Potent and prolonged hypoglycemic activity of an oral insulin--Tat mixture in diabetic mice.

Trans-activator of transcription (Tat) is a cell penetrating peptide which can translocate and carry macromolecular cargoes through cell membranes. This study investigated the hypoglycemic activity of orally delivered insulin - Tat mixture in alloxan-induced diabetic mice. The mixtures of insulin and Tat at 1:1, 1:3 and 1:6 molar ratios were given orally at the insulin doses ranging from 1-200 IU/kg. The fasting blood glucose (FBG) levels were measured at initial, 1, 2, 4, 6, and 12 h after administration. At 1:3 molar ratio of the mixture and after 12 h of administration, insulin at 200 IU/kg showed the highest with prolonged hypoglycemic activity of 74.0±10.3% FBG reduction (2.18 folds of subcutaneously injected (SC) insulin). Free insulin administered orally did not show any hypoglycemic activity. The mixtures at the insulin doses of 100 and 50 IU/kg also showed potent FBG reduction of 73.8±8.2 and 71.3±16.9% at 12 h after administration (2.18 and 2.10 folds of SC insulin, respectively). After incubation with Mono-Mac-6 cells, only the -mixtures but not the free insulin showed intra-cellular insulin uptake, indicating the insulin penetration through the cell membranes via Tat. In simulated gastric fluid, the insulin content in the mixture was not found, demonstrating the degradation of insulin in the gastric environments. Insulin may be absorbed at upper gastrointestinal tract facilitated by Tat. The potent and prolonged hypoglycemic activity of insulin co-administered orally with Tat can be further developed as an effective oral insulin delivery system.

Stability of luciferase plasmid entrapped in cationic bilayer vesicles.

Characteristics and physical stability of luciferase plasmid (pLuc) entrapped in cationic bilayer vesicles prepared from various molar ratios of amphiphiles (DPPC, Tween61 or Span60), cholesterol (Chol) and cationic charge lipid (DDAB) were investigated. The cationic liposomes were composed of DPPC/Chol/DDAB in the molar ratio of 7:2:1. The cationic (Tween61 or Span60) niosomes were composed of Tween61/Chol/DDAB or Span60/Chol/DDAB in the molar ratio of 1:1:0.05. The maximum loading of pLuc was 15.29, 22.70, and 18.92 microg/mg of the total lipids or surfactants of liposomes, Tween61 and Span60 niosomes, respectively. The morphology of the vesicles showing multilamellar structure was characterized by transmission electron microscope (TEM). The particle sizes of the vesicles in nanosize range (160-850 nm) were determined by Photon Correlation Spectroscopy (PCS). Gel electrophoresis and gel documentation were modified to determine the entrapment efficiency of pLuc in cationic bilayer vesicles. The cationic bilayer vesicles gave the pLuc entrapment efficiency of 100%. The pLuc entrapped in cationic liposomes exhibited higher stability than pLuc in solution and pLuc entrapped in cationic Tween61 or Span60 niosomes, when stored at 4, 30 and 50 degrees C for 8 weeks. After 8 weeks at 4 degrees C, pLuc contents remained in cationic liposomes was 2 and 3 times higher than cationic Span60 and Tween61 niosomes, respectively. After 3 weeks, 50 and 2% of pLuc was remained in cationic liposomes at 30 and 50 degrees C respectively, whereas all pLuc in cationic Span 60 and Tween61 niosomes were degraded within 2 and 1 week, respectively. At 30 and 50 degrees C, pLuc in an aqueous solution or in bilayer vesicular formulations were not in a stable supercoil form. This study has indicated that the stability of pLuc can be enhanced by entrapping in cationic liposomes more than in niosomes. Higher temperature with increase storage time can affect the stability of pLuc even entrapped in bilayer vesicles.

Improving natural principles with genetic engineering: TNK-tissue plasminogen activator.

The fibrinolytic system plays an important role in the physiological maintenance of blood flow and the dissolution of thrombi. Administration of fibrinolytic agents in indications such as myocardial infarction, pulmonary embolism, deep vein thrombosis or stroke, therefore, offers a rational means to dissolve pathological thrombi and restore vascular patency. The functional domains of the physiological tissue plasminogen activator (t-PA) provide fibrin specificity and serine protease activity for plasminogen cleavage and binding to liver receptors which gives the molecule a short half-life. In order to combat acute thromboembolic events such as myocardial infarction, the structure of the natural t-PA molecule was genetically modified to prolong its half-life, to increase its fibrin-specificity and to improve its resistance to plasminogen activator inhibitor. These features of TNK-t-PA allow bolus administration in emergency situations, early reperfusion of the blood vessel and a low rate of bleeding complications, thus improving the overall benefit to patients.

