Keywords: Amino acid, cholesterol, vitamins.

The culture of mammalian cells represents without a doubt one of the areas of greater development and technological impact of biotechnology today. Development in recent years has been considerable in three fundamental areas: (1) the identification and development of new drugs, (2) the development of new therapeutic methods (genetic therapy, tissue engineering and artificial organs) and (3) the production, at commercial level, of therapeutic proteins, diagnostic kits and vaccines. Mammalian cell is the only technology available to produce such molecules with the required structure and biological activity to perform their function. Major challenges in this area are: (1) the increase of the productive capacity of the biopharmaceutical industries in order to fulfil the increasing demand, (2) the media formulation at low cost, serum or protein free (defined chemically), as strongly enforced by the regulatory agencies and (3) the development of high performance and high throughput processes of purification. In this research we used CHO (Chinese Hamster Ovary) cell line because of its relevance for the production of recombinant proteins used in human therapy, such as growth factors, anti-thrombolytic agents and monoclonal antibodies. In fact, CHO cell lines are widely used for being highly stable expression systems for heterologous genes (those from a different organism), and for its relatively simple adaptation to adherence-independent growth in serum and protein free media. This work refers to the improvement of the serum-free, low protein BIOPRO1 culture medium (BioWhitaker Europe, Belgium), for the production of t-PA (tissue plasminogen activator) with CHO TF 70R cells by the addition of vitamins, cholesterol, fatty acids and specific amino acids, with the purpose to determine the effect of medium supplementation on cell growth, t-PA production and biological functionality. t-PA is a thrombolytic agent -clot-busting drug approved for use in certain patients having a heart attack or stroke.

In vitro mammalian cell cultivation requires a complex combination of nutrients, considering glucose and glutamine as main carbon, energy and nitrogen sources. Mineral salts, amino acids and vitamins are also required; other essential nutrients, like growth factors, hormones, and receptor and transport proteins are required in small quantities as well. These latter compounds are usually provided by foetal bovine serum (FBS). Vitamins are required as cofactors in several reactions related to amino acid metabolism and protein functionality. Because of its complexity and high cost, optimization of media formulation is a key aspect for bioprocess development in animal cell cultivation. Suppression of FBS is nowadays enforced because of the well-reported drawbacks of its use, which precludes the approval of its use for the production of therapeutics. Besides, its considerable protein load severely hampers the purification of the product and its high cost can have a negative impact on the economy of the process. However, serum-free media implies a case to case design based on the particular supplements required for each strain. Chemically defined serum-free media have been extensively studied and several reviews have been published on the subject. Hormones and growth factors requirements are specific for each type of cell. Albumin preparations, which are sometimes added instead of FBS, have proven to be effective for cell cultivation; its function is usually related to fatty acids and lipids transport and protection against cell damage due to shear stress. Being so, the direct addition of fatty acids and lipids could be a better option to formulate a defined medium with low protein content. Recombinant CHO TF 70R cells producing t-PA, as those used in this work, have been cultivated in DMEM: Ham's F12 50:50 medium with and without serum addition, showing that cell growth was somewhat lower in the serum free medium. In this work the basal medium used was a proprietary serum-free and low protein medium denominated BIOPRO1, kindly provided by BioWhitaker Europe (Verviers, Belgium). BIOPRO1 was supplemented with vitamins, chemically defined lipids concentrate containing fatty acids and cholesterol, proline, serine and asparagine, as indicated in Table 1 for each culture (Ci). The medium was also supplemented with glucose and glutamine in the different experiments. Eight cultures (C1 to C8) were carried out in the three series of experiments (S1 to S3) as shown in Table 1. In each series, a control culture (CC) was performed to consider eventual variations associated to the physiological condition of the inoculum cells.

The results obtained in the first series of experiments show that C1 allowed a maximum viable cell concentration (Xv) significantly higher than that obtained in CC. Similar results were obtained in C2 when folic acid, cobalamine, biotin and benzoic acid was supplemented, which is relevant in terms of cost. The specific growth rate (µ) was similar in C1 and C2 and around 14% higher than CC. On the other hand, despite its positive effect on cell growth, the increase in vitamin concentration did not affect the t-PA specific production rate (qtPA) nor its specific biological activity (atPA), and the maximum levels of t-PA obtained in the different cultures were of the same order of magnitude than those reported in the literature for this cell line.

