Expanded bed adsorption process for protein recovery from whole mammalian cell culture broth.

An expanded bed process for the recovery of soluble protein from whole mammalian cell culture broth by adsorption on specially-designed cation exchange resin is described. A bed of large, dense resin particles in a 5 cm ID column was expanded 2.45 times by the upward flow of three-fold dilute broth. The void space was sufficient to permit cells to flow through the column as the protein was adsorbed. The liquid flow in the column was distributed to minimize back-mixing and create a multiplate adsorption process. During elution, the liquid flow was reversed and the resin bed was packed. In contrast to conventional filtration, the expanded bed process combines clarification, product recovery and concentration into a single-step process. Residence time distribution analysis showed a small degree of axial dispersion and the generation of 22 theoretical plates in the expanded bed (170 ml resin with an unexpanded height of 8.6 cm). Two pilot runs of the process were done with 26 and 36 liters of whole broth at a linear velocity of 135 cm h-1. More than 95% adsorption of the antibody product was achieved without breakthrough in a single pass through the column. Elution recovered 70-85% of the antibody at a concentration as much as 39 times higher than in the broth. The antibody was purified seven-fold in the recovery process mainly because adsorption conditions prevented the binding of 80% of the undesired protein. Because it is less labor-intensive, the expanded bed process is potentially more economical than the filtration recovery process. Although processing time with the expanded bed is considerable, it does not require constant monitoring as does filtration.

Inclined sedimentation for selective retention of viable hybridomas in a continuous suspension bioreactor.

The continuous separation of nonviable hybridoma cells from viable hybridoma cells by using a narrow rectangular channel that is inclined from the vertical has been investigated experimentally. The effectiveness of the settler in selectively retaining viable hybridomas in the bioreactor while permitting the removal of nonviable hybridomas has been shown to depend on the flow rate through the settler. Intermediate flow rates through the settler have been found to provide the highest removal of nonviable hybridomas relative to viable hybridoma retention. At high dilution rates through the chemostat, over 95% of the viable cells could be partitioned to the bottom of the settler while over 50% of the nonviable cells are removed through the top of the settler. This successful separation is due to the significantly larger size of the viable hybridomas than the nonviable ones. A continuous perfusion experiment was performed in which an external inclined settler was used to retain virtually all of the viable hybridomas in the culture, while selectively removing from the culture approximately 20% of the nonviable cells that entered the settler. A stable viable cell concentration of 1.0 x 10(7) cells/mL was achieved, as was an antibody productivity of over 50 micrograms/(mL.day). These represent 3- and 6-fold increases, respectively, over the values obtained from a chemostat culture without cell retention.

Verification of immune response optimality through cybernetic modeling.

An immune response cascade that is T cell independent begins with the stimulation of virgin lymphocytes by antigen to differentiate into large lymphocytes. These immune cells can either replicate themselves or differentiate into plasma cells or memory cells. Plasma cells produce antibody at a specific rate up to two orders of magnitude greater than large lymphocytes. However, plasma cells have short life-spans and cannot replicate. Memory cells produce only surface antibody, but in the event of a subsequent infection by the same antigen, memory cells revert rapidly to large lymphocytes. Immunologic memory is maintained throughout the organism's lifetime. Many immunologists believe that the optimal response strategy calls for large lymphocytes to replicate first, then differentiate into plasma cells and when the antigen has been nearly eliminated, they form memory cells. A mathematical model incorporating the concept of cybernetics has been developed to study the optimality of the immune response. Derived from the matching law of microeconomics, cybernetic variables control the allocation of large lymphocytes to maximize the instantaneous antibody production rate at any time during the response in order to most efficiently inactivate the antigen. A mouse is selected as the model organism and bacteria as the replicating antigen. In addition to verifying the optimal switching strategy, results showing how the immune response is affected by antigen growth rate, initial antigen concentration, and the number of antibodies required to eliminate an antigen are included.

A structured kinetic modeling framework for the dynamics of hybridoma growth and monoclonal antibody production in continuous suspension cultures.

A structured kinetic model is developed to describe the dynamics of hybridoma growth and the production of monoclonal antibodies and metabolic waste products in suspension culture. The crucial details of known metabolic processes in hybridoma cells are incorporated by dividing the cell mass into four intracellular metabolic pools. The model framework and structure allow the dynamic calculation of the instantaneous specific growth rate of a hybridoma culture. The steady state and dynamic simulations of the model equations exhibit excellent agreement with experimentally observed trends in substrate utilization and product formation. The model represents the first to include any degree of metabolic detail and structure in describing a hybridoma culture. In so doing, it provides the basic modeling framework for incorporating further details of metabolism and can be a useful tool to study various strategies for enhancing hybridoma growth as well as viability and the production of monoclonal antibodies in suspension cultures.