This NSF award by the Chemical and Biological Separations program supports work to characterize the principal impurities to be removed in the purification of monoclonal antibodies (mAbs) produced in cell culture by Chinese hamster ovary (CHO) cells, which are used ubiquitously in industrial practice. We will identify the impurity proteins that persist in the product stream, with the main source being proteins produced by the cells that make the mAbs, hence the name host cell protein (HCP). An additional class of impurities to be characterized are product-associated impurities, i.e., proteins that bind to the mAb product and are therefore difficult to remove from the process stream during the purification steps. These analyses to identify the principal impurities to be removed in a given mAb process and can serve as the basis for optimizing the strategies for separating them from the mAb product.

Complementary to characterizing these two classes of impurities is characterization of their behavior in the important class of separations involving liquid chromatography. Knowledge of the chromatographic behavior of the different proteins, both mAb product and impurity, under different conditions will allow identification of the conditions most suitable for removing the impurities from the product stream.

The results will be applied to representative efforts to optimize particular mAb separations. Capabilities such as those to be developed in this project can be instrumental in streamlining industrial process development methods for the large and growing class of mAb therapeutics. The research will also serve as an effective vehicle for student education and research training, via the students who will perform most of the research.

Project Report

Abraham M. Lenhoff and Kelvin H. Lee University of Delaware Currently, there are two classes of medicines that are FDA-approved to treat diseases. One class of medicines is made up of small molecules that are manufactured using chemical synthesis. A second class of medicines, also known as biologics, includes larger molecules. The most common type of biologics are therapeutic proteins, more specifically "monoclonal antibodies" that are biomanufactured by taking advantage of the ability of cells to synthesize the monoclonal antibody. The market for therapeutic antibodies approaches $100 billion per year in the United States and most of these molecules are biomanufactured in cells known as Chinese hamster ovary (aka CHO) cells. A major challenge in the biomanufacturing of these medicines is the need to purify the molecule of interest (the monoclonal antibody) from the other proteins made by the CHO cells. Elimination of these "host cell proteins" helps ensure that the final drug is pure and reasonably safe for human use. This project explored the range and nature of CHO host cell proteins that likely co-purify with monoclonal antibodies. Because each monoclonal antibody may have a different set of host cell proteins that co-purify with it, we tested four different monoclonal antibodies to look for which host cell proteins co-purify with more than one of the antibodies. We developed and optimized methods to screen host cell proteins and then used those methods to characterize the co-eluting proteins. We discovered dozens of proteins that appear to co-purify with monoclonal antibodies and that therefore pose a challenge to the biomanufacturing of medicines. We also discovered that the range of host cell proteins made by CHO cells changes over time. As a result, one must be cautious developing ways to purify medicines because the contaminants may change over time based on the cells used. These are all novel findings. In fact, the most recent findings were important enough that a female graduate student was awarded a best poster award at the most prestigious international scientific conference on this topic. Also, one major biotechnology company hosted another graduate student for an internship and offerered him a permanent position. Some biotechnology companies are now pursuing similar studies to understand how these findings relate to their specific medicines.

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University of Delaware
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