A. V. Carvalhal

Cell Growth Arrest by Nucleotides, Nucleosides and Bases as a Tool for Improved Production of Recombinant Proteins

Arresting cell growth and thus decreasing cell division potentially lessens the chance for genetic drift in the cell population; this would be of utmost importance for the consistent production of biopharmaceuticals during long periods. The drawback of the addition of well‐known synchronizing agents, such as chemotherapeutics, is that they cause a disproportionate accumulation of cellular constituents, leading to cell death. The use of compounds that are naturally synthesized by the cell, as is the case of nucleotides, nucleosides, and bases (Nt/Ns/B), is shown in this work to be a promising tool. The addition of purines and pyrimidines was tested using a CHO cell line producing the secreted form of the human placental alkaline phosphatase enzyme (SEAP). From the chemical alternatives tested, AMP was the most promising compound for protein production improvement; it reduced cell growth and maintained the culture with high cell viability for long periods, while increasing SEAP specific productivity 3‐fold. The use of CHO and BHK mammalian cells producing Factor VII and the use of a insect cell line (Sf9) showed that the effect of AMP addition seems to be independent of the r‐protein and cell line. With the addition of AMP, accumulation of cells at the S phase was accompanied by an increase of the protein specific productivity. Addition of known synchronizing drugs (aphidicolin and doxorubicin) and application of environmental cell growth arrest strategies (depletion of nutrients and byproduct accumulation) showed also to effectively arrest CHO cell growth. A careful look onto cell cycle distribution in the different scenarios created, shows whether it is important to consider r‐protein expression dependency upon cell cycle in process optimization and operation strategies.

Metabolic changes during cell growth inhibition by p27 overexpression

The overexpression of p27, a cyclin-dependent kinase (CDK) inhibitor, has been shown to effectively inhibit cell growth at the G1-phase of different cell lines, potentiating a valid genetic strategy for cell proliferation control. In order to characterize the energy requirements after p27 overexpression in CHO cells expressing SEAP (secreted form of the human alkaline phosphatase enzyme), key metabolic parameters were evaluated. Cell growth inhibition led to a significant increase in cell size concomitant with a 2-fold increase in cell protein content. The simultaneous increase of the intracellular proteolytic activity with protein content suggests higher protein synthesis. A general 2-fold increase in oxygen, glutamine and glucose consumption rates, coupled with an increase in lactate and ammonia production was observed. p27 overexpression led to a significant increase in the intracellular pool of AMP (8.5-fold), ADP (6-fold) and, more uncommonly, ATP (4.5-fold). Nevertheless, cells were able to maintain the equilibrium among the three adenine nucleotides since both the ATP/ADP ratio and the energy charge values remained similar to those observed with non-growth inhibited cells. This work shows that the observed 4-fold increase in SEAP specific productivity after cell growth inhibition by p27, occurred concomitantly with a higher expenditure of cell energy. This characterization of cell metabolism becomes important in demonstrating the applicability of growth inhibition systems.

Purification and Characterization of an Anti-Apoptotic Protein Isolated from Lonomia obliqua Hemolymph

Previously it was reported that supplementation of insect cell culture with Lonomia obliquahemolymph could extend culture longevity (Maranga et al. Biotechnol. Prog. 2003, 19, 58–63). In this work the anti‐apoptotic properties of this hemolymph in Spodoptera frugiperda (Sf‐9) cell culture were investigated. The presence or absence of apoptotic cells was characterized by light microscopy, flow cytometry, and agarose gel electrophoresis. Hemolymph was fractionated by several ion exchange and gel filtration chromatographic steps for identification of the compounds responsible for this effect. Fractions exhibiting a potent anti‐apoptotic effect were isolated and tested in cell culture. A protein of about 51 kDa was identified, isolated, and tested for apoptosis inhibition. Addition of this purified protein to Sf‐9 cultures was able to prevent apoptosis induced by nutrient depletion as well as by potent apoptosis chemical inducers such as Actinomycin D. This work confirms that the enhanced culture longevity obtained by supplementation with L. obliquahemolymph is due to the presence of potent anti‐apoptotic factors.