Secretion of active recombinant human tissue plasminogen activator derivatives in Escherichia coli.

The DNA fragment coding for kringle 2 plus serine protease domains (K2S) of tissue plasminogen activator (tPA) was inserted into a phagemid vector, pComb3HSS. In the recombinant vector, pComb3H-K2S, the K2S gene was fused to gpIII of PhiM13 and linked to the OmpA signal sequence. The resulting gene, rK2S-gpIII, was inducibly expressed in Escherichia coli XL-1 Blue. The protein was presented on the phage particle. To stop the expression of gpIII, a stop codon between K2S and the gpIII gene was inserted by site-directed mutagenesis. This mutated vector, MpComb3H-K2S, was transformed in XL-1 Blue. After induction with IPTG (isopropyl-beta-D-thiogalactopyranoside), rK2S was found both in the periplasm as an inactive form of approximately 32% and in the culture supernatant as an active form of approximately 68%. The secreted form of rK2S was partially purified by ammonium sulfate (55%) precipitation. The periplasmic form was isolated from whole cells by chloroform extraction. The fibrin binding site of kringle 2 was demonstrated in all expressed versions (phage-bound, periplasmic, and secreted forms) using the monoclonal anti-kringle 2 antibody (16/B). Only the secreted form of rK2S revealed a fibrinogen-dependent amidolytic activity with the specific activity of 236 IU/microg. No amidolytic activity of rK2S was observed in either the periplasmic or the phage-bound form. The secretion of rK2S as an active enzyme offers a novel approach for the production of the active-domain deletion mutant tPA, rK2S, without any requirements for bacterial compartment preparation and in vitro refolding processes. This finding is an important technological advance in the development of large-scale, bacterium-based tPA production systems.

Monitoring of intracellular ribonucleotide pools is a powerful tool in the development and characterization of mammalian cell culture processes.

Efficient cell culture process development for the industrial production of recombinant therapeutics is characterized by constraints which pertain to issues such as costs, competitiveness and the meeting of project timelines. These constraints require tools which can help the developer learn as much as possible as quickly as possible about the cell at hand and identify features of a particular culture which are amenable to improvement. Current on- and off-line monitoring parameters, however useful, provide only late indications (cell concentration, viability) and circumstantial evidence (lactate, ammonia, etc.) with regard to the physiologic status of cells at the time of sampling. The relative intracellular content of purine to pyrimidine nucleotide triphosphates as well as the ratio of UTP to UDP-N-acetylhexosamines have been previously described as sensitive indicators of a cell's metabolic status, growth potential, and overall physiological condition. The sensitivity of such nucleotide ratios and their usefulness in commercially relevant process development and characterization were tested at Boehringer Ingelheim Pharma KG in a large number of fermentations (>80) with a variety of culture modes, cells, and products in scales up to 10,000 litres. Monitoring of these intracellular parameters allows a timely and reliable assessment of cell state and growth potential, which is possible neither by classical cell number and viability measurements nor by a variety of fermentation data typically monitored. The view inside the cell afforded by nucleotide monitoring enables prediction of the behavior of a culture up to 2 days before any hint of physiological changes is given by cell number and viability estimation. In this paper, data relating the growth behavior of CHO and hybridoma cell lines to their nucleotide pools are shown. Two very different processes for the production of recombinant tPA in 10,000-litre bioreactors are compared and characterized with respect to their nucleotide profiles. Examples from industrial process development cases in which intracellular nucleotide information is used to advantage are also presented and discussed.

Transgenic manufactured biopharmaceuticals: a new method of drug manufacturing.

The limit to product yield in biotechnology is reached at 500 - 800 mg/l fermentation broth. However, if large quantities of proteins are required for products which have to be marketed as quickly as possible, biotechnology remains the technology of choice. Challenging biotechnology, transgenic manufacturing processes use animals or plants with a productivity of up to 20 g/l. Whereas animal transgenic technology still risks potential contaminantion with prions, transgenic plants offer a rather safer manufacturing technology. Since glycosylation of the protein is specific to the production organism from the onset of product development, it must be decided whether the therapeutically active protein should be produced by mammalian cell culture, transgenic animal or transgenic plant. This, however, requires a decision on the manufacturing technology, prior to the knowledge of dose and potential market.

