John Tharakan

Development of an immunoaffinity process for factor IX purification.

Abstract. An immunoaffinity process based on monoclonal antibody (MAb) to factor IX (FIX) has been developed. Initially, vitamin‐K‐dependent proteins from cryoprecipitate‐poor plasma are isolated on DEAE‐Sephadex. The eluate is applied to an immunoaffinity column that utilizes a divalent metal‐ion‐dependent MAb directed against FIX. After washing the column with high salt in the presence of magnesium ion, the FIX is eluted using a citrate‐ or EDTA‐containing buffer. Coagulation assays and Western blots show no detectable amounts of any contaminating proteins. Purity of the FIX product is established using reduced and nonreduced Coomassie‐stanined SDS‐PAGE and HPLC. The N‐terminal 20 amino acids of the single peak of the HPLC were shown to be identical to those reported for FIX. The process shows no detectable leakage of monoclonal antibodies (MAb), efficient utilization of MAb, and provides yields greater than 95%. The use of solvent/detergent treatment as a potential viral inactivation method is incorporated in the process. Studies with tritiated Triton X‐100 indicate that the detergent can be washed out of the MAb column so that less than 1 ppm (total) Triton X‐100 coelutes with the FIX.

Physical and biochemical characterization of five commercial resins for immunoaffinity purification of factor IX

The American Red Cross has developed an immunoaffinity chromatography method to purify human coagulation factor IX (FIX) to homogeneity using monoclonal antibodies (MAb) that bind FIX in the presence of divalent cations. The MAb is immobilized on Sepharose CL2B, a soft gel with a low pressure tolerance as well as poor large-scale performance characteristics, including low reusability, and resin crumbling and deterioration. In this study, we examined several commercially available resin supports. Aside from Sepharose CL2B, we studied two other cross-linked agaroses, as well as two synthetic polymer supports. Immobilization chemistries included cyanogen bromide activation of agarose, 2-fluoro-2 methylpyridinium toluene-4-sulfonate activation of one of the synthetic polymer as well as aldehyde group reduction by NaCNBH3 to form secondary amine linkages on one of the cross-linked agaroses. To determine the feasibility of using the resins in large-scale immunoaffinity chromatographic purification of FIX, we studied physical and biochemical properties of the resins. The physical characteristics studied included the crushability of the resins under pressure as well as ability to support increasing flow-rates at increasing pressures. The biochemical examination of the various resins focused on efficiency of antigen capture by the immobilized antibody ligand and the effect of flow-rate on MAb efficiency, where we found that very low flow-rates slightly increased the capacity of the MAb. The results demonstrate a straightforward method of assessing the feasibility of using particular resins in large-scale affinity purification.

Hybridoma growth and antibody secretion in serum-supplemented and low protein serum-free media

Hybridoma cell growth and monoclonal antibody secretion were studied with three murine hybridomas, SS1.1, NS6.3 and 455, propagated in four low protein serum-free media and various serum-supplemented media. Cell metabolism, as indicated by cell growth and glucose uptake, and antibody production rates were measured. The analysis focused on the secretion of monoclonal antibodies as a function of the medium makeup. Although cell densities achieved were generally higher for serum-supplemented media, glucose uptake rates did not vary significantly, and antibody secretion rates measured at peak cell density were higher for serum-free media with all three hybridomas. Decreasing the serum concentration had opposite effects for the IgM and IgG secretors, NS6.3 and SS1.1; secretion rates measured at peak cell density were higher for the former and lower for the latter.

Cametabolic biotransformation of trinitrotoluene (tnt) supported by aromatic and non-aromatic cosubstrates

Abstract A microbial consortia was isolated from trinitrotoluene (TNT) contaminated soil and cultured in media supplemented with structurally similar (aromatic) or non-aromatic cosubstrates and TNT to examine their impact on cometabolic TNT biotransformation. A microbial inoculum with 105 CFU/ml with TNT concentrations of 5, 10, 30 and 50 ppm in separate triplicate cultures revealed maximum TNT biotransformations of 23, 21, 9 and 3%, respectively, after 9 days. A larger inoculum (l07CFU/ml) resulted in transformation of TNT concentrations of 8, 15, 22 and 30 ppm by 71, 54, 50 and 38%, respectively. Cultures supplemented with benzene, nitrobenzene or toluene revealed no enhancement of TNT biotransformation rates or extents over those in culture with TNT as sole carbon source. With glucose and citric acid at 30 mg/l, TNT biotransformations of 56 and 51%, respectively, were obtained. With glucose or citric acid at 3000 mg/l, 100% transformation of 30ppm TNT was realized in 24 hours and of 100ppm TNT in 72hrs. Metabolites identified included 2-amino-4,6-dinitrotoluene, 4-amino-2,6-dinitrotoluene, 2,6-diamino-4-nitrotoluene, and 2,4-dinitrotoluene; analysis also demonstrated that TNT disappearance paralleled glucose utilization and cell growth.

