FDA Researchers Explore Fundamental Chemical Reaction that Could Threaten the Quality of Therapeutic Protein Products

Spotlight on CDER Science

V. Ashutosh Rao, Ph.D., Chief, Laboratory of Biochemistry, Division of Biotechnology Review and Research III, Office of Biotechnology Products, Office of Pharmaceutical Quality, Center for Drug Evaluation and Research

The potential for biotechnology to transform medicine remains immense, with several therapeutic protein products already having come into widespread clinical use and hundreds of proteins under clinical investigation. For the FDA, the regulatory oversight of therapeutic protein development poses a great challenge, not only because of the increasing number of products, but also because proteins, by their very nature, are highly variable, and compared to small-molecule drugs, more likely to undergo chemical reactions over time. Chemical reactions that occur in these proteins (for example, during storage in vials before administration to patients) can have a significant impact on protein function. For FDA scientists in the CDER Office of Biotechnology Products (OBP), these reactions are of great interest because they can directly impact the quality, safety, and efficacy of protein products.

For decades—before the biotechnological revolution and the rise of therapeutic proteins—FDA drug reviewers focused primarily on small-molecule drugs. Aspirin, for example, contains only nine carbon atoms, whereas the modern protein product bevacizumab contains well over 6,000 carbon atoms. In general, proteins also contain sulfur atoms (bevacizumab has 44!), and biochemists have long known that sulfur-containing molecules are prone to undergo reactions related to the presence of unstable oxygen and other atoms in our environment. (Proteins are not the only molecules that undergo such oxidative reactions. The rusting of iron tools and statues, where iron atoms interact with oxygen atoms in the air and water, is also oxidative.) In some instances, oxidized proteins can be damaged in various ways, which could in turn trigger an unwanted immune response in patients. This feature is a unique concern with therapeutic proteins.

Recently, biochemists have begun to investigate the exact locations of oxidative reactions within large intact protein molecules. Laboratory researchers in OBP, for example, have published important new information (see References) concerning protein carbonylation, which entails the addition of a single atom of oxygen, originating from the environment, to discrete carbon atoms (rather than sulfur atoms) within protein molecules. Although protein carbonylation has been recognized within the context of disease and age-related conditions, its occurrence during the manufacture, storage, use, and transport of therapeutic proteins is a relatively new area of study. For pharmaceutical quality scientists, key issues include: understanding the different tendencies of various protein products to undergo oxidative carbonylation; identifying the role that containers and chemicals that contact proteins may have in these reactions; and discovering reliable methods for specifically and consistently detecting and controlling carbonylation.

To detect protein carbonylation, the OBP team has refined an antibody-binding assay that takes advantage of the reactivity of the “carbonyl group,” which is the name given to the carbon- and oxygen-atom grouping that occurs upon protein carbonylation. Carbonyl groups that form in proteins can make the entire protein molecule less stable and lead to damage after degradation or aggregation. By exposing the carbonyl groups that are formed in proteins to a laboratory reagent known as DNPH, the OBP team found that different proteins undergo carbonylation at different levels and at specific sites. The rate and site of carbonylation can depend on temperature, time, and other variables, such as the presence of small amounts of metals that accelerate protein oxidation reactions. The laboratory also investigated the tendency for certain additives in drug formulations to start or speed up these reactions. The methods developed by the team allow many samples, under a variety of conditions, to be studied in a short period of time. Moreover, the method is sensitive enough to detect as little as a single carbonylation modification within a large protein molecule.

The findings of the OBP team may help protein drug developers produce therapeutic proteins under optimized manufacturing conditions, potentially with structural alterations or by adding stabilizing additives that could prevent harmful carbonylation reactions. More stable protein drugs could offer a longer shelf life, reduced risk of quality problems, and more predictable clinical performance. Understanding reactions such as protein carbonylation may result in improved versions of current drugs or new drugs with superior stability, purity, and potency. Above all, the goal is to have safe and effective high-quality protein products available for patients.


Uehara, H and Rao, VA. Metal-meditated protein oxidation: applications of a modified ELISA-based carbonyl detection assay for complex proteins. Pharm Res (2015) 32:691-701.

Kryndushkin, D, Wu, WW, Venna, R, Norcross, MA, Shen, RF, and Rao, VA. Complex nature of protein carbonylation specificity after metal-catalyzed oxidation. Pharm Res (2017) 34:765-779.

Rao, VA. Perspectives on engineering biobetter therapeutic proteins with greater stability in inflammatory environments. In Biobetters (eds A Rosenberg and B Demeule) Springer 2015.

Page Last Updated: 06/12/2018
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