Design of a bubble-swarm bioreactor for animal cell culture

Gudermann F, Lütkemeyer D, Lehmann J. Design of a bubble-swarm bioreactor for animal cell culture. Cytotechnology. 1994;15(1-3):301-309.A stationary bubble-swarm has been used to aerate a mammalian cell culture bioreactor with an extremely low gas flow rate. Prolonging the residence time of the gas bubbles within the medium improved the efficiency of the gas transfer into the liquid phase and suppressed foam formation. An appropriate field of speed gradients prevented the bubbles from rising to the surface. This aeration method achieves an almost 90% transfer of oxygen supplied by the bubbles. Consequently, it is able to supply cells with oxygen even at high cell densities, while sparging with a gas flow of only 0.22 x 10(-3) -1.45 x 10(-3) vvm (30-200 ml/h). The reactor design, the oxygen transfer rates and the high efficiency of the system are presented. Two repeated batch cultures of a rat-mouse hybridoma cell line are compared with a surface-aerated spinner culture. The used cell culture medium was serum-free, either with or without BSA and did not contain surfactants or other cell protecting agents. One batch is discussed in detail for oxygen supply, amino acid consumption and specific antibody production

Rapid high-performance liquid chromatographic quantification of recombinant human antithrombin III during production and purification

Büntemeyer H, Tebbe H, Lütkemeyer D, Lehmann J. Rapid high-performance liquid chromatographic quantification of recombinant human antithrombin III during production and purification. Journal of Chromatography, B: Biomedical Sciences and Applications. 1994;662(2):209-216.For monitoring of recombinant human antithrombin III during cell culture processes and subsequent purification steps a rapid method for quantitative determination was developed. The need for the introduction of this rapid method came from the limited availability of a quantitative enzyme-linked immunosorbent assay (ELISA) and the very time-consuming ELISA procedure. The developed method is based on reversed-phase high-performance liquid chromatography using a C 4 column. The separation by gradient elution using water and acetonitrile takes less than 20 min even when complex samples, such as serum containing cell culture samples, have to be analyzed. Automation and a high sample throughput are possible with this reliable method. If necessary, insulin, transferrin and albumin can also be quantified with minor changes of the elution profile

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

Riese U, Lütkemeyer D, Heidemann R, Büntemeyer H, Lehmann J. Re-use of spent cell culture medium in pilot scale and rapid preparative purification with membrane chromatography. Journal of Biotechnology. 1994;34(3):247-257.Based on experiments in bench scale, a recycling of spent cell culture medium was performed in a 100-l 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

Continuous optical in-line glucose monitoring and control in CHO cultures contributes to enhanced metabolic efficiency while maintaining darbepoetin alfa product quality

Great efforts are directed towards improving productivity, consistency and quality of biopharmaceutical processes and products. One particular area is the development of new sensors for continuous monitoring of critical bioprocess parameters by using online or in-line monitoring systems. Recently, we developed a glucose biosensor applicable in single-use, in-line and long-term glucose monitoring in mammalian cell bioreactors. Now, we integrated this sensor in an automated glucose monitoring and feeding system capable of maintaining stable glucose levels, even at very low concentrations. We compared this fed-batch feedback system at both low (< 1 mM) and high (40 mM) glucose levels with traditional batch culture methods, focusing on glycosylation and glycation of the recombinant protein darbepoetin alfa (DPO) produced by a CHO cell line. We evaluated cell growth, metabolite and product concentration under different glucose feeding strategies and show that continuous feeding, even at low glucose levels, has no harmful effects on DPO quantity and quality. We conclude that our system is capable of tight glucose level control throughout extended bioprocesses and has the potential to improve performance where constant maintenance of glucose levels is critical. © 2021 The Authors. Biotechnology Journal published by Wiley-VCH Gmb

Investigating the dynamics of recombinant protein secretion from a microalgal host

Lauersen KJ, Huber I, Wichmann J, et al. Investigating the dynamics of recombinant protein secretion from a microalgal host. Journal of Biotechnology. 2015;215:62-71

The Super-Spinner: a low cost animal cell culture bioreactor for the CO 2 incubator

Heidemann R, Riese U, Lütkemeyer D, Büntemeyer H, Lehmann J. The Super-Spinner: a low cost animal cell culture bioreactor for the CO 2 incubator. Cytotechnology. 1994;14(1):1-9.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(R) 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 (OTR(max)) of the Super Spinner was detected. For this purpose one spinner flask was equipped with an oxygen electrode. The OTR(max) 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

Heidemann R, Lütkemeyer D, Büntemeyer H, Lehmann J. 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. CYTOTECHNOLOGY. 1998;26(3):185-197.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

Effects on growth behavior in continuous hybridoma cell cultures: The role of viral contamination

Hawerkamp A, Lütkemeyer D, Gudermann F, Falkenhain A, Büntemeyer H, Lehmann J. Effects on growth behavior in continuous hybridoma cell cultures: The role of viral contamination. CYTOTECHNOLOGY. 1998;28(1/3):19-29.This article describes the retrovirus expression with optimal nutrient supply and its potential growth inhibition effects in continuous hybridoma cell cultivation. A special reactor setup with total cell retention was developed to examine growth inhibition effects. Using this fermentation strategy we observed a decrease of viability cell rate which occurred at a defined state of the process despite sufficient nutrient supply. Therefore we assume that inhibitory substances are responsible for these effects. The molecular weight range of the inhibitory substances and the possible retrovirus cooperation of these growth inhibition effects were examined. To determine the molecular weight range we used the following methods: ultrafiltration, gelfiltration, ultracentrifugation and gel electrophore sis. Furthermore, RT-PCR and western-/immunoblot are used to detect retrovirus particles in the supernatant and to show a retrovirus participation on growth inhibition effects. The possible growth modulation was tested in a biological assay (MTT-assay)