glucagon stimulates liver cells to convert glycogen to glucose, measurements of the rat cells’ glucose release indicate the extent of the product’s biologic activity. The potency can be calculated with statistical methods and comparisons detailed in other USP chapters. In order to meet the requirements of the bioidentity test, the glucagon sample must have a potency of not less than 0.80 USP rGlucagon Unit/mg.
INSULIN AND GLUCAGON BIOASSAY TESTING AT PACIFIC BIOLABS
Having over 18 years of experience in performing Insulin Bioassays, Pacific BioLabs expert panel of scientists – have extensive experience dealing with several product pipelines and have been actively involved with providing inputs for the writing of USP chapter <121> and <123>.
Our aim is to provide our clients with a combination of knowledge, rigor in our quality systems, personalized attention that is hard to find in the contract research world and help bring your product to market. You can also learn more about our capabilities at our Compendial Bioassays and our In Vivo Bioassays pages.
1. Herman WH, Zimmet P. Type 2 diabetes: An epidemic requiring global attention and urgent action. Diabetes Care. 2012 [accessed 2016 July 15];35:943-944. http://care.diabetesjournals.org/content/35/5/943
2. 2016 U.S. Pharmacopoeia-National Formulary [USP 39 NF 34]. Volume 1. Rockville,Md: United States Pharmacopeial Convention, Inc; 2015. <121> Insulin assays;123.
3. 2016 U.S. Pharmacopoeia-National Formulary [USP 39 NF 34]. Volume 1. Rockville,Md: United States Pharmacopeial Convention, Inc; 2015. <123> Glucagon bioidentity tests; 198.