Biomedical subjects

H Büntemeyer

Publications and source records attributed to H Büntemeyer.

15 recordsLinked to original sources

PubMed2001

An automatic system for the assessment of complex medium additives under cultivation conditions.

Complex medium additives such as yeast extract or peptone are often used in industrial cell culture processes to prolong cell growth and/or to improve product formation. The quality of those supplements is dependent on the preparation method and can differ from lot to lot. To guarantee consistent production these different lots have to be tested prior to use in fermentation processes. Because a detailed qualitative and quantitative analysis of all components of such a complex mixture is a very difficult task, another assessment method has to be chosen. The best way to evaluate the effect of such supplements is to monitor cell activity during real cultivation conditions with and without the added supplement lot. A bioreactor-based test system has been developed to determine the oxygen requirement of the cells as a response to the addition of a supplement to be tested under standardized conditions. Investigations were performed with a mouse-mouse hybridoma cell line and yeast extracts as an example for complex medium additives. The results showed differences in the impact between different extract lots and between different concentrations of an extract.

Animals↗

PubMed2000

Cultivation of immortalized human hepatocytes HepZ on macroporous CultiSpher G microcarriers.

Cultivation of the new immortalized hepatocyte cell line HepZ was performed with a 1:1 mixture of DMEM and Ham's F12 media containing 5% FCS. The cells were grown in their 40th passage in 100 mL and 1 L volumes in spinner flasks and in a bioreactor, respectively. For the production of adherently growing HepZ cells macroporous CultiSpher G gelatin microcarriers were used in various concentrations from 1 to 3 g/L. The cells were seeded in a density of 2 x 10(5) cells/mL when using a microcarrier concentration of 1 g/L and 5 x 10(5) cells/mL at a microcarrier concentration of 3 g/L. After 7 days of cultivation a maximum cell concentration of 4.5 x 10(6) cells/mL was obtained in the spinner culture using a microcarrier concentration of 1 g/L. With bubble-free aeration and daily medium exchange from day 7, 7.1 x 10(6) cells/mL were achieved in the bioreactor using a microcarrier concentration of 3 g/L. The cells exhibited a maximum specific growth rate of 0.84 per day in the spinner system and 1.0 per day in the bioreactor, respectively. During the growth phase the lactate dehydrogenase (LDH) activity rose slightly up to values of 200 U/L. At the end of cultivation the macroporous carriers were completely filled with cells exhibiting a spherical morphology whereas the hepatocytes on the outer surface were flat-shaped. Concerning their metabolic activity the cells predominantly consumed glutamine and glucose. During the growth phase lactate was produced up to 19.3 mM in the spinner culture and up to 9.1 mM in the bioreactor. Maximal oxygen consumption was 1950 nmol/(10(6) cells. day). HepZ cells resisted a 4-day long chilling period at 9.5 degrees C. The cytochrome P450 system was challenged with a pulse of 7 microgram/mL lidocaine at a cell density of 4.5 x 10(6) cells/mL. Five ng/mL monoethylglycinexylidide (MEGX) was generated within 1 day without phenobarbital induction compared to 26 ng/mL after a preceded three day induction period with 50 microgram/mL of phenobarbital indicating hepatic potency. Thus, the new immortalized HepZ cell line, exhibiting primary metabolic functions and appropriate for a mass cell cultivation, suggests its application for a bioartificial liver support system.

Amino Acids, Branched-Chain↗

PubMed1999

Cultivation and characterization of a new immortalized human hepatocyte cell line, HepZ, for use in an artificial liver support system.