Appropriate mammalian expression systems for biopharmaceuticals.

Process development for biopharmaceuticals is dictated by product quality, drug safety and economy of the manufacturing process. Not surprisingly, these factors also play a key role in the evaluation of mammalian cell expression systems to be used in the production of pharmacologically active glycoproteins. To date, the most prominent candidates for efficient expression of glycoproteins are mammalian cell lines such as mouse fibroblast cells (C 127-BPV), Chinese hamster ovary cells (CHO-DHFR, CHO-NEOSPLA, CHO-GS), mouse myeloma cells (NSO-GS) as well as transgenic animals carrying c-DNA or genomic DNA which codes for the protein of interest. The expression titer in the case of glycoproteins is mainly determined by the promoter construct, the site of integration into the chromosome, the copy number and the type of protein in question. Based on expression titer, CHO-NEOSPLA and NSO-GS expression systems are most effective in the production of monoclonal antibodies and, to a lesser extent, of recombinant DNA derived proteins. However, based on overall product yield, expression of recombinant DNA derived proteins in transgenic animals is by far the most promising system. Therefore, for proteins required in large quantities, transgenic expression systems offer an attractive choice. However, cost of goods for products for which the dosage or the overall annual quantities are low, is dominated by downstream processing, filling, lyophilization and packaging and not by the fermentation process. Such proteins are preferentially produced by classical mammalian cell culture systems. Concerns which have to be addressed with respect to drug safety in the transgenic animal approach are the size of the herd, genetic stability from animal to animal, variation in productivity and in impurity profiles during lactation periods, microbial, viral, mycoplasma and prion contaminants, the dependence on health status and the life span of the animal. In a number of cases glycosylation of the protein is relevant for the prevention of immunogenicity of the protein, the pharmacological activity, the pharmacokinetic profile, solubility and stability against proteolysis. The glycosylation pattern, depending on protein structure, is influenced by the enzymatic system of the host cell as well as by fermentation conditions. Therefore, selection of host cells and culture conditions must take into account the requirement for a specific and stable glycosylation pattern. For the assessment of glycovariants, a number of protein analytical methods such as peptide mapping, isoelectric focusing, oligosaccharide mapping, MALDI-TOF (matrix assisted laser desorption mass spectrometry-time of flight), capillary electrophoresis and specific potency assays are available. In our experiments, glycosylation of proteins expressed in CHO cells was demonstrated to be very stable. Only extreme process times, cultivation methods and ammonium ion concentrations had an influence on the glycosylation profile. Among the three products investigated--tissue plasminogen activator (t-PA), interferon omega and soluble intercellular adhesion molecule (s-ICAM)--t-PA expressed the most stable glycosylation pattern. Only at extreme ammonium concentrations an increase of mannose-5 structures was observed, whereas biantennary complex structures were reduced. On the other hand, interferon omega and s-ICAM showed greater susceptibility to increased ammonium concentrations and to adherent cultivation. Such conditions induced quantitative changes to the glycosylation pattern favoring the appearance of higher branched structures. Short cultivation times resulted in more heterogenous oligosaccharide structures. Since the glycosylation of the three proteins is different in the same host cell, the amino acid sequence of the protein apparently influences the glycosylation pattern and its sensitivity to culture conditions. In NSO-mouse myeloma cells, production of s-ICAM is two times as high as in CHO cells

Innovative and economic potential of mammalian cell culture.

Innovations for economic optimization of manufacturing processes of mammalian cell culture processes address new expression systems, optimized cell culture media and feeding systems, economy of scale, efficient harvest systems for viable cell separation, redesign of downstream processing and reduction of the overall number of quality control assays. A very efficient expression system in Chinese hamster ovary cells is the NEOSPLA expression system yielding 60-100 micrograms monoclonal antibodies per cell and day. Efficient supplements in nutrient feeding are insulin and amino acids which contribute to a high extent to the productivity of the mammalian cell culture process. Large scale manufacturing processes lower cost of goods by reduction of turn around cost for cleaning and steaming in place, media preparation, number of batches for annual campaign, in-process and quality control. In downstream processing the number of process steps and the step yield are responsible for the economics. Process control systems in a computer assisted manufacturing plant increase success rate, reduces man power and minimizes shifts. In the innovative process also alternative technologies such as transgenic animals should be considered to improve the economy of the manufacturing processes.

