The determination of biological potency plays a key role in the development, registration, and control of biological and biotechnology-derived
products. Advances in science have affected both the way these assays are carried out experimentally and how they are analyzed
statistically. During the past several years, the United States Pharmacopeial Convention (USP) has focused on public standards
to modernize and expand the bioassay requirements in the United States Pharmacopeia (USP). This work has been carried out in several expert committees of the Council of Experts, USP's standards-setting body composed
of volunteer scientists from many countries of the world.
Tina S. Morris, PhD
CELL-BASED ASSAYS TO REPLACE ANIMAL TESTING
In the area of assay modernization, USP activities have focused on the replacement of animal-based assays with cell-based
assays. The Biologics & Biotechnology: Proteins and Polysaccharides Expert Committee currently is evaluating several proposals
for product-specific, cell-based assays with allied reference material. The intent is to place these new assays in general
chapters along with the already official, animal-based tests to allow compendial users a transition without compliance challenges.
By using the bioassay procedure, the cell system, and the allied reference material, manufacturers, regulatory officials,
and many others will soon have a comprehensive approach to ensure up-to-date measurement of the potency of biological and
biotechnology-derived products. This potency is measured in units/mass of drug substance and should be consistent for a manufacturer's
product over time, across manufacturers making the same product, and when feasible, calibrated to World Health Organization
The following chapters are expected to be included in Pharmacopeial Forum and USP's Journal of Standards Development and Compendial Review during 2009: Glucagon Bioassays <123> and Somatropin Bioassays <126>. In both cases, USP will support the chapters with a reference cell line that will be available for users to license.
In the case of glucagon, the primary liver cell assay is supplemented with a modern assay that is based on a recombinant cell
line that expresses the human glucagon receptor in large numbers. For somatropin, USP is adding a cell-proliferation assay
that uses an engineered mouse pro-B cell line that was donated to USP by a pharmaceutical manufacturer and the assay is currently
being established in the USP Biologics & Biotechnology Laboratory in USP's Applied Compendial Research Department. A collaborative
study is planned to establish the cell line as a reference standard.
The first application of a USP general chapter to elaborate a product-specific bioassay was insulin. The USP general chapter Insulin Assays <121> describes the use of the rabbit glucose test both as a quantitative and as a bioidentity test. Pharmaceutical manufacturers
have submitted a revision request to USP to add a cell-based insulin assay to <121>. The assay is based on embryonic fibroblast
3T3L1 cells that are triggered into differentiation to form fully mature, lipid-bearing adipocytes, and glucose uptake in
the cells is measured by residual glucose determination in the surrounding medium. The Proteins and Polysaccharides Expert
Committee has decided to publish the new assay first as a stimulus article in Pharmacopeial Forum 35(3) [May–June], while concurrently conducting additional evaluation studies in the USP Biologics & Biotechnology laboratory
and with collaborating manufacturer laboratories. Interested parties can contact the USP laboratory to participate in collaborative
GENERAL BIOASSAY GUIDANCE
In addition to advances in modern bioanalytical procedures, rapid progress in computational analysis, assay design tools,
and statistical understanding has required revision of USP's core compendial bioassay, the general chapter Design and Analysis of Biological Assays <111>. The comprehensive revision of the chapter was proposed recently in Pharmacopeial Forum and has initiated important public comment.1,2 Some key issues are: 1) the evaluation of curve similarity, 2) the use of equivalence testing as an effective statistical
method in several areas of bioassay data analysis, and 3) the best means for combining data from multiple assays. In its next
iteration, the text will be re-proposed as Analysis of Biological Assays <1034> and will become part of a suite of bioassay guidance chapters. The current <111> will eventually be shortened and
reserved for topics appropriate for a chapter below <1000>. Validation of Biological Assays <1033> will be proposed in the March–April 2009 issue of Pharmacopeial Forum, and Design of Biological Assays <1032> currently is in development. The entire suite of four chapters (<111>, <1032>, <1033>, and <1034>) will be accompanied
by a roadmap chapter that will include a unifying glossary of terms. It is USP's goal that the chapters be accompanied by
example data sets that would be made available on the web. USP ultimately may make the General Chapter suite available for
download from the USP web site.