Process transfer and optimization of Chinese hamster ovary cell cultivation for monoclonal antibody production

Biopharmaceutical market has been progressively expanding, and moving away from small molecular drugs to biotechnologically produced therapeutics such as recombinant proteins. Genetically modified mammalian cells, such as Chinese Hamster Ovary cells, are being used extensively for the production of these proteins. However, a challenge faced by the biopharmaceutical industry is to attain maximum product within a limited culture volume due to volume constraints in the bioreactor. This is overcome by intensifying the process and understanding the fundamentals of cell growth, which varies across different cell strains. The main objective of this study was to successfully transfer and scale the process in different types of bioreactors with a working volume ranging from 15 mL to 50 L. Process attributes, namely, viable cell concentration and final titre quantity were used to evaluate the scalability of the process. It was shown that the process was robust and scalable across different types of bioreactors. The second part of the project was to optimize the cultivation process in terms of testing the process parameters that control cultivation, primarily, the dissolved oxygen (DO) concentration and pH. We identified that reducing the DO to 40% and maintaining the pH at 7.1 not only decreased the requirement of pure oxygen in production scale bioreactors, but also reduced the damage to cultivated cells caused by oxygen driven free radicals. The next part of process optimization was conducted by varying the concentrations of ingredients in production medium and feed media used for the fed batch process. Concentration of carbon (glucose) and nitrogen (glutamine and glutamate) sources in the production medium were altered and the impact on the viable cell concentration and protein production was studied. The results showed that the production medium can be further improved by altering the initial concentration of glutamine and glucose to range between 0.6 to 1.2 g/L and 6 to 12 g/L, respectively. Glutamate was essentially used for protein production and was supplemented to the culture through the feed medium. Therefore, was not required to be added additionally in the production medium. In order to optimize the percentage of feed medium, different concentrations of the two feed media (FMA and FMB) were added to the cell culture. It was shown that increasing the concentration of the FMA beyond 6.52% and FMB beyond 0.62 % of the total working volume had a detrimental effect on the cell growth and protein production. Along with the above mentioned tests, the amino acid consumption across different scales of bioreactor was also studied. The amino acids were divided into two groups: amino acid required for cell growth (glutamine, tyrosine, phenylalanine and isoleucine) and protein production (the remaining essential and non-essential amino acids). This provided an insight into the function of amino acids within the cells

Improved Membrane Filtration For Water And Wastewater Using Air Sparging And Backflushing

Thesis (Ph.D.) University of Alaska Fairbanks, 2005The goal of this research was to investigate methods and techniques that enhance mass transfer through the membranes. Two general types of fluids were investigated: synthetic wastewater treated in a membrane bioreactor (MBR) and natural and simulated river water. For both fluids, a wide range of solid concentrations (up to 18 g/L) were tested. The membranes investigated were all tubular modules at pilot scale between 0.75 and 1.20 m length, with tubular diameters of 5.5--6.3 mm, 0.2 mum pore size, and membrane surface areas of 0.036--0.1 m2. For flux enhancement, two techniques were applied: air sparging (AS), and backflushing (BF). Both techniques were compared with the sponge ball cleaning method. The experimental temperature ranged between 10 and 30�C, cross-flow velocities (CFV) ranged between 0.5 and 5.2 m/s, and transmembrane pressure (TMP) ranged between 30 and 350 kPa. Research results showed, that AS was able to enhance the conventional flux over weeks to months up to factor of 4.5 for river water and a factor of 3 for wastewater. At modest CFV of 1.5--2 m/s, AS was as successful as BF. If higher CFV (up to 5.2 m/s) were supplied for BF, this technique could enhance the wastewater flux by factor 4.5. The supply of AS and BF combined was superior to the single application even at moderate CFV. The major finding of this research was that cake thickness on the membrane surface was decreased by AS, contrary to research by other authors. AS can be used as substitute aeration in MBRs, without impairing the degradation performance. The combination of AS and BF generated the least filter cake, but the lowest fouling was observed for AS. An empirical equation was proposed to calculate the viscosity in a sidestream MBR depending on reactor temperature and mixed liquor suspended solids (MLSS)

A theoretical and empirical investigation into the growth of ultralong carbon nanotubes

