The CHO g2p project is possible due to the Research Infrastructure Improvement Track-2 Focused EPSCoR (Established Program to Stimulate Competitive Research) Collaborations (RII Track-2 FEC) award and aims to bring together Clemson University, University of Delaware, Tulane University, and Delaware State University in order to develop new biological approaches to better understand the Chinese hamster ovary (CHO) cell line, which is used to manufacture most biopharmaceuticals. CHO cells are used to manufacture more than 50% of biopharmaceuticals, and products from CHO cells have global sales over $70 billion per year. Some biopharmaceuticals produced using this cell line include infliximab to treat Crohn's disease and erythropoietin (EPO) to treat severe anemia. CHO cell lines provide a unique opportunity to quantitatively address the complex interactions between the genome and phenome because these cells can be cultured in very tightly controlled environments (bioreactors) to generate variable phenomes due to genome instability.

A lack of understanding of the fundamental link between genome stability and the phenome significantly limits the ability to improve cell lines and ultimately increase product yields. This project will generate foundational knowledge linking the genome (what is in the chromosome or DNA) to what is observed (the phenome) and insight into the basis for genomic instability in CHO cells. This award provides the opportunity for investigators with complementary expertise to collaborate in advance of developing bio-manufacturing knowledge. Additionally, the project will mentor four early-career tenure-track underrepresented minority (URM) faculty and train postdoctoral scholars and graduate and undergraduate students.

As a critical part of this project, In June of 2018, Dr. Harcum’s lab acquired the first ambr® 250, a high throughput, single-use, multiparallel bioreactor, at a US academic institution. This advanced piece of equipment will provide a high precision environment that supports the demands of mammalian cultures, specifically CHO cells. With the ambr® 250, the team will be able to explore a wide range of CHO cell culture conditions with scalability through both benchtop and large stirred tank bioreactors used in industry (10,000-L).

The project’s overall vision is to: 1) expand the research infrastructure in participating institutions; 2) build individual and collective competence graduate students in genome-phenome knowledge and CHO cell cultivation and engineering; 3) share genome-phenome knowledge and tools that could be applicable to any other organism whether or not it has issues with genome stability; 4) broaden and foster the participation of tenure-track early-career underrepresented minority (URM) faculty members; and 5) provide research experiences for URM students and establish student exchanges among the participating EPSCoR jurisdictions.

Click here to learn about the CHO g2p project.

This material is based upon work supported by the National Science Foundation under Grant No. OIA-1736123. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation.

In collaboration with Johns Hopkins University, the University of Delaware, and the University of Massachusetts-Lowell, Dr. Harcum and Clemson University are in the planning phase of developing an NSF- Industrial/University Cooperative Research Center (I/UCRC) entitled the Advanced Mammalian Biomanufacturing Innovation Center (AMBIC.org). AMBIC is focused on reducing the time and cost to develop biopharmaceuticals.

Over the past two decades, the biopharmaceutical industry has emerged as one of the major manufacturing industries and engines of growth in the US economy. Biomanufacturing represents nearly 2% of the total US GDP and its fraction is expanding. Much of biomanufacturing involves the use of cells to make medicines. Examples of these types of medicines include cancer medicines and vaccines. A key element in developing these medicines is the need to establish complex manufacturing processes. AMBIC will implement engineering innovations to enhance the capabilities of our nation to manufacture these important life-extending and life-saving medicines. Such improvements will improve the competitiveness of US biomanufacturing in coming decades leading to more economic investment by these companies and more jobs for American workers.

The mission of AMBIC is to develop enabling technologies, knowledge, design tools and methods that apply and integrate high-throughput and genome-based technologies to fast-track advanced biomanufacturing processes. AMBIC is the first I/UCRC dedicated to mammalian cell culture upstream development focusing on Chinese hamster ovary (CHO) cells, the principal biopharmaceutical production host of industry. AMBIC will bring together leading academic and industrial biotechnologists focused on mammalian cell culture manufacturing at a pre-competitive research level to address the complex problems in biopharmaceutical manufacturing. This multi-university center will allow AMBIC to leverage the skills and the expertise of many faculty members across the Sites. AMBIC will be a critical catalyst towards maintaining national excellence in biopharmaceutical production by conducting research in: 1) Understanding Industrially-Relevant Biology (e.g., all -omics, bioinformatics, process and product quality, etc.); 2) Process Monitoring & Control (e.g., analytics, instrumentation, data mining and modeling); 3) Consensus and Standardization Issues (e.g., standards, simple fingerprints, raw material issues, regulatory issues, forensic bioprocessing, clonality). Through systems-level biology analysis, novel cell line development, bioreactor optimization, and advanced analytics, AMBIC will provide transformative solutions that can lower biomanufacturing costs and improve bioprocessing efficiency. Most importantly, these advances may ultimately serve to make more biopharmaceuticals available to patients that need them and lower overall health care costs for consumers. In addition, AMBIC will establish and maintain a pipeline of educated and motivated students at multiple levels for careers in biopharmaceutical manufacturing and development. Collaborations with corporate partners will enable the students to work on the most pressing problems that the industry faces. Furthermore, this center will serve to engage and excite students from under-represented minority populations to pursue a career in life sciences, engineering, or related STEM fields. An important part of the AMBIC activities and a committed goal of the PI's is to increase the participation of women and under-represented minorities in STEM disciplines by energizing students from all backgrounds about the exciting opportunities to help others through STEM careers in biotechnology and biomedicine.

