Takuya Sugahara

Partial purification and characterization of immunoglobulin production stimulating factor derived from Namalwa cells.

SummaryWe screened for immunoglobulin (Ig) production stimulating factor (IPSF) which enhanced Ig production of human-to-human hybridomas in serum-free culture, and found that culture supernatant and lysate of human lymphoblastoid Namalwa cells stimulated proliferation and Ig production of human-to-human hybridoma HB4C5 cells. The IPSF in Namalwa lysate was partially purified with DEAE-Toyopearl 650M, hydroxylapatite and Superose 6HR 10/30 column chromatographies. The partially purified IPSF was a macromolecule of about 500 000 dalton containing 72 000 dalton protein as a major component. The activity was stable at pH 6 to 12, but inactivated partially by heating over 40° C (60% decrease) and completely by trypsin digestion. These results suggest that the IPSF activity is due to its protein and heat-stable components. The Namalwa IPSF stimulated proliferation of human-to-human hybridomas but not that of mouse-to-mouse hybridomas. The IPSF also stimulated Ig production of human-to-human hybridomas derived from NAT-30 cells, but not that of other human-to-human or mouse-to-mouse hybridomas. NAT-30 is a human fusion partner derived from Namalwa cells. These results suggest that the Namalwa IPSF is an autocrine factor that stimulates proliferation and Ig production of hybridomas derived from NAT-30 cells.

Stimulation of monoclonal antibody production by human-human hybridoma cells with an elevated concentration of potassium or sodium phosphate in serum-free medium

Potassium or sodium phosphate was found to stimulate the production of human monoclonal antibody by human-human hybridoma HB4C5. The addition of 15 mM Na-phosphate (pH 7.4) into serum-free culture medium increased the antibody production up to 4-fold, when seeded at cell density of 1×105 cells/ml in dishes. At the higher cell density of 5×105 cells/ml, K-phosphate was more effective than Na-phosphate, at the same concentration. In large-scale continuous culture, the addition of 10 mM Na-phosphate into serum-free culture medium stimulated antibody production by HB4C5 cells 6-fold.

Purification and characterization of immunoglobulin production stimulating factor derived from human B lymphoblastoid HO-323 cells.

An immunoglobulin (Ig) production stimulating factor (IPSF) for hybridomas was found in spent medium of the human B lymphoblastoid cell line, HO-323. The IPSF was purified by serial use of DEAE chromatography, ultrafiltration, gel filtration and HPLC-DEAE chromatography. Purified IPSF was estimated to be a 410 k macro molecule by gel filtration, and contained three types of isomers which were separated from each other by native polyacrylamide gel electrophoresis. All of the isomers were, however, assumed to have the same protein components by SDS polyacrylamide gel electrophoresis.The IPSF was effective for human-human and mouse-mouse hybridomas producing IgM, but not for IgG producers in the experimental condition used here. Human-human hybridoma HF10B4, cultured in IPSF-containing medium, produced 20 times more IgM than in IPSF-free medium under serum-free conditions. The IPSF showed very little proliferation stimulating activity on HF10B4 cells.

Purification and characterization of immunoglobulin production stimulating factor-IIβ derived from Namalwa cells

Two immunoglobulin production stimulating factors (IPSF) have been found in human Burkitts lymphoma Namalwa cells. One IPSF named IPSF-IIα was purified and identified as glyceraldehyde-3-phosphate dehydrogenase as previously reported. We report here purification, identification and characterization of IPSF-IIβ. IPSF-IIβ was purified by the serial use of ammonium sulfate fractionation, hydrophobic interaction column chromatography, anion-exchange column chromatography and gel filtration. The IPSF-IIβ was estimated as a 46 KD monomeric polypeptide by gel filtration and SDS-PAGE. Partial amino acid sequence of the 46 KD protein was analyzed for 26 amino acid residues. The sequence very closely coincided with enolase (EC 4.2.1.11) derived from various origins and, it was completely homologous with that of human enolase α-chain. Rabbit muscle enolase stimulated IgM production of hybridoma lines, and IPSF-IIβ had the enzymic activity. These results suggested that IPSF-IIβ was α-enolase or its isozyme. IPSF activities of IPSF-IIβ was stable in alkaline conditions whereas the enzymic activity was rapidly lost in alkaline conditions. Though IPSF-IIβ stimulated IgM production of both human-human and mouse-mouse hybridoma lines in serum-free condition, it partially suppressed IgE production of mouse-mouse hybridoma lines.

Purification of immunoglobulin production stimulation factor II α derived from Namalwa cells

An immunoglobulin production stimulating factor (IPSF) in human lymphoblastoid Namalwa cells was purified by the serial use of ammonium sulfate fractionation, hydrophobic interaction chromatography and gel filtration, and named IPSF-II α. IPSF-II α was estimated as a 112 KD protein composed of a 40 KD polypeptide and two 36 KD polypeptides. The 36 KD protein extracted from SDS-polyacrylamide gel showed IPSF activity, but not the 40 KD protein. The IPSF activity was reasonably stable in alkaline but unstable in acidic solution and heat-unstable. In a serum-free medium, IPSF-II α stimulated IgM production of human-human and mouse-mouse hybridomas 4–15 and 2-fold, respectively, although its growth stimulatory effect on hybridomas was negligible. The factor did not stimulate IgG production in either human or mouse hybridomas in the same serum-free medium. These results suggested that IPSF-II α was a new cellular factor for stimulating IgM productivity of hybridomas.

