Dhinakar S. Kompala

Metabolic burden in recombinant CHO cells: effect ofdhfr gene amplification andlacZ expression.

Foreign protein production levels in two recombinant Chinese hamster ovary (CHO) cell lines were compared in cells transfected with different expression vectors. One vector pNL1 contained the gene for neomycin resistance (neor) and thelacZ gene which codes for intracellular β-galactosidase, with both genes controlled by the constitutive simian virus (SV40) promoter. The other vector CDβG contained the amplifiabledhfr gene andlacZ gene, controlled by the constitutive SV40 and cytomegalovirus (CMV) promoters, respectively. Cell growth and β-galactosidase expression were compared quantitatively after cells were selected in different concentrations of the neomycin analog G418 and methotrexate, respectively. A 62% reduction in growth rate occurred in recombinant CHO cells in which thelacZ anddhfr genes were highly amplified and expressed. In contrast, the combined effects of the unamplifiedneor gene andlacZ gene expression on the growth kinetics were small. Any metabolic burden caused bylacZ gene expression, which was evaluated separately from the effect ofneor gene expression, must be negligible, as higher expression of β-galactosidase (1.5×10−6 units/cell) occurred in unamplified cells compared to the cells in whichlacZ was amplified by thedhfr-containing vector (3×10−7 units/cell). Thus, the main factor causing severe growth reduction (“metabolic burden”) in cells containing the amplifieddhfr gene system was not overexpression of β-galactosidase butdhfr andlacZ gene co-amplification anddhfr gene expression.

Effect of amplification of dhfr and lac Z genes on growth and β-galactosidase expression in suspension cultures of recombinant CHO cells

Studies were conducted to characterize the effect of gene amplification and foreign gene expression on recombinant CHO cell growth. Chinese hamster ovary (CHO) cells were transfected with an expression vector containing the gene for dihydrofolate reductase (dhfr) and the gene for human β-interferon (β-IFN) or thelac Z gene which codes for β-galactosidase (β-gal). The recombinant genes in these CHO cells were amplified stepwise by growth in 0, 10−7, and 10−6 M methotrexate (MTX), and the β-gal expressing cells were adapted to suspension culture. Flow cytometric methods (FCM) were used to measure the distribution of amplifieddhfr gene content and foreign β-gal gene expression in the cell populations. A biochemical assay for β-gal was also used. Beta-gal expression was found to increase with increasing gene amplification. The growth rate of recombinant CHO cells at 10−7 M MTX was found to be 20% lower than that of recombinant CHO cells in MTX-free medium, and the cell growth rate at 10−6 M MTX was 20% lower than that of recombinant CHO cells at 10−7 M MTX. There was no effect of 10−5 M MTX on the growth of CHO-DG44 (dhfr-) cells. The reduction of growth rate in recombinant CHO cells is therefore thought to be mainly due to the effect ofdhfr and foreign gene amplification and increased β-galactosidase expression.

Effect of Production Method and Gene Amplification on the Glycosylation Pattern of a Secreted Reporter Protein in CHO Cells

We have investigated the independent effects of selective gene amplification (using the dhfr amplifiable selection marker) and culture operating strategy (batch vs repeated fed‐batch vs semicontinuous perfusion) on the glycosylation of a recombinant reporter protein (secreted alkaline phosphatase, SEAP) produced in transfected Chinese hamster ovary (CHO) cells. HPLC analyses coupled with susceptibility to various exoglycosidases were used to determine the N‐glycosylation profile of SEAP samples. The dhfr amplified cell line yielded an almost 10‐fold increase in specific productivity as compared to that of the unamplified cell line. The glycosylation pattern of the reporter protein produced in batch bioreactor cultures of the amplified cell line showed only slight differences as compared to the glycosylation pattern of the protein from batch bioreactor cultures of the unamplified cell line. In contrast, analysis of SEAP glycosylation structures from the protein isolated from semicontinuous perfusion cultures indicated that both relative glycan content and extent of sialylation were increased as compared to samples isolated from repeated fed‐batch cultures. These results suggest that the slow growing perfusion cultures produce more completely glycosylated proteins than the faster growing repeated fed‐batch cultures.