Serum free fed batch production of IgM

SummaryFed batch cultivation of a murine hybridoma secreting IgM in a serum free medium was successfully attempted. Cell growth and IgM productivity remained high for over a month, and compared well to a similar fed batch culture in medium supplemented with 10% fetal calf serum. The average specific secretion rates of antibody in both media were the same. Sufficient inoculum cell density was crucial to the establishment of a viable culture in serum free medium for the cell line, NS6.3, used.

Effect of feed flow-rate, antigen concentration and antibody density on immunoaffinity purification of coagulation factor IX

A simple physical model of immunoaffinity chromatography (IAC) demonstrates that immobilized monoclonal antibody (MAb) capacity in IAC purification will be a function of many parameters, including feed flow-rate and antigen concentration, and MAb density (mg MAb immobilized/ml resin). We studied IAC of factor IX, and examined the effect of parameter variation on MAb capacity. MAb capacity (1) was not affected by feed flow-rate or antigen concentration, and (2) decreased as MAb density increased. (1) Suggested that diffusion of factor IX into the resin bead was not limiting. Characteristic diffusion, convection and reaction times were calculated and used in dimensional analysis to compare their relative magnitudes. If MAb was assumed to be localized to the outer 10% of the bead volume, this analysis concluded that diffusion was not limiting, consistent with the suggestions of our experimental data. (2) Suggests that high MAb densities make MAb less accessible.

IgG production kinetics in serum free media

SummaryA murine hybridoma (455) was cultured in four different serum free media formulations, and a newborn calf serum supplemented medium was used as a basis of comparison. The serum supplemented medium supports a higher cell growth rate and results in a higher IgG titer. However, the antibody secretion rate on a per cell basis is higher in the serum free media, indicating that serum could be inhibitory to antibody secretion. The results identify the possibility of a least eliminating serum during the monoclonal antibody production phase.

Ligand efficiency in axial and radial flow immunoaffinity chromatography of factor IX

Abstract Radial flow (RF) columns are attractive for process chromatography primarily because larger throughputs and lower pressure drops are achievable in such columns. Large scale immunoaffinity processes using soft resins can benefit most from this configuration. In this study, we compared immobilized ligand efficiency in axial flow (AF) and RF columns using monoclonal antibody against factor IX as the immobilized ligand and a coagulation factor IX complex as the source material. We examined the effects of flow-rate, total protein loading, feed antigen concentration and direction of flow (centrifugal or centripetal for RF, and downward or upward for AF) on immobilized antibody capacity (measured as mg antigen bound per mg antibody). Our results corroborate earlier work, and suggest that none of the factors, in the ranges examined, significantly altered the efficiency of the monoclonal antibody (MAb) in binding factor IX. We also investigated the efficiency of the immobilized antibody upon reuse and found that, over twenty cycles, there was no significant decrease in antibody efficiency. Our results demonstrated that efficiencies obtainable in AF columns can be achieved in RF columns with the same bed thickness, suggesting that radial dispersion, mass transfer and intraparticular diffusion may not have a significant influence on immunoaffinity chromatography efficiency in RF and AF columns.

Introducing Survival Ethics into Engineering Education and Practice

Given the possibilities of synthetic biology, weapons of mass destruction and global climate change, humans may achieve the capacity globally to alter life. This crisis calls for an ethics that furnishes effective motives to take global action necessary for survival. We propose a research program for understanding why ethical principles change across time and culture. We also propose provisional motives and methods for reaching global consensus on engineering field ethics. Current interdisciplinary research in ethics, psychology, neuroscience and evolutionary theory grounds these proposals. Experimental ethics, the application of scientific principles to ethical studies, provides a model for developing policies to advance solutions. A growing literature proposes evolutionary explanations for moral development. Connecting these approaches necessitates an experimental or scientific ethics that deliberately examines theories of morality for reliability. To illustrate how such an approach works, we cover three areas. The first section analyzes cross-cultural ethical systems in light of evolutionary theory. While such research is in its early stages, its assumptions entail consequences for engineering education. The second section discusses Howard University and University of Puerto Rico/Mayagüez (UPRM) courses that bring ethicists together with scientists and engineers to unite ethical theory and practice. We include a syllabus for engineering and STEM (Science, Technology, Engineering and Mathematics) ethics courses and a checklist model for translating educational theory and practice into community action. The model is based on aviation, medicine and engineering practice. The third and concluding section illustrates Howard University and UPRM efforts to translate engineering educational theory into community action. Multidisciplinary teams of engineering students and instructors take their expertise from the classroom to global communities to examine further the ethicality of prospective technologies and the decision-making processes that lead to them.

Survival Ethics in the Real World: The Research University and Sustainable Development

We discuss how academically-based interdisciplinary teams can address the extreme challenges of the world’s poorest by increasing access to the basic necessities of life. The essay’s first part illustrates the evolving commitment of research universities to develop ethical solutions for populations whose survival is at risk and whose quality of life is deeply impaired. The second part proposes a rationale for university responsibility to solve the problems of impoverished populations at a geographical remove. It also presents a framework for integrating science, engineering and ethics in the efforts of multidisciplinary teams dedicated to this task. The essay’s third part illustrates the efforts of Howard University researchers to join forces with African university colleagues in fleshing out a model for sustainable and ethical global development.