Process scale-up issues: Relics of the past or continues to cause major headaches

Since the inception of biopharmaceutical industry, scale up issues hampered the success of process transfers and product commercialization. In the last 30 years, the field evolved significantly, we have gained significance experience, broadened our understanding of cell culture, and resolved issues such as shear, mixing, and aeration. Requirement of maintaining geometric similarity and scale-up parameters (power per volume or tip speed) was proven not to impact the performance of the bioreactors significantly. Today’s processes could be scaled up from micro-bioreactors to full-scale production bioreactors rather easily. This talk will discuss the factors that played a significant role for this accomplishment. While we can claim we solved most of the scale-up issues, new areas of concern, mostly peripheral issues, prevent us from claiming victory. These points will be discussed with examples

Fascin expression in colorectal carcinomas

PURPOSE: The purpose of this study was to investigate the significance of fascin expression in colorectal carcinoma. METHODS: This is a retrospective study of 167 consecutive, well-documented cases of primary colorectal adenocarcinoma for which archival material of surgical specimens from primary tumor resections were available. We chose a representative tissue sample block and examined fascin expression by immunohistochemistry using a primary antibody against "fascin". We calculated the "immunohistochemical score (IHS)" of fascin for each case, which was calculated from the multiplication of scores for the percentage of stained cells and the staining intensity. RESULTS: Fascin immunoreactivity was observed in 59 (35.3%) of all cases with strong reactivity in 24 (14.4%), moderate reactivity in 25 (14.9%) and weak reactivity in 10 (6.0%) cases. Strong/moderate immunoreactivities were mostly observed in invasive fronts of the tumors or in both invasive and other areas. Fascin immunoreactivity scores were significantly higher in tumors with lymph node metastasis (p:0.002) and advanced stage presentation (p:0.007). There was no relation between fascin expression and age, gender, depth of invasion, distant metastasis or histological grade (p>0.05). There was a higher and statistically significant correlation between fascin immunoreactivity in the invasive borders of tumors and lymph node metastasis (r:0.747, p:0.005). In stage III/IV tumors, two-year survival was 92.2% in tumors without fascin immunoreactivity, and only 60.0% in tumors with a fascin IHS>10 (p:0.003). CONCLUSION: These findings suggest that fascin is heterogeneously expressed in approximately one third of colorectal carcinomas with a significant association with lymph node metastasis, tumor stage and location. Moreover, these results indicate that fascin may have a role in the lymph node metastasis of colorectal carcinoma

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