Recombinant Cell Culture Research Articles

Recombinant protein synthesis and plasmid instability in continuous cultures of Escherichia coli JM103 harboring a high copy number plasmid

Escherichia coli JM103[pUC8] was employed as a model to investigate the behavior of a recombinant microbial system harboring a plasmid at high copy numbers. Experiments with batch and continuous cultures of recombinant and plasmid-free cells were conducted in a well-controlled bio-reactor. In batch experiments, plasmid copy number varied typically from an average of 500 during the exponential growth phase to as high as 1250 during the stationary phase. While the segregational plasmid instability was negligible in batch experiments, severe segregational instability occurred in continuous experiments conducted over a range of dilution rates, resulting in complete loss of plasmid-bearing cells from the continuous cultures within few residence times after transition to continuous operation. The profound differences in the specific growth rates and mass yields of the plasmid-free and plasmid-bearing cells resulting from the extra metabolic burden on the plasmid-bearing cells mainly due to excessive plasmid DNA content was the major cause for the plasmid instability. Plasmid multirnerization was detected in batch and continuous cultures and was found to have significant influence on the effective copy number and was partially responsible for the severe segregational instability in continuous cultures. A quasi-steady state representative of plasmid-bearing cells was established in the initial portion of each continuous culture experiment. Due to the profound growth rate differential between the two types of cells, transients of considerable duration were observed in each continuous culture experiment (initiated with a pure culture of plasmid bearing cells) following the slow accumulation of plasmid-free cells near the end of the quasi-steady state. Significant variations in various culture parameters (including a rapid decline in the plasmid-bearing fraction of the total cell population) occurred during this period, leading ultimately to a steady state for a culture dominated entirely by plasmid-free cells. In continuous cultures, plasmid copy number during the quasi-steady states increased with decreasing dilution rate from 50 (at 0.409 h(-1)) to 941 (at 0.233 h(-1)). Production of the plasmid-encoded protein (beta-lactamase) in these experiments was maximized at an intermediate dilution rate, corresponding to an optimum copy number of about 450. A similar optimum copy number was observed in batch cultures. Significant excretion of beta-lactamase was observed at both low and high dilution rates.

Effects of Promoter Induction and Copy Number Amplification on Cloned Gene Expression and Growth of Recombinant Cell Culturesa

Effects of Promoter Induction and Copy Number Amplification on Cloned Gene Expression and Growth of Recombinant Cell Cultures^a

Cellular plasmid content and cloned‐gene expression: Some useful equations

Cellular plasmid content determines the yield of closed-gene product, affects plasmid replication, and influences the behavior of the culture by dictating the extent of metabolic burden on the host cell and plasmid segregation at cell division. Hence, it is a variable of primary importance in the study of recombinant cell cultures. In this article, equations are developed to enable the conversion of experimental determinations of cellular plasmid content into theoretically important units. The importance of different units in characterizing plasmid effect on cell response is highlighted. Also, equations that relate cellular plasmid content to cloned-gene expression are developed and successfully tested on several sets of data from literature.

Purification and characterization of pre-S-containing hepatitis B surface antigens produced in recombinant mammalian cell culture.

Heterogeneous, pre-S-rich HBsAg particles were expressed in recombinant mammalian cell culture and purified to near homogeneity. The purification process comprises: concentration of cell culture medium, protein precipitation by poly(ethylene glycol), gel filtration column chromatography, isopycnic ultracentrifugation by KBr and sucrose density gradient ultracentrifugation. The resulting HBsAg product was greater than 98% pure, and contained much of pre-S1 and pre-S2 components. Scanning densitometry analysis of the silver-stained HBsAg product showed approximately 70-80% S protein, approximately 10-20% pre-S2 protein, and approximately 5-15% pre-S1 protein. It was estimated that the amount of HBV-specific DNA present the final product was less than 7 pg mg-1 HBsAg. Further biochemical analysis has demonstrated that the HBsAg particles are very heterogeneous in charge and density. Charge heterogeneity was quite random among the particles, but density heterogeneity could be related to the different amounts of pre-S2 component in the particles.

Adaptive estimation of bioreactors: monitoring plasmid instability

An adaptive state-estimator is presented, for the detection of revertants in a recombinant cell culture. It consists of a Linear Kalman Filter whose parameters are simultaneously identified by a Recursive Prediction Error algorithm. This approach is applied, in simulation, using total biomass and total rate of growth as the measurements. Two predictor structures are tried for parameter identification. The first uses the state-space model, performs well when both cell populations are present and can track time-varying parameters. The second uses a model relating the outputs only and works well whether revertants are present or not, but its tracking ability is poor. Despite these reservations, both lead to state-estimators sufficiently robust and easy to tune for real-time implementation. For monitoring plasmid instability, the second is to be preferred. The generality of this approach is briefly discussed.

Microcarrier culture of recombinant Chinese hamster ovary cells for production of human immune interferon and human tissue-type plasminogen activator

Recombinant Chinese hamster ovary cells were successfully cultured semi-continuously on microcarriers of gelatin or modified dextran under non-selective conditions for up to three weeks. High and constant production rates for human immune interferon and tissue-type plasminogen activator were obtained. For cells that produced interferon, the highest cell concentration and interferon production was obtained with gelatin microcarriers though the specific production when grown in the presence of 0.2% fetal calf serum was slightly higher for cells cultured on dextran microcarriers (0.12 U/cell day versus 0.11 U/cell day). For cells that produced plasminogen activator, a slightly higher cell concentration was obtained for cells grown on dextran microcarriers (9x105 cells/ml versus 7x105 cells/ml). However, the specific and total production rates were significantly higher for cells cultured on gelatin microcarriers (6.7 pg/cell day versus 2.1 pg/cell day). The maximum cell concentration and specific production rate could be increased to 2.3x106 cells/ml and 3.4 pg/cell day for dextran microcarriers by adding 6-aminohexanoic acid to the medium. For gelatin microcarriers, the addition of 6-aminohexanoic acid increased the specific production rate to 14.4 pg/cell day. Cell growth, however, was inhibited.

Purifying hepatitis B surface antigen from recombinant cell cultures by separating cells, fractional protein precipitation of the filtrate and purification on an anion exchanger

Purifying hepatitis B surface antigen from recombinant cell cultures by separating cells, fractional protein precipitation of the filtrate and purification on an anion exchanger

Human hepatitis B vaccine from recombinant yeast.

The worldwide importance of human hepatitis B virus infection and the toll it takes in chronic liver disease, cirrhosis and hepatocarcinoma, make it imperative that a vaccine be developed for worldwide application. Human hepatitis B vaccines are presently prepared using hepatitis B surface antigen (HBsAg) that is purified from the plasma of human carriers of hepatitis B virus infection. The preparation of hepatitis B vaccine from a human source is restricted by the available supply of infected human plasma and by the need to apply stringent processes that purify the antigen and render it free of infectious hepatitis B virus and other possible living agents that might be present in the plasma. Joint efforts between our laboratories and those of Drs W. Rutter and B. Hall led to the preparation of vectors carrying the DNA sequence for HBsAg and antigen expression in the yeast Saccharomyces cerevisiae. Here we describe the development of hepatitis B vaccine of yeast cell origin. HBsAg of subtype adw was produced in recombinant yeast cell culture, and the purified antigen in alum formulation stimulated production of antibody in mice, grivet monkeys and chimpanzees. Vaccinated chimpanzees were totally protected when challenged intravenously with either homologous or heterologous subtype adr and ayw virus of human serum source. This is the first example of a vaccine produced from recombinant cells which is effective against a human viral infection.