Upstream and downstream processing in line
As it has done in 2009, Trenzyme GmbH is also planning to further expand its downstream processing capacities in 2010. For the biotechnology company located in Constance, Germany, protein purification usually comes as part of a complete service package, ranging from the cloning of a gene to the expression of the protein and its final purification and analysis. As Dr. Christoph Glanemann, project manager and head of the company’s Downstream Processing Unit, tells us in the following interview, the optimised linkage of up- and downstream processes brings cost- and time savