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Bioprocessing Exploiting Cells' Electrical Behavior Angelo DePalma, Ph.D. The "Analytical and Monitoring" track at the recent "European Society for Animal Cell Technology" was notable for three presentations on advanced techniques based on the electrical capacitance of cells in media. SAFC's raw material characterization program encourages the screening of raw At the conference, Katrin Braasch, a doctoral student at the University of Manitoba, presented data on fve independent methods for determining cell density and/or viability. Her techniques include particle counting, image analysis with trypan blue exclusion, on-line capacitance, off-line fow cytometer apoptosis kits, and a novel dielectrophoretic (DEP) cytometer. Developed at U. Manitoba (Thomson and Bridges group), the DEP cytometer analyzes individual CHO cells through radiofrequency actuation in a narrow capillary. This technique, which relies on shifts in dielectric properties corresponding to loss of cell viability, is capable of identifying sub-populations of cells during apoptosis, which may be further characterized through fuorescence markers and capacitance. During the measurement with the DEP prototype, a cell suspension diluted in lowconductivity medium is passed through a microfuidic channel, where individual cells pass over an electrode array that detects and vertically displaces the cells. The degree of displacement directly relates to the cell's po- larizability, which in turn correlates with the cells' metabolic health and viability. "Our results show that the various onand off-line techniques gave similar values during exponential phase and that measurements diverged only at the point of highest cell density," Braasch said. She found that the intermediate-stage apoptosis assay agreed with data from the bulk capacitance probe, while early-stage apoptosis measurements correlated with the DEP cytometer. Both bulk capacitance and DEP cytometry allow for earlier detection of apoptosis than trypan blue exclusion, the standard apoptosis assay in use today. Earlier detection prompts investigators to harvest at that stage, or alter the feeding strategy to extend culture life. "While trypan blue viability assays remain standard, it only picks up cells at the end stage of apoptosis, well after the loss of cell viability is detected by the bulk capacitance probe and the DEP cytometer," Braasch explained. "This makes the DEP cytometer a potential low-cost, online viability measurement tool." materials using near-infrared and Raman sensing devices. DEP allows Braasch to map the trajectory of viability in CHO cells, including identifying subpopulations associated with various stages of apoptosis, and to distinguish viable from nonviable cells. It is also possible to identify subpopulations of apoptosis in a bioprocess using fow cytometer assays. However, fow cytometry assays are expensive and time consuming because of incubation times required for staining protocols. DEP works without staining. Despite the trend toward online and inline monitoring, offine methods such as cell counting, and at-line semi-automated techniques still dominate biomass detection. Yet NEWS Bioprocessing > Thermo Plants Dry Powder Me- ly; it is also a cGMP manufacturing plant. Thermo Fisher Scientifc is opening a new manufacturing plant in Singapore to produce dry powder media (DPM), a cell culture raw material used to manufacture biologics including vaccines and anticancer therapeutics. Thermo says the facility is the frst-ever cell culture DPM production site in Singapore and is designed to address the increased global requirements from biopharmaceutical companies to mitigate critical raw material supply risks. The company says the 30,800-squarefoot (2,861-square-meter) facility, located in Tuas, will provide critical manufacturing redundancy of DPM to help ensure a secure and uninterrupted supply to biopharmaceutical customers global- > Double Manufacturing dia Production Flag in Singapore Deals for Sanof Two Sanofi companies have entered biomanufacturing deals with Vivus and Gallus Biopharmaceuticals. Sanofi subsidiary Sanofi Chimie has made an agreement with Vivus to manufacture and supply the active pharmaceutical ingredient (API) for erectile dysfunction drug avanafil (known as Stendra™ in the United States and Spedra™ in the European Union) on an exclusive basis in the U.S. and other territories and on a semi-exclusive basis in the European Union and Latin America. Meanwhile, Genzyme, another Sanofi company, entered a multiyear development and manufacturing supply agreement with Gallus Biopharmaceuticals in which Gallus will provide process and method development, scale-up, clinical supply, and preparation for commercial manufacturing for a protein-based therapy targeting Niemann-Pick type B. > ScinoPharm Changshu and Sundia Form CRO Team in China ScinoPharm (Changshu) Pharmaceuticals has formed an alliance with Sundia MediTech to seek more opportunities for collaboration on contract research and manufacturing services (CRAMS) in mainland China. The collaboration will combine Sundia's know-how in new drug R&D; with ScinoPharm's process R&D; and production advantages, especially in the field of highly potent oncological APIs. n both approaches are time consuming and are incapable of monitoring a process continuously. One emerging "electrical" technique, radiofrequency impedance (RFI) detection, is capable of in situ viable biomass detection and is well established in biopharmaceutical processing, but only in traditional glass and stainless steel bioreactors. "Biomass characterization and measurement is one of the most requested parameters in bioprocessing," said John Carvell, Ph.D., sales and marketing director at Aber Instruments. "Knowledge of the biomass progress during a fermentation provides deeper process knowledge and control, and helps to defne induction, harvest, or infection points." RFI detection exploits the fact that healthy living cells act like electrical capacitors, accumulating then releasing electrical charges. The phenomenon applies to both suspended and attachment-dependent cells on microcarriers. "Compromised cells lose that capacity," Dr. Carvell told GEN. RFI detection correlates well with trypan blue staining until cells begin to enter their death phase. Several recent papers explain the differences between RFI and standard live-cell counting methods. The Aber sensor matches the Guava ViaCount assay during the death phase, but detects the onset of apoptosis earlier than the trypan blue. So which test is more accurate? "Unfortunately, there is no gold-standard test for live-cell concentration," Dr. Carvell explained. Carvell's work becomes increasingly important as more processes become "born and bred" in plastic. The RFI probes must be compatible with single-use equipment's agitation, aeration, and capacity for gamma sterilization, and work through signal fuctuations See Monitoring on page 40 38 | September 1, 2013 | GENengnews.com | Genetic Engineering & Biotechnology News