A symposium on “Cell Factories of the Future” was held at the Laupheim-based company Rentschler Biotechnologie on 14th and 15th October 2010. The event attracted around 70 scientists from academia and industry and will now take place every two years. The symposium focused on new biomanufacturing developments and technologies, and included lectures on (animal) cells, cell factories and the future of recombinant protein production.
Dr. Nikolaus Rentschler, CEO of Rentschler Biotechnologie explained: “The idea behind this new symposium is to enhance discussions and networking at the cutting edge of development in cell technology and use this to advance biomanufacturing.”
Hermann Katinger gave the keynote lecture in which he referred to the last 30 years of cell line development in animal cells, in a humorous but at the same time authoritative way. Katinger is an Austrian cell culture pioneer who did his doctoral thesis in 1971, at a time when biology was still rather descriptive, and before it developed into an exact science based on new molecular findings. Katinger, who was previously director of the Institute of Applied Microbiology at the Vienna University of Natural Resources and Life Sciences, is the founder and CEO of the Austrian company Polymun Scientific GmbH.
There are many reasons why nearly 70 per cent of all recombinant protein therapeutics are produced in Chinese hamster ovary (CHO) cells. One such reason relates to the safety concerns of the authorities, which has potentially led to this wide use of CHO cells. Katinger explains why: virologists from the big pharmaceutical company Sandoz flatly rejected the use of CHO cells due to their viral safety. Katinger sees this as key evidence as to why CHO cells have had a successful career as biopharmaceutical production cells.
Alfred Pühler gave a presentation on the sequencing of the hamster genome. Pühler who studied physics before going on to do his PhD in biology and his habilitation in genetics, is senior research professor at the Centre for Biotechnology (CeBiTec) at the University of Bielefeld. He is regarded as one of the most influential biotechnologists in Germany. The CeBiTec owns two high-throughput sequencers and a bioinformatics platform with high computer and storage capacities, which enables researchers to sequence and analyse genomes and transcriptomes of any size.
In 2009, researchers from the University of Bielefeld began working in collaboration with colleagues from the Vienna University of Natural Resources and Life Sciences to analyse the transcriptome and genome of different CHO cell lines with the hope of enhancing biopharmaceutical production using CHO cells.
The genome researchers were faced with the problem that only a small proportion of the hamster genome was encoded in genes. By combining the sequence data of numerous sequencing runs, the Bielefeld researchers obtained 36,000 contigs, which are overlapping DNA fragments with a length of 500 bp. The researchers analysed 28,000 active genes in CHO cells. CHO cell lines are characterised by high genomic variation. Additional CHO cell lines containing the CHO genome need to be analysed and further sequencing is required to specify the function of the genes.
Volker Sandig, CSO of Berlin-based ProBiogen AG, reported on systems that can be used for the production of recombinant proteins, antibodies and viruses. ProBiogen AG sees itself as a development partner for cell lines. “Certain proteins are able to impair the metabolism of the host cells. These proteins can be modified or degraded in the cell and can be prone to misfolding or aggregations,” said Sandig.Sandig also spoke about difficulties that arise when switching from one production platform to another, a process that is very time consuming and is avoided whenever possible. Sandig showed ways in which these difficulties can be overcome by optimising protein folding, adding alternative cell substrates or using suitable selection or screening methods. Sandig’s presentation about using Muscovy Duck cells as alternative to chicken cells for the production of vaccines provoked astonishment and interest among the audience. He emphasised that the duck cells have a similar pattern to human cells.
Despite all the progress that has been made, the development of mammalian cell-based fermentation processes is still an empirical procedure based on experience and statistical experiment design because of the lack of quantitative understanding of intracellular processes. Thomas Noll from the Institute of Cell Culture Technology at the University of Bielefeld envisages that the combination of the former lead disciplines of cell biology, molecular biology and bioprocess engineering with functional genome analysis and bioinformatics also makes biopharmaceuticals less expensive to produce.Noll and his team focus on the influence of process conditions on the cellular and molecular level by combining differential proteomic and intercellular metabolomic analyses. He pointed out that proteomics and metabolomics allow the most direct measurement of a cell’s physiological activity and that they have proven their potential for microbial cell line and process development. The proteomic analysis of mammalian cells such as CHO is rendered difficult due to incomplete CHO sequencing data, while the analysis of the metabolome is hampered by the cells’ mechanical instability and cellular compartmentalisation, which requires technical improvements and specialised solutions.