Click the links to learn more about NSF-I/UCRCs and AMBIC (AMBIC.org)

Chinese hamster ovary (CHO) cells are used to manufacture the majority of all the therapeutic proteins on the market today. CHO cells are able to express recombinant glycoproteins, including monoclonal antibodies, which require complex post-translational modifications. Even though CHO cells have become the new "Escherichia coli" workhorse cell line, there are still many engineering challenges that need to be addressed.

Protein aggregation is particularly problematic for therapeutic proteins and glycoproteins, as the protein aggregates can lead to decreased activity, decreased solubility, increased immunogenicity, and other adverse reactions in the patient. There is evidence that protein aggregation first begins intracellularly and all biological systems have an evolved mechanism for protein quality control through molecular chaperones and proteolytic devices; although a comprehensive understanding has yet to be delivered. Traditional approaches for studying protein aggregation have relied on mechanistic methods without encompassing gene expression studies and elucidating an effective intervention system is unlikely without the below approaches. The proposed research will integrate gene expression and mechanistic studies to gain a better understanding of protein aggregation in CHO cell cultures. We expect to identify genes that modulate and are regulated by protein aggregation, while greatly enhancing the ability of researchers to study CHO cells at the gene expression level by providing well annotated gene sequences to the research community. This research is funded by an NSF EAGER grant.

Regenerative medicine-based therapies hold the power to dramatically enhance treatments. At the forefront of regenerative therapies waits the untapped potential of stem cells; however, for stem cell-based therapies to be feasible, the challenge of generating sufficient numbers of undifferentiated stem cells remains.

Twenty years ago, mammalian cell densities of one-half million cells/mL were commonly obtained. Today, researchers routinely achieve cell densities of over ten million cells/mL. Improved control of bioreactors coupled with a deeper understanding of how the bioreactor environment affects cell physiology allowed this improvement. Significantly increasing throughputs and yields for therapeutic stem cell production by improving the robustness of bioreactor controls along with an improved understanding of stem cell physiology in bioreactors remains the long-term goal.

Major transformative changes in bioreactor control are needed.  Our innovative approach combines current mammalian cell production methods with existing mathematical biological models and real-time parameter estimation into a single robust platform that can greatly improve stem cell yields.

The first step in this project was to eliminate the need for elevated CO₂ gas sparging for pH control.  This change allows for independent oxygen and pH control.  A custom media buffer was developed that allowed the stem cells to be culture in atmospheric air.  As of Summer 2012, we have achieved equivalent cell growth for mesenchymal stem cells (MSCs) grown in the custom buffered media in atmospheric air.  The MSCs grown in atmospheric air also maintained multipotency and were able to differentiate along the osteogenic, adipogenic, and chondrogenic lineages.  Work continues to combine these media changes with improve control algorithms.

In the medicine and pharmaceutical industry, E. coli cells can be used to produce many different types of proteins. E. coli cells have key advantages, such as rapid growth rates and robustness to environmental factors. Additionally, DNA can be easily modified in this organism; thus, we can control which proteins are produced. Maximizing the productivity of E. coli cultures is a big challenge. Many different stimuli can stress the cells and reduce their growth rates and their protein production rates. Insoluble inclusion bodies (usually in the form of misfolded proteins) are one such stressor. Unfortunately, insoluble inclusion bodies can sometimes be difficult to purify. By understanding how these inclusion bodies affect the actual gene expression of E. coli, we can take steps to modify the DNA accordingly and make the cells less sensitive to these stressors.

As the non-renewable sources like natural gas, petroleum etc. keep depleting, the demand for an alternative source of energy is growing at rapid rate. One of the most widely researched alternative fuels is ethanol. One of the major challenges faced is that the production of ethanol is not an economical process. This is primarily due to the inability of yeast organisms, mainly Saccharomyces species - the most widely used microorganisms for this process, to consume xylose – one of the major sugars present in lignocellulosic hydrolysates, apart from glucose. Hence a novel two-stage fermentation process was developed, in our lab, to improve the bioethanol production. In the first stage, glucose present in the hydrolysate is consumed by Saccharomyces pastorianus to produce bioethanol. Then, the mixture of ethanol and unconverted xylose is separated. In the second stage, E. coli is allowed to grow on the unconverted xylose. The E. coli cells obtained are heat-killed and fed to first stage yeast fermentation. This new cyclic process has resulted in increased bioethanol productivity. We have filed for a patent on this process.