Measurement of ammonia and glutamine in cell culture media by gas sensing electrodes

This paper describes the use of a commercially available off-line gas sensing electrode for determination of ammonia and glutamine in cell culture media. The measurement technique was tested in different media preparations with different serum concentrations. The glutamine decomposition was studied as a function of pH for cell culture medium and the results were compared to those obtained by conventional methods, i.e. , HPLC. Finally, glutamine and ammonia metabolism were studied during the cultivation of a hybridoma cell line.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/42489/1/10542_2005_Article_BF01876051.pd

Targeted Mutagenesis of a Therapeutic Human Monoclonal IgG1 Antibody Prevents Gelation at High Concentrations

A common challenge encountered during development of high concentration monoclonal antibody formulations is preventing self-association. Depending on the antibody and its formulation, self-association can be seen as aggregation, precipitation, opalescence or phase separation. Here we report on an unusual manifestation of self-association, formation of a semi-solid gel or “gelation”. Therapeutic monoclonal antibody C4 was isolated from human B cells based on its strong potency in neutralizing bacterial toxin in animal models. The purified antibody possessed the unusual property of forming a firm, opaque white gel when it was formulated at concentrations \u3e40 mg/mL and the temperature wa

Kinetic characterization of hybridoma growth, metabolism, and monoclonal antibody production rates.

Monoclonal antibodies produced by hybridoma cells are one of the most important products of biotechnology. Optimal design and development of bioreactors require a quantitative understanding of cell growth, metabolism, and antibody production rates. This thesis is a comprehensive investigation of the influence of culture environment on these biological variables. Both the extent of cell growth and the final antibody concentrations were influenced by the inoculum size, but specific growth, metabolic, and antibody production rates were less sensitive to initial cell density. Short-term exposure to new serum concentrations influenced the growth rate in a Michaelian fashion, but did not alter the cell metabolism and antibody production rate. When cells were cultured in low serum-containing media for prolonged periods of time (6 months), they adapted and both growth and antibody titer were improved. However, for one cell line, adaptation to low serum resulted in a gradual loss of antibody productivity. We have determined that this loss is due to the appearance of a sub-population that has lower internal and surface antibody content. Cell growth was inhibited at 100% air saturation and at very low dissolved oxygen concentrations leading to an optimal range between 25 and 50% air saturation. We have also demonstrated that the cells used in this study could grow and produce antibody under total anaerobic conditions, which has important implications for the design of high density cultures. The antibody production rate was unaffected by the dissolved oxygen concentration. Cell growth and antibody production were optimal at pH 7.2 while the specific antibody production rate, though unaltered under alkaline conditions, was 2-3 fold higher under acidic conditions. Elevated media osmolarity also influenced the specific antibody production rate. Both ammonia and lactate inhibit growth, but do not accelerate cell death. Cell metabolism was influenced by lactate and ammonia levels. However, the specific antibody production rate was unaffected. It is hoped that the results presented in this thesis will contribute significantly to a better understanding of cell physiology in bioreactor environments, and provide coherent design principles for the optimization of mammalian cell culture technology.Ph.D.Chemical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/105413/1/9023613.pdfDescription of 9023613.pdf : Restricted to UM users only

Examination of serum and bovine serum albumin as shear protective agents in agitated cultures of hybridoma cells

A murine hybridoma cell line (167.4G5.3) was cultured in batch mode using IMDM containing different serum concentrations and bovine serum albumin (BSA). Cell growth and death, metabolism and antibody production were studied in these cultures. The cells were more susceptible to shear in the stationary and in the decline phase of growth as evidenced by higher death rates. Cell growth was best at high serum concentrations with high specific growth and low specific death rates. When BSA was used instead of serum in IMDM, no protective effect was observed. Cell metabolism and monoclonal antibody production rates were not influenced by the level of serum or by BSA. The use of serum in commercial serum-free media (OPTI-MEM) also resulted in no change in both growth and death rates.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/29384/1/0000455.pd

Effect of medium osmolarity on hybridoma growth, metabolism, and antibody production

No Abstract.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/37906/1/260371015_ftp.pd

Effect of initial cell density on hybridoma growth, metabolism, and monoclonal antibody production

A murine hybridoma cell line (167.4G5.3) was cultivated in batch mode with varying inoculum cell densities using IMDM media of varying fetal bovine serum concentrations. It was observed that maximum cell concentrations as well as the amount of monoclonal antibody attainable in batch mode were dependent on the inoculum size. Specifically, cultures with lower inoculum size resulted in lower cell yield and lower antibody concentrations. However, in the range of 102 to 105 cells per ml, the initial cell density affected the initial growth rate by a factor of only 20%. Furthermore, specific monoclonal antibody production rates were independent of initial cell density and the serum concentration. Glutamine was the limiting nutrient for all the cultures, determining the extent of growth and the amount of antibody produced. Serum was essential for cell growth and cultures with initial cell concentrations up to 106 cells per ml could not grow without serum. However, when adapted, the cells could grow in a custom-made serum-free medium containing insulin, transferrin, ethanolamine, and selenium (ITES) supplements. The cells adapted to the ITES medium could grow with an initial growth rate slightly higher than in 1.25% serum and the growth rate showed an initial density dependency-inocula at 103 cells per ml grew 30% slower than those at 104 or 105. This difference in growth rate was decreased to 10% with the addition of conditioned ITES medium. The addition of conditioned media, however, did not improve the cell growth for serum-containing batches.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/28331/1/0000090.pd

Letter to the Editor

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/41536/1/11095_2004_Article_306008.pd

Dissolution of lonizable Drugs in Buffered and Unbuffered Solutions

The dissolution kinetics of ionizable drugs (weak acids or bases) are analyzed with a mathematical model derived from the theory of mass transfer with chemical reaction. The model assumes that the overall process is diffusion limited, that all the reactions are reversible and instantaneous, and that dissolution and reaction are limited to the stagnant fluid film adjacent to the solid phase. Dissolution into buffered and unbuffered aqueous solutions are considered separately, with covenient analytical solutions obtained in both cases. In addition, equations for the time to partial and complete dissolution are derived. The dissolution rate is shown to be dependent on the p K a and intrinsic solubility and the medium properties, i.e., pH, buffer capacity, and mass transfer coefficient. Equations of a form analogous to the nonionized case are derived to account explicitly for all these factors, with dissolution rates expressed in terms of the product of a driving force (concentration difference) and resistance (inverse of mass transfer coefficient). The solutions are in an accessible analytical form to calculate the surface pH and subsequently the surface concentrations driving the drug dissolution. Numerical examples to illustrate dissolution into unbuffered and buffered media are presented and the results are shown to be in accord with experimental data taken from the literature.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/41523/1/11095_2004_Article_306188.pd

On the Use of the Quasi-Equilibrium Assumption for Drug Dissolution

The quasi-equilibrium assumption is often used to simplify the analysis of reaction diffusion problems, including those that arise in drug dissolution and ionization processes. This approximation often makes the governing equations tractable, and analytical solutions may then be obtained. However, the application of the quasi-equilibrium assumption may lead to simplified solutions that (1) are apparently inconsistent with stated boundary conditions and (2) have a physical interpretation that is different from those of the original problem statement. Herein we discuss these two issues as they arise in the modeling of drug dissolution processes. In spite of the different conceptualizations, the concentration profiles and dissolution fluxes obtained from the full and approximate solutions converge as the reaction response times exceed those of diffusion, thus supporting the applicability of the quasi-equilibrium assumption for ionized drug dissolution processes.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/41545/1/11095_2004_Article_305737.pd