Rüdiger Heidemann

The use of peptones as medium additives for the production of a recombinant therapeutic protein in high density perfusion cultures of mammalian cells.

Protein hydrolysates as substitutes for serum havebeen employed by many in cell culture mediumformulation, especially with the shift to low proteinor protein-free media. More recently, vegetablehydrolysates have also been added as nutritionalsupplements to fortify the amino acid content in smallpeptide form for batch and fed-batch fermentations. Several of these new hydrolysates (peptones of soy,rice, wheat gluten etc.) were tested as protein-freemedium supplements for the production of a recombinanttherapeutic protein. Multiple peptone-supplemented,continuous perfusion bioreactor experiments wereconducted, varying dilution rates and basal mediumcomposition over the various runs. Cell specificrates and product quality studies were obtained forthe various peptones and compared with peptone-freemedium. The potential for peptones to decreaseintrinsic and proteolytic degradation of the productwas also investigated.It was found that peptones confer a nutritionalbenefit, especially at low dilution rates, for therecombinant BHK cell line used in this investigation.The specific productivity increased 20–30% comparedto the peptone-free controls. However, this benefitwas also fully delivered by using fortified medium inplace of the peptone-enriched media. Therefore, whilepeptones may be considered as useful medium additiveswhen development time is limited, their addition maybe avoided by systematic medium development ifpermitted by the time line of the project.

Metabolic flux analysis of CHO cells in perfusion culture by metabolite balancing and 2D [13C, 1H] COSY NMR spectroscopy.

The physiological state of CHO cells in perfusion culture was quantified by determining fluxes through the bioreaction network using (13)C glucose and 2D-NMR spectroscopy. CHO cells were cultivated in a 2.5L perfusion bioreactor with glucose and glutamine as the primary carbon and energy sources. The reactor was inoculated at a cell density of 8 x 10(6)cells/mL and operated at approximately 10 x 10(6)cells/mL using unlabeled glucose for the first 13 days. The second phase lasted 12 days and the medium consisted of 10% [U-(13)C]glucose, 40% labeled [1-(13)C]glucose with the balance unlabeled. After the culture attained isotopic steady state, biomass samples from the last 3 days of cultivation were considered representative and used for flux estimation. They were hydrolyzed and analyzed by 2D [(13)C, (1)H] COSY measurements using the heteronuclear single quantum correlation sequence with gradients for artifacts suppression. Metabolic fluxes were determined using the 13C-Flux software package by minimizing the residuals between the experimental and the simulated NMR data. Normalized residuals exhibited a Gaussian distribution indicating good model fit to experimental data. The glucose consumption rate was 5-fold higher than that of glutamine with 41% of glucose channeled through the pentose phosphate pathway. The fluxes at the pyruvate branch point were almost equally distributed between lactate and the TCA cycle (55% and 45%, respectively). The anaplerotic conversion of pyruvate to oxaloacetate by pyruvate carboxylase accounted for 10% of the pyruvate flux with the remaining 90% entering the TCA cycle through acetyl-CoA. The conversion of malate to pyruvate catalyzed by the malic enzyme was 70% higher than that for the anaplerotic reaction catalyzed by pyruvate carboxylase. Most amino acid catabolic and biosynthetic fluxes were significantly lower than the glycolytic and TCA cycle fluxes. Metabolic flux data from NMR analysis validated a simplified model where metabolite balancing was used for flux estimation. In this reduced flux space, estimates from these two methods were in good agreement. This simplified model can routinely be used in bioprocess development experiments to estimate metabolic fluxes with much reduced analytical investment. The high resolution flux information from 2D-NMR spectroscopy coupled with the capability to validate a simplified metabolite balancing based model for routine use make (13)C-isotopomer analysis an attractive bioprocess development tool for mammalian cell cultures.

The Super-Spinner: A low cost animal cell culture bioreactor for the CO2 incubator

The production of small quantities of monoclonal antibodies and recombinant proteins was carried out using a new low cost production system, the Super Spinner. Into a 1 1 standard Duran® flask a membrane stirrer equipped with a polypropylene hollow fiber membrane was installed to improve the oxygen supply by bubble-free aeration. The aeration was facilitated by using the CO2 conditioned incubator gas, which was pumped through the membrane stirrer via a small membrane pump. The maximal oxygen transfer rate (OTRmax) of the Super Spinner was detected. For this purpose one spinner flask was equipped with an oxygen electrode. The OTRmax was measured by the dynamic method. The ratio of membrane length to culture volume was adapted corresponding to the oxygen uptake rate of the cells according to the desired cell density. A balanced nutrient supply resulted in an optimal formation and yield of products.

Effects of dissolved oxygen levels and the role of extra- and intracellular amino acid concentrations upon the metabolism of mammalian cell lines during batch and continuous cultures

The effects of dissolved oxygen and the concentration of essential amino acids upon the metabolism of two mammalian cell lines (rCHO producing human active (t-PA) and a mouse-mouse hybridoma) were investigated in batch, chemostat, and perfusion cultures. Intracellular amino acid concentrations were measured for both cell lines during repeated batch cultures and the KS-values for the essential amino acids were calculated using Monod equations via computer simulation. The KS-values were in the range of 10 mmol L−1 and the pool of most intracellular amino acids remained constant at about 10–100 fold higher in concentration than in the medium. No significant differences were observed between the hybridoma and CHO cell. The specific nutrient uptake rates corresponded with the cell specific growth rate and the effects of reduced dissolved oxygen concentrations only became evident when the DO dropped below 5% of air saturation (critical concentration below 1%). Nevertheless, a correlation between nutrient concentration and specific oxygen uptake was detected.

Production of recombinant human antithrombin III on 20-L bioreactor scale: Correlation of supernatant neuraminidase activity, desialylation, and decrease of biological activity of recombinant glycoprotein

Chinese hamster ovary (CHO) cells producing the recombinant glycoprotein human antithrombin III (rhAT III) were batch cultivated in a 20-L bioreactor for 13 days. Neuraminidase activity in cell-free supernatant was monitored during cultivation and free sialic acid was determined by HPLC. Neu5Acalpha(2-->3)Gal-specific Maackia amurensis and Galbeta(1-->4)GlcNAc-specific Datura stramonium agglutinin were used for determination of sialylated and desialylated rhAT III, respectively. A commercial test kit was used for evaluation of functional rhAT III activity. Supernatant neuraminidase as well as lactate dehydrogenase activity increased significantly during batch growth. The enhanced number of dead cells correlated with increased neuraminidase activity, which seemed to be principally due to cell lysis, resulting in release of cytosolic neuraminidase. Loss of terminally alpha(2-->3) linked sialic acids of the oligosaccharide portions of rhAT III, analyzed in lectin-based Western blot and lectin-adsorbent assays, correlated with a decrease of activity of rhAT III produced throughout long-term batch cultivation. Thus, structural oligosaccharide integrity as well as the functional activity of recombinant glycoprotein depend on the viability and mortality of the bioreactor culture, and batches with a high number of viable cells are required to guarantee production of glycoproteins with maximum biological activity. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 441-448, 1997.

Re-use of spent cell culture medium in pilot scale and rapid preparative purification with membrane chromatography

Based on experiments in bench scale, a recycling of spent cell culture medium was performed in a 100-1 pilot scale bioreactor. The cell cultivation has been done as a repeated batch procedure after the initial batch in the following four repeated batches spent medium from the previous batch was partially re-used. After microfiltration and ultrafiltration a part of the filtrate was mixed with a concentrate of amino acids and glucose, sterile filtered and subsequently filled back into the bioreactor. Up to 65% of the harvested cell- and product-free spent medium was re-used in each repeated batch. This procedure results in a saving of pure and waste water volume and saving of supplemented proteins as transferrin, insulin and lipoproteins and, therefore, also in a reduction of the production costs. A strongly acidic membrane ion exchanger was evaluated for the ability to purify the monoclonal antibodies from the pilot scale cultivation. Within minutes, gram quantities of product could be purified in a high flux system, especially developed for this purpose, achieving purities of 80%. The capacity of the acidic membrane ion exchanger was found in former investigations to be 1 mg cm-2 with recoveries up to 96%. Final purification was carried out by gel column filtration.

The Use of a Mobile Robot for Complete Sample Management in a Cell Culture Pilot Plant

A mobile robot system was developed that is capable of automating the complete sample management in a biotechnological laboratory. The robot consists of a wheeled platform and a mounted industrial robot arm with a gripper tool attached to it. The proper interaction with the biotechnological devices is given by the use of a colour camera for object recognition, a force/torque sensor to prevent damages and laser scanners for localisation. Furthermore necessary changes to the environment of the robot are kept to a minimum. By providing a scripting language, the robot system can be easily adapted to new devices and further tasks. By the use of this autonomous robot operating distinct devices, a fully automated sample management system was established which is available day and night.

Lysis-free separation of hybridoma cells by continuous disc stack centrifugation.

The non-destructive removal of hybridoma cells from fermentation broth with an improved disc stack centrifuge (CSA1, Westfalia Separator AG, Oelde, Germany) was investigated. The centrifuge was equipped with a hydrohermetic feed system, which allowed a gentle, shearless acceleration of the cells inside the bowl. No significant cell damage was observed during the separation of hybridoma cells from repeated batch fermentation in 100 liter scale. In the clarified liquid phase there was no increase in Lactate-Dehydrogenase (LDH) activity. Consequently, there was no increased exposure of the product to intracellular components.Due to continuous operation with a periodic and automatic discharge of sediment, a high throughput was achieved without any considerable loss of product. The clarification for mammalian cells was in the range of 99% to 99.9%, depending on the operating conditions. The content of cell debris and other small particles decreased about 30 to 50%, depending on the particle load in the feed stream. The centrifuge was fully contained; cleaning and sterilizing in place possible. Therefore, the decice could be integrated easily into the fermentation process.

The Use of Peptones as Medium Additives for High-Density Perfusion Cultures of Animal Cells

This paper describes the test of several new vegetarian hydrolysates (peptones of soy, rice, wheat gluten etc.) as protein-free medium supplements for the production of a recombinant therapeutic protein. Multiple peptone-supplemented, continuous perfusion bioreactor experiments were conducted. Cell specific rates and product quality studies were obtained for the various peptones and compared with peptone-free medium. It was found that peptones confer a nutritional benefit, especially at low dilution rates, for a recombinant BHK cell line used in this investigation. The specific productivity increased 20–30% compared to the peptone-free controls. However, this benefit was also fully delivered by using fortified medium in place of the peptone-enriched media.

The Effect of Bioreactor pH and Temperature on Protein Glycosylation in Perfusion Cultures of Mammalian Cells

Bioreactor pH, ranging from 6.7 to 7.2, was evaluated in several 2L perfusion bioreactors. Prolonged exposure to pH between 6.7 and 6.9 has no impact on alpha (1–3)-galactose levels for a rBHK cell line. However, prolonged exposure to high pH (pH > 7.0) significantly increased the alpha-gal level. Interestingly, the recombinant protein alpha-gal level correlated qualitatively with the cell surface alpha-gal levels measured by FACS and the gene expression of alpha-1,3- galactosyltransferase measured by RT-PCR. This indicates that the effects of bioreactor culture pH on alpha-gal levels are not specific to the recombinant protein. Furthermore lowering the cultivation temperatures from 35.5°C (std. conditions) to 34.0°C significantly reduced the terminal sialic acid content by about 10%, while the overall glycosylation profile was unchanged.