Heino Büntemeyer

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.

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.

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 Hams 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.

Sialidase activity in culture fluid of Chinese hamster ovary cells during batch culture and its effect 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 α(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 Daturastramoniumagglutinins for specific determination of Neu5Acα(2→3)Gal‐ and Galβ(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 concentrations is highly recommended to avoid product desialylation.

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.

Fast filtration for metabolome sampling of suspended animal cells

A new method for sampling suspended animal cells by fast filtration is presented that allows rapid quenching of cellular metabolism and efficient separation of the cells from culture medium. Compared to sampling with a microstructure heat exchanger or centrifugation without prior quenching, the adenylate energy charge and the measured concentrations especially of metabolites with a high turnover rate or of metabolites early in metabolic pathways were substantially higher. No leakage of ATP from the cells was observed when using iso-osmotic NaCl solution in the washing step. The combination of fast filtration and cold methanol extraction is therefore suitable for intracellular metabolomic studies of suspended animal cell cultures and superior to other methods currently applied.

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.

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.