The new human hepatocyte cell line HepZ was investigated with regard to use it for a mass cell cultivation. The cells were originally derived from a human liver biopsy and immortalized through lipofectamine-mediated transfection of albumin-promotor-regulated antisense constructions against the negative controlling cell cycle proteins Rb and p53 (pAlb asRb, pAIb asp53). Furthermore, plasmids including genes coding for the cellular transcription factor E2F and D1 cyclin (pCMV E2F, pSV2neo D1) were cotransfected to overcome the G1-restriction point. Cell cultivation was performed in a 2-liter bioreactor with a working volume of 1 liter. With CultiSpher G microcarriers used in a concentration of 3 g/l a maximal density of 7.1 x 10(6) cells/ml was achieved in a cultivation period of 20 days. The cells exhibited a maximal specific growth rate of 1.0 per day in the first 4 days. After 9 days of cultivation the stationary growth phase was reached with an average cell density of 5.5 x 10(6) cells/ml. The viability status of the culture was determined indirectly by measuring of the lactate dehydrogenase activity (LDH) at 37 degrees C. During the growth phase the activity rose slightly up to a value of 200 U/l. The cells were flat after first attachment on the gelatine microcarriers and spherical after growing into the three-dimensional inner matrix--both of which characteristics were verified by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The liver-specific cytochrome P450 activity was challenged with a pulse of 7 micrograms/ml lidocaine at a cell density of 4.5 x 10(6) cells/ml. After an induction period of 3 days with 50 micrograms/ml of phenobarbital, 26 ng/ml MEGX were generated within one day compared to 5 ng/ml without induction. The new cell line HepZ has proven to retain liver-specific qualities and to be appropriate for mass cell cultivation for bioartificial devices.

Albumins↗

PubMed1994

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

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 C4 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.

Animals↗

PubMed1994

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.

Animals↗

PubMed1994

Evaluation of membranes for use in on-line cell separation during mammalian cell perfusion processes.

In this study two microporous hollow fibre membranes were evaluated for their use as cell retention device in continuous perfusion systems. A chemically modified permanent hydrophillic PTFE membrane and a hydrophilized PP membrane were tested. To investigate the filtration characteristic under process conditions each membrane was tested during a long term perfusion cultivation of a hybridoma cell line. In both cultivations the conditions influencing membrane filtration (e.g. transmembrane flux) were kept constant. Filtration behaviour was investigated by monitoring transmembrane pressure and protein permeability. Transmembrane pressure was measured on-line with an autoclavable piezo-resistive pressure sensor. Protein permeability was determined by quantitative evaluation of unreduced, Coomassie stained SDS-PAGE. The membrane fouling process influences the filtration characteristic of both membranes in a different way. After fermentation the PP membrane was blocked by a thick gel layer located in the big outer pores of the asymmetric membrane structure. The hydraulic resistance was higher but the protein permeability was slightly better than of the PTFE membrane. For this reason the PP membrane should be preferred. On the other hand, transmembrane pressure decreases slower when the PTFE membrane is used, which favours this membrane for long term cultivations, especially when low molecular weight proteins (< 30 KD) are produced.

Animals↗

PubMed1994

A direct computer control concept for mammalian cell fermentation processes.

In the last 10 years, new assignments and the special demands of mammalian cells to the culture conditions caused the development of complex small scale fermentation setups. The use of continuous fermentation and cell retention devices requires appropriate process control systems. An arrangement for control and data-acquisition of complex laboratory-scale bioreactors is presented. The fundamental idea was the usage of a standard personal computer, which is connected to pumps, valves and sensors via ADA-transformation. The possibility of free programming allowed the development of user-oriented software, especially designed for the far-reaching requirements of a university laboratory in the field of animal cell culture. Control of aeration, pumps, data-acquisition and data-storage are combined within one program, which allows the automation of standard operations like measurement of kLa- or OTR-values. Pump control algorithms for all common fermentation strategies (batch, fed batch, chemostat, perfusion) are included and can be selected any time during cultivation. Oxygen partial pressure and pH are controlled via direct digital control (ddc), providing simple adaption of control parameters and set points to current fermentation conditions.

Animals↗

PubMed1994

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.

Animals↗

PubMed1993

Structural characterization of gangliosides from resting and endotoxin-stimulated murine B lymphocytes.

