Paul M. Hayter

Chinese hamster ovary cell growth and interferon production kinetics in stirred batch culture.

SummaryRecombinant human interferon-λ 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.

Application of a statistical design to the optimization of culture medium for recombinant interferon-gamma production by Chinese hamster ovary cells.

SummaryThe importance of serum-free medium components on the growth of Chinese hamster ovary (CHO) cells and production of recombinant human interferon(IFN)-gamma was investigated. The complexity of the medium led to the adoption of a statistical optimization approach based on a Plackett-Burman design. From this analysis a set of nutritional components was identified as important for cell growth and recombinant protein production. Glycine was identified as an important determinant of specific growth rate, whereas for cell production bovine serum albumin (BSA), phenylalanine and tyrosine were also identified as important. BSA, sodium pyruvate, glutamate, methionine, proline, histidine, hydroxyproline, tyrosine and phenylalanine were shown to be important for IFN-gamma production. Other medium components, such as insulin, arginine, aspartate and serine produced an inhibitory effect on both cell growth and IFN-gamma production. The effect of the stimulatory nutrients as a whole group was tested by increasing their concentration in the medium. A significant improvement in specific cell growth rate, cell production and IFN-gamma production (up to 45%) was achieved on both shake-flask and fermentor cultures. An increase in the medium concentration of the negative variables had only a small inhibitory effect (approximately 10%) on the same parameters. Analysis of the effects of the group of stimulatory amino acids and BSA on CHO cell growth showed that the effect of the former was independent of BSA.

The Macroheterogeneity of Recombinant Human Interferon-Gamma Produced by Chinese- Hamster Ovary Cells is Affected by the Protein and Lipid-Content of the Culture-Medium

The culture environment exerts a major effect on the glycosylation pattern of recombinant human interferon‐gamma (IFN‐gamma) produced by Chinese‐hamster ovary (CHO) cells. The recombinant IFN‐gamma is heterogeneous and consists of a mixture of fully (2N), partially (1N) and non‐glycosylated (0N) glycoforms, and throughout batch cultures there is a decline in the proportion of fully glycosylated IFN‐gamma. Glucose and glutamine, nutrients that are depleted early in such cultures, were prima facie candidates for causing such a shift in glycoform profile. Batch feeding of these nutrients did not prevent the decline in 2N glycoform, but the glycosylation pattern of IFN‐gamma was affected by the initial glutamine concentration in the culture. Under different serum‐free environments the extent of IFN‐gamma glycosylation was affected by (1) the concentration of BSA, (2) the quality of BSA, (3) the lipid composition of the culture medium and (4) the presence of surfactants. Moreover, the inclusion of serum in cultures caused changes in the molecular masses of the major glycoforms, that was indicative of cleavage of the core polypeptide. The results reported emphasize the necessity of considering the effects of culture media on product quality as well as on product quantity during process optimization.

CHO cell growth and recombinant interferon-γ production: Effects of BSA, Pluronic and lipids

The role of bovine serum albumin in mammalian cell cultures and the possibility of its substitution by other components in a serum-free medium has been investigated. In this study, BSA was shown to be important for growth and product formation in CHO cells expressing recombinant human interferon-γ. There were indications that its stimulating growth effect was dependent on the source of BSA used and probably was related to the purification procedure used for the production of the desired albumin fraction. Cell growth did not occur in the absence of BSA but at low concentration (1 mg ml−1) it was stimulated by the addition of a combination of a commercial lipid mixture plus Pluronic F68. However, under the latter conditions IFN-γ production was adversely effected. The importance of individual lipid components was investigated using a statistical approach based on a Plackett-Burman design. Linoleic acid was identified as a positive variable for cell growth while cholesterol was identified as a negative variable for both cell growth and IFN-γ production. When a combination of linoleic acid plus Pluronic F68 was included in the formulation of low BSA medium, cell growth was similar to that at high BSA concentration (5 mg ml−1) but the IFN-γ concentration was significantly reduced (ca. 45%).