Immunoglobulin production stimulating factor-IIα (IPSF-IIα) is glyceraldehyde-3-phosphate dehydrogenase like protein

Amino acid sequence of the 36 KD protein which is the active subunit of immunoglobulin production stimulating factor-IIα (IPSF-IIα) derived from Burkitts lymphoma Namalwa cells was analyzed for the 20 amino acids from N-terminus. The N-terminal amino acid sequence of this protein coincided very closely with glyceraldehyde-3-phosphate dehydrogenase (GPD; EC 1.2.1.12) derived from various origins. Especially, it was completely homologous with that of human liver GPD. Several GPDs derived from human erythrocytes, rabbit muscle and Bacillus stearothermophilus also stimulated IgM production of hybridomas, as well as IPSF-IIα. Conversely, IPSF-IIα had GPD enzymic activity as strong as rabbit muscle and B. stearothermophilus, and stronger than human erythrocytes GPD. These results suggested that 36 KD subunit of IPSF-IIα was a GPD, or GPD like protein. The level of mRNA for IgM was not enhanced by IPSF-IIα in hybridoma cells, though the IgM productivity of the cell was remarkably stimulated by the protein, indicating that IPSF-IIα does not stimulate immunoglobulin production by enhancement of transcription.

Stimulation of proliferation and immunoglobulin M production by lactoferrin in human-human and mouse-mouse hybridomas cultures in serum-free conditions

The effects of growth factors, such as insulin, transferrin, lactoferrin, ethanolamine, and selenium, on proliferation and IgM production of human-human hybridomas HB4C5 cells in a serum-free enriched RDF (eRDF) medium were studied. Among them, lactoferrin markedly stimulated proliferation and IgM production of the cells. Another iron-binding protein, transferrin, stimulated proliferation of HB4C5 cells as well as lactoferrin, but its stimulatory effect on IgM production was negligible. The proliferation and IgM production of HB4C5 cells gave some detectable delays in conventional ITES-eRDF medium at low cell densities, but the delays were avoided by the addition of lactoferrin. However, eRDF medium supplemented with lactoferrin could not support proliferation and IgM production of the cells at high cell densities. For proliferation and IgM production of HB4C5 cells, eRDF medium supplemented with 25 μg/ml lactoferrin, 10μM ethanolamine, 35 μ/ml transferrin, and 2.5 nM selenium (LETS-eRDF) gave the best result. Lactoferrin stimulated proliferation of human-human and mouse-mouse hybridomas producing IgG or IgM, but stimulation of Ig production was detected only in IgM producers. These results suggest that cell proliferation, IgM production, and IgG production of hybridomas are regulated by different mechanisms.

Virus Containment Continuous Culture of Animal Cells

Human-human hybridoma HB4C5 cells were cultured in a high-density continuous culture system SHC-1 (SHIMADZU) equipped with a 500 ml culture vessel at the density of 2 × 107 cells/ml. Cultured medium were filtrated by the BMM hollow fiber module which can eliminate virus particles. This module was able to filtrate over 20 L of cultured medium over 30 days without decrease in the recovering rate of the cultured medium. The high density continuous culture system installed with the BMM hollow fiber can be used for virus containment in the vessel and producing virus-free products by continuous culture.

Removability and Permeability of DNA in Protein Solution Using BMM

We intended to show the removability and/or permeability of the contaminating DNA in protein solution produced from cell culture by filtration using the BMM virus removal filter and correlate the dispersion state of DNA with its permeability. We employed the cell culture solution containing monoclonal antibody (MAb) produced from the hybridoma cell culture. Dead-end filtration was performed under a constant transmembrane pressure (TMP) of 200 mm Ilg. The concentration of DNA was detected by Threshold total DNA assay system. In the case of BMM with a mean pore size of 35nm (BMM35), the permeability of MAb was 0.84 and that of DNA was 0.18. DNA in protein solution was more effectively removed than naked DNA in buffer solution. We concluded that DNA passed through BMM was naked DNA and DNA removed by BMM was complex with protein or cell fragment and packaged in microbe.

PURIFICATION AND CHARACTERIZATION OF IMMUNOGLOBULIN PRODUCTION STIMULATING FACTOR IIα DERIVED FROM NAMALWA CELLS

Immunoglobulin production stimulating factor (IPSF) IIα was purified from Namalwa cell lyzate by ammonium sulfate precipitation, hydrophobic interaction chromatography and gel filtration. The purified IPSF was estimated a 106 KD protein which was composed of a polypeptide of 40 KD and two polypeptides of 33KD by gel filtration and SDS polyacrylamide gel electro-phoresis. The 33KD protein extracted from SDS polyacrylamide gel showed IPSF activity, but not the 4 0 KD protein. The IPSF activity was fairly stable in alkaline but unstable in acidic solution. In a serum-free medium, the IPSF-IIα stimulated IgM production of human-human and mouse-mouse hybridomas 4–15 and 2-fold, respectively. However, IgG production of neither human nor mouse was stimulated by the factor in the same serum-free medium.