Critical Analytical Measurements for Bioreactor Optimization. controlling an organism s chemical environment leads to consistent and
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1 Critical Analytical Measurements for Bioreactor Optimization Mettler-Toledo Ingold, Inc., Bedford, MA Abstract Most bioreactor processes share a basic principle; optimizing and controlling an organism s chemical environment leads to consistent and enhanced product yield. The required conditions do not remain constant throughout the various stages of cell growth, and therefore, must be closely monitored and controlled. Among the critical analytical measurements are ph, dissolved oxygen (D.O.), dissolved carbon dioxide (CO 2 ), and cell density. Maintaining ideal solution ph is critical for proper cell development and growth. During aerobic fermentation, controlling dissolved oxygen concentration assures adequate supply of oxygen for respiration. In keeping with the principles of the Process Analytical Technology (PAT) initiative, the addition of the measurement of dissolved carbon dioxide in solution provides a more complete picture of the respiratory cycle, and helps prevent CO 2 toxicity. In this paper, emphasis is given to the use of this new CO 2 measurement device. Measurement and control of the above parameters optimizes the likelihood of a successful fermentation. Monitoring the resulting cell mass using optical density provides immediate feedback confirming the process progression. Details of each measurement methodologies are presented. Specific system demands of the biotechnology industry including hygienic and sterilizable designs and agency conformance are discussed.
2 Outline Overview ph Dissolved Oxygen Dissolved Carbon Dioxide Cell Density/Turbidity Industry Requirements Overview The biochemical reactions taking place within a reactor are very dynamic. There exists a delicate balance of proper environmental conditions such as temperature and mixing, and more complicated interactions with nutrient source, respiration conditions, and microorganism growth. As seen in the table below, the tolerance window may be very narrow, and growth rate and viability may drop off precipitously. Batch reactors may run from 3-5 days to as long as 3-5 months. The analytical measurements must remain reliable and accurate throughout this period, and maintain the most demanding requirements for sterility. Deviations from control conditions may impact the growth rate, product purity, processing time, and ultimately profitability. Influence of ph, po 2, pco 2 on Melanoma Cell Growth Parameter Parameter Value Growth Rate (d -1 ) Percent Viability ph po 2 (mbar) pco 2 (mbar)
3 ph Measurement Reliable ph control in fermentation processes ph is a critical parameter to monitor during biotechnology fermentation processes because it has a profound influence on the growth characteristics of the microorganisms. Also, ph and CO 2 are interrelated, further increasing the significance of ph control. Most process ph measurements today are performed with a combination ph electrode. The anatomy of a combination electrode is illustrated on the right. Combination ph Electrode Anatomy ELECTRICAL CONNECTOR REFERENCE ELECTROLYTE REFERENCE LEAD ph LEAD REFERENCE JUNCTION ph GLASS
4 Some of the critical electrode design features are as follows. The heart of a ph electrode is the ph sensitive glass. While there are many different glass formulations available, for bioreactor applications, it is critical that the glass be able to withstand multiple steam sterilization cycles at up to 140 C, with negligible shift in performance. In addition to the specialized glass formulation, the reference element must resist stripping of the critical silver chloride coating which is brought on by the temperature cycling introduced by sterilization. The Argenthal system effectively maintains a constant concentration of silver chloride at the reference silver wire, providing stable and repeatable reference voltages. Also, with high protein concentration often encountered, keeping the reference junction from fouling presents a challenge. A constant flowing reference electrolyte helps extend the life of the electrode. Further, use of an internal silver-ion trap eliminates fouling by proteins or silver sulfide precipitates by permitting the use of a silver-free outer electrolyte in contact with the sample. The form of these electrodes take many shapes as illustrated for a liquidfilled (left) and gel-filled (right) electrode.
5 Dissolved Oxygen Oxygen; essential for life Oxygen is essential for most of the life on earth. Because oxygen is such a necessary component in biological processes, it is a key parameter in bioreactor control. Aerobic microorganisms require a source of oxygen for respiration. Inadequate supply will restrict cell growth, result in undesirable metabolic products, or ultimately kill the cells. Bubbling too much air or oxygen can cause excess foaming as well as waste utilities. Measurement and control of dissolved oxygen in solution balances these extremes. The Clark measurement principle for dissolved oxygen is a polarographic method. Dissolved oxygen in the measuring solution diffuses through a gas permeable membrane in the sensor cap into an internal electrolyte (see Figure). At the platinum cathode surface, this oxygen is reduced according to the following equation: O H 2 O + 4 e - 4 OH - At the silver anode, the following reaction takes place: 4 Ag + 4 Cl - 4 AgCl + 4 e - The resulting current is directly proportional to the oxygen concentration. The oxygen sensor is designed for easy replenishment of the internal electrolyte and periodic replacement of membrane cap as illustrated below. Dissolved Oxygen Sensor Principle
6 Dissolved CO 2 CO 2 is a critical parameter influencing product growth during the fermentation process. (oxidation) heat CO 2 + H 2 O The oxidation of carbohydrates to CO 2 and water is the basis for aerobic forms of life. ph and DO are widely established as required measurement parameters in bioreactors around the world. The impact of dissolved carbon dioxide in the cultivation media has, however, drawn little attention in the past. In recent years dissolved carbon dioxide is increasingly also becoming a parameter of interest. Examining oxygen uptake and CO 2 release provides a detailed understanding of the respiratory cycle of living cells. ADP ATP heat Energy of metabolism Carbon dioxide is a product of the respiratory and fermentative metabolism of microorganisms. Depending on the concentration or partial pressure, carbon dioxide may either positively or negatively influence the growth and metabolism of micro-organism. Until recently, CO 2 measurements have been mainly made through the use of off-line grab sample analysis, using blood gas analyzers (BGA). This is very labor intensive yet only provides periodic snapshots of the conditions within the reactor. Attempts to obtain continuous CO 2 information by infrared measurement of the off-gas, result in expensive, high maintenance equipment, substantial lag times, and only an inferred indication of what is happening in the liquid phase. Another well proven technique for determining dissolved CO 2 with far less investment is potentiometric carbon dioxide electrodes, using the Severinghaus principle. The principle of the measurement is shown in the figure below. Dissolved CO 2 in the liquid sample, diffuses through a gas permeable membrane. The CO 2 reacts with the internal electrolyte, resulting in a change of ph of the electrolyte. CO 2 + H 2 O HCO H +
