Biological Potency Assays are Key to Assessing Product Consistency

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 international units.

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 testing (mra@usp.org, 301.816.8181).

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.

OPEN SCIENTIFIC DISCOURSE

USP held an open public discussion of the new bioassay approaches in a Bioassay Workshop in August 2008 at USP Headquarters in Rockville, MD. USP will hold a similar workshop from August 12–13, 2009, at the same venue. The 2009 meeting will seek to further engage stakeholders by expanding and building on the progress made on USP's developing collection of bioassay guidance chapters. Attendees will have an opportunity to discuss chapters <1033> and <1034> with the expert drafting group and will hear an update about the development of chapter <1032>. In addition, the workshop will again provide a forum for scientific presentations and dialog among bioassay experts in the bioassay community. Special product-specific focus areas this year include bioassays for monoclonal antibodies and vaccines. To prepare a scientifically challenging and productive event, USP would like to hear from members of the bioassay community. What topics should appear on the agenda for this August? Stakeholders can submit ideas by email to tsm@usp.org. Please use the subject line: "Ideas: Bioassay Workshop 2009." Information regarding registration and meeting logistics will be available from meetings@usp.org.

CONCLUSION

As ongoing advances in biochemical and biophysical characterization continue to illuminate the molecular features of biological molecules, the measurement of biological potency will help establish structure–function correlations, assist in determining of immunologic response, and elucidate the molecule's biological identity. Most importantly, stability in potency results assures practitioners and patients that they are receiving a product with consistency in anticipated therapeutic outcomes batch-after-batch over many years following market access. For multiple products under the same name, the bioassay should also assist in ensuring comparable potency across manufacturers making the same ingredient or product. To this end, USP continues to work with stakeholders to provide state-of the art bioanalytical tools for compendial scientists.

Tina S. Morris, PhD, is vice president of Biologics & Biotechnology, Robert Singer is chair, Bioassay Ad Hoc Advisory Panel and founding Member of Biometry Associates LLC, Union City, CA, Michael R. Ambrose, PhD, is director, Biologics & Biotechnology Laboratory, and Walter W. Hauck, PhD, is senior scientific fellow, all at United States Pharmacopeia, Rockville, MD, 301.816.8377, tsm@usp.org.

REFERENCES

1. Hauck WW, et al. Summary of planned revisions to design and analysis of biological assays <111>. Pharm Forum. 2007:33(3);580–81.

2. <111> Design and Analysis of Biological Assays. Pharm Forum. 2008 34(3):685–735.