The results of a study done by scientists at the U.S. Food and Drug Administration (FDA) suggest that small segments of DNA-like molecules called aptamers can be used in an efficient and practical method to compare the conformations of a therapeutic protein during drug development and quality control testing.
One class of aptamers comprises short strings of DNA building blocks called nucleic acids that fold up into specific shapes. The shape of an aptamer depends on the type of nucleic acids it represents and the order in which they stack in the string. By identifying which aptamer binds tightly to a specific part of a protein, scientists can predict the shape of that part of the protein, like predicting the shape of a lock by knowing the shape of the key that fits into it. It is relatively easy to develop pools which contain over a trillion random DNA sequences, which in turn provide a starting point for a very large number of uniquely shaped probes that can help identify the shape of a given protein.
Therapeutic proteins comprise a wide variety of products, such as those used to treat hemophilia (excessive bleeding), prevent abnormal blood clotting, treat the collapse of air sacs in the lungs, or enhance a weakened immune system. They are produced in living cells in a laboratory by using complex genetic engineering methods. Proteins made in these cellular “factories” sometimes have minor changes in their three-dimensional structure that reduce or eliminate their effectiveness and can sometimes trigger harmful immune system reactions. Current techniques used to test whether such proteins have the correct three-dimensional structure require laborious and time-consuming methodologies.
The FDA study is important because it showed that the aptamer technique has potential for not only improving quality control testing of new therapeutic proteins, but also for facilitating the development, manufacture, and regulatory approval of so-called biosimilars. Biosimilars are biological products that are highly similar to a reference product (an already approved biological product), notwithstanding minor differences in clinically inactive components and for which there are no clinically meaningful differences between the biosimilar and the approved biological product in terms of the safety, purity, and potency of the product.
Manufacturers of biosimilars must demonstrate to FDA regulators that their biological products are very similar to the original products, just as makers of generic versions of chemically synthesized drugs must demonstrate that their products can effectively substitute for brand name products.
Immune responses to altered three-dimensional sites on the surface of biosimilar therapeutic proteins are a significant impediment to the development of these products. The FDA study suggests that aptamer–based testing has the potential to improve quality control testing, and provides an efficient method to compare the shape of a biosimilar with the reference product in the solution phase.
The FDA scientists showed that specific aptamers were able to detect small differences among protein products called thrombin made by various manufacturers. Thrombin controls a key part of the body’s complicated blood clotting process and is used to treat people with bleeding diseases.
In addition, the scientists subjected thrombin proteins to a type of heat treatment used in the manufacture of an approved product that was previously associated with a dangerous immune response even though protein’s therapeutic activity was not affected. While a standard quality control test using antibodies failed to detect conformational (shape) changes due to heat treatment, the aptamers did detect them, even though the changes were likely to be very small.
The use of aptamers instead of antibodies for quality control testing would eliminate the need to raise specific types of antibodies against particular targets on proteins. Antibodies are made using laboratory animals such as mice, a process that can take weeks and requires harvesting the antibodies from the animals and purifying them. In addition, the quality of these antibodies can vary from batch to batch. In contrast, aptamer production is relatively easy and inexpensive. Therefore, aptamers have the potential to make quality control testing not only more reliable, but also less cumbersome and expensive.
“Aptamers as a Sensitive Tool to Detect Subtle Modifications in Therapeutic Proteins”
2012; 7(2): e31948.
Published online 2012 February 27.
Ran Zichel, Wanida Chearwae, Gouri Shankar Pandey, Zuben E. Sauna (Laboratory of Hemostasis, Division of Hematology, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD)
Basil Golding (Laboratory of Plasma Derivatives, Division of Hematology, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD)