(75f) Microfluidic Scale-Down of Upstream Biopharmaceutical Production

The production of therapeutic proteins is the fastest growing segment of the pharmaceutical industry. The post-translational addition of carbohydrate groups to the protein product has been shown to alter the efficacy, immunogenicity, clearance time in the body, and solubility of therapeutic proteins.  Chinese Hamster Ovary (CHO) cells represent the preferred expression system for biopharmaceutical production because of the cell’s inherent ability to perform post-translational modification within the secrationary pathway as the protein is transported in the endoplasmic reticulum (ER). As a result, approximately 70% of biopharmaceuticals in the market are produced in CHO.  Glycosylation of proteins is sensitive to pH, temperature, dissolved oxygen (DO), dissolved CO₂, shear, and specific metabolite concentrations.  Shear sensitivity (due to morphological deformation of the ER) is what distinguishes CHO cell culture from microbial expression systems.   Shear stresses as low as 0.10 Pa have been shown to suppress productivity by 34.2% and smaller variations of shear have been shown to alter the glycosylation of proteins at an energy dissipation rate (EDR) of 6.0 x 10⁴ W/m³. Microscale mammalian culture has been explored previously, however either small volumes or laborious sampling have greatly limited in-line and off-line measurement techniques. In Amanullah et.al., 700uL volumes are insufficient for both in-line characterization of the process (e.g. metabolite profiling [RX Daytona]) or product characterization (e.g.glycosylation profiling [HPAEC-PAD]).  

The device shown in Figure 1 contains a 2mL micro-bioreactor for batch and fed-batch culture of CHO cells with online sensors to monitor pH, dissolved oxygen (DO), dissolved carbon dioxide (DCO₂), optical density (OD) and temperature in real time. The pH is controlled by varying the partial pressure of carbon dioxide in the gas headspace during the early stages of the culture and by injecting liquid base during the latter part of the culture.  The maximum shear stress experienced by cells has been modeled and the effects of shear stress on the growth and productivity of the CHO cells are experimentally measured for different mixing rates. The gas transfer rate, k_La, is measured for oxygen (O₂) and carbon dioxide (CO₂) gas, which can be independently controlled and adjusted till the k_La for both O₂ and CO₂ are similar to that of large scale bioreactors. A batch culture of CHO (Invitrogen CHO-S) cells is grown in the micro-bioreactor and the growth rate and productivity of cells grown in the micro-bioreactor are compared with shake flask cultures. Periodic samplings for offline measurements are performed to validate the online sensors and track the osmolarity increase of the culture medium.  The cell viability and growth rate are monitored throughout the culture.