Kazuaki Kitano

Production of human monoclonal antibodies by heterohybridomas

SummaryMouse human-human heterohybridomas secreting human monoclonal antibodies (MoAb) against tetanus toxoid and hepatitis B virus surface antigen were effectively cultivated in a medium containing a serum substitute called GFS, a 55% to 70% ammonium sulphate fraction of serum from adult cattle. A perfusion culture system using a jar fermentor equipped with a cell sedimentation column with a double jacket was developed and applied to produce human MoAb. In this fermentor, maximum cell density of a heterohybridoma reached 1.2×107 cells/ml and MoAb was continuously accumulated at a constant rate for at least 40 days; this led to the production of more than one gram of human MoAb using a culture vessel with a 1-1 working volume.

Polyethylene glycols for promoting the growth of mammalian cells

SummaryPolyethylene glycol (PEG), a well known fusogen for mammalian cells and microbial or plant protoplasts, markedly stimulated the growth of mammalian cells at low concentration under serum-free or low serum-conditions. Polyvinyl alcohol and polypropylene glycol also stimulated cell growth. A serum-free medíum containing PEG, PEG-86-1, has been established for cultivating a variety of mammalian cell lines. It is also useful for producing an anti-tetanus toxoid human monoclonal antibody by a mouse · human-human heterohybridoma.

Intracellular degradation of recombinant proteins in relation to their location in Escherichia coli cells

Abstract Recombinant interferon-αA (rIFN-αA) accumulated in Escherichia coli 294 was rapidly degraded in vivo when glucose or oxygen in the medium was limited, whereas recombinant interferon-γ (rIFN-γ) and recombinant interleukin-2 (rIL-2) were stable under the same conditions. The degradation in vitro of rIL-2 by E. coli proteases was as rapid as that of rIFN-αA. On the other hand, rIFN-γ maintained the same activity after proteolysis in vitro. Western blot analysis of the reaction mixture, however, showed that the rIFN-γ molecule was converted into a smaller protein of about 15 kDa by losing the COOH-terminal portion of the peptide. Electron microscopic observations showed that rIFN-γ and rIL-2 accumulated and formed inclusion bodies. No clear inclusion bodies were found in the cells accumulating rIFN-αA, which is rapidly degraded in vivo. These results indicate that intracellular location and protein structure are related to the stability in vivo of recombinant proteins. rIFN-αA was also degraded rapidly in a lon− mutant of the same organism, suggesting that protease La does not participate in this degradation process.

Effective production of a human monoclonal antibody against tetanus toxoid by selection of high productivity clones of a heterohybridoma

A mouse.human-human heterohybridoma, N12-16.63, has been described which produces an anti-tetanus toxoid human monoclonal antibody (MoAb). A clone, N12-16.63.49.19, which produces eight times as much MoAb as that produced by the original cell line, was selected by repeating the recloning and selection twice. Two clones, N12-16.63.49.19.69 and N12-16.63.49.19.127, further selected from this clone produced almost 20 times more than that produced by the original cell line. Though the production of MoAb by these clones gradually decreased with repeating transfers, they still produced a large amount of human MoAb even after 3 months of transfer. Human MoAb (IgM) was isolated from the culture supernatants of the original and high productivity clones and the products were confirmed to be identical. Human MoAb was effectively produced by batch culture on the 20 liter scale or a perfusion culture on the 1 liter scale using these high productivity clones.

Efficient production of anti-(hepatitis B virus) antibodies and their neutralizing activity in chimpanzees

For industrial production of human monoclonal antibodies (hmAb) against hepatitis B virus surface antigen (HBsAg), we scaled-up a short-term perfusion culture in serum-free medium, which was chosen as the most suitable culture method, to a 50–1 fermentor equipped with a rotating shear filter. Using hydrophobic chromatography as the initial step of hmAb purification, the mAb HBW4, HBW6 and W471 were isolated in good quality from the respective culture broths in yields of approximately 75%. Each of the three purified hmAb alone, and a cocktail of the three, protected chimpanzees against HB virus, when injected intravenously 3 h after viral challenge, as long as the serum antibody levels were significant. A pharmacokinetic study using cynomolgus monkeys demostrated that the hmAb have a long plasma half-life and bioavailability of approximately 76% upon intramuscular injection in primates. Thus, anti-HBsAg hmAb produced by an industrial process are expected to be successfully used in clinical fields.

Comparison of culture methods for human-human hybridomas secreting anti-HBsAg human monoclonal antibodies

Human-human hybridomas which secrete a human monoclonal antibody (h-MoAb) against hepatitis B virus surface antigen showed growth associated production kinetics. The rate of h-MoAb production rapidly decreased after cell growth was arrested in a perfusion culture, even if the perfusion rate was increased. A continuous suspended-perfusion culture, in which both culture broth and culture supernatant are continuously harvested and the same volume of fresh medium is continuously fed into the reactor, was developed to maintain continuous growing conditions during cultivation. In this culture system, the production of h-MoAb continued for more than 50 days with an average productivity of 5.0 mg/l of working volume/day. A semicontinuous immobilized-perfusion culture in which parts of the cells are repeatedly removed from the immobilized reactor was another useful technique for the long term cultivation of these h-h hybridomas. As an average h-MoAb production rate, 62 mg/l of immobilized-bed volume/day was achieved for 65 days of cultivation using a ceramic matrix reactor, and 327 mg/l/day was achieved over 47 days of cultivation using a hollow fiber reactor equipped with Cultureflo MTM Thus, the antibody productivity per reactor volume per day by the semicontinuous immobilized-perfusion culture was much higher than that of the continuous perfusion culture in an agitation reactor.

