Mapping and partial characterization of proteases expressed by a CHO production cell line.

The proteolytic activities expressed by a Chinese hamster ovary (CHO) cell line in the cultivation supernatant during the production of recombinant factor VIII were mapped with a broad spectrum protease assay and a series of different types of protease inhibitors. The destabilizing effect on the product emanated from a metalloproteinase, which could be effectively blocked by chelating agents to lead to product stabilization. Amino acid sequences of the isolated metalloproteinase were found to have sequence homology with matrix metalloproteinases (MMPs) MMP3, MMP10, and MMP12. Several species with metalloproteinase activity were characterized and found to be related to each other. The results indicate that an MMP pro-enzyme of >/=200 kDa was released from the CHO cells during the production phase. The enzyme expressed collagenase/gelatinase activity when activated. Due to autoproteolysis, a number of smaller, less specific MMPs were formed with the smallest form, a 19.4 kDa protein, being the most active. These results may be of particular relevance for other production processes using CHO cells for the expression of recombinant proteins.

[Continuously perfused cultivation of genetically-engineered CHO cells producing prothrombin in a modified Super-Spinner].

A Super-Spinner was Modified by mounting a stainless steel filter(pore size 75 microns) to the impeller shaft to retain cells while fresh nutrient is perfused. Using Macroporous microcarrier Cytopore 1, continuously perfused cultivation of a recombinant CHO cell line, CHO2DS producing prothrombin was performed with the perfusion of a protein-free medium DF6S. The cell retention rate was more than 90% during the 24 days continuously perfused cultivation. The viable cell density of CHO2DS and prothrombin concentration reached 4.62 x 10(6)(cells.m/L) and 11.3(mg/L) respectively after 9 days culture.

Capture of proteins from mammalian cells in pilot scale using different STREAMLINE adsorbents.

This presentation compares three different expanded bed matrices. STREAMLINE rProtein A, STREAMLINE SP-XL and STREAMLINE Chelating were monitored in respect to their ability to clarify the broth, to concentrate and to purify the distinct target protein. The capture of a mouse IgG1 and a recombinant prothrombin (PT) was carried out in pilot scale using a 100-l bioreactor and STREAMLINE 100 and 200 columns, respectively. The robustness of the process was also estimated monitoring the expansion behaviour and the cell and debris concentrations during the load and in the eluat. In all cases the capture of the target proteins was comparable to conventional chromatographic systems. The purification success was mainly dependent on the selectivity of the ligand used. The affinity process resulted in a highly purified product. The ion exchanger and chelating material mainly concentrated the product. In all three cases 100 l of cell broth were successfully processed in one run. The robustness of the ion exchanger process was poor, because of strong cell matrix interaction. However, for the chelating and especially for the affinity matrix a highly reproducible process was obtained.

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.

First steps in robot automation of sampling and sample management during cultivation of mammalian cells in pilot scale.

The robot automation of sampling and the subsequent treatment and storage of aliquots during mammalian cell cultivations was investigated. The complete setup, the development and testing of the sampling device, the robot arm, and the cell imaging system are described. The developed sampling device is directly coupled to a pilot bioreactor. It allows the computerized sterile filling of cell broth into 50 mL sample tubes. After each sampling the whole tubing system is steam sterilized. For further off-line treatment a robot takes the sample to the different devices. This robot is equipped with a camera and a force/torque sensor. A color-based object recognition guides the arm in a complex surrounding with different illumination situations, enabling the robot to load the sampling device with tubes and take the sample to further devices. For necessary pipetting and refilling we developed a computerized device. Cells are automatically stained and counted using an imaging system. The cell number and viability is automatically saved in a process control system together with the on-line parameters. During several cultivations in 20 and 100 L scale these main components of the automation strategy were successfully tested.

Influence of alterations in culture condition and changes in perfusion parameters on the retention performance of a 20 mum spinfilter during a perfusion cultivation of a recombinant CHO cell line in pilot scale.

Since 1969 much attention has been devoted to the useof spinfilter systems for retention of mammalian cellsin continuous perfusion cultivations. Previousinvestigations dealt with hydrodynamic conditions,fouling processes and upscaling. But hydrodynamicconditions and fouling processes seem to have asecondary importance in spinfilter performance duringauthentic perfusion cultivations. Obviously,alterations in culture condition are more relevantespecially during long-term processes. Therefore, ourpratical approach focussed on the performance qualityof a commercially available 20 mum spinfilterduring a perfusion cultivation of a recombinant CHOcell line in pilot scale regarding the followingissues: 1) retention of viable cells in thebioreactor; 2) removal of dead cells and cell debrisfrom the bioreactor; 3) alterations in culturecondition; and 4) changes in perfusion mode.Furthermore, we tested the performance of 20 mumspinfilters in 2 and 100 l pilot scale using solidmodel particles instead of cells. Our investigationsshowed that retention of viable cells in pilot scalewas independent of spinfilter rotation velocity andperfusion rate; the retention increased from 75 to 95%corresponding to operation time, enlarging celldiameter and enhanced formation of aggregates in theculture during the perfusion cultivation. By means ofthe Cell Counter and Analyzer System (CASY) anoperation cut off of 13 mum was determined forthis spinfilter. Using solid model particles in 2 lscale, optimal retention was achieved at a tip speedof 0.43 m s(-1) (141 rpm) - furtherenhancement of spinfilter rotation velocity up to0.56 m s(-1) (185 rpm) decreased the retentionrapidly. In pilot scale best retention performance wasobtained with tip speeds of 0.37 m s(-1)(35 rpm) and 1.26 m s(-1) (120 rpm). Hence,significant retention in pilot scale could already beachieved with low agitation. Therefore, the additionof shear force protectives could be avoided so thatthe purification of the target protein from thesupernatant would be facilitated.