Identification and development of new biopharmaceuticals.

The 34 protein based biopharmaceuticals which are presently on the market reached a total sale of 10 Mio $ in 1996. Out of 500 biopharmaceutical under investigation, 200 are already in clinical trial programs. Although biopharmaceuticals have good records in efficacy and drug safety, biologics are still competitive due to economic reasons. In future in some cases gene therapy or oligonucleotide antisense therapy as modern technologies for in vivo protein synthesis regulation will be an alternative to protein replacement therapy. All three therapeutic approaches will be supported by the human genome project which will give new hints on genetic disorders related to specific diseases. The identification of new therapeutic developments will be on the DNA-, RNA- and protein level. The decision whether the therapeutic principle will be gene therapy or whether the differential genomic analysis will provide just a research tool for high through-put screening of small molecules will be decided upon the evaluation of the results of the genomic projects and the research strategy of the company.

Investigations on oxygen limitations of adherent cells growing on macroporous microcarriers.

Macroporous microcarriers are commonly applied to fixed and fluidized bed bioreactors for the cultivation of stringent adherent cells. Several investigations showed that these carriers are advantageous in respect to a large surface area (Griffiths, 1990; Looby, 1990a).When growing a rC-127 cell line on Cytoline 2 (Pharmacia Biotech), no satisfactory product yield could be achieved. A possible limitation in the supply of nutrient components was investigated to explain these poor results. No significant concentration gradients could be detected. Nevertheless, fluorescence staining revealed a decreasing viability, particularly inside the macroporous structure. Therefore, oxygen transfer to and into the carriers was examined by means of an oxygen microprobe during the entire process. Additional mathematical modeling supported these results.The maximum penetration depth of oxygen was determined to be 300 µm. A critical value influencing the oxygen uptake rate of the rC-127 cells occured at a dissolved oxygen concentration of 8% of air saturation. A significant mass transfer resistance within a laminar boundary film at the surface of the carrier could be detected. This boundary layer had a depth of 170 µm. The results showed that even a 40% air saturation in the bulk liquid could not provide an efficient oxygenation of the surface biofilm during the exponential growth phase. Fluorescent staining reveals a poor viability of cells growing inside the carrier volume. Thus, oxygen supply limits the growth of rC-127 cells on macroporous microcarriers. Poor process performance and low product yield could be explained this way.

Monitoring the glycosylation pattern of recombinant interferon-omega with high-pH anion-exchange chromatography and capillary electrophoresis.

The enzymatic glycosylation of certain asparagine residues in the protein structure has a profound influence on their physico-chemical properties. Many recombinant DNA derived glycoprotein pharmaceuticals for therapeutic use are glycosylated. Their oligosaccharides are important with regard to stability, solubility, in vivo activity and antigenicity. Sophisticated analytical methods that allow a high resolution of synthesized carbohydrate structures are therefore necessary to ensure a constant product quality. To elucidate the robustness of interferon-omega glycosylation regarding process modifications. Chinese Hamster Ovary (CHO)-cells expressing human interferon-omega were cultivated under different fermentation conditions. The most significant glycosylation alterations resulted from the varied parameters such as initial ammonia concentration in the production medium, cultivation mode (adherent versus suspended) or process time. These are detectable with HPAEC (high-pH anion-exchange chromatography) oligosaccharide mapping as well as with capillary electrophoresis.

Safe biotechnology. 7. Classification of microorganisms on the basis of hazard. Working Party "Safety in Biotechnology" of the European Federation Biotechnology.

The current systems for classifying human pathogens on the basis of hazard are well developed and their basic criteria are in general agreement one with another. Of more importance, the safety practices based on these classifications have generally been successful. They have enabled extensive research activities, medical practice and industrial production to be conducted on an ever-increasing scale, involving dangerous microorganisms (e.g. in vaccine production and treatment of infected patients) with a very low incidence of adverse effects on the workers involved and the general public. Although the EU has adopted a harmonised list of agents in groups 1-4 there is as yet no complete agreement among member states and individual microbiologists. The purpose of this paper is to present a historical survey and to discuss the current processes for identifying and classifying the hazards posed by the use of microorganisms in research and technology. This is essential in the design of appropriate methods of counteracting potential risks.