Secretion of active human lysozyme by Acremonium chrysogenum using a Fusarium alkaline protease promoter system

We constructed expression vectors for Acremonium chrysogenum using a Fusarium alkaline protease promoter region and tested their potential as secretion systems for foreign proteins using the human (h)-lysozyme gene as an indicator. The gene encoding h-lysozyme was linked to the coding region of (1) the carboxy terminal of the alkaline protease pre peptide, (2) the carboxy terminal of the prepro peptide, (3) three amino acids of the mature protein preceded by the prepro peptide and (4) the carboxy terminal of chicken lysozyme signal peptide, inserted into the genomic DNAs of A. chrysogenum and expressed under the control of the alkaline protease promoter. The transformants of A. chrysogenum with each of these plasmids secreted enzymatically active h-lysozyme. A maximum yield in excess of 40 mg l-1 was obtained when h-lysozyme was linked to the carboxy terminal of alkaline protease prepro peptide. The majority of the amino terminal sequence of the purified h-lysozyme from the culture supernatant was identical with that of authentic h-lysozyme, but it showed some heterogeneity.

Controlled expression of human basic fibroblast growth factor mutein CS23 in Escherichia coli under a bacteriophage T7 promoter

A DNA sequence coding for human basic fibroblast growth factor (hbFGF) mutein CS23 was inserted downstream of a T7 promoter in pBR322. The plasmid was introduced into Escherichia coli MM294 lysogenized with a bacteriophage λ having the T7 RNA polymerase gene under the control of the lacUV5 promoter. When 0.4 mM IPTG was added to induce gene expression in this system, the accumulation of hbFGF mutein CS23 was very rapid at the beginning, but it quickly stopped. Therefore, the total amount was low, and most of the product was inactive due to the formation of inclusion bodies. By controlling the expression of the T7 RNA polymerase at an appropriate level, we succeeded in accumulating a large amount of the mutein in the cells, all in a soluble and active form. By adding 0.04 mM IPTG, more than 1.5 g per liter of hbFGF mutein CS23 was accumulated under optimum culture conditions.

Effective production of anti-tetanus toxoid and anti-HBsAg human monoclonal antibodies by serum-free culture of hybridomas.

Two hybridoma systems, mouse·human-human (m·h-h) heterohybridoma and human-human (h-h) hybridoma, have been established, and hybridomas secreting anti-tetanus toxoid and anti-HBsAg human monoclonal antibodies (MoAbs), both having a neutralizing activity have been obtained. Cell-line improvement was shown to be an efficient method for improving the productivity in a cell culture process. Two kinds of serum-free media, GFS (a serum substitute)-containing media and polyethylene glycol (PEG)-containing media, have been established to produce human MoAbs. m·h-h Heterohybridomas could be cultivated for a long period by perfusion culture in an agitation vessel, but h-h hybridomas could not. We found that h-h hybridomas show growth-associated antibody production kinetics and established two kinds of long-term cultivation systems: continuous perfusion culture and semicontinuous immobilized perfusion culture. We also scaled up batch culture and short-term perfusion culture to 200-L and 50-L fermentors, respectively. Processes for large-scale purification from the culture supernatants of both GFS- and PEG-containing serum-free media have also been developed.Two hybridoma systems, mouse.human-human (m.h-h) heterohybridoma and human-human (h-h) hybridoma, have been established, and hybridomas secreting anti-tetanus toxoid and anti-HBsAg human monoclonal antibodies (MoAbs), both having a neutralizing activity have been obtained. Cell-line improvement was shown to be an efficient method for improving the productivity in a cell culture process. Two kinds of serum-free media, GFS (a serum substitute)-containing media and polyethylene glycol (PEG)-containing media, have been established to produce human MoAbs. m.h-h Heterohybridomas could be cultivated for a long period by perfusion culture in an agitation vessel, but h-h hybridomas could not. We found that h-h hybridomas show growth-associated antibody production kinetics and established two kinds of long-term cultivation systems: continuous perfusion culture and semi-continuous immobilized perfusion culture. We also scaled up batch culture and short-term perfusion culture to 200-L and 50-L fermentors, respectively. Processes for large-scale purification from the culture supernatants of both GFS- and PEG-containing serum-free media have also been developed.

Factors affecting the synthesis of the N-terminal methionine-free molecule of recombinant human interleukin-2 by Escherichia coli

Abstract Escherichia coli harboring the gene encoding human interleukin-2 (IL-2) produces a mixture of two recombinant IL-2 species: one with the amino-terminal alanine (rIL-2) and the other having an additional methionine residue at the amino terminus (Met-rIL-2). Ways to increase the amount of rIL-2 and its proportion to the total IL-2 were tried. Among E. coli K-12 derivatives, N4830 was an effective producer of recombinant IL-2. The production of the mixture was greatly increased by optimizing the medium ingredients or culture conditions. However, the percentage of rIL-2 in the product decreased almost linearly with an increase of the total production of recombinant IL-2 and was less than 10% under optimal culture conditions. By adding 4.1 × 10 −5 M maganese and 7.4 × 10 −5 M ferric ions to the medium, we succeeded in raising the percentage of rIL-2 to 50% without any decrease of the total production.