Estimation of cell damage in bench- and pilot-scale affinity expanded-bed chromatography for the purification of monoclonal antibodies.

The first step in downstream processing of mammalian cell culture includes the separation of the cells without cell damage to avoid the release of intracellular enzymes, which could potentially cause proteolytic degradation of the target protein and may increase the impurities for further chromatographic steps. This especially includes the reduction of host DNA for therapeutic proteins. The aim of this investigation was to examine the extent of cell damage at the bench and pilot scale using a stabilized fluidized bed (expanded bed) for direct recovery of IgG from cell culture broth. For this purpose, Streamline-25 and -200 columns containing 75 mL and 5 L of rProtein A matrix, respectively, were used. The repeated batch cultivations resulted in high cell viabilities of about 90% prior purification. The pH was gently adjusted to pH 8 before the broth was applied to the gel. In bench scale, 1 to 6 L of unclarified feed was applied to the Streamline-25 column. In pilot scale, up to 95 L was processed using the Streamline-200 column. The antibodies from 95 L of unclarified feed were recovered after approximately 1.5 h. The possible cell damage, caused either by the equipment or by the cells' passage through the expanded bed, was detected by the following assays: microscopic count of the cells using trypan blue dye exclusion to determine viability; monitoring of intracellular components (i.e., DNA concentration); activity of lactate dehydrogenase (LDH); and, finally, the particle load in the flow through and the eluate. Despite the sensitivity of hybridoma cells to shear forces, neither the high flow rate (300 to 450 cm/h) nor the passage of the cells through the expanded bed caused any relevant cell damage or clogging of the gel. Excellent DNA depletion was observed.

Pilot scale recovery of monoclonal antibodies by expanded bed ion exchange adsorption.

The aim of the investigations was to estimate the scale up properties of an efficient chromatographic first capture step for the recovery of murine IgG1 from undiluted and unclarified hybridoma cell culture broth using an ion exchange matrix in expanded bed mode. The tested new sulfopropyl-based ion exchange matrix (Streamline SP XL, Amersham Pharmacia Biotech) stands out due to its enhanced capacity compared to its precursor (Streamline SP). Defining the working pH in preliminary electrophoretic analyses (titration curve, SDS-PAGE) and small-scaled chromatographic binding studies showed, that the optimal value for the IgG purification was pH 4.6, where a co-chromatography of the medium supplement albumin (500 mg l-1, pI = 4.8) could not be avoided. Further scouting experiments dealt with the dynamic capacity of the matrix, which was evaluated by frontal adsorption analysis. In packed bed mode no break-through of the target protein was achieved even after 6.5 mg IgG per ml matrix were applied. These results could not be reproduced in expanded bed mode with cell-free supernatant, where the dynamic capacity was found to be only 1.5 mg IgG/ml SP XL. Processing cell-containing broth resulted in an additional decrease of the value down to 0.5 mg ml-1, presumably caused by the remarkable biomass adsorption to the matrix. The search for the reasons led to the examination of the hydrodynamic conditions. Buffer experiments with a tracer substance (acetone) pointed out, that the flow in expanded bed was significantly more influenced by back-mixing effects and channel formations than in packed bed. These effects could be compensated with an enhanced viscosity of the liquid phase, which was achieved by the addition of glucose. As a result of the improved hydrodynamic conditions in the expanded bed, the dynamic capacity could be increased from 0.5 to more than 4.5 mg IgG/ml matrix for the processing of cell culture broth with 400 mM glucose. Finally, the scale up from a Streamline 25 to a Streamline 200 column was performed under conditions, which proved to be optimal: 100 L of unclarified hybridoma broth were concentrated with a binding rate of 95% in less than 3.5 hours. Loading the column no break-through of the target protein was achieved. However, the eluate still contained debris and cells, which points out the major disadvantage of the method: the biomass attachment to the matrix.

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

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 electrophoresis. 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).

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 K(S)-values for the essential amino acids were calculated using Monod equations via computer simulation. The K(S)-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.

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.

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.

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 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.

Design of a bubble-swarm bioreactor for animal cell culture.

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.

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.

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.