B lymphocytes from CBA/J mice were stimulated in splenocyte cultures for 72 h with various endotoxins. Bisphosphoryl lipid A from Escherichia coli had the highest stimulatory effect followed by LPS of Citrobacter freundii and Salmonella minnesota as measured by [3H]thymidine uptake. Gangliosides of stimulated B cells (metabolically labeled with D-[1-14C]galactose and D-[1-14C]glucosamine) and unlabeled gangliosides from resting B cells (prepared from spleens without stimulus) were analyzed by high-performance TLC, DEAE anion-exchange HPLC, and immunostaining procedures. Contents of ganglioside-derived sialic acids, quantified by HPLC as their fluorescent derivatives, decreased from stimulated to resident B lymphocytes in the following order: LPS S. minnesota > LPS C. freundii > bisphosphoryl lipid A E. coli > resting B cells. Gangliosides of resting B cells contained more N-glycolyl- than N-acetylneuraminic acid, whereas inverse ratios were found in activated cells, indicating a shift from N-glycolyl- to N-acetylneuraminic acid due to stimulation. Furthermore, a higher disialoganglioside content was characteristic for activated B cells. Fast atom bombardment mass spectrometry was performed with permethylated mono- and disialoganglioside fractions of LPS S. minnesota and LPS C. freundii stimulated B cells. Major gangliosides were GM1a and GD1a beside minute amounts of GD1b. The structural heterogeneity in the gangliosides was caused by (a) N-substitution of the sialic acids with either acetyl or glycolyl groups, (b) variation in the long-chain base (sphingosine, sphinganine), and (c) substitution of the ceramide moiety by fatty acids of different chain length and degree of unsaturation (C16:0, C24:0,24:1).2+ p6

Animals↗

PubMed1993

Membrane chromatography for rapid purification of recombinant antithrombin III and monoclonal antibodies from cell culture supernatant.

The task of purifying monoclonal antibodies (MAbs) and human recombinant antithrombin III (rATIII) from cell culture supernatant was carried out using two different approaches, both based on the use of membraneous matrices. The first approach employed a strongly acidic and a strongly basic membrane ion exchanger, which were evaluated for their ability to purify monoclonal antibodies and the human active recombinant antithrombin III from cell culture supernatant. Within minutes gram amounts of product could be purified in a high-flux system, specially developed for this purpose, achieving purities of 80% for MAbs and 75% for rATIII, respectively. The capacity of the acidic membrane ion exchanger for MAbs was found to be 1 mg/cm2 with recoveries up to 96% and that of the basic membrane ion exchanger for rATIII was 0.15 mg/cm2 with recoveries up to 91%. The second approach consisted of using heparin, a mucopolysaccharide with a strong affinity towards ATIII, coupled to amine-modified or epoxy-activated membranes by reductive amination, for the purification of rATIII. The ATIII binding capacities of the membranes were found to be 91 micrograms/cm2 for the amine-modified and 39 micrograms/cm2 for the epoxy-activated membrane, achieving purities of 75%. The coupling proved to be fairly stable over a period of 5 months and the membranes remained operable even after steam sterilization and treatment with sodium dodecyl sulphate. Final purification in both instances was carried out by gel filtration.

Animals↗

PubMed1992

Optimal medium use for continuous high density perfusion processes.

For maintenance of high cell density in continuous perfusion processes not only feeding with substrates but also removal of inhibitors and toxic waste products are of special interest. High perfusion rates cause large volumes of product containing medium which have to be processed in product isolation. In order to minimize these volumes concentrated feed solutions of optimized medium are used. On the other hand, such media may cause high concentrations of toxic or inhibitory metabolites which can negatively influence cell growth and product formation. Especially, if the spent medium (or special parts of it) is used again after product isolation, the removal or even better the control of inhibitor production is of highest importance. We have developed a continuous fermentation concept and system (continuous medium cycle bioreactor, cMCB) in which both limitation and inhibition effects can be generated to identify special substances as limiting or inhibitory components. With the results from those experiments it was possible to lower the total perfusion rate during serum-free perfusion cultures of hybridoma cells and to obtain an optimal substrate utilization. The advantages for decreasing the production costs (for media, special supplements and product isolation) are obvious. The other aim of this study was to identify secreted metabolic waste products as inhibitor or toxic metabolite.

Animals↗

PubMed1991

The medium cycle bioreactor (MCB): monoclonal antibody production in a new economic production system.

The perfusion mode of a continuous cell culture bioreactor was modified to establish a closed loop system. Eighty percent of the spent medium was re-used twice. The medium cycle bioreactor unit was operated sterile and uncomplicated without a technical retention system for the high molecular weight substances. Therefore, only 20% of the actual medium was necessary to run the recycling process. During seven days culture time in a two liter scale 5 grams of IgG1 type monoclonal antibody was produced. During that period the cell specific productivity was constant. Renewal of proteins was omitted because the protein content in the system persisted at a high level. Therefore, self-conditioning substances of the cells were retained in the system as well as the expensive medium components (proteins with catalytic or stimulating function). Seventy to 80% of medium costs and medium quantity were saved for each medium recycling step. Only cheap metabolites that are consumed by the cells had to be supplemented. Uptake rates of glucose and amino acids were calculated to establish a suitable supplementation mixture for the recirculated medium.

