Search

One of the most pertinent concerns of using differentiated cells derived from human embryonic stem cells (hESC) is the presence of residual undifferentiated hESC, because they carry a risk of teratoma formation. A new cell-cell separation approach that eliminates teratoma-forming hESC in order to ensure safer cell therapy was developed. By combining antibodies (IgMs or IgGs) for the selective removal of undifferentiated hESC using magnetic activated cell sorting (MACS) followed by selective killing of residual hESC with the unique cytotoxic antibody mAb 84, the required purity of differentiated hESC can be achieved. The applicability and robustness of this separation strategy is shown here in a case study using pools of undifferentiated hESC and human fibroblast cells at different ratios (5%-50% hESC) to reflect the different scenario of contaminating hESC in a differentiated cell population. Notably, 97.2%-99.7% of the hESC were removed after the MACS step and 99.1%-100%, after the mAb 84 treatment step, which was confirmed by double-staining flow cytometry and RT-qPCR analysis. These in vitro findings were further validated in an in vivo severe combined immunodeficiency (SCID) mouse model. Importantly, we observed the absence of teratoma formation in eight out of nine SCID mice 28 weeks postinjection of cells after the MACS step, whereas teratomas were observed in all of the controls. Thus, the combination of MACS with the unique cytotoxic antibody mAb 84 constitutes an indispensible tool for successful and safe cell therapy.

The substantial progress in embryonic stem cell (ESC) research could lead to new possibilities in the treatment of various diseases. Currently, applications of ESC for cell therapy are impeded by the presence of potentially teratoma-forming undifferentiated ESC. Thus, a selective and quantitative removal of undifferentiated ESC from a pool of differentiated and undifferentiated cells is essential before cell therapy. We evaluated the highly selective magnetic activated cell sorting (MACS) method for the quantitative removal of undifferentiated ESC. We found that the clearance rates for undifferentiated ESC decreased with decreasing amount of undifferentiated ESC in the cell pool. Using a simplified model calculation we could predict that, assuming an initial purity of 60%, an estimated 31 steps are required to achieve less than 10(-1) cell per 10(9) cells. Thus, a log clearance rate of 10, which would be necessary for a therapeutically application, is hard to achieve. Our work clearly indicates that the current MACS technology is insufficient to meet the purification needs for cell therapy.

A two-step purification strategy comprising of polyethylene glycol (PEG) precipitation and anion-exchange chromatography was developed for a panel of monoclonal immunoglobulin M (IgM) (pI 5.5-7.7) produced from hybridoma cultures. PEG precipitation was optimized with regards to concentration, pH and mixing. For anion-exchange chromatography, different resins were screened of which Fractogel EMD, a polymer grafted porous resin had the highest capacity. Despite its significantly slower mass transfer, the binding capacity was still higher compared to a convection driven resin (monolith). This purification strategy was successfully demonstrated for all 9 IgMs in the panel. In small scale most antibodies could be purified to >95% purity with the exception of two which gave a lower final purity (46% and 85%). The yield was dependent on the different antibodies ranging from 28% to 84%. Further improvement of recovery and purity was obtained by the digestion of DNA present in the hybridoma supernatant using an endonuclease, benzonase. So far this strategy has been applied for the purification of up to 2l hybridoma supernatants.

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.

For biotechnological research, development, and production various analytical methods are required to determine the quality of the target product. In this context, the determination of isoforms is state-of-the-art; however, the majority of applied techniques are more qualitative than quantitative. To address this fact, we evaluated different post- and pre-electrophoretic staining dyes for their applicability on linear IPG gels using recombinant human erythropoietin as a model protein. Each evaluated dyes was able to detect all isoforms reproducibly, but CyDyes were found to be the most promising. Using CyDyes, up to three samples can be focused on the same lane under identical electrophoretic conditions, thus, a fast, reproducible, sensitive and quantitative isoform determination can be performed. To illustrate the practical relevance, quantitative CyDye technique was used for the characterization of our model protein, recombinant human Epo-Fc. This method makes it possible to determine the isoform pattern of nonpurified supernatants as well as purified proteins. We conclude that quantitative pre-electrophoretic staining IEF using CyDyes is a fast, simple, accurate method to determine isoforms, which can be used in research, development, and manufacturing for product quality analysis, e.g., clone screening, process optimization, and purification monitoring.

