Development of in vitro Assays for Proteins in Hemostasis

Principal Investigator: Timothy Lee, PhD
Office / Division / Lab: OBRR / DH / LH

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Overview

Public Health Issue: Various hereditary bleeding disorders require replacement therapy to treat the disease, such as the use of Factor VIII for hemophilia A, and von Willebrand Factor for certain types of von Willebrand disease. Whether these therapeutic proteins are derived from human plasma or recombinant DNA technology, assays are an important part of characterization used to assure the consistency of the manufacturing process and the quality of these products.

Regulatory Contribution: 1) Provide capability for testing of lot release samples and conformance lots, (2) Development of an international standard for thrombin with a unified unit, (3) Participation in calibration studies for International Standards of FVIII and FIX, and (4) Development of assays for currently licensed and emerging products to facilitate their characterization.

Research Approach: We collaborate with institutions, such as NIBSC, to organize studies for the calibratiion of reference standards, and participate in field studies to evaluate the uniformity of commonly used assays. Mostly, we follow established procedures to validate the assay procedure in our lab.

Mission Relevance & Outcomes: The primary responsibility of the Division of Hematology is to evaluate biologic products related to blood. For the Laboratory of Hemostasis, the focus is on coagulation factors. Many coagulation factors have multiple reactivity to various ligands. With the emergence of recombinant products as alternatives to their plasma-derived counterparts, it is imperative to examine the various functionality of a given protein to assure that the final product will be efficacious in the human system. In this regard, multiple assays are needed to assess the integrity and biological activity of the product in question. For example, proteins such as Fibrinogen and Factor VIII whose intact quaternary structures are essential for their functions need to be examined with pertinent methods to establish their correct assembly in their production systems. Even for plasma-derived proteins, more sensitive assays are required to ascertain their structural integrity since they are now subjected to more vigorous procedures for viral inactivation. Development of these in vitro assays will further our understanding of how these coagulation factors work, and facilitate the evaluation of products we regulate. An aspect closely related to the evaluation of proteins involved in hemostasis is the establishment of standards. The FDA is responsible for supplying manufacturers with reference standards for coagulation proteins, and for performing lot release on some of these products. The development of an international standard for thrombin with a unified unit fulfills this responsibility.

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Publications:

Thromb Haemost 2005 Feb;93(2):261-6
A reunification of the US ("NIH") and International Unit into a single standard for Thrombin.
Whitton C, Sands D, Lee T, Chang A, Longstaff C.