Carbon nanotubes (CNTs) were first discovered and named as such by Iijima in 1991. Various institutes and researchers have since widely conducted ongoing research on carbon nanotube growth. The exceptional properties of CNTs, including their electrical and mechanical properties, aim to revolutionise the applications of electronics and devices in the future such as transmission power lines and lightweight high-strength carbon nanotube fibres. Therefore, understanding the mechanisms of growing ultra-long carbon nanotubes (UL-CNTs) that can increase the length to more than a centimetre long can unlock the full potential of the CNTs. This PhD project will have three parts: (Ⅰ) the growth experiments using different types of monometallic & bimetallic iron based catalysts for growing carbon nanotubes; (Ⅱ) the computational simulation of flow fields around carbon nanotube geometry in a micro-scale; (Ⅲ) the applications of carbon nanotubes produced from waste plastics, such as Ethernet & audio cables, and public engagement events about the research. In the growth experiment topic, the primary objective of this research is to study the catalyst activities on the rate of carbon nanotube growth using monometallic (Fe) & bimetallic catalysts (Fe-Cu, Fe-Co, Fe-Ni, Fe-Sn, Fe-Ga, Fe-Mg & Fe-Al) dissolved in deionised water, and find which catalysts have the potential to grow the longest carbon nanotubes with improved characteristics, such as G/D (graphene/ disorders) ratio. As we know, the carbon source gas flow rate and reactor temperature profiles can affect the length of carbon nanotubes from the literature; an effective way to optimise experimental conditions to grow UL-CNTs is to use computational fluid dynamics (CFD) modelling methods. So far, there has been little research on the growth of ultra-long carbon nanotubes under a non-continuous flow environment on a nanoscale. Most computational modelling studies have only focused on the continuity of flow in a traditional approach. This research uses the BGK-Boltzmann equation and molecular collision models to investigate flow behaviours at the nanoscopic scale. Thus, this study provides an exciting opportunity to advance the knowledge of growing ultra-long carbon nanotubes (UL-CNTs) of centimetre length or higher and may be used in applications including the carbon nanotube Ethernet and audio cables as mentioned in this project

Investigation of novel ammonia production options using photoelectrochemical hydrogen

Hydrogen and ammonia are two of the most significant clean fuels, energy carriers and storage media in the near future. Production of these chemicals are desired to be environmentally friendly. Renewable energy, in particular solar energy-based hydrogen and ammonia production technologies bring numerous attractive solutions for sustainable energy production, conversion and utilization. The energy of the sun is endless and the water is a substance which is always accessible and renewable. Ammonia is currently one of the mostly used chemicals throughout the world due to many applications, such as fertilizers, cooling agents, fuel, etc. The Haber-Bosch process is the most dominant ammonia synthesis process which requires very high temperatures and pressures to operate and consumes massive amounts of fossil fuels mainly natural gas leading a non-sustainable process in the long-term. Therefore, alternative methods for ammonia production are in urgent need of development. This study theoretically and experimentally investigates the photoelectrochemical production of hydrogen and electrochemical synthesis of ammonia in a cleaner and integrated manner. In this respect, the main objective of this thesis is to develop a novel solar energy based ammonia production system integrated to photoelectrochemical hydrogen production. The hybrid system enhances the utilization of sunlight by splitting the spectrum and combining the photovoltaic and photoelectrochemical processes for electricity, hydrogen and ammonia production. The photoelectrochemical reactor is built by electrodeposition of the photosensitive semiconductor (copper oxide) on the photocathode. The characterization of the reactor under solar simulator light, ambient irradiance and concentrated light is accomplished. Furthermore, an electrochemical ammonia synthesis reactor is built using molten salt electrolyte, nickel electrodes and iron-oxide catalyst. The electrochemical synthesis of ammonia is succeeded using hydrogen and nitrogen feed gases above 180??C and at ambient pressures. The photoelectrochemically produced hydrogen is then reacted with nitrogen in the electrochemical reactor to produce clean ammonia. The comprehensive thermodynamic, thermoeconomic, electrochemical and life cycle models of the integrated system are developed and analyses are performed. The results obtained through models and experiments are comparatively assessed. The spectrum of solar light can be separated for various applications to enhance the overall performance of energy conversion from solar to other useful commodities such as electricity, fuels, heating and cooling. The results of this thesis show that under concentrated and split spectrum, the photoelectrochemical hydrogen production rates and efficiencies are improved. The overall integrated system exergy efficiencies are found to be 7.1% and 4.1% for hydrogen and ammonia production, respectively. The total cost rate of the experimental system for hydrogen and ammonia production is calculated to be 0.61 $/h from exergoeconomic analyses results. The solar-to-hydrogen conversion efficiency of the photoelectrochemical process increases from 5.5% to 6.6% under concentrated and split spectrum. Similarly, the photovoltaic module efficiency can be increased up to 16.5% under concentrated light conditions. Furthermore, the maximum coulombic efficiency of electrochemical ammonia synthesis process is calculated as 14.2% corresponding to NH3 formation rate of 4.41??10-9 mol s-1 cm-2