Animals↗

PubMed1991

Optimization of serum-free fermentation processes for antibody production.

Serum free fermentation procedures of cell cultures have got a wide application in production of biochemicals. But, cells cultured in serum free media in general are more sensitive to changes in culture condition, especially to nutrient limitation. There are no substances from serum which can support the cells when conditions are changing. In this study special attention is directed to amino acid utilization of mouse hybridoma in batch, chemostat and perfusion fermentations. Detailed data are presented which show the considerable difference of amino acid consumption rates in different fermentation modes. Already, in batch mode there are differences of the two investigated mouse hybridoma cell lines, although they are derived from the same myeloma line. In chemostat running at a dilution rate representing maximal growth rate most of the consumption rates are significant higher than in batch. On the other hand, in perfusion mode the rates are lower than in batch. This indicates clearly the different conditions of the fermentation modes. Therefore, it is necessary to develop serum free processes under the desired production conditions. An accurate analysis of the process is strongly recommended.

Amino Acids↗

PubMedDate not supplied

Development of serum-free bioreactor production of recombinant human thyroid stimulating hormone receptor.

For the detection of autoantibodies to thyroid stimulating hormone receptors (TSH-R) in Graves' disease based on a novel coated tube assay system, human TSH-R is needed in large amounts. Whereas expression of TSH-R in bacteria, yeast, or insect cells results in nonfunctional, denaturated receptor, mammalian cells such as COS, CHO, and HeLa are able to express functional TSH-R, but only in very low amounts. Furthermore, for all of these cultivations expensive standard media containing 10% fetal calf serum are needed to obtain functional receptor. Here we report on the development of a serum-free production-scale process based on a stable transformed and highly productive human leukemia cell line K562 (1). Starting with K562-TSH-R cells growing in medium containing 10% fetal calf serum the cell line was adapted to serum-free medium. The adaptation medium was optimized in regards to amino acid and protein concentrations, since the use of unadjusted medium caused cell death after 2 days. The adapted cells were stable and could be cultivated without antibiotics for more than 50 cell doublings without losing their productivity. The obtained receptor showed improved TSH binding. The process development was based on cultivations in a 2-L bench-scale bioreactor. Cultivations in batch mode and chemostat mode and perfusion cultivation with the usage of an internal microfiltration device and a spin-filter device were compared. After process optimization a continuous process using spin-filter was set up and run in a 20 L-pilot-scale bioreactor. The presented results were the prerequisite for the production of the novel assay for the diagnosis of autoantibodies to TSH-R in Graves' disease.

Bioreactors↗

PubMedDate not supplied

Sialidase activity in culture fluid of Chinese hamster ovary cells during batch culture and its effects on recombinant human antithrombin III integrity.

Sialidase activity in cell-free supernatant of batch-cultivated Chinese hamster ovary (CHO) cells producing human recombinant antithrombin III (rhAT III) was monitored during cultivation using 4-methylumbelliferyl substrate and HPLC for free sialic acid determination. Supernatant sialidase as well as lactate dehydrogenase activity increased significantly during batch growth. The enhanced number of dead cells correlated with increasing sialidase activity which seemed to be principally due to cell lysis, resulting in release of cytosolic sialidase. Loss of terminally alpha (2-->3) bound sialic acids of the oligosaccharides of rhAT III was analyzed in lectin-based Western blot and enzyme-linked lectin assays, using Maackia amurensis and Datura stramonium agglutinins for specific determination of Neu5Ac alpha (2-->3)Gal- and Gal beta (1-->4)-GlcNAc-terminated glycoproteins, respectively. Results show a remarkable loss of terminal sialic acids of rhAT III along with decrease in CHO cell viability and concomitant increase of dead cells throughout long-term batch cultivation. To avoid this degradation effect, process parameters forcing high viability are essential and harvesting of culture at an early time even at suboptimal recombinant protein concentration is highly recommended to avoid product desialylation.

Animals↗