A process for the batch culture of animal cells comprises culturing the cells in nutrient medium characterised in that during the culture the medium is supplemented with a combined feed of one or more energy sources and one or more amino acids, and culturing is continued into the decline phase of the culture to provide the product(s) of the cells. The process is particularly applicable to genetically modified cells, especially hybridoma cell cultures to produce monoclonal antibodies. Preferably, the supplemental feed is fed to the culture at a slow rate over a prolonged period. Very significant enhancement of overall product yield may be obtained.

Animal cell culture.

Although studies with primary lymphocytes are almost always conducted in CO2 incubators maintained at atmospheric oxygen levels (atmosO2; 20%), the physiological oxygen levels (physO2; 5%) that cells encounter in vivo are 2–4 times lower. We show here that culturing primary T cells at atmosO2 significantly alters the intracellular redox state (decreases intracellular glutathione, increases oxidized intracellular glutathione), whereas culturing at physO2 maintains the intracellular redox environment (intracellular glutathione/oxidized intracellular glutathione) close to its in vivo status. Furthermore, we show that CD3/CD28-induced T cell proliferation (based on proliferation index and cell yield) is higher at atmosO2 than at physO2. This apparently paradoxical finding, we suggest, may be explained by two additional findings with CD3/CD28-stimulated T cells: (i) the intracellular NO (iNO) levels are higher at physO2 than at atmosO2; and (ii) the peak expression of CD69 is significantly delayed and more sustained at physO2 that at atmosO2. Because high levels of intracellular NO and sustained CD69 tend to down-regulate T cell responses in vivo, the lower proliferative T cell responses at physO2 likely reflect the in vitro operation of the natural in vivo regulatory mechanisms. Thus, we suggest caution in culturing primary lymphocytes at atmosO2 because the requisite adaptation to nonphysiological oxygen levels may seriously skew T cell responses, particularly after several days in culture.

https://www.pnas.org/content/pnas/104/11/4547.full.pdf

Importance of culturing primary lymphocytes at physiological oxygen levels.

The cellulase enzyme system consists of cellobiohydrolase, endoglucanase, and beta-glucosidase and has been extensively studied with respect to its biosynthesis, properties, mode of action, application, and, most recently, secretion mechanisms. A knowledge of the factors governing the biosynthesis and secretion of these enzymes at the molecular level will be useful in maximizing enzyme productivity in extracellular fluid. Among other topics, the regulatory effects of sorbose (a noninducing sugar which is not a product of cellulose hydrolysis) on cellulase synthesis and release are described. Cellulase genes have recently been cloned into a number of microorganisms with a view to understanding the gene structure and expression and to obtaining the enzyme components in pure form. The factors governing biosynthesis and secretion of cellulases in recombinant cells are also discussed. Cellulases are known to be glycoproteins, therefore, the role of O- and N-linked glycosylation on enzyme stability and secretion is also detailed.

Regulatory aspects of cellulase biosynthesis and secretion.

Mechanisms and kinetics of monoclonal antibody synthesis and secretion in synchronous and asynchronous hybridoma cell cultures.

The filamentous fungus Trichoderma reesei is the predominant industrial producer of cellulolytic enzymes by secreting an enzyme system capable of degrading crystalline cellulose, which consists of several cellobiohydrolases, endoglucanases and β-glucosidases. All of these enzymes occur in multiple forms. A critical appraisal of the methods used to assess cellulase multiplicity is presented. By the aid of gene technology, advanced protein analytics and immunology, “true” isoenzymes and proteolytic fragments of all of these enzymes could be identified, and their structure and properties are described. Also, the recent elucidation of the three-dimensional domain structure of cellulases, their active center, and the role of both in the hydrolysis of cellulose are dealt with. Particular emphasis is presented on the differences in the enzymatic reaction mechanisms of cellobiohydrolase I and II, and their synergism.

The cellulase proteins of Trichoderma reesei: Structure, multiplicity, mode of action and regulation of formation

Factors that affected the production of monoclonal antibodies by a mouse-mouse hybridoma cell line, propagated in vitro in stirred vessels, were investigated The purpose of the research was to estimate the efficiency of this system for large scale production of monoclonal antibodies The antibody produced by these hybridoma cells was an IgG2a, specific for a surface antigen on Rhizobium japonicum NR-7 cells Antibody content in the culture supernatant was determined by a radial-immunodiffusion assay using rabbit anti-mouse IgG antibodies in the immobile phase and mouse IgG (the monoclonal antibody) as the antigen in the mobile phase This method was found to be more reproducible and reliable compared with an ELISA method Cells were adapted to grow in an inexpensive, low protein content medium based on Dulbecco's Modified Eagle Medium (DMEM) supplemented with 025% Primatone RL, 001% Pluronic polyol F-68 and fetal bovine serum as low as 1% Doubling time for the cells averaged 24 hrs, and final yields reached 2 X 10(6) cells per ml The hybridoma cells were grown in the newly developed medium in 3 liter fermentors Monoclonal antibody was produced during the early growth phase (3 days), however, most of the antibody was produced during a later growth phase (3-10 days) when 30 to 90% of the cells were dead Final antibody yields were estimated to be 100-200 micrograms/ml A low level of dissolved oxygen (25% air saturation) in the culture was found to increase the amount of antibody produced as compared with cells propagated at 60% air saturation (the optimal level for cell propagation) since the cells were kept alive for longer periods at the lower dissolved oxygen concentration Using a fed-batch propagation method we were able to keep cells alive for long periods (up to 1 month) at a concentration of about 1 X 10(6) cells per ml, and thus to increase further monoclonal antibody production Yields of 300-400 micrograms/ml were obtained

Production of monoclonal antibodies in culture.

Since polyclonal antibodies to cellobiohydrolase sometimes cross-react with endoglucanases, monoclonal antibodies (McAb) to the exoglucanase were prepared and evaluated with the objective of developing a specific assay for the enzyme. Fusion of myeloma cells with spleen lymphocytes from mice immunized with cellobiohydrolase I (CBH I) from Trichoderma reesei resulted in several hybrid lines that secreted McAb to that enzyme. The McAb from one cell line was studied for its interaction with proteins both in crude culture broth and in purified CBH I from T. reesei using immunoblotting techniques. McAb reacted with the major protein in CBH I (mol. wt. 66 kd, pI 4.2), and also with several additional proteins. These other cross-reacting proteins were not endoglucanases ors-glucosidases and can represent multiple forms or degradation products of CBH I arising through differences in glycosylation or proteolytic modification. Most importantly, the McAb should be useful as a probe for CBH I in a direct assay for this enzyme.

F. Riske

Papers

Production of monoclonal antibodies in culture.

Monoclonal antibodies against cellobiohydrolase from trichoderma reesei