Optimal induction of protein synthesis in recombinant bacterial cultures.

The yield of recombinant biologicals depends not only on the functional capability of the host cell, but also on the biosynthetic capacity of the cell. Many researchers have alluded to the importance of this capacity.’-’ Indeed, the elimination of biological bottlenecks and the directed metabolic flow through any given pathway is being pursued by many researchers for many products, recombinant proteins notwithstanding. In recent years, significant progress has been made towards characterizing, circumventing, and eliminating plasmid instability in recombinant bacterial systems.6 For example, by inserting a gene into the expression plasmid that encodes the synthesis of an essential enzyme, whose activity has been deleted in the host cell, we can prevent growth of plasmid-free cells born by uneven partitioning. Many researchers have proposed methods for estimating and potentially ensuring plasmid or culture stabilitY ;’-I0 however, few have addressed the capacity of a cell to produce a recombinant product. Our previously reported model for recombinant E . colil’ contains sufficient metabolic detail to determine the influence of rDNA and its concomitant activity on the metabolism of the host so that the biosynthetic capacity of the host can be evaluated. This work uniquely and explicitly calculates the specific growth rate of a bacterial cell mass as a dynamic function of the metabolic burdens of foreign protein translation and plasmid replication. We illustrate its power here by predicting the influence that induced protein synthesis and induced “runaway” replication have on the growth rate and, in turn, the influence that the change in growth rate has on the product expression and eventual yield in batch cultures.

Production of a secreted glycoprotein from an inducible promoter system in a perfusion bioreactor.

The primary advantage of an inducible promoter expression system is that production of the recombinant protein can be biochemically controlled, allowing for the separation of unique growth and production phases of the culture. During the growth phase, the culture is rapidly grown to high cell density prior to induction without the extra metabolic burden of exogenous protein production, thus minimizing the nonproductive period of the culture. Induction of the culture at high cell density ensures that the volumetric production will be maximized. In this work, we have demonstrated the feasibility of overexpressing a reporter glycoprotein from the inducible MMTV promoter in recombinant Chinese hamster ovary (CHO) cells cultured in a high cell density perfusion bioreactor system. Retention of suspension‐adapted CHO cells was achieved by inclined sedimentation. To maximize volumetric production of the culture, we have demonstrated that high cell density must be achieved prior to induction. This operating scheme resulted in a 10‐fold increase in volumetric titer over the low density induction culture, corresponding directly to a 10‐fold increase in viable cell density during the highly productive period of the culture. The amount of glycoprotein produced in this high cell density induction culture during 26 days was 84‐fold greater than that produced in a week long batch bioreactor. Long‐term perfusion cultures of the recombinant cell line showed a production instability, a phenomenon that is currently being investigated.

Characterization of heterologous and native enzyme activity profiles in metabolically engineered Zymomonas mobilis strains during batch fermentation of glucose and xylose mixtures.

Zymomonas mobilis has been metabolically engineered to broaden its substrate utilization range to include D-xylose and L-arabinose. Both genomically integrated and plasmid-bearing Z. mobilis strains that are capable of fermenting the pentose D-xylose have been created by incorporating four genes: two genes encoding xylose utilization metabolic enzymes (xylA/xylB) and two genes encoding pentose phosphate pathway enzymes (talB/tktA). We have characterized the activities of the four newly introduced enzymes for xylose metabolism, along with those of three native glycolytic enzymes, in two different xylose-fermenting Z. mobilis strains. These strains were grown on glucose-xylose mixtures in computer-controlled fermentors. Samples were collected and analyzed to determine extracellular metabolite concentrations as well as the activities of several intracellular enzymes in the xylose and glucose uptake and catabolism pathways. These measurements provide new insights on the possible bottlenecks in the engineered metabolic pathways and suggest methods for further improving the efficiency of xylose fermentation.

