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Cell Culture in Process Development Andrea Cortez What to expect from this Presentation? Overview of Cell Culture Process Cell Culture Maintenance and Monitoring with process development considerations Insights for Future Technicians What are Cells? Cells are the smallest discrete elements of living organisms. Composition : 90% water, the remainder is half protein, 15% each carbohydrates and nucleic acids, and 10% lipids, with the last tenth representing trace compounds and elements such as vitamins and minerals. What is Cell Culture? Basic : It’s like farming or animal husbandry on a very small scale. It’s about the feeding and care of living things so that they will provide valuable products. Technical : Propagation of cells outside the organism, in-vitro, (microorganisms, plant or animal) Biotechnology : Process utilizing mammalian (e.g., CHO) cells to make proteins that are similar to those made in humans for therapeutic or research purposes What is Fermentation? Basic :A chemical process in which an agent causes an organic substance to break down into simpler substances; the anaerobic breakdown of sugar to CO2 and alcohol by yeast; the breakdown of carbohydrates by microorganisms to produce energy. Biotechnology : Large scale cultivation of microorganisms, i.e., manufacturing or production scale. In some cases, it is a process utilizing bacterial (e.g., E. coli) cells to produce proteins. What is Process Development? Improvement or change in any process towards an ideal point of maximum capability, potential, quality or usefulness Propagation of Cell Cultures Adherent Monolayer-Cells adhere to the substrate in order to survive and proliferate; hard to scale-up; used for R&D purposes only. Suspension Cultures – cells grow floating in medium; cells survive and proliferate without attachment, e.g., transformed cell lines, or cells from malignant tumors (hybridoma cells); Preferred method for scaling up cultures, i.e., large-scale manufacturing. Propagation of Cell Culture . . . In order to maintain cell culture, the cells must be sub-cultured (splitting or passaging). Subculture by dilution – the process of transferring the required number of cells in media suspension from an old flask (source) into another, at a new, lower density, then topping off spinner with new fresh medium. Medium Exchange – cells are separated from the media by centrifugation, old media are removed and new fresh media are added back to the cell pellets. Cell Metabolism To stay alive, an organism or cell needs a continuous supply of energy in usable form. Metabolism is a catch-all word to describe the energyrelated chemical processes occurring within an organism or cell. Cellular Respiration is the process by which the chemical bonds of energy-rich molecules such as glucose are exploited for energy. Some of that energy is trapped in the form of a nucleotide called adenosine 5’-triphosphate (ATP). CO2 and water are the waste products. Glycolysis is the breakdown of glucose to pyruvic acid (pyruvate), does not require oxygen and is performed by all living things. Cell Metabolism In cell culture, it is literally the medium through which we provide a benign physical environment for the cells not only to thrive, but also to produce. We want to provide them the kind of environmental conditions that will make them happy and free of stress and mostcost effective. Phases of Cell Growth Phases of Cell Growth Lag Phase. Immediately after inoculation of the cells into fresh medium, the population remains temporarily unchanged. Although there is no apparent cell division occurring, the cells may be growing in volume or mass, synthesizing enzymes, proteins, RNA, etc., and increasing in metabolic activity. Exponential (log) Phase. The exponential phase of growth is a pattern of balanced growth wherein all the cells are dividing regularly. The cells divide at a constant rate depending upon the composition of the growth medium and the conditions of incubation. This is the ideal phase for experiments. Phases of Cell Growth Stationary Phase. Exponential growth cannot be continued forever in a batch culture (e.g. a closed system such as a test tube or flask). Population growth is limited by one of three factors: 1. exhaustion of available nutrients; 2. accumulation of inhibitory metabolites or end products; 3. exhaustion of space, in this case called a lack of "biological space". Death Phase. If incubation continues after the population reaches stationary phase, a death phase follows, in which the viable cell population declines. During the death phase, the number of viable cells decreases geometrically (exponentially), essentially the reverse of growth during the log phase. Microbial Nutrients Requirements Carbohydrates: For bacteria and CHO, glucose is the most readily metabolized sugar; most fungi can use disaccharides Lipids: Some bacteria and fungi can use longchain fatty acids such as linoleic and oleic acids. Steroids other than cholesterol are generally unnecessary. Ergosterol is a nutritional requirement for all fungi Purines and Pyrimidines: Generally only in bacteria have cases of purine and pyrimidine metabolism been reported. Algae do not use them at all. Microbial Nutrients Requirements Vitamins and Growth Factors: There is a considerable species variation in the requirements of vitamins and related factors by microorganisms. Generally vitamins A, C, D, & K are unnecessary for growth. Amino Acids: Useful to most industrial microorganisms, w/ the exception that yeasts have no use for critrulline. Some bacteria can metabolize forms of amino acids that eukaryotes cannot. Nitrogen Sources: Few popular species require or use N2 compounds, but some species have been shown to use them. Fungi requires ammonia, nitrate, and nitrite. Microbial Nutrient Requirements Sulfur Sources: Some yeasts can use elemental sulfur and sulfate but not want sulfur-containing organic compounds. Bacteria require glutathione and thioacetic acid, whereas yeasts will take sulphonic acid amides, thioacetate, thiocarbonate, thioglycolate, and