Bioprocessing and monoclonal antibody (mAb) production in Chinese hamster ovary (CHO) cells

Monoclonal antibodies and other therapeutic proteins and biomolecules have revolutionized modern medicine, providing lifesaving and life-changing treatments for a wide range of conditions including cancer, autoimmune disorders and infectious disease. The Sharfstein laboratory has focused on a number of bioengineering aspects of production of therapeutic proteins, working extensively with academic and industrial collaborators including biopharmaceutical manufacturers and small businesses.

 

Proteomic and Transcriptomic Analysis of CHO Cells

We have extensively characterized a collection of Chinese hamster ovary (CHO) cells, all producing the same therapeutic antibody with varying productivities to understand the factors that control productivity cells. In particular, we have investigated proteomic and transcriptomic differences between high and low productivity clones, identifying ribosomal machinery, metabolism, the proteosome and tRNA biosynthesis as important cellular changes and pathways.

 

 

 

 

 

 

DNA Methylation of Promoter Sequences

Promoter methylation can play a significant role in controlling transcription. We compared the methylation of the cytomegalovirus (CMV) promoter in four different cell lines with varying productivities and found significant differences in DNA methylation at critical sites.

Effects of tRNA Production on Productivity

tRNA production, modification, and utilization can change dramatically during cell culture. Working with Codomax, we are evaluating the effects of cell line and culture conditions on RNA biology and its impact on antibody productivity.  

Control of Protein Glycosylation on Monoclonal Antibodies Produced in CHO Cells

Protein glycosylation affects biological activity, immunogenicity and half-life of therapeutic proteins and antibodies which is particularly critical for biosimilars (off patent versions of biopharmaceuticals). Working with sxRNA Technologies, we developed a novel cell line selection approach and used it to evaluate variation in glycan profiles from different clones and the effects of culture conditions on glycan structures.

Production of a Bioengineered Heparin

Heparin is the world’s most widely used anticoagulant drug with ~100 metric tons used annually. Heparin is currently a slaughterhouse product, produced largely from pig intestines in China. To generate a safer, bioengineered heparin, we have worked with other academic labs and TEGA Therapeutics on cell line engineering and bioprocessing to improve heparin production.

Effect of Product Sequence on Productivity for mAbs Produced in CHO Cells

Subtle changes in antibody sequence can dramatically affect productivity. Building on previous studies in collaboration with UCB Celltech, we are currently working with DeepSeq.AI, to explore how that occurs mechanistically and to design better therapeutics that can be produced more easily.   

Tissue Engineering and Regenerative Medicine

Salivary Gland Engineering

The salivary gland produces saliva, which is critical for oral health and digestion. In response to aging, disease and radiation treatment, salivary gland disfunction can lead to dry mouth compromising overall health. Working with Professor Mindy Larsen in biology, we are exploring novel engineering approaches for improving salivary gland function and for screening new therapies.

Development of a Human Trabecular Meshwork Model for Drug and Genetic Screening

Glaucoma is a leading cause of irreversible blindness, affecting over 80 million people worldwide with limited therapeutic treatments. We developed a novel scaffold using photolithography to culture trabecular meshwork cells (the critical ocular tissue) [US Patent 9506907 B2] in a three-dimensional, flow configuration. This technology resulted in a spinout company, Humonix Biosciences, led by one of our graduate students. Working collaboratively with Humonix, our current studies are focused on extending the model into gene therapy and developing novel sensing technologies.

Use of a Novel Bioreactor for Expansion and Differentiation of Pluripotent and Progenitor Cells

A critical aspect of cell manufacturing for tissue engineering and regenerative medicine is expansion of cells to generate ~109 – 1012 cells needed for various applications. Working with Sepragen corporation, we are exploring a novel low-shear, high mass transfer bioreactor for expansion and differentiation of pluripotent stem cells, particularly into pancreatic progenitors.

Future Manufacturing of Electronic and Photonic Devices Integrated into Mammalian Cell Culture Systems

“Through the convergence of such fields as robotics, artificial intelligence, biotechnology and materials research, future manufacturing will create revolutionary products with unprecedented capabilities, produced sustainably in facilities across the country by a diverse, newly trained workforce” -- NSF Director Sethuraman Panchanathan

Our vision is to develop manufacturable systems to interrogate and direct the behavior of in vitro cell culture systems by addressing the barriers that limit the development of manufacturable biosensing and cell manipulation technologies and to engage and prepare a workforce capable of operating at the interface between biomanufacturing and device design and fabrication (Picture 11). Bringing together expertise from Professor Nate Cady, Professor Yubing Xie, The Neural Stem Cell Institute, and AIM Photonics we aim to create new devices and manufacturing methods to integrate electronics and photonics with mammalian cell culture systems.

 

Development of Conformal Microelectrode Arrays for Sensing Three Dimensional Cultures

Three-dimensional (3D) culture systems are of increasing interest to replicate in vivo physiology and pathology. However, interrogation of 3D systems, particularly neural organoids, is challenging due to a lack of conformal microelectrode arrays to wrap around the organoids. To address this issue, we have fabricated a range of microelectrode arrays on the flexible substrate polyimide and are exploring their biocompatibility, electrical properties and their ability to interrogate neural organoids.

 

Use of Photonics for Biosensing in Culture Systems

Environmental parameters play an important role in cell physiology as do secreted cell signaling molecules. While electrochemical sensors do exist for many of the common neurotransmitters, acetylcholine, glycine, glutamate, dopamine, norepinephrine, epinephrine, serotonin, histamine, and γ-aminobutyric acid (GABA)53,54, electrochemical sensors suffer from a number of limitations including low sensitivity and interference from the culture medium and similar molecules. Photonic sensors can be easily operated in a reagentless mode, either using the spectroscopic signature of the target molecule, or an attached capture molecule. Using chemical vapor deposition (CVD), we fabricated silicon nitride (SiN) photonic “chiplets” containing a number of passive photonic devices, Bragg gratings, Mach-Zehnder interferometers and ring resonators and are currently evaluating the use of aptamers to detect dopamine using ring resonators.

Novel Approaches to Pressure and Strain Sensing in Mammalian Cell Cultures

Hydrogels are widely used in biological systems to provide biological components such as extracellular matrix materials with their attendant attachment and signaling molecules (e.g. collagen, fibronectin) as well as for their mechanical properties to create physical compliance similar to native tissues in vivo. Recently, there has been increased interest in using hydrogels as photonic materials and in exploiting hydrogel-based optical fibers to measure strain. We are proposing to investigate neural crest (NC) differentiation on hydrogel microfibers and migration detection by strain sensing using structural color hydrogels.

EMG-controlled Robots for Outreach and Workforce Development

To engage with middle school students about interactions between people and electronics, we developed a robotic car controlled by electromyography (EMG-electrical currents generated in muscles during its contraction representing neuromuscular activities) signals. We have used the car with multiple summer science camps (Tech Valley High Innov@tion, 15-Love, Flying Cloud Girls Science Camp) to expose student to the technology. With a second robotic car built in summer of 2024, robot races are next!