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The physiology of a recombinant Chinese hamster ovary cell line in glucose-limited chemostat culture was studied over a range of dilution rates (D = 0.008 to 0.20 h(-1)). The specific growth rate (micro) deviated from D at low dilution rates due to an increased specific death rate. Extrapolation of these data suggested a minimum specific growth rate of 0.011 h(-1) (micro(max) = 0.025 h(-1)) The metabolism at each steady state was characterized by determining the metabolic quotients for glucose, lactate, ammonia, amino acids, and interferon-gamma (IFN-gamma). The specific rate of glucose uptake increased linearly with mu, and the saturation constant for glucose (K(s)) was calculated to be 59.6 microM. There was a linear increase in the rate of lactate production with a higher yield of lactate from glucose at high growth rates. The decline in the rate of production of lactate, alanine, and serine at low growth rate was consistent with the limitation of the glycolytic pathway by glucose. The specific rate of IFN-gamma production increased with mu in a manner indicative of a growth-related product. Despite changes in the IFN-gamma production rate and cell physiology, the pattern of IFN-gamma glycosylation was similar at all except the lowest growth rates where there was increased production of nonglycosylated IFN-gamma.

A Chinese hamster ovary (CHO) cell line expressing recombinant human interferon-gamma (IFN-gamma) was grown under glucose limitation in a chemostate at a constant dilution rate of 0.015 h(-1) with glucose feed concentrations of 2.75 mM and 4.25 mM. The changes in cell concentration that accompanied changes in the glucose feed concentration indicated that the cells were glucose-limited. The cell yield on glucose remained constant, but there was a decline in residual glucose concentration and a reduced lactate yield from glucose in the latter stages of the culture. The consumption rates for many of the essential amino acids were increased later in the culture. The volumetric rate of interferon-gamma production was maintained throughout the course of this culture, indicating that IFN-gamma expression was stable under these conditions. However, the specific rate of IFN-gamma production was significantly lower at the higher glucose feed concentration. Under glucose limitation, the proportion of fully glycosylated IFN-gamma produced by these cells was less than that produced in the early stages of batch cultures. The proportion of fully glycosylated IFN-gamma increased during transient periods of glucose excess, suggesting that the culture environment influences the glycosylation of IFN-gamma.

Recombinant human interferon-gamma production by Chinese hamster ovary cells was restricted to the growth phase of batch cultures in serum-free medium. The specific interferon production rate was highest during the initial period of exponential growth but declined subsequently in parallel with specific growth rate. This decline in specific growth rate and interferon productivity was associated with a decline in specific metabolic activity as determined by the rate of glucose uptake and the rates of lactate and ammonia production. The ammonia and lactate concentrations that had accumulated by the end of the batch culture were not inhibitory to growth. Glucose was exhausted by the end of the growth phase but increased glucose concentrations did not improve the cell yield or interferon production kinetics. Analysis of amino acid metabolism showed that glutamine and asparagine were exhausted by the end of the growth phase, but supplementation of these amino acids did not improve either cell or product yields. When glutamine was omitted from the growth medium there was no cell proliferation but interferon production occurred, suggesting that recombinant protein production can be uncoupled from cell proliferation.

Recombinant human interferon-gamma (Hu-IFN-gamma) produced by Chinese-hamster ovary (CHO) cells was analysed by immunoprecipitation and SDS/PAGE. Up to twelve molecular-mass variants were secreted by this cell line. Three variants were recovered after enzymic removal of all N-linked oligosaccharides or when glycosylation was inhibited by tunicamycin. The presence of three polypeptide forms rather than a single form suggested that proteolytic cleavage had occurred at two sites in both the glycosylated and non-glycosylated forms. Proteolytically cleaved IFN-gamma was more prevalent in cell lysates than in the secreted glycoprotein. In common with naturally produced IFN-gamma, both fully glycosylated IFN-gamma (asparagine residues 28 and 100 occupied) and partially glycosylated product (thought to be substituted at position Asn28) were secreted. This was deduced from the Mr of the glycosylated products and the relative amounts of sialic acid expressed by each variant. In contrast with naturally produced IFN-gamma, non-glycosylated IFN-gamma was also secreted by the transfected CHO cells. When the cells were grown in batch culture in serum-free medium under pH and dissolved-oxygen control, the proportion of non-glycosylated IFN-gamma increased from 3 to 5% after 3 h, to 30% of the total IFN-gamma present after 195 h. This change in the proportion of glycosylated protein produced was not seen when metabolically labelled IFN-gamma was incubated for 96 h with cell-free supernatant from actively growing CHO cells. This implied that an alteration in intracellular glycosylation was occurring rather than a degradation of oligosaccharide side chains after secretion. The decrease in IFN-gamma glycosylation was independent of the glucose concentration in the culture medium, but could be related to specific growth and IFN-gamma production rates, as these declined steadily after 50 h of culture, in line with the increased production of non-glycosylated IFN-gamma.

Using an adoptive transfer system in mice, an allotype-specific suppression has been induced by a monoclonal IgG3 anti-Igh-1b (Hyb 5.7) reagent. Suppression was specific for IgG2a (Igh-1b) and led to a compensatory increase of the Igh-1a response in irradiated mice reconstituted with allotype heterozygous (Igha/b) spleen cells. Suppression, which was not antigen-specific, lasted for at least 1 month after anti-allotype treatment.

It has been reported (Contemp. Top. Immunobiol. 1974. 3:41) that allotype-specific T suppressor cells can be induced after monoclonal anti-allotype treatment of neonatal (BALB/c X SJL)F1 (Igha/b) mice. Here we show that (BALB/c X CB20)F1 adult-derived spleen cells (SC) are, by contrast, potently suppressed by monoclonal allotype-specific reagents, (when transferred into irradiated BALB/c recipients) in the absence of primary T suppressor cell induction. Such suppression is only induced in activated B cells [exposed to lipopolysaccharide or sheep red blood cells (SRBC)], and is probably dependent on the isotype of the anti-allotype sera administered. For example, two independently produced IgG1 monoclonal reagents raised against the Igh-1b allotype were poorly suppressive or nonsuppressive, whereas an IgG3 and an IgG2a monoclonal antibody induced a 90% suppression of the target allotype in transferred adult SC. It was found that suppression was not due to a depletion of antigen-specific T cell help since: (a) the addition of SRBC-educated T cells did not break suppression and (b) suppressed SC were as good a source of T cell help as normal SC, in the response of virgin or memory B cell (Thy-1-depleted) responses to SRBC in vivo. Suppression was maintained in suppressed cells which had been rechallenged with SRBC after transfer into a second irradiated recipient, but was not induced in normal SC when these were admixed with an equal number from this suppressed SC population. These findings point to a possible mechanism for the regulation of B cell expression, through the formation of an antibody-Ig receptor complex at the surface of the B lymphocyte. After complexing the target cell is either deleted or inactivated. The response to SRBC was reduced or ablated for at least 70 days after treatment with a single dose of anti-allotype serum.