7 This ph change is detected using an internal ph electrode, and the ph value directly correlates with the CO 2 concentration of the sample. This new CO 2 system delivers precise, real-time data to better manage critical fermentation and cell culture processes. This data provides valuable insight into cellular metabolism and other changes within the bioreactor. The in-situ sensor measures exactly the same partial pressure as the cells experience. CO 2 Measurement Principle CO 2 electrode exploded view One of the major trends in biotechnology today is the increasing use of mammalian cell lines including human, monkey, mouse and bovine cells. One of the most important requirements for optimal cell growth in a bioreactor is continuous monitoring and control of critical parameters which include po 2, ph, CO 2 and temperature. Reliable measurement of CO 2 is essential for successful large-scale operation as the accumulation of CO 2 becomes more problematic as viable cell concentrations rise. High CO 2 concentrations can inhibit cell growth and product formation in mammalian cells. By maintaining low and constant levels, the production rate of pharmaceuticals, proteins and antibodies can be significantly increased.
8 7 Good Reasons for In-situ CO 2 Control in Fermentation Processes: Mammalian cells require certain CO 2 levels for proper metabolic function. High CO 2 concentration inhibits further growth Extremely high CO 2 concentration can be toxic to mammalian cells CO 2 levels provide information on biomass concentration and substrate consumption. In-situ measurement enables fast and accurate CO 2 control in the reactor. Only using in-situ measurement does the sensor see the same CO 2 level as the cells. The formation of products and by-products in the fermentation frequently depends on the CO 2 concentration. InPro 5000 CO 2 vs. Blood Gas Analyzer
9 Optical Density Optical density systems for in-line biomass concentration measurement Proper control of ph, DO, and CO 2 promotes active cell growth. The progress of this growth can be tracked using optical density/turbidity. Many medicines containing purified antigens are produced through classical aerobic bacteria fermentation. For these fermentations, cell growth can be tracked from an initially fully clear and particle-free medium, through to the thick, cell-laden broth using an optical turbidity system. Optical density, or turbidity, is measured using backscattered light technology. A light source is directed into the sample. Particles within the sample will reflect a portion of the light directly back toward the source. The more particles present, the more light reflected back. The intensity of the backscattered light is proportional to the concentration of particles in the medium. Light Source Reflected Light
10 Many processes start with clear solutions. Following inoculation, as cell mass increases, it results in an increase in turbidity. The use of backscattered optical density provides an instantaneous indication of the progression of cell growth (see below). Batch cycle time can be controlled based on the progression of cell growth leading to greater efficiency and throughput. 40 Optical Density Application - Fermentation biomass [g/l] time [h]
11 Requirements of the Industry Hygiene and sterilization in pharmaceutical and biotechnology production Accurate measurement and control of ph, dissolved oxygen, dissolved carbon dioxide, and optical density is particularly important with many manufacturing processes to maximize yield and assure product quality. Continuous inline acquisition of these values allows distinctly improved process reliability as compared to grab samples which are analyzed in the laboratory. To function, however, inline sensors are required to meet stringent demands of the industry. An important requirement in biotechnological processes is absolute aseptic reliability of the equipment employed. Dr. Werner Ingold laid the cornerstone for this success through his development of the first sterilizable ph electrode for the pharmaceutical industry in Other sensor design considerations include the following: Sterility: Following insertion into the vessel, the sensor must withstand aggressive Clean-In-Place (CIP) and Sterilize-In-Place (SIP) conditions without impacting ability to accurately measure the media. Surface Finish: Industry is moving to finer surface finishes to minimize grooves where organics and/or microorganisms can adhere (see Figure). Long-term stability: Since it is often not acceptable to remove and reinsert a sensor from a vessel once the process has begun, it is important that the sensor provide very stable and reliable readings over the entire duration of the process. No media interference: The high protein concentrations found can often result in precipitates and/or sensor coatings that can result in sensor failure of inappropriate sensor designs. Agency conformance: Increasing industry demands include traceability of steel components (3.1 B), o-ring compliance with FDA or USP 6, certificate of cleanability such as EHEDG (European Hygienic Equipment Design Group).
12 Surface Finish Comparison
13 Conclusion Competitive and financial pressure is increasing forcing the biotechnology industry to look for more efficient, and therefore more profitable methods of production. ph and dissolved oxygen have been relied upon for decades to optimize conditions for consistent and maximum biomass growth. Today s improved dissolved CO 2 sensors permit insitu measurement of this critical parameter. The growth curve of the resulting biomass is then monitored by use of cell density/turbidity. Combined, ph, DO, CO 2, and cell density systems, provide accurate long-term stability under challenging conditions while meeting stringent regulatory agency demands. Careful control of these parameters leads to prime growth conditions for microorganisms producing more consistent and enhanced product yield.
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