Metabolic changes during cell growth inhibition by the IRF-1 system

Abstract A genetic approach based on the activation of interferon-regulated-factor-1 (IRF-1) has been applied to arrest BHK cell growth in spinner flasks through the presence of estradiol in the culture medium, leading to the activation of the constitutively expressed IRF-1/estrogen receptor fusion protein (IRF-1-hER). Two days after estradiol addition cell proliferation was inhibited; cell concentration being kept for the next 6 days, although a decrease in cell viability was observed. IRF-1 activation alters the cell energetic metabolism, as there is extra metabolic activity with higher glucose, glutamine and oxygen consumption rates. Although the proteolytic activity is higher than in the control cells, the cell protein content increases significantly after IRF-1 activation, suggesting an overall increase in protein synthesis. A significant increase in lactate dehydrogenase activity and a higher reduction of MTT (3-(4,5-dimethylthiazol-2-yl)-2–5-diphenyl tetrazolium bromide) were also observed, showing an increase in cell activity during cell growth inhibition. Changes in ATP and in cellular phospholipid content as well in glucose metabolism were detected, in real time by 31 P and 13 C nuclear magnetic resonance spectroscopy (NMR). The levels of ATP were unaffected but changes in cellular phospholipid content were observed. Although an increase on ADP and AMP content was observed 48 to 72 h after IRF-1 activation, both the ATP content and the energy charge (EC) remained constant. It can be concluded that the IRF-1 activation leads to a significant increase of the cell activity until the cell is no longer able to maintain the same energetic metabolism and starts to lose viability.

Strategies to modulate BHK cell proliferation by the regulation of IRF-1 expression

Activation of the constitutively expressed interferon-regulatory-factor-1/estrogen receptor fusion protein (IRF-1-hER) in BHK cells was accomplished through the addition of estradiol to the culture medium, which enabled IRF-1 to gain its transcriptional activator function and inhibit cell growth. With the addition of 100 nM estradiol at the beginning of the exponential phase of a cell suspension culture, IRF-1 activation led to a rapid cell growth inhibition but also to a significant decrease in cell viability. To apply this concept in industry, a reduction of the time span of estradiol exposure is required. Cycles of estradiol addition and removal were performed in 2-l stirred tank bioreactors operated under perfusion, where an initial step addition of 100 nM estradiol was performed, followed, after 48-72 h, by a slow dilution with estradiol-free fresh medium (perfusion rate varying between 0.7 and 1.4 per day). Cell growth inhibition was successfully achieved for three consecutive cycles. Diluting the estradiol by perfusing medium without estradiol to concentrations lower than 10 nM led to cell growth and viability recovery independently of the perfusion rate used. These observations permitted the definition of operational strategies for regulated IRF-1 BHK cell growth by pulse estradiol addition, followed by a period of 48 h in the presence of estradiol and by fast perfusion to estradiol concentrations lower than 10 nM. Cell growth response to IRF-1 activation and following estradiol removal by perfusion was also evaluated with an IRF-1-hER regulated clone expressing constitutively Factor VII, where the time of estradiol exposure and perfusion rate were varied. This clone presented a stronger response to IRF-1 activation without an increase in Factor VII specific productivity after cell growth inhibition; this clearly indicates that the stationary phase obtained is clone dependent. This work proves that it is possible to modulate the IRF-1 effect for cell growth control by the manipulation of cycles of addition and removal of estradiol, potentially representing a new generation of culture procedures for controlled growth production purposes.

Manipulation of culture conditions for BHK cell growth inhibition by IRF-1 activation

The activation of interferon-regulatory-factor-1 (IRF-1) hasbeen applied to regulate the cell growth of BHK cells. Theconstitutively expressed IRF-1-estrogen receptor fusion protein(IRF-1-hER) activated by the addition to the culture medium ofan estrogen analogue (estradiol), enabled IRF-1 to gain itstranscriptional activator function. By using a dicistronicstabilised self-selecting construct it was possible to controlcell proliferation. With the addition of 100 nM of estradiol at the beginning of the exponential phase, the IRF-1 activationled to a rapid cell growth inhibition. Two days after estradioladdition cell concentration was still maintained but a decreasein cell viability was observed. This cell response isindependent on clone (producer and non-producer) and culturesystem (static and stirred cultures). Specificrecombinant-protein productivity of the producer clone was notsignificantly altered. Control experiments confirmed that IRF-1activation effect was not due to the addition of estradiol per se, estradiol solvent or serum concentration. The extent ofcell growth inhibition is dependent on estradiol concentrationand estradiol addition time, although a decrease in cellviability was always observed. Reducing the time span ofestradiol exposure allowed the decrease in the cell viability tobe controlled and the stationary inhibited phase to be extended:when the time of contact between the cells and estradiol isreduced cell viability increases, archieving values similar tothose obtained if no estradiol is added. During this recoveryphase the cells passed two different phases: first a stationaryphase extension where cell growth was still inhibited, followedby an increase of cell concentration. The IRF-1 system isreversible. This pattern can be repeated for an extended period when estradiol addition and removal are repeated, showing acyclic response. Thus, it is possible to modulate the IRF-1effect by manipulating cycles of addition/removal of estradioland in this way the stationary phase can be maintained.

