Wolfgang Noe

Safety and economic aspects of continuous mammalian cell culture.

Mammalian cell cultures are the most appropriate host cells for recombinant DNA derived products if complex protein structures have to be synthesized in their native form. Due to their physiological behaviour they grow either adherent or in suspension. For the attachment of adherent cells, microcarriers or wire springs can be applied to increase the internal surface of the bioreactor. Both systems provide a simplified media exchange but, however, show some limitations in scale up. In contrast, suspension culture systems as homogeneous systems independent of any carrier have not shown any limitation in scale up. Because most cell lines which are of commercial interest grow in suspension, this technology is best advanced and used in batch and continuous mode. Although mammalian cell cultures are sensitive to hydrodynamic shear forces, technologies for deep tank production are developed which allow stirrer tip speed of up to 1.5 m s-1 sufficient for oxygen uptake, suspension of cells and homogeneous supply with nutrients. For long term bioprocesses without selection pressure it has to be considered that transformed cell lines might show genetic instability due to their variations of chromosomes. In addition, sterile technology becomes an important factor in long term bioprocesses. The decision as to which cell culture system should be chosen, whether batch or continuous processes should be applied essentially is based on the capital investment, the amount of material to be produced, genetic stability of the production cell line, reliability of sterile technology and the flexibility required in the production plant. Under the assumption that 20 kg of a protein have to be produced per year and the same product concentrations in the harvest fluid are reached in the batch process and for instance in the chemostat, it can be considered that the capital investment for one 10,000 l batch process and a 2 x 2,000 l continuous process, necessary to produce the amount of material, is comparable. Risk of microbial contamination or technical failure can be considered to be fairly low in the batch process. The economic risk for long term bioprocess in the chemostat can be expected to be medium and high in the perfusion system which is in the large scale not technically fully satisfactory. In addition, due to the longer down time period after contaminations and the start up of the continuous process, the annual yield of the batch process can be considered to be higher.(ABSTRACT TRUNCATED AT 400 WORDS)

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 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 cells 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.

Immunobased elution assay for process control

Abstract A flow-injection analysis (FIA) system with an integrated immunobased elution assay was developed for the on-line determination of biotechnological products. This paper describes its application for tissue-type plasminogen activator (rt-PA) and recombinant antithrombin III (r-AT III). The short response time of the immunochemical system allows its use for measurement and control of biotechnological production processes. Immobilized polyclonal antibodies were found to bind rt-PA in the cartridge better than monoclonal antibodies. Sepharose-4B was found to be the best support for the antibodies. The cartridge with immobilized antibodies can be prepared easily, has a high reliability and is stable for more than two weeks. The total time necessary for one assay cycle is in the range of 6–8 min, depending on the conditions. The whole procedure is controlled by a personal computer. The FIA system can be used for automatic analysis as a stand-alone version with a frequency of 7 samples per hour. The standard operation range is from 500 μg/ml down to 2 μg/ml. A comparison with the conventional microtiter ELISA assay shows that this assay can be applied for an accurate determination of undiluted cultivation samples.

Automated imunoanalysis systems for monitoring mammalian cell cultivation processes

Two different automated immunoanalysis systems are presented. Both are based on the principles of flow-injection analysis and were developed to provide reliable, rapid monitoring of relevant proteins in animal cell cultivation processes. One system uses a turbidimetric analysis, and the other employs a heterogeneous chemistry with immobilized immunocomponents. For both systems, the analysis time is in the range of a few minutes, and a complete analysis cycle, including triplicate analyses and various washing steps, is in the range of 20–30 minutes. Samples from cultivation processes can be analyzed directly without dilution. Quantitation of proteins such as rt-PA or monoclonal antibodies can be performed over an analyte concentration range of 1–1000 mg/L. Both systems were compared to conventional ELISA assays on microtiter plates. The turbidimetric analysis system also included a biosensor for simultaneous glucose determination.

Optimization of vaccine production for animal health

Vaccines on the basis of mammalian cell cultures are of major importance for human and animal health. Therefore efforts are undertaken for the improved production of more effective vaccines. Of course, the main purpose of all these approaches is to save lives and improve the quality of life for human beings. However, there is also some remarkable effort in the food industry and the associated animal production, especially in the case of some Flaviviridal viruses (BVD), where>80% of all cattle herds are found to be infected. These viruses can cause tremendous economic losses of calfs and embryos (Ames, 1990). Because of these facts, there is a continuous endeavour for improving the manufacturing of therapeutics or preventing agents such as vaccines for the treatment of cattle. The competitive economic situation and the specific market demands still require effective and high yield production methods, especially in the case of one of the most widespread viral diseases in cattle like BVD (Ames, 1990).We have succeeded in establishing an improved method for the production of BVD on the basis of a continuous fermentation mode, that consist of modifications of the corresponding process and media improvements.

Monitoring and control of large — scale vaccine production via dynamic oxygen uptake rate measurement

For large — scale production of BVD (Bovine Viral Diarrhea) antigen on adherent MDBK target cells we have established a 300 L airlift reactor with stainless steel wire springs as carrier material.

Glycosylation patterns of recombinant therapeutic proteins produced in two mammalian cell lines

To elucidate the quality of glycosylation patterns when changing the host cell, the desired protein or the extracellular environment, we expressed human therapeutic glycoproteins in Chinese Hamster Ovary (CHO) and mouse myeloma (NSO) cells under different fermentation conditions. sICAM consistencies produced in both cell lines were shown to differ significantly. Their dissimilar Oligosaccharide structures became apparent with all applied electrophoretic and Chromatographic methods. In the case of CHO-expressed proteins, the altered sICAM, t-PA and IFN-ω glycosylation patterns resulting from varied fermentation conditions were rather small and limited on quantitative changes of preexisting Oligosaccharide structures. These changes in product consistencies were therefore difficult to evaluate with conventional carbohydrate analytical methods such as the HPAEC / PAD mapping technique. However, differences became evident after capillary electrophoretic separation and mapping of aminobenzamide (AB)-labeled Oligosaccharides.

Model-Based Monitoring and Control of a Monoclonal Antibody Production Process

A structured mathematical model describing the dynamics of hybridoma growth and monoclonal antibody (mAb) production in suspension cultures is presented. The model fits well to experimental data obtained from batch, fed-batch, and continuous cultures with hybridoma cells. Applications of the model for process control are demonstrated. 1. An extended Kalman filter (EKF) was designed to estimate the state of the process. The oxygen consumption rate of the cell culture is monitored on-line and is used as the only measurement information for the EKF. This EKF is able to provide good estimates for living and dead cell densities and the medium composition. 2. The mathematical model can be applied for the optimization of fed-batch cultures.

The Real Meaning of High Expression

There exist some common misconceptions about the application of high expression systems (HES) to an industrial setting. This paper intends to sensitize the developer and even more importantly the end-user of HES towards generic complications encountered during the industrial application of such systems, complications which, if overlooked, can render the term high expression meaningless.