glutathione. Trace Elements and Inorganic Ions: Mineral nutrients required by microorganisms are species dependent, but consist generally of Fe, K, Mg, and Mn. Sometimes S, N, Ca, Co, Cu, P, and/or Zn are required. Basic Steps in Cell Culture Formulation of Media Sterilization of Media (use of filter) Note: Pre-warm media prior to use (inoculation) Sterilization of Equipment (use of autoclave) Creation (inoculation) of an active culture Growth of the organisms Daily monitoring of cell culture (morphology, physical environment, color of medium, cell density, feed/glucose requirement) Induction/extraction of the product Disposal of effluent Cells Physical Environment pH (7.2 – 7.4) Use buffers: bicarbonate when culture is acidic, CO2 when too alkaline Use pH probe measurement (on-line/off-line) Temperature (37.00C) Use heating blanket Use RTD for measurement (on-line) Osmotic Pressure (260-320 mOsm/kg) Salt may need to be added or removed to accommodate cell’s needs Use Osmometer for measurement (on-line/off-line) Dissolved Oxygen (Physiologic 20-80% air sat) Air sparge/O2 supply, agitator Use dO2 probe (on-line/off-line) Measurement of Cell Growth and Viability % PCV (Packed Cell Volume)-use a special graduated centrifuge tube to measure cell density (alive and dead cells) VCD (Viable Cell Density) – use hemocytometer to determine cell density based on live cells/mL % Viability – use hemocytometer or trypan blue staining method is used to count dead cells that are stained blue Other Parameters Measured Nutrients – Glucose and Glutamine Waste Product Lactate: produced by the oxidation of pyruvate; inefficient use of glucose; decreases pH and increases osmolality of medium; inhibits cell growth above critical concentration Ammonia : by-product of glutamine; can inhibit cell growth and production Physical Environment : pH, Temperature, dO2, osmolarity, Na. Purpose of Cell Culture in Process Development Improve product quality (safe drugs) Increase the amount of final product Robust process before transfer to manufacturing Validation studies Optimization studies Assessment of new technology Assessment of media sources FDA approval Purpose of Cell Culture in Process Development Majority of the experiments are for data generation -one experiment is about 18 x 2.0L cell cultures Non-GMP (Good Manufacturing Practices) Environment Overview of a Cell Production Process Inoculum Train (Non-Selective Medium) Production (Non-Selective Medium + Feed) Harvest Increasing Cell Density Cell Source Cell Mass “Maintenance” “Accumulation” Process Manipulations Cell Growth and “Productivity” E. Coli vs. CHO Cells Doubling times : E.Coli @ 37 0C 1 hour E.Coli @ 33 0C 2 hours CHO @ 37 0C 16-24 hours CHO @ 330C 30 hours Final Fermentation/Cell Culture Times: E. Coli 1-2 days CHO 1-2 weeks Growing E. Coli vs. CHO Cells E. Coli Flask in Shaker Rapid growth rate OD for counts Constant air supply Generates heat Continuous Glucose Feed pH control with NH3 Produces Acetic acid CHO Spinner w/ agitator in a spinner base Slower growth rate PCV for counts dO2 control air/O2 No heat built up Glucose in medium or feed (may be added in the entire experiment depending on need) pH control with HCO3 Produces Lactic acid Standard cell culture configuration for a 3 Liter BioNet Bioreactor Sensor Package: pH Electrode (220 mm Length) pH Cable (6 foot Length) Dissolved Oxygen Sensor (220 mm Length) Dissolved Oxygen Cable (6 foot Length) Temperature Sensor (220 mm Length) Source : Broadley James Corporation (www.broadleyjames.com) Bioreactor Configuration Continuation . . . Standard Configuration for Cell Culture: Headplate (18 ports) Autoclavable Glass Vessel Lipseal Stirrer Assembly Scoping Marine Impeller pH Electrode Holder Dissolved Oxygen Sensor Holder Triple Port Thermowell Sparger Pipe Air Overlay Pipe Septum Holder Adjustable Sample Pipe Blind Caps (8 each) Mill Nuts Stand Source : Broadley James Corporation (www.broadleyjames.com) Bioreactor Configuration Continuation . . . Motor Package: 40 mm Brushless Motor Digital Motor Controller Motor Interface Cable Additional Options: Level/Foam Sensor and Cable Air Outlet Condenser Assorted Impellers Consult Factory for Custom Configurations Heating Blanket Source : Broadley James Corporation (www.broadleyjames.com) Headplate Configuration Sparger Pipe Line pH Electrode dO2 Sensor Baffle Agitator Baffle Thermowell Blind Cap Air Overlay Triple Port (for base, vent) Harvest Tube Septum Holder Feed Line Port Sample line Source : Broadley James Corporation (www.broadleyjames.com) Typical Operations of Technicians Prepping the bioreactors (sometimes fast turnarounds) Set-up and DCU programming Daily Counts Inoculation of 2.0L/10.0L/20.0L Harvest of Cell Cultures Passaging of spinners Solera or passaging of 20L Support Tasks (maintenance of machines, etc. Main Goal : To execute the experiments and produce good data. Technician Skills Necessary in Cell Culture Laboratory Technical Skills: Aseptic technique – Contamination is a major problem-happens even to the best!!! Sterile techniques and practices are very important when performing cell culture. Use of laminar Flow Hood Use of filter or sterilizer Use of disinfectant Single use flask Basic Math – Conversions and measurements -Mass Balance Equation Input = Output C1V1 = C2V2 Good Documentation - Must be permanent, clear, timely, correct, traceable, complete Certifies that written procedures have been followed Provides complete history of product Provides means to investigate problems that may arise Technician Skills Continuation . . . General Skills: Communication – Good verbal and written communication Teamwork –Participates in and contribute to a team environment of mutual respect and high performance Accountability – Takes accountability, responsibility and initiatives in building commitment and driving results. Detail-Oriented – Follow procedure/SOP in detail. Always thinking about safety on the floor, improvement of operation, success of each project Acknowledgement Paul Bezy Tara Yokomizo Gokhan Abali Hardat Prashad Scott Broadley