Development of an IRF-1 Based Proliferation Control System

Mammalian cell cultures are the preferred production system for secreted pharmaceutical proteins (Hauser, 1997). Life of a mammal begins with fertilization of the oocyte that divides and proliferates until the animal reaches its mature size. Further cell growth is tightly controlled. Proliferation and cell death are balanced to keep the total cell mass essentially constant, while the synthesis and secretion of cellular products continues. Despite rapid progress in the understanding of growth regulatory mechanisms, tumorigenic growth due the loss of proliferation control of a single body cell initially is still a leading cause of disease and death.

Extracellular purine and pyrimidine catabolism in cell culture

The presence of purines and pyrimidines bases, nucleosides, and nucleotides in the culture medium has shown to differently affect the growth of a Chinese hamster ovary (CHO) cell line producing the secreted form of the human placental alkaline phosphatase enzyme (SEAP; Carvalhal et al., Biotech Prog. 2003;19:69‐83). CHO, BHK, as well as Sf9 cell growth was clearly reduced in the presence of purines but was not affected by pyrimidines at the concentrations tested. The knowledge about the mechanisms by which nucleotides exert their effect when present outside the cells remains very incomplete. The catabolism of both extracellular purines and pyrimidines was followed during the culture of CHO cells. Purines/pyrimidines nucleotides added at a concentration of 1 mM to the culture medium decreased to negligible concentrations in the first 2 days. Purine and pyrimidine catabolism originated only purinic and pyrimidic end‐products, respectively. The comparison between AMP catabolism in serum‐free cultures (CHO cells expressing Factor VII and Sf9 cells) and in cultures containing serum (CHO cells expressing SEAP and BHK cells expressing Factor VII) showed that AMP extracellular catabolism is mediated by both cells and enzymes present in the serum. This work shows that the quantification of purines and pyrimidines in the culture medium is essential in animal cell culture optimization. When using AMP addition as a chemical cell growth strategy for recombinant protein production improvement, AMP extracellular concentration monitoring allows the optimization of the multiple AMP addition strategy for a prolonged cell culture duration with high specific productivity.

Cell Growth Inhibition by the IRF-1 System

A genetic approach, based on the growth regulatory protein interferon-regulated-factor- 1 (IRF-1), activated by the addition of estradiol, has been applied to regulate BHK cell growth. With the addition of 100 nM of estradiol 48 hours after inoculation, growth inhibition occurs within 24 hours but is followed by a significant decrease in cell viability, whereas rec-protein specific productivity is not significantly altered. Viability decrease is not due to estradiol effect and is independent upon the culture system. In order to define strategies to extend the stationary growth phase, several parameters have been studied: estradiol concentration, time post inoculation for estradiol addition and time span of estradiol exposure. When the time of contact between the cells and estradiol is reduced the cell viability increases, achieving similar values of the control, without the estradiol, and leading to a stationary growth phase extension.

Cell Growth Arrest by Nucleotides as a Tool for Improved Production of Biopharmaceuticals

Arresting cell growth and thus decreasing cell division potentially lessens the chance for genetic drift in the cell population: this would be of utmost importance for the consistent production of high quality products. Cell growth arrest of two industrially relevant animal cell lines (CHO and BHK cells) has been successfully achieved by the overexpression of a cyclin-dependent kinase inhibitor (p27) and of a tumor suppressor gene (IRF-1) (genetic strategy), purines addition (chemical strategy) and by the depletion of essential nutrients from the culture medium (environmental strategy). Adenosine and AMP addition, as well as the overexpression of p27 led to a 3 to 4-fold increase in the specific productivity of two recombinant proteins: the secreted form of the human placental alkaline phosphatase (SEAP) and the coagulation Factor VII. AMP addition showed to be the most promising tool for biopharmaceuticals production: fine tuning of drug addition was required. All the tested cell growth al-rest strategies interfered not only with cell cycle but also with cell metabolism. A careful look onto cell cycle distribution in the different scenarios created, shows whether it is important to consider recombinant protein expression dependency upon cell cycle in process optimization and operation strategies.