[New horizons for medicine on the basis of genetic technology]. / Neue Horizonte für die Medizin auf Basis der Gentechnik.

In case of a single gene defect a number of appropriate gene probes are available for prenatal diagnosis. Knowledge of the genetic disorders enables in some cases early onset of therapy or the option for abortion. However, gene technology which enables the diagnosis should not be viewed from an ethical point of view but rather the action taken when diagnostic results are available. Gene therapy for a single gene defect still is at the early stage of development. Only few patients have been treated in various indications. Difficult to be overcome are the low frequency and unspecific integration of inserted DNA into the chromosome, lack of sufficient transcription control and short half-lives of the integrated gene. From an ethical perspective gene therapy complies with the therapeutic concept of medicine. Antisense oligonucleotides are under clinical development for blockage of the synthesis of oncogenes and viral proteins. Stability of obligonucleotides as well as selectivity for specific cells will have to be overcome for broader application. Its therapeutic application is in accordance with the ethical principles of medicine. Substitution therapy with recombinant DNA derived human proteins is in therapeutic application to replace their counterparts from native source by human pharmacologically active proteins which cannot be isolated from their natural source. For recombinant DNA derived proteins where the mode of action is known short development times frames can be expected allowing an early return on investment. The expected market potential for recombinant DNA derived pharmaceuticals in 1995 will reach 4.4 billion DM.

Gene technology: chances for diagnosis and therapy.

In the case of a single gene defect, a number of appropriate gene probes are available for prenatal diagnosis. In some cases, knowledge of the genetic disorders enables early onset of therapy or the option for abortion. However, gene technology which enables the diagnosis should not be viewed from an ethical point of view but rather the action taken when diagnostic results are available. Gene therapy for a single gene defect still is at the early stage of development. Only a few patients have been treated in various indications. Difficult to overcome are the low frequency and unspecific integration of inserted DNA into the chromosome, lack of sufficient transcription control, and short half-life of the integrated gene. From an ethical perspective gene therapy complies with the therapeutic concept of medicine. Antisense oligonucleotides are under clinical development for blockage of the synthesis of oncogenes and viral proteins. Stability of oligonucleotides as well as selectivity for specific cells will have to be overcome for broader application. Its therapeutic application is in accordance with the ethical principles of medicine. Substitution therapies with recombinant DNA derived human proteins are in therapeutic application to replace their counterparts from native source in a safer way or for human pharmacologically active proteins which cannot be isolated from their natural source. For recombinant DNA derived proteins where the mode of action is known, short development time frames can be expected allowing for an early return on investment. The expected market potential for recombinant DNA derived pharmaceuticals in 1995 will reach 4,400 million DM. Due to their specificity, monoclonal antibodies are used for tumor imaging when labeled by 99mtechnetium or for tumor therapy when labeled by rhenium or yttrium. Both concepts are under clinical evaluation. Vaccines derived from recombinant DNA technology offer the chance of producing safer vaccines consisting of the antigen determinant only. In general, recombinant DNA technology and biotechnology offer the opportunity of providing new diagnostic and therapeutic principles of high ethical value. The biotechnical manufacturing processes used for this purpose are friendly to the environment by using raw material from renewable sources, low energy consumption, and producing biodegradable products only. In almost all cases, host cells used for manufacturing belong to the safety category 1, in which no danger is expected for the operator, the public, and the environment.(ABSTRACT TRUNCATED AT 400 WORDS)

Pentapeptide identified as a monoclonal antibody binding site in the serine-protease domain of t-PA.

The first defined sequential epitope of the tissue plasminogen activator (t-PA) was determined by a monoclonal antibody against a synthetic peptide segment corresponding to peptide sequence 341-354 of t-PA. This segment was selected by computer assisted epitope prediction. Balb/c mice were immunized with catalase-peptide and tripalmitoyl-S-glyceryl-cys-teinyl-seryl-peptide conjugates. A monoclonal antibody derived from this immunization was reactive with native recombinant t-PA (rt-PA) and reduced carboxymethylated recombinant t-PA (RCM rt-PA). The sequential epitope was detected by Pepscan method using overlapping octa- and nonapeptides. By fine epitope mapping with tetra-, penta-, hexa- and heptapeptides the epitope was minimized to the pentapeptide EEEQK (347-351). Replacement set analysis confirmed the importance of this amino acid sequence, especially of the amino acid E348, for antibody binding. Functional assays of rt-PA were not affected by this antibody indicating that the epitope has no influence on the enzymatic center and the binding site of the inhibitor. The analysis demonstrates that the predicted recognition site of the monoclonal antibody 17-134/11 is exposed on the surface of the native rt-PA molecule.