Engineering Chinese hamster ovary (CHO) cells to achieve an inverse growth – associated production of a foreign protein, β-galactosidase

Protein synthesis in mammalian cells can be observed in two strikingly different patterns: 1) production of monoclonal antibodies in hybridoma cultures is typically inverse growth associated and 2) production of most therapeutic glycoproteins in recombinant mammalian cell cultures is found to be growth associated. Production of monoclonal antibodies has been easily maximized by culturing hybridoma cells at very low growth rates in high cell density fed- batch or perfusion bioreactors. Applying the same bioreactor techniques to recombinant mammalian cell cultures results in drastically reduced production rates due to their growth associated production kinetics. Optimization of such growth associated production requires high cell growth conditions, such as in repeated batch cultures or chemostat cultures with attendant excess biomass synthesis. Our recent research has demonstrated that this growth associated production in recombinant Chinese hamster ovary (CHO) cells is related to the S (DNA synthesis)-phase specific production due to the SV40 early promoter commonly used for driving the foreign gene expression. Using the stably transfected CHO cell lines synthesizing an intracellular reporter protein under the control of SV40 early promoter, we have recently demonstrated in batch and continuous cultures that the product synthesis is growth associated. We have now replaced this S-phase specific promoter in new expression vectors with the adenovirus major late promoter which was found to be active primarily in the G1-phase and is expected to yield the desirable inverse growth associated production behavior. Our results in repeated batch cultures show that the protein synthesis kinetics in this resulting CHO cell line is indeed inverse growth associated. Results from continuous and high cell density perfusion culture experiments also indicate a strong inverse growth associated protein synthesis. The bioreactor optimization with this desirable inverse growth associated production behavior would be much simpler than bioreactor operation for cells with growth associated production.

Analysis of foreign protein overproduction in recombinant CHO cells. Effect of growth kinetics and cell cycle traverse.

Intracellular foreign protein (beta-galactosidase) expression in recombinant CHO cell lines in continuous culture was analyzed by developing a mathematical model that includes the effects of metabolic burden and cell cycle dependence of intracellular foreign protein expression. This combined growth kinetic and cell cycle model, assuming S- or G1-phase-dependent expression, was stimulated to predict productivity on a single-cell and culture-volume basis in continuous cultures. In the case of S-phase-dependent expression, the intracellular foreign protein level increases monotonically, but in the case of G1-phase-dependent expression it decreases monotonically with increasing dilution rate. Also, the trends of foreign protein concentration in the culture volume differ significantly between S- and G1-dependent expression kinetics. Thus, the cell cycle dependency of foreign protein expression should be included in process optimization concepts and operating strategies of continuous bioreactors.

Foreign protein expression from S phase specific promoters in continuous cultures of recombinant CHO cells

Foreign protein expression from the commonly used SV40 promoter has been found to be primarily during the S-phase of the cell cycle. Simple mathematical models with this cell cycle phase dependent expression of foreign protein suggest that the specific production rate will be proportional to the cell growth rate, which is particularly disadvantageous in high cell density fed-batch or perfusion bioreactors. In this study we investigate this predicted relationship between the production rate and growth rate by culturing recombinant CHO cells in a continuous suspension bioreactor. One CHO cell line, GS-26, has been stably transfected with the plasmid pSVgal, which contains the E. coli lac Z gene under the control of the SV40 promoter. This GS-26 cell line was grown in suspension cultures over a range of specific growth rates in batch and continuous modes. The intracellular β-galactosidase activity was assayed using a standard spectrophotometric method after breaking the cells open and releasing the enzyme. A strong growth associated relationship is found between the intracellular β-galactosidase content and the specific growth rate in batch and continuous cultures, as predicted.

Stability in continuous cultures of recombinant bacteria: A metabolic approach

Continuous-culture population dynamics of recombinant bacteria are predicted with a structured kinetic model. The instantaneous specific growth rates of the plasmid-bearing and plasmidfree cells are explicitly calculated from their metabolic activities. The resultant growth-rate differential (between plasmid-bearing and plasmid-free cells) is dynamic and changes over the course of a fermentation. Further, the growth-rate differential is a function of dilution rate. We present the experimental determination of model constants governing plasmid replication and foreign protein expression for a host/vector systemE. coli RR1 [pBR329]. For a different experimental system, we estimate the increased polypeptide expression from a DNA insert solely from the instability population dynamics. Stability predictions agree quite well with experimental observations from the literature and our lab.