Statistical Design of Culture Medium for Recombinant Interferon-γ Production

A statistical optimization approach based on a Plackett-Burman design was used to identify important nutrient components for both CHO cell growth and IFN-γ production. Nutrient components of the serum-free culture medium were first subjected to this analysis. BSA was shown to be one of the major medium components; preliminary studies indicated that its stimulating growth effect was a nutritional one. Cell growth in BSA-reduced cultures supplemented with a combination of a commercial lipid mixture plus Pluronic F68 was similar to control cultures, but IFN-γ production was adverselly affected. A second statistical analysis was then carried out in order to investigate the importance of individual lipid components on those cultures. Linoleic acid was identified as a positive variable for cell growth while cholesterol was identified as a negative variable for both cell growth and IFN-γ production. When a combination of linoleic acid plus Pluronic F68 was included in the formulation of BSA-reduced medium, cell growth was similar to cultures with high BSA concentration. However, IFN-γ concentration was depressed to a large extent (ca. 45%). No accumulation of IFN-γ was found within the cell.

THE CULTURE ENVIRONMENT AFFECTS RECOMBINANT PROTEIN PROCESSING IN CHO CELLS

ABSTRACT The effect of the culture environment on the expression of recombinant human interferon-gamma (IFN-γ) in Chinese hamster ovary cells (CHO) was investigated. In typical batch cultures IFN-γ production appeared to be growth-related, however it was possible to manipulate the environment such that IFN-γ production continued in the absence of cell growth. This suggests that IFN-γ production was influenced by the metabolic state of the cell rather than the specific growth rate per se. The degree of IFN-γ glycosylation also varied with the physiological state of the cell with a gradual increase in the proportion of under glycosylated IFN-γ produced by the cells towards the end of the growth phase.

Chemostat Culture Studies on CHO Cells Producing Interferon-γ

The physiology of a recombinant Chinese hamster ovary cell line in glucose-limited chemostat culture has been studied over a range of dilution rates (D=0.008-0.020h-1). There was a deviation of the specific growth rate (µ) from D at low dilution rates due to an increased specific death rate. Extrapolation of these data suggested a minimum specific growth rate of 0.011h-1 (µmax=0 0.025h-1). The specific rate of glucose uptake increased linearly with µ and KS was calculated to be 59.6µM. There was also a linear increase in the rate of lactate production with a higher yield of lactate from glucose at high growth rates. The specific rate of IFN-γ production increased with µ in a manner indicative of a growth related product. Despite changes in the IFN-γ production rate and cell physiology, the pattern of IFN-γ glycosylation was similar at all except the lowest growth rates where there was a decrease in the proportion of fully glycosylated IFN-γ.

Effects of Environmental Parameters on CHO Cell Growth in Batch and Chemostat Cultures and on Human IFN-γ Synthesis and Fidelity

The work reported in this paper is directed at optimizing the production of recombinant human therapeutic glycoproteins in animal cell cultures. Two particular problems are of interest to us in this context: (1) product yield, and (2) product authenticity. Both of these parameters are profoundly affected by the physiological state of the host cell and the choice of host cell per se. It is crucial, therefore, to acquire a detailed understanding of the effects of culture environment on cell physiology and on recombinant protein production kinetics. The system which we have chosen for these investigations is a Chinese hamster ovary (CHO) cell line producing human interferon-γ (IFN-γ).

ANALYSIS OF RECOMBINANT HUMAN IFN-GAMMA PRODUCED IN CHO CELLS:EFFECTS OF GLYCOSYLATION AND PROTEOLYTIC PROCESSING ON PRODUCT HETEROGENEITY.

Recombinant human IFN-gamma (Hu-IFN-δ) secreted by CHO cells was analysed in static and stirred batch cultures. After immunoprecipitation and SDS-gel analysis twelve molecular weight variants could be separated. After complete deglycosylation or tunicamycin treatment, only three IFN forms were seen, indicating that (a) variation in glycosylation, and (b) processing of the IFN-δ polypeptide, was occurring. In common with natural IFN-δ, both fully-glycosylated and partially-glycosylated IFN-δ were secreted. When the cells were grown in suspension culture the proportion of non-glycosylated IFN-δ rose steadily, with a corresponding decrease in the fullyglycosylated form which was independent of the glucose concentration present in the medium. The shift in glycosylation may be in response to changing culture environment in line with the decline in specific growth and IFN production rates after 50 hours of culture.