Investigation of High Temperature Steam Gasification of Biomass Char

Solar thermal gasification of biomass is a promising route to renewable fuel production. In order to design efficient solar biomass gasifiers, the kinetic rate of the char gasification step must be determined. The key advantages of solar thermal gasification are the ability to operate at temperatures significantly higher than those used in traditional gasifiers and to operate with steam instead of oxygen to produce a product stream with higher energy content. High temperature steam gasification kinetics are rarely studied in the literature, and the methods that are commonly used to measure low temperature gasification kinetics are often not applicable at high temperatures, for example due to heat and mass transfer limitations. The work presented in this thesis comprises studies designed to advance the state of the art in high temperature steam gasification by investigating char gasification kinetics, incorporating those kinetics into a CFD model, and facilitating gasification studies in aerosol flow through the development of a novel particle feeding system. A primary goal of this work was to develop a low-cost method to obtain an empirical rate expression for steam-char gasification. A modified fixed bed reactor was used to limit the effects of heat transfer, steam consumption, and hydrogen inhibition in order to ensure that the rate was measured at known conditions. After minimizing the above effects within the constraints of our laboratory system, the reaction rate was so rapid that our factory configured non-dispersive infrared analyzer could not provide high enough temporal resolution. In analyzing the data, we observed that the outlet flow meter could respond very quickly to changes in the gasification rate. After further analysis and testing, it was determined that the flow meter alone could be used to measure the rate of gasification within the fixed bed. This gas flow measurement technique was able to provide high resolution data with a very low cost and simple to use flow meter. Using the gas flow measurement technique, data were collected over a range of temperatures, steam concentrations, hydrogen concentrations and degrees of conversion. The results were used to develop an empirical rate expression based on the Random Pore Model for the dependence on conversion and a Langmuir-Hinshelwood type expression for the dependence on the reactor conditions. To demonstrate the power of an accurate kinetic rate expression, a simplified CFD model of a small fixed bed gasifier was developed using the commercially available Ansys Fluent software package. Validation experiments were performed in a laboratory scale fixed bed reactor. The model was able to accurately predict the overall reaction rate throughout time, and to lend insight into steam gasification in a fixed bed configuration. In addition to the investigation of high temperature steam char kinetics, a novel particulate feeding system was developed to aid in aerosol flow studies. Aerosol flow reactors are a valuable tool for measuring extremely fast reaction rates with very small particles, but they require the feedstock to be delivered pneumatically at a very consistent rate. The feeding system developed in this thesis is capable of feeding a variety of organic and inorganic particles with a diameter of less than 150 µm, and has successfully fed milled biomass containing a high fraction of hard to feed, high aspect ratio particles. It has been successfully used in several studies in our lab to measure gasification kinetics with a variety of feedstocks

Feed System Innovation for Gasification of Locally Economical Alternative Fuels (FIGLEAF)

The Feed System Innovation for Gasification of Locally Economical Alternative Fuels (FIGLEAF) project was conducted by the Energy & Environmental Research Center and Gasification Engineering Corporation of Houston, Texas (a subsidiary of Global Energy Inc., Cincinnati, Ohio), with 80% cofunding from the U.S. Department of Energy (DOE). The goal of the project was to identify and evaluate low-value fuels that could serve as alternative feedstocks and to develop a feed system to facilitate their use in integrated gasification combined-cycle and gasification coproduction facilities. The long-term goal, to be accomplished in a subsequent project, is to install a feed system for the selected fuel(s) at Global Energy's commercial-scale 262-MW Wabash River Coal Gasification Facility in West Terre Haute, Indiana. The feasibility study undertaken for the project consisted of identifying and evaluating the economic feasibility of potential fuel sources, developing a feed system design capable of providing a fuel at 400 psig to the second stage of the E-Gas (Destec) gasifier to be cogasified with coal, performing bench- and pilot-scale testing to verify concepts and clarify decision-based options, reviewing information on high-pressure feed system designs, and determining the economics of cofeeding alternative feedstocks with the conceptual feed system design. A preliminary assessment of feedstock availability within Indiana and Illinois was conducted. Feedstocks evaluated included those with potential tipping fees to offset processing cost: sewage sludge, municipal solid waste, used railroad ties, urban wood waste (UWW), and used tires/tire-derived fuel. Agricultural residues and dedicated energy crop fuels were not considered since they would have a net positive cost to the plant. Based on the feedstock assessment, sewage sludge was selected as the primary feedstock for consideration at the Wabash River Plant. Because of the limited waste heat available for drying and the ability of the gasifier to operate with alternative feedstocks at up to 80% moisture, a decision was made to investigate a pumping system for delivering the as-received fuel across the pressure boundary into the second stage of the gasifier. A high-pressure feed pump and fuel dispersion nozzles were tested for their ability to cross the pressure boundary and adequately disperse the sludge into the second stage of the gasifier. These results suggest that it is technically feasible to get the sludge dispersed to an appropriate size into the second stage of the gasifier although the recycle syngas pressure needed to disperse the sludge would be higher than originally desired. A preliminary design was prepared for a sludge-receiving, storage, and high-pressure feeding system at the Wabash River Plant. The installed capital costs were estimated at approximately $9.7 million, within an accuracy of {+-}10$12.40 per wet ton of municipal sludge delivered. This is based on operation with petroleum coke as the primary fuel. Similarly, with coal as the primary fuel, a minimum tipping of $16.70 would be required. The availability of delivered sludge from Indianapolis, Indiana, in this tipping-fee range is unlikely; however, given the higher treatment costs associated with sludge treatment in Chicago, Illinois, delivery of sludge from Chicago, given adequate rail access, might be economically viable