For the following series of cultures (S2), BIOPRO 1 supplemented with folic acid, cobalamine, biotin and benzoic acid was chosen to evaluate the enrichment with lipids concentrate since BIOPRO 1 has a limited spectrum of lipids. This solution was added to obtain a cholesterol concentration in the medium of 20 mg/ml and 30 mg/ml in C3 y C4 respectively (see Table 1). In addition, at those two levels, vitamin supplement (folic acid, cobalamine, biotin and benzoic acid)was further increased by doubling its amount (C5 and C6) as shown in Table 1. Most relevant results of S2 show that the addition of lipids promoted a substantial increase in cell concentration (Xv) and cell viability (CV), best results being obtained in C4, which was the one with the highest content of lipids though not of vitamins. In this case, 65% increase in µ and 85% in Xv were obtained with respect to CC and growth phase was prolonged by 30 hrs. Doubling the vitamin supplement (C6), produced instead a 10% decrease in maximum Xv with respect to C4, probably because some of the vitamins in this preparation reached a level that is inhibitory or toxic to the cells. The beneficial effect of lipids and cholesterol supplement on cell growth is related to the creation of a less aggressive environment for the culture by decreasing shear stress, strengthening the plasmatic membrane and providing complex structural blocks that represent energy savings for the cell. Higher t-PA concentration was obtained in the culture in which cell growth was higher (C4), with an increase of 31% with respect to CC. In this case, the addition of lipids had opposite effects on t-PA production, increasing atPA while decreasing qtPA. A possible explanation is that cholesterol stiffens the plasmatic membrane, which increases its mechanical strength and therefore confers a higher viability to the cells. This implies a lesser protease release by cell lysis and increased product stability. However, this membrane stiffening may hinder the excretion of t-PA, which would explain the decrease in qtPA. This could be tested by measuring intracellular t-PA concentration. This aspect was not considered here but it is worthwhile studying in the future. Therefore no further increase in lipid supplementation was considered.

To design the S3 experiments, the residual concentration of amino acids in C4 was analyzed. Apart from glutamine, the lower residual amino acid concentrations corresponded to serine, asparagine and proline. Serine and asparagine are not considered essential amino acids for CHO cells; however, high consumption rates have been determined in other CHO cell lines. Therefore, in the S3 experiments the BIOPRO1 medium used in C4 was supplemented with 0.75 mmol of serine and 0.45 mmol of proline per liter of medium in C7 and with 0.75 mmol of serine, 0.45 mmol of proline and in addition 0.65 mmol of asparagine per liter of medium in C8, to assess the specific effect of asparagine. This effect is not apparent, since this amino acid could be limiting (it was consumed almost completely) while contributing to increase the level of ammonia.

The results show that proline and serine addition (C7) allowed an increase in cell concentration higher than 130% with respect to CC, which represents an additional increase of 45% to that already obtained in C4. Increase of t-PA concentration was 42% and increase in specific activity was 36% with respect to CC. Best results were obtained in C8, where cell concentration increased over threefold with respect to CC. This allowed a 58% increase in t-PA concentration while maintaining a high level of specific activity (34% higher than in CC). Although there is no clear explanation for this behavior, which can only be attributed to the increase in asparagine concentration, stimulatory effect of specific amino acids on protein production has been reported in the case of arginine in the production of thrombopoietin with CHO cells. Even though the medium was enriched in amino acids not considered essential for CHO cell growth, its supplement allowed the cells to take up these compounds from the medium instead of spending energy in its synthesis, which has a beneficial impact on cell growth. Besides, amino acids are not exclusively used for protein synthesis, but its degradation products converge to the TCA cycle furnishing energy or intermediary metabolites for the synthesis of other biomolecules like DNA and RNA.

Fortification of BIOPRO1 medium with specific vitamins and amino acids, fatty acids and cholesterol allowed to increase Xv significantly and to prolong the exponential growth phase in batch cultures of CHO TF 70R cell line. As a consequence, a much higher concentration of t-PA (over 80%) and specific activity of t-PA (a_tPA, over 35%) were obtained, which is certainly a very relevant asset for t-PA production. Furthermore, it is a strategy which can have a very general use in other CHO cell lines.