In order to improve the current techniques of cell cultivation in the absence of serum, we have developed a protein-free transfection protocol for CHO cells, based on the Nucleofector technology. After starting with a heterogeneous pool of primary transfectants which express the fusion protein EpoFc, we isolated single clones and compared them with parallel clones generated by lipofection in serum-dependent cultivation. Our intensive characterization program was based on determination of specific productivity (q(p)) and analysis of genetic parameters. In two nucleofection experiments, transfection with 5 microg of DNA resulted in best productivities of the primary cell pools. After subcloning, the q(p) could be raised up to 27 pg x cells(-1) x day(-1). While the serum-dependent transfectants exhibited specific productivities up to 57 pg x cells(-1) x day(-1) in serum-dependent cultivation, a significant decrease that resulted in the range of q(p) of the protein-free transfectants was observed after switching to protein-free conditions. Investigation of genetic parameters revealed higher mRNA levels and gene copy numbers (GCN) for the protein-free adapted serum-dependent transfectants. Therefore, we assume that problems during protein-free adaptation (PFA) lead to a less efficient translation machinery after serum deprivation. We describe the generation of stable-producing recombinant CHO clones by protein-free transfection of a protein-free adapted host cell line, which reduces the risk of adverse clonal changes after PFA. The main advantage of this approach is the earlier predictability of clone behavior, which makes the generation of production clones by protein-free transfection, a viable and highly efficient strategy for recombinant cell line development., ((c) 2006 Wiley Periodicals, Inc.)

We have generated a recombinant CHO cell line expressing the fusion protein EpoFc. After selection and screening, protein expression, gene and mRNA copy numbers were analysed in order to gain more information on the influence of genetic parameters on the productivity and stability of production cells. Results from semi-quantitative blot methods were compared to quantitative PCR (qPCR) analyses, whose advantage mainly lies in their higher sensitivity, and the cheaper and faster methodology. We developed stable and high producing clones with low gene copy numbers, in contrast to other cell lines where multiple steps of methotrexate amplification have lead to hundreds of copies of inserts with the risk of karyotypic instabilities and decreased growth rates that overcome the benefits of increased productivities. When comparing genetic parameters to productivity, a good correlation of mRNA levels with specific productivity was observed, whereas high gene copy numbers were not always accompanied by high protein expressions. Based on our data derived from a typical example of a cell line development process, genetic parameters are useful tools for the selection of scalable production clones. Nevertheless, a wider range of cell lines has to be investigated in order to implement genetic analyses into a screening process.

One challenge in biotechnology industry is to produce recombinant proteins with prolonged serum half-life. One strategy for enhancing the serum half-life of proteins includes increasing the molecular weight of the protein of interest by fusion to the Fc part of an antibody. In this context, we have expressed a homodimer fusion protein in CHO cells which consists of two identical polypeptide chains, in which our target protein, recombinant human erythropoietin (rhEpo), is N-terminally linked with the Fc part of a human IgG(1) molecule. In the present study, culture supernatant of a stable clone was collected and purified by affinity chromatography prior characterization. We emphasized product quality aspects regarding the fusion protein itself and in addition, post-translational characterization of the subunits in comparison to human antibodies and rhEpo. However, overproduction of recombinant proteins in mammalian cells is well established, analysis of product quality of complex products for different purposes, such as product specification, purification issues, batch to batch consistency and therapeutical consequences, is required. Besides product quantification by ELISA, N-acetylneuraminic acid quantification in microtiterplates, quantitative isoform pattern and entire glycan profiling was performed. By using these techniques for the characterization of the recombinant human Epo-Fc (rhEpo-Fc) molecule itself and furthermore, for the separate characterization of both subunits, we could clearly show that no significant differences in the core glycan structures compared to rhEpo and human antibody N-glycans were found. The direct comparison with other rhEpo-Fc fusion proteins failed, because no appropriate data were found in the literature.

