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Growth of Hybridoma HB121 (producing anti IgE)

Growth of Hybridoma HB121 (producing anti IgE). Antibodies. The market for a single particular antibody is usually less than 1 kg/yr. Most antibodies are produced in hybridoma cells, though other mammalian cell systems and even bacterial systems have been used. HB121.

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Growth of Hybridoma HB121 (producing anti IgE)

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  1. Growth of Hybridoma HB121(producing anti IgE)

  2. Antibodies • The market for a single particular antibody is usually less than 1 kg/yr. • Most antibodies are produced in hybridoma cells, though other mammalian cell systems and even bacterial systems have been used.

  3. HB121 • HB121 is an example of a hybridoma cell line which happens to produce mouse anti IgE • The cell line can be purchased from ATCC • The ATCC murine hybridoma cell line HB121 used in these studies was derived from a P3 63Ag8.653 myeloma parent line hybridized with splenocytes of BALB/c mice.

  4. Growth of HB121 • HB121 was banked over liquid nitrogen and routinely subcultured in Dulbecco’s modified Eagles medium (DMEM) with 1% fetal bovine serum (FBS) and 9% horse serum (HS) without the use of antibiotics. DMEM from JRH BioSciences (Lenexa, KS) was formulated from powder, and growth kinetics were routinely tested against previous batches. Fetal bovine serum and horse serum were screened and purchased from Hyclone Laboratories (Logan, UT); the same serum lots were used for all studies.

  5. Cell line and maintenance The mouse-mouse hybridoma • cell line, MAK, which produces IgG monoclonal antibody and the • medium used have been described previously (23, 24). Cells are • maintained in a defined, serum-free medium consisting of a 1 : 1 • mixture of Dulbecco’s modified Eagle’s medium (DMEM) and • Ham’s F 12 medium supplemented with transferrin (5 mg/l), 2-mer- • captoethanol (1.7 $/I), ethanolamine (0. I ml/Z), L-ascorbic acid • (0.11 mM), sodium selenite (29 nM), putrescine (6.2 PM) and Plu- • ronic F68 (1 g/l). The concentrations of glucose and glutamine • used were 17.51 mM (3.15 g/r) and 4 mM (0.615 g/l), respectively. • pre-column derivatization with orthophthaldialdehyde (OPA).

  6. Hybridoma cells were cultivated in a chemically defined medium in continuous cultures. These • cultures reached different steady states marked by distinctive cell metabolism depending on the • culture conditions leading to the steady state. Those steady states with different metabolism are • characterized by different stoichiometric ratios of lactate production to glucose consumption • (AL/AC). The specific consumption rates of glucose, glutamine and other amino acids are reduced • when ALlAG reduces. Those steady states do not have a few discrete values of ALlAGs, rather they • span from a high U/AC state (~1.0) to an intermediate state (0.1 SALlAGS l.O), and reduces even • further at a low ALlAG state (x0.1). Metabolic flux analysis was performed to compare energy • metabolism of cells in cultures representing these three distinct metabolic states. The material • balance on carbon and nitrogen was facilitated by the use of chemically defined medium. The for- • mation of biomass was systematically estimated. It was revealed that all glycolysis and TCA cycle • fluxes are reduced as AL/AC decreases. At the low AL/AC state, a reduction in amino acid specific • consumption rate is accompanied by a reduction in all the fluxes around pyruvate. The analysis • also shows that the oufflux from the TCA cycle to form pyruvate, which contributes to lactate for- • mation, is possibly linked to the higher consumption rate of amino acids at the high ALlAG state. • Taken together the results suggest the amino acid metabolism plays an important role in reducing • lactate production in mammalian cell culture.

  7. Things to look at • Chemically defined media • Dulbeccos medium • Lactate production vs glucose consumption • Antibody titer • G1 phase • Amino acid production/consumption

  8. Antibody titer • How much antibody is produced per liter of culture? • Monoclonal Antibody Assay. The anti-IgE monoclonal antibody produced by hybridoma HB121 was detected by an enzyme-linked immunosorbant assay composed of an antimouse IgG (Fab specific) goat antibody coated on the surface of NUNC Immulon I 96 well plates and an antimouse IgG (Fc specific) goat antibodyhorseradish peroxidase conjugate. All immunochemicals were purchased from Sigma Chemical Co.

  9. Methods • Perfusion • Ca Alginate • Suspension culture

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