Diaminomaleonitrile H2Nx
/NH 2
c=c x
NC '
CN
IMIDAZOLES &
PYRAZINES N^.C02H
Pyrazinecarboxylic acid
N^.C02H
2,3-Pyrazinedicarboxylic acid
N^X0NH2
^y Jv
3-Aminocarbonylpyrazinecarboxylic acid
hr
CO2H
CI
^ N \
CN
CI
^N^
CN
// H2N
N
y\
X
N Η
^ /X \
5.6-Dichloro-2,3pyrazinedicarbonitrile
\
CN
Optimizing bioreactor antibody production
lmidazole-4.5-dicartaonJtrile
Approaches to running bioreactors containing hybridoma cultures in ways that could lead to the highest productivity of monoclonal antibodies have been suggested by chemical engineering research at Rensselaer Polytechnic Institute. The work involved fluorescence-activated cell sorting and investigated productivity as a function of the culture medium's cell concentration and of its antibody concentration. The research was described in New York City at the recent 4th Chemical Congress of North America in a Division of Biochemical Technology-sponsored poster session by Rensselaer's Katherine L. McKinney, Robert Dilwith (now at the New York State Department of Health's Wadsworth Center for Laboratories & Research), and chemical engineer-
Η C0NH2
lmidazole-4,5-dicarboxamide S
N^ Η
"C0NH2
C02H
//
\ N
1H-lmidazole-4.5-dicarboxylic acid
X ^C0 2 H
Η
For more information, check the reply card.
Pharmaceutical Dept.
N I P P O N S O D A C O M P A N Y LIMITED 2-2-1, OHTEMACHI, CHIYODA-KU, TOKYO, JAPAN TEL: (03)3245-6181 TELEX : 0222-2379 NISSO J NISSO AMERICA INC. 220 EAST 42TH STREET, SUITE 3002, NEW YORK, N.Y. 10017, U.S.A. TEL : 212-490-0350 FAX : 212-972-9361 TELEX : AAB-238136 NZA UR
32
ETS-4 readily adsorbs water and oxygen, but sulfur dioxide is com pletely excluded. After exchange with the calcium form, the sulfur di oxide is readily adsorbed, implying that the effective pore diameter can be altered by ion exchange. None of the ETS-4 samples tested adsorbed methane. This suggests that, in the as-synthesized form, ETS-4 has a maximum pore diameter of 3.7 Â, which can be increased to at least 4.0 Â with appropriate ion exchange. In the case of ETS-10, the effective diameter is typical of the large-pored zeolites, such as types X and Y. Molecules used to probe the structure included n-hexane (4.3 Â), triethylamine (7.8 Â), and 1,3,5-trimethylbenzene (8.1 Â). Adsorption and desorption of these molecules were rapid and reversible, leading to the conclusion that the limiting pore sizes of the new materials are comparable to those of zeolites and can be varied by ion exchange. Chemical analysis indicates that each octahedral titanium atom imparts a net negative divalent charge to the framework. This requires that either two monovalent ions or a single divalent ion be present to maintain charge neutrality. Joseph Haggin
2-Amino-4,5dicyano-1 H-imii imidazole
"f C N
/
Science/Technology
CIRCLE 23 ON READER SERVICE CARD September 30, 1991 C&EN
ing professor Georges Belfort. McKinney, a graduate student, explains that the work involved a number of different experiments with mouse hybridoma cells, HB124, producing an IgG2a antibody against insulin. McKinney notes that in the experiments, surface antibody fluorescence was found to be correlated with specific antibody secretion rate. It was possible, she says, to use a fluorescence-activated cell sorter to select for high secretors. These were found to retain their high specific antibody secretion rate and lower growth rate in fed-batch culture. Since the researchers were unable to correlate surface antibody with intracellular concentration, they concluded that secretion is the rate-limiting step. The experiments also determined that surface fluorescence and specific antibody secretion rate are inversely correlated with initial cell-seeding density. Higher seeds produce higher antibody titer, according to McKinney, although it is at a slower rate per cell than for lower seeds. Hence in actual production operations, periodically testing for changes in the culture secretion rate could ensure that faster growing but low-producing cells are not overgrowing the culture. Also, artificially increasing the medium's antibody concentration had a profound effect on specific antibody secretion rate, McKinney says, with the rate falling to zero at about the maximum concentration normally found in HB124 cell culture, 27 to 36 mg per mL. This suggests a high-rate perfusion bioreactor could be used to maintain a relatively low IgG medium concentration so as to obtain the highest specific antibody secretion rate and minimal antibody degradation rate. A further experiment sought to determine the effect of ammonium chloride and sodium chloride solute stress on antibody production. It was found that sodium chloride stress caused an increase in surface fluorescence with increasing medium osmolarity. Sodium chloride stress might thus be useful if cells are initially grown to desired cell density and then the osmolarity of the medium increased to boost antibody secretion rates. fames Krieger