Currently, one of the most used techniques for the determination of isoform pattern analysis is isoelectric focusing. Routinely, this is performed by immunoblotting. Blotting of proteins after isoelectric focusing on IPG gels may cause several problems, such as protein loss by the blotting itself and band broadening, in some cases the immunostaining with antibodies might be problematic. In the present study, an alternative isoform prestaining method with CyDye fluors is presented. For this approach, a highly glycosylated fusion protein, Epo-Fc, was used consisting of two recombinant human erythropoietin attached to the Fc part of a human IgG(1) molecule. By using CyDye fluors, up to three samples can be focused on the same lane under identical electrophoretic conditions. A fundamental benefit of this technique is the ability to perform quantitative isoform pattern analysis directly from serum-free culture supernatant.

The impact of process environment changes on process performance is one of the most crucial process safety issues when cultivating mammalian cells in a bioreactor. In contrast, directed shifting of process parameters can also be used as an optimization tool providing higher cell and product yields. Compared to other strategies that also aim on the regulation of cell growth and protein expression process parameter shifts can be easily performed without reagent addition or even genetic modification of the host cell line. However, a successful application of changing process conditions implies a profound understanding of the provoked physiological changes within the cells. In a systematic approach we varied the dissolved oxygen tension (DOT), pH, and temperature of CHO cultures in controlled bioreactors and investigated the impact on growth, productivity, metabolism, product quality and cell cycle distribution using a recombinant CHO cell line expressing the highly glycosylated fusion protein Epo-Fc. We found the reduction of cultivation temperature and the reduction of (external) pH to exert the most significant effects on process performance by mainly reducing cell growth and metabolism. With respect to the cell line used we identified a set of parameters capable of affecting cell proliferation in favor of an increased specific productivity and total product yield. The well directed alteration of the process environment has emerged as a tool adequate for further process optimization applying a biphasic cultivation strategy., (Copyright 2006 Wiley Periodicals, Inc.)

Regulation of cell growth and protein expression potentially results in a sustainable enhancement of the volumetric productivity in a fermentation process. Following a biphasic cultivation strategy the process initially passes through a cell proliferation phase to generate a sufficiently high viable cell mass. In the subsequent production phase cells are maintained viable and productive without significant cell proliferation leading to increased viable cell days and product yields. In a previous work we have shown that the well directed alteration of the process environment based on process parameter shifting is a promising tool to regulate cell growth and protein expression. In continuation of this work we investigated process parameters which have been identified to affect cell proliferation in favor of an increased specific productivity and total product yield in a series of biphasic batch cultivation experiments. In most of these processes the integral of viable cells and the specific productivity were increased leading to a significant improvement of both final product concentration and volumetric productivity. In addition, combined parameter shifts (pH 6.90/30 degrees C and pH 6.90/33 degrees C) exerted a synergistic effect on product quality. The loss of product sialylation which occurred at reduced temperatures was prevented by simultaneously reducing the external pH. In conclusion, biphasic cultivation based on combined shifting of process parameters is a suitable tool for controlling cell proliferation and protein expression of mammalian cells in a batch bioreactor leading to enhanced volumetric productivities and therefore offers an enormous potential for bioprocess optimization., (Copyright 2006 Wiley Periodicals, Inc.)

Recombinant CHO cell lines have integrated the expression vectors in various parts of the genome leading to different levels of gene amplification, productivity and stability of protein expression. Identification of insertion sites where gene amplification is possible and the transcription rate is high may lead to systems of site-directed integration and will significantly reduce the process for the generation of stably and highly expressing recombinant cell lines. We have investigated a broad range of recombinant cell lines by FISH analysis and Giemsa-Trypsin banding and analysed their integration loci with regard to the extent of methotrexate pressure, transfection methods, promoters and protein productivities. To summarise, we found that the majority of our high producing recombinant CHO cell lines had integrated the expression construct on a larger chromosome of the genome. Furthermore, except from two cell lines, the exogene was integrated at a single site. The dhfr selection marker was co-localised to the target gene.