Monitoring of the production of monoclonal antibodies by hybridomas. Part I: Long-term cultivation in hollow fibre bioreactors using serum-free medium.

The long-term cultivation of hybridoma cells in hollow fibre bioreactors using serum-free medium, was monitored with respect to quantitative and qualitative aspects of the produced mAbs, cell viability, LDH and proteolytic activity. During the culture periods of hybridoma cells producing mAb OT-1C and 3A, the mAb concentration showed a decreasing trend with a concomitant increase of IgG fragments. The major IgG fragments did not bind the antigen and the molecular weights were significantly different from the corresponding IgG heavy and light chains. In addition, a good correlation was found between cell lysis, the presence of acid protease(s) and IgG fragments. The physicochemical and immunochemical properties of the "intact" mAbs (such as molecular weights, IEF patterns and affinity) did not change significantly during the culture period.

A hollow fibre dialysis system for the in vitro production of monoclonal antibodies replacing in vivo production in mice.

Haemodialysis units have been used as bioreactors for the in vitro production of monoclonal antibodies. Mouse hybridomas were grown in the extra-capillary compartment of dialysis modules with a constant supply of protein-free medium through the hollow fibres. It was found that addition of serum to the media had no stimulatory effect on the production of monoclonal antibodies. For the determination of monoclonal antibodies a sol particle immunoassay (SPIA) has been used. Monoclonal antibodies were harvested from the extra-capillary compartment at two-week intervals over periods of several weeks at concentrations comparable to the harvests of ascitic fluids from mice. The production capacity of one haemodialysis unit is equivalent to that of at least 50 mice. This in vitro system enables a yearly production of 50 grams per unit of monoclonal antibodies of high purity and devoid of contaminants from mouse origin or serum. It has been shown that this system can be successfully used to produce a constant supply of monoclonal antibodies for a variety of applications, e.g. reagents for diagnostic tests and materials for immuno-chromatographic purification. As a consequence this in vitro production of monoclonal antibodies has replaced the many thousands of mice required for in vivo production.

Antibody engineering, a strategy for the development of monoclonal antibodies.

Antibody engineering is the selection process enabling the isolation of hybridoma clones, each of which produces an antibody with predefined qualities. The state of the art of hybridoma technology is reviewed with emphasis on the results obtained by antibody engineering in our laboratories for the development of monoclonal antibodies for specific use in diagnostic tests. The perspective for in vitro monoclonal antibody production as well as the application of monoclonal antibodies for diagnostic reagents, industrial purification and therapeutic use are indicated.

Development, screening and quality specifications of hybridomas synthesizing anti-polypeptide hormone antibodies to be used for diagnostic tests and in-process control.

Hybridomas were developed for the production of monoclonal antibodies against human chorionic gonadotrophin (hCG) and against bovine and porcine (pro)insulins. Screening tests were devised to select those hybridomas synthesizing antibodies with predefined antigen-specificity. Cell lines, which produce anti-hCG antibodies with a low cross-reactivity against luteinizing hormone (hLH) and which are suitable for agglutination tests, were isolated. In addition hybridomas were obtained with various specifications for porcine and bovine (pro)insulins. Their monoclonal antibodies can be used for quantitative enzyme immunoassays of insulin and proinsulin mixtures. Specifications of hybridomas for production purposes have been defined with regard to growth requirements, antibody production capacity and absence of mycoplasma contamination.

Monoclonal antibodies for diagnostic tests and affinity chromatography: a first step to antibody engineering.

The hybridoma technology for obtaining murine monoclonal antibodies has been developed for the production of antibodies to human gonadotrophins and viral antigens. Various aspects of the hybridomna selection strategy and the cloning to continuous and stable antibody producing cell lines are reported. This leads to production of antibodies of predefined characteristics (antibody engineering). The application of monoclonal antibodies for diagnostics and for isolation of products in industrial processes are indicated.

Continuous flow centrifugation of cell cultures under asceptic conditions.

A disposable pyrogen-free centrifuge bowl is described for continuous asceptic separation of cells from cultures up to 50 litres.

The activity of deoxyribonucleic acid polymerase in some species of algae.

1. The activities of DNA polymerase preparations from the algae Euglena gracilis, Chlamydomonas reinhardtii, Chlorella pyrenoidosa, Anabaena variabilis and Anacystis nidulans were measured. The blue-green algae Anabaena and Anacystis contain a 5-20-fold higher activity of the enzyme than do the green algae. DNA polymerases from the blue-green algae show a pH optimum of 9 and prefer a relatively low Mg(2+) concentration (1-3mm). DNA polymerases from the green algae, however, display a pH optimum between 7.5 and 8.5 and an optimum Mg(2+) concentration of 8mm. With all algae, a higher polymerase activity was obtained with denatured salmon sperm DNA as template than with native DNA. All four deoxyribonucleoside 5'-triphosphates must be present for full activity of the polymerases. 2. With one exception, the deoxyribonuclease activities in the preparations, measured under conditions of the DNA polymerase assay, are low compared with corresponding preparations from Escherichia coli. Chlamydomonas extracts contain a high deoxyribonuclease activity. 3. After purification on columns of DEAE-cellulose, the polymerase activity was linear over a wide range of protein concentrations, except for Chlamydomonas preparations, where the observed deviation from linearity was probably attributable to the high nuclease activity. 4. DNA polymerases from all these algae bind strongly to DNA-cellulose; 6-40-fold purifications of the enzyme were obtained by chromatography on columns of DNA-cellulose. 5. The partially purified polymerases of Euglena and Anacystis are heat-labile but become much more heat-stable when tested in the presence of DNA.