The production of recombinant therapeutic proteins in mammalian cells usually involves the transient and stable expression of transgenic proteins. For long-term production, only stable expression cell lines are used, as transient expression only leads to low quantities of protein with short life cycles. However, Bernd Rehberger, Head of Protein Expression at Rentschler Biotechnologie, believes that this approach has some disadvantages. He pointed out that stably expressed production cell lines are generated by integrating an expression plasmid into the genomic DNA of the host cell. However, the integration of the vector DNA into the genome is a random event, which does not enable the prediction of the resulting productivity. As a result, time-consuming and complex selection and screening procedures are necessary to find the most suitable cell line for production.For this reason, the use of different technologies to direct and control the integration of vectors for recombinant protein production into the host cell DNA have been tested for some time. Rehberger presented an alternative, which involves S/MAR (scaffold/matrix attachment region) minicircles. Minicircles are circular, superhelical DNA vector molecules that result from recombinase-induced homologous recombination of “normal” parental plasmids. The advantage is that they do not contain unwanted bacterial traces or elements originating from the parental plasmid that would compromise the expression of the transgene. If these minicircles are combined with S/MAR elements (short DNA fragments that bind to the nuclear matrix and separate active from inactive DNA regions), stable transgene expression can be achieved by episomal replication of the minicircle, which is coupled to cell and nuclear division through S/MAR-induced nuclear matrix interactions rather than through the integration of the vector into the genome.
Aziz Cayli, CEO of Laupheim-based Cellca GmbH talked about his fondness for simplicity. The biotechnologist believes that the development of culture media still has a “trial and error” character despite the fact that high-performance media already enable the batch production of as many as ten billion cells or more per millilitre. Early media contained serum, which was replaced in the 1980s to reduce the risk of animal-related contamination. In addition, it was necessary to develop media that were free of hydrolysates. Nowadays, many cell culture media consist of clearly identifiable single chemicals. Cayli also reported that complex ingredients (e.g., hydrolysate, serum) were omitted, so single chemicals had to be added to the media to achieve a similar performance. As a consequence, the recipes of modern media contain more ingredients than those published in the 1960s. The number of trace elements in particular increased over time.He went on to point out that it is still not understood in detail what cells’ needs and preferences are. Cayli believes that future challenges will revolve around more rational approaches and around the search for an understanding of the cellular demand. He believes that once cell metabolism is understood, it will be possible to steer cell growth, protein production and quality in the preferred direction by supplying the cells with predetermined nutritional cocktails.
Sabine Geisse, Senior Scientific Expert at the Novartis Institutes for Medical Research, focused on two questions in her presentation: How to generate a ‘happy protein' in sufficient quantity and quality and, how does one find the best fit between the individual nature of the target vs. the variety of options for expression?
She presented diverse problematic aspects that compromise protein expression and discussed potential reasons for this. In addition, drawing on her practical experience with signal peptides, tags and different host cells, she presented potential solutions and discussed whether multi-parallel expression experiments might have an advantage over more standardised procedures.
Johannes Grillari from the University of Natural Resources and Life Sciences in Vienna (Austria) talked about microRNAs (miRNAs) and their role in the ageing of CHO cells. “There are still no markers for the biological ageing of humans. We have recently discovered miRNAs in human serum whose quantity increases with age. One of these miRNAs seems to inhibit the formation of osteoblasts, which might impair osteogenic differentiation and cellular proliferation (osteoporosis).”Grillari and his colleagues have been able to show that miRNA signatures represent effective markers for the state of CHO cells. “We consider that miRNAs of CHO cells represent novel markers during CHO cell factory design as well as novel tools and targets for the development of cell lines.”
Stefan Schlatter presented Boehringer Ingelheim’s proprietary protein expression technology platform that enables the fast-track development of biopharmaceutical production processes from CHO cells. Schlatter believes that the concept, which is a combination of several state-of-the-art technology platforms, meets demands for shorter development times leading to first use of a new biological entity in humans.He went on to talk to the audience about the need to focus on “commercial scale potential, product quality, safety and comparability” very early during the production process in order to avoid unnecessary costs and delays at a later stage. Schlatter explained that Boehringer Ingelheim’s platform (BI-HEX) combines various technologies, including selection strategies, automated high-throughput screening, yield optimisation, the introduction of optimised or novel genetic elements and the implementation of disposable technologies. Boehringer Ingelheim uses the BI-HEX platform at its Biberach production site to produce cells with specific productivities of more than 100 picogrammes (one billionth of a gramme) per cell. Cumulated fed-batch processes are thus able to achieve yields of more than eight grammes per litre without compromising product quality and the bioactivity of the recombinant antibodies.