Mammalian cell cultures. Part III: Safety and future aspects.

Long standing experience with mammalian cell cultures demonstrates that they per se do not constitute any hazard to healthy man. However, permanent cell lines may cause transplantable tumors in immune-suppressed animals and man but not in healthy species. A potential hazard associated with mammalian cell cultures might be caused by endogenous viruses which can be harboured in the mammalian cells or adventitious agents associated with mammalian cells. Especially a corresponding validation for removal of viruses has to be established in the manufacturing process. Free nucleic acids or recombinant nucleic acid sequences are regarded as not being biologically active and therefore do not cause any hazard. Because mammalian cell cultures which are ranking lowest in terms of safety measures are able to synthesize reliably complex glycoprotein structures, the economy of mammalian cell culture processes has to be approved for future prospects on the background of the competitive situation with microorganisms and the overall cost limitation of the health insurance systems. High yielding expressing systems are required including detoxification genes for cytotoxic metabolites. Shorter doubling times of the host cell, higher productivity and higher cell densities would be further goals. Also new aspects of mammalian cell cultures, not only their use as factories for protein synthesis, but also in gene therapy as carrier for the desired genetic information or in enhancing the wound healing process, will gain future importance.

Mammalian cell cultures. Part II: Genetic engineering, protein glycosylation, fermentation and process control.

For expression of human genes in mammalian cell culture regulatory sequences such as promotor or terminator region of viral origin are required. The most widely used expression system uses dihydrofolic acid reductase (DHFR) as a selection marker in conjunction with a DHFR deficient Chinese hamster ovary (CHO) cell using methotrexate as selection pressure. Alternatively the glutamine synthetase amplification system seems to be one of the most efficient expression systems using methionine sulphoximine (MSX) as selection pressure. Folding and glycosylation takes place in mammalian cell cultures at the sites of endoplasmatic reticulum and the Golgi apparatus and is comparable to synthesis in human cells. Most large scale manufacturing processes for products derived from mammalian cell cultures are fed batch suspension culture processes up to 15,000 l. Important factors for productivity are media composition and feeding strategies. Sterility of the entire system during the fermentation period is a decisive factor for success rate. Because mammalian cell cultures reacting very sensitive to small changes in temperature, pH, osmolality and agitation the fermentation system requires a reliable process control system. Validation of the entire manufacturing process is required to ensure consistent product quality which meets predetermined specifications and to provide a basis for an economic process. In a joint effort equipment qualification, process validation, in-process controls and quality controls provide the basis for product consistency from batch to batch.

Mammalian cell cultures. Part I: Characterization, morphology and metabolism.

Primary cell cultures are obtained by trypsinization from tissue cultures usually as a monolayer culture. The absence of fetal calf serum will support suspended growth behaviour of spontaneously transformed cells. After several passages the cell line becomes more stable and gives rise to a continuous cell line. Such continuously growing cell lines are a prerequisite for production of recombinant DNA derived proteins. Mammalian cells are 10-100 times larger in diameter than microorganisms. They have no cell wall and express therefore a higher sensitivity to hydrodynamic sheer forces. One of the most stringent problems in mammalian cell culture are "silent" contaminants with mycoplasma which might change cell growth. Mammalian cell cultures show a complex metabolism where regulation of metabolites and catabolites are not fully understood. Glucose is the main carbohydrate source. Also three groups of intercorrelated amino acids are known. Lactate as the primary metabolite of glucose and ammonia as a metabolite of glutamine are expected to be cytotoxic for mammalian cells. Although in some experiments even the addition of ammonia has no significant effect on the viability of hybridoma cells. Adherent cells can be cultivated attached to surfaces such as microcarrier or wire springs. Suspended cells are grown in stirred bioreactors with a comparable technology to fermentation of microorganism. Parameters such as pH, temperature, stirring tip speed and osmolality have to be well controlled in order to obtain high cell viability and cell density.