Friedemann Hesse

A novel cultivation technique for long-term maintenance of bloodstream form trypanosomes in vitro

We used an axenic cultivation system to grow African trypanosomes in vitro. Long-term cultivation for more than 60 days has been achieved by replacing the culture medium at regular intervals between 6 and 48 h. In contrast to a control culture without medium replacement, increasing amounts of maximum cell concentrations have been obtained, ranging from 5 x 10(6) to 2 x 10(7) trypanosomes ml-1, whereas the generation doubling time remained constant (about 6 h). Higher cell concentrations have only been obtained by total medium replacement; neither addition of fresh medium nor serum led to a higher cell yield, suggesting that a trypanosome-derived factor or metabolite accumulated in the medium rather than medium was depleted of an essential nutrient. Most interestingly, however, successive waves have been obtained which eventually led to a damped oscillation curve with a constant high population density after about 40 days of cultivation. Cultures were started with a homogeneous population of the long-slender form. As judged by light microscopy, cells showed a stumpy morphology during the declining phase and became slender again in the following growth phase. At later time points, when cells remained in a stationary phase at high population density, many different morphological stages have been observed, similar to those described by early authors as intermediate forms [Ormerod, W. E. (1979) In: Biology of the Kinetoplastida, Vol. 2, pp. 340-393], although many dividing forms are still present at that time. In contrast, identically treated procyclic cultures were unable to produce cyclic growth waves. Based on these results, a novel concept considering a possible differentiation mechanism is discussed.

Functional analysis of the broadly neutralizing human anti-HIV-1 antibody 2F5 produced in transgenic BY-2 suspension cultures

We report the production of an important human therapeutic antibody in plant cell suspension cultures and the functional analysis of that antibody, including a comparison with the same antibody produced in CHO cells. We established transgenic tobacco BY2 suspension cell cultures expressing the human monoclonal antibody 2F5, which shows broadly neutralizing activity against HIV‐1. The antibody was directed to the endoplasmic reticulum of the plant cells and was isolated by cell disruption, followed by protein A chromatography. The plant‐derived antibody was shown to be largely intact by SDS‐PAGE and immuno‐blot. Antigen binding activity was investigated by elec‐trophoretic mobility shift assay and quantitatively determined by ELISA and Biacore biosensor technology. Ligand binding properties were analyzed using the ectodomain of human FcγRI for kinetic analysis. The plant‐derived antibody showed similar kinetic properties and 89% of the binding capacity of its CHO‐derived counterpart, but was only 33% as efficient in HIV‐1 neutralization assays. Our results show that plant suspension cultures can be used to produce human antibodies efficiently and that the analysis methods used in this study, including biosensor technology, provide useful functional data about antibody performance. This highlights important issues raised by the use of plant systems to produce human biologics.—Sack, M., Paetz, A., Kunert, R., Bomble, M., Hesse, F., Stiegler, G., Fischer, R., Katinger, H., Stoeger, E., Rademacher, T. Functional analysis of the broadly neutralizing human anti‐HIV‐1 antibody 2F5 produced in transgenic BY‐2 suspension cultures. FASEB J. 21, 1655–1664 (2007)

Developments and improvements in the manufacturing of human therapeutics with mammalian cell cultures.

During recent years, biopharmaceutical products manufactured by processes that use mammalian cell cultures have gained increasing importance. At the same time, a strong awareness of the importance of the safety and quality of such products has also emerged. This has led to improvements in cultivation and production technology, validation procedures and process organization.

How can measurement, monitoring, modeling and control advance cell culture in industrial biotechnology?

This report highlights the potential of measurement, monitoring, modeling and control (M(3) C) methodologies in animal and human cell culture technology. In particular, state-of-the-art of M(3) C technologies and their industrial relevance of existing technology are addressed. It is a summary of an expert panel discussion between biotechnologists and biochemical engineers with both academic and industrial backgrounds. The latest ascents in M(3) C are discussed from a cell culture perspective for industrial process development and production needs. The report concludes with a set of recommendations for targeting M(3) C research toward the industrial interests. These include issues of importance for biotherapeutics production, miniaturization of measurement techniques and modeling methods.

Transcriptional profiling of phenotypically different Epo‐Fc expressing CHO clones by cross‐species microarray analysis

Chinese hamster ovary (CHO) cells exhibit large variabilities regarding growth, recombinant protein production and post‐translational processing during cell line development and clone selection. To accelerate the development of stable high quality cell factories, new efficient strategies for cell screening and clone selection are required. In our work, we combined phenotypic characterisation of recombinant CHO clones during early cell line development with transcription profile analysis using cross‐species microarrays. The objective was to identify genes or gene patterns that correlate with clone specific alterations in terms of productivity, sialylation capacity and stress resistance. In all high producer clones transcriptional profiling revealed a common enrichment of gene ontology categories related to protein metabolism, transcription, nucleus and nucleolus, whereas no common genes were differentially regulated in clones showing higher sialylation capacities. Furthermore, we identified predictive stress‐related marker genes that were up‐regulated in one clone without showing the corresponding phenotype at an early stage of development. Thus, we successfully applied gene expression profiling to allocate transcriptomal differences to specific phenotypes that changed during cell line development. These promising results will further increase our efforts to develop CHO specific microarrays that deliver information about the suitability of a clone candidate for industrial production.

miR-2861 as novel HDAC5 inhibitor in CHO cells enhances productivity while maintaining product quality.

Histone deacetylase (HDAC) inhibitors have been exploited for years to improve recombinant protein expression in mammalian production cells. However, global HDAC inhibition is associated with negative effects on various cellular processes. microRNAs (miRNAs) have been shown to regulate gene expression in almost all eukaryotic cell types by controlling entire cellular pathways. Since miRNAs recently have gained much attention as next‐generation cell engineering tool to improve Chinese hamster ovary (CHO) cell factories, we were interested if miRNAs are able to specifically repress HDAC expression in CHO cells to circumvent limitations of unspecific HDAC inhibition. We discovered a novel miRNA in CHO cells, miR‐2861, which was shown to enhance productivity in various recombinant CHO cell lines. Furthermore, we demonstrate that miR‐2861 might post‐transcriptionally regulate HDAC5 in CHO cells. Intriguingly, siRNA‐mediated HDAC5 suppression could be demonstrated to phenocopy pro‐productive effects of miR‐2861 in CHO cells. This supports the notion that miRNA‐induced inhibition of HDAC5 may contribute to productivity enhancing effects of miR‐2861. Furthermore, since product quality is fundamental to safety and functionality of biologics, we examined the effect of HDAC inhibition on critical product quality attributes. In contrast to unspecific HDAC inhibition using VPA, enforced expression of miR‐2861 did not negatively influence antibody aggregation or N‐glycosylation. Our findings highlight the superiority of miRNA‐mediated inhibition of specific HDACs and present miR‐2861 as novel cell engineering tool for improving CHO manufacturing cells. Biotechnol. Bioeng. 2015;112: 2142–2153.

A study on the temperature dependency and time course of the cold capture antibody secretion assay.

The cold capture assay as described by Brezinsky et al. [Brezinsky, S.C.G., Chiang, G.G., Szilvasi, A., Mohan, S., Shapiro, R.I., MacLean, A., Sisk, W., Thill, G., 2003. A simple method for enriching populations of transfected CHO cells for cells of higher specific productivity. J. Immunol. Methods 277, 141-155] stands out as the most simple of single cell secretion assays which can be used to sort for high productivity in recombinant cell lines. At low temperatures the process of protein release from transport vesicles is assumed to be delayed as both vesicle fusion and product release is slowed, so that secreted proteins can be stained on the cell surface using a fluorescent antibody. Typically, the fluorescent signal obtained correlates to the cell specific production rate of the analysed cell. In the present study we compared staining of human antibody producing CHO cells performed at different temperatures and we observed the fluorescent signal over 24h. We found that the staining temperature did not influence signal intensity. The fluorescent signal was stable for 24h at 4 degrees C, decreased to 80% at room temperature (21 degrees C), while it decreased significantly already after 2h at 37 degrees C. Initially, the fluorescent signal was observed on the cell surface, however, at later stages it was found in compartments in the cytoplasm. Finally we compared differences in signal stability depending on whether the antibody used for staining bound to the light or heavy chain of the product and on whether the fluorescent label was a relatively stable protein (phycoerythrin) or a pH-dependent small molecule (FITC). Our results indicate that the secreted product is trapped by the staining antibody on the cell surface at all temperatures. Subsequently these aggregates are endocytosed by the cells, a process which is slowed down at low temperatures.

Identification of a novel temperature sensitive promoter in cho cells

BackgroundThe Chinese hamster ovary (CHO) expression system is the leading production platform for manufacturing biopharmaceuticals for the treatment of numerous human diseases. Efforts to optimize the production process also include the genetic construct encoding the therapeutic gene. Here we report about the successful identification of an endogenous highly active gene promoter obtained from CHO cells which shows conditionally inducible gene expression at reduced temperature.ResultsBased on CHO microarray expression data abundantly transcribed genes were selected as potential promoter candidates. The S100a6 (calcyclin) and its flanking regions were identified from a genomic CHO-K1 lambda-phage library. Computational analyses showed a predicted TSS, a TATA-box and several TFBSs within the 1.5 kb region upstream the ATG start signal. Various constructs were investigated for promoter activity at 37°C and 33°C in transient luciferase reporter gene assays. Most constructs showed expression levels even higher than the SV40 control and on average a more than two-fold increase at lower temperature. We identified the core promoter sequence (222 bp) comprising two SP1 sites and could show a further increase in activity by duplication of this minimal sequence.ConclusionsThis novel CHO promoter permits conditionally high-level gene expression. Upon a shift to 33°C, a two to three-fold increase of basal productivity (already higher than SV40 promoter) is achieved. This property is of particular advantage for a process with reduced expression during initial cell growth followed by the production phase at low temperature with a boost in expression. Additionally, production of toxic proteins becomes feasible, since cell metabolism and gene expression do not directly interfere. The CHO S100a6 promoter can be characterized as cold-shock responsive with the potential for improving process performance of mammalian expression systems.

Comparison of a Production Process in a Membrane-Aerated Stirred Tank and up to 1000-L Airlift Bioreactors Using BHK-21 Cells and Chemically Defined Protein-Free Medium

The applicability of a protein‐free medium for the production of recombinant human interleukin‐2 with baby hamster kidney cells in airlift bioreactors was investigated. For this purpose, a BHK‐21 cell line, adapted to grow and produce in protein‐free SMIF7 medium without forming spheroids in membrane‐aerated bubble‐free bioreactors, was used as the producer cell line. First, cultivation of the cells was established at a 20‐L scale using an internal loop airlift bioreactor system. During the culturing process the medium formulation was optimized according to the specific requirements associated with cultivation of mammalian cells under protein‐free conditions in a bubble‐aerated system. The effects of the addition of an antifoam agent on growth, viability, productivity, metabolic rates, and release of lactate dehydrogenase were investigated. Although it was possible to establish cultivation and production at a 20‐L scale without the use of antifoaming substances, the addition of 0.002% silicon‐oil‐based antifoaming reagent improved the cultivation system by completely preventing foam formation. This reduced the release of lactate dehydrogenase activity to the level found in bubble‐free aerated stirred tank membrane bioreactors and led to a reduction in generation doubling times by about 5 h (17%). Using the optimized medium formulation, cells were cultivated at a 1000‐L scale, resulting in a culture performance comparable to the 20‐L airlift bioreactor. For comparison, cultivations with protein‐containing SMIF7 medium were carried out at 20– and 1000‐L scales. The application of protein supplements did not lead to a significant improvement in the cultivation conditions. The results were also compared with experiments performed in a bubble‐free aerated stirred tank membrane bioreactor to evaluate the influence of bubbles on the investigated culture parameters. The data implied a higher metabolic activity of the cells in airlift bioreactors with a 150% higher glucose consumption rate. The results of this study clearly demonstrate the applicability of a protein‐free chemically defined medium for the production of recombinant proteins with BHK cells in airlift bioreactors.

miRNA profiling of high, low and non-producing CHO cells during biphasic fed-batch cultivation reveals process relevant targets for host cell engineering

Fed-batch cultivation of recombinant Chinese hamster ovary (CHO) cell lines is one of the most widely used production modes for commercial manufacturing of recombinant protein therapeutics. Furthermore, fed-batch cultivations are often conducted as biphasic processes where the culture temperature is decreased to maximize volumetric product yields. However, it remains to be elucidated which intracellular regulatory elements actually control the observed pro-productive phenotypes. Recently, several studies have revealed microRNAs (miRNAs) to be important molecular switches of cell phenotypes. In this study, we analyzed miRNA profiles of two different recombinant CHO cell lines (high and low producer), and compared them to a non-producing CHO DG44 host cell line during fed-batch cultivation at 37°C versus a temperature shift to 30°C. Taking advantage of next-generation sequencing combined with cluster, correlation and differential expression analyses, we could identify 89 different miRNAs, which were differentially expressed in the different cell lines and cultivation phases. Functional validation experiments using 19 validated target miRNAs confirmed that these miRNAs indeed induced changes in process relevant phenotypes. Furthermore, computational miRNA target prediction combined with functional clustering identified putative target genes and cellular pathways, which might be regulated by these miRNAs. This study systematically identified novel target miRNAs during different phases and conditions of a biphasic fed-batch production process and functionally evaluated their potential for host cell engineering.