Hybridomas systems

Table 8.5 Kinetic parameters for specific uptake rates in hybridoma systems equations are valid for both substrates - GLC and GLN...

A hybridoma can live indefinitely in a growth medium that includes salts, glucose, glutamine, certain amino acids, and bovine serum that provides essential components that have not been identified. Serum is expensive, and its cost largely determines the economic feasibihty of a particular ciilture system. Only recently have substitutes or partial replacements for serum been found. Antibiotics are often included to prevent infection of the culture. The pH, temperature and dissolved oxygen, and carbon dioxide concentration must be closely controlled. The salt determines the osmotic pressure to preserve the integrity of the fragile cell. 

The shear forces are mainly in the range of 1 to lONm. This exposure causes cell death between 20 and 80% depending on the exposure duration which is between a few seconds and several hours. Studies performed in a bioreactor have an exposure duration of several days. The results are partly contradictory. Tramper et al. [30] found a critical stress level of 1.5 Nm" for insect cells, whereas Oh et al. [31] could not show an influence on hybridoma cells even at high stirrer speed. This shows that each cell line reacts different and that there is a necessity for defined stress systems if the results is to be comparable. 

Shen, B., Greenfield, P., and Reid, S., Calcium Alginate Immobilized Hybridomas Grown Using a Fluidized-Bed Perfusion System with a Protein-Free Medium, Cytotechnol., 14 109 (1994)... 

In order to compare the specific activity of plant-derived C5-1 to that of the hybridoma-derived antibody, the antigen-binding capacity of antibodies produced in each system was assayed by enzyme-linked immunosorbent assay (ELISA). As shown in Table 1.2, antibodies from both sources demonstrated similar binding characteristics against human IgGs [8]. Furthermore, the stability of alfalfa-derived C5-1 in the blood stream of Balb/c mice was comparable to that of the hybridoma-derived IgG [8]. 

Over half of all biopharmaceuticals thus far approved are produced in recombinant E. coli or S. cerevisiae. Industrial-scale bacterial and yeast fermentation systems share many common features, an overview of which is provided below. Most remaining biopharmaceuticals are produced using animal cell culture, mainly by recombinant BHK or CHO cells (or hybridoma cells in... 

A rat anti-mouse VEGFR2 (Flkl) monoclonal antibody (DClOl) was developed by ImClone Systems (New York, NY, www.imclone.com) using conventional hybridoma technique [194] to conduct proof-of-concept studies. 

Hybridomas—Fusion of a tumor cell and a specialized cell of the immune system, able to produce a large amount of the same immune system binding protein. 

Over half of all biopharmacuticals thus far approved are produced in recombinant E. coli or S. cerevisiae. Industrial-scale bacterial and yeast fermentation systems share many common features, an overview of which is provided below. Most remaining biopharmaceuticals are produced using animal cell culture, mainly by recombinant BFIK or CFiO cells (or hybridoma cells in the case of some monoclonal antibodies Appendix 1). While industrial-scale animal cell culture shares many common principles with microbial fermentation systems, it also differs in several respects, as subsequently described. Microbial fermentation/animal cell culture is a vast speciality area in its own right. As such, only a summary overview can be provided below and the interested reader is referred to the Further Reading section. 

Optimal fermentation parameters have been well established and air-lift, stirred tank, and hollow fibre systems have all been used. At commercial scale, fermentation volumes in excess of 1000 litres can be used, which can yield 100 g or more of final product. While hybridoma growth is straightforward, production levels of antibody can be quite low compared with ascites-based production systems. Typically, fermentation yields antibody concentrations of 0.1-0.5 mg/ml. Removal of cells from the antibody-containing media is achieved by centrifugation or filtration. An ultrafiltration step is then normally undertaken in order to concentrate the filtrate by up to 20-fold. 

Krebber, A., S. Bornhauser, J. Burmester, A. Honegger, J. Willuda, H.R. Bosshard, and A. Pluckthun, Reliable cloning of functional antibody variable domains from hybridomas and spleen cell repertoires employing a reengineered phage display system. J Immunol Methods, 1997. 201(1) 35-55. 

An artificial immune system was constructed based on phage display to make antibodies without using rodents and hybridoma technology.23,26,27 The gene III... 

Lane, D. P. and Lane, E. B. (1981) A rapid antibody assay system for screening hybridoma cultures. J. Immunol. Methods 47, 303—307. 

Cell Fusion Unlike antibody-secreting cells, myeloma cells, malignant tumor cells of the immune system, can be cultured continuously. Kohler and Milstein (1975) developed a method to fuse (hybridize) B-lymphocytes from the mouse spleen with mouse myeloma cells, so that the fused cell, hybrid-myeloma (or hybridoma) cell, can have the characteristic of the both cell lines that is, the production of specific antibodies and the immortality. Since the hybridoma is derived from a single B-lymphocyte, it produces only one kind of antibody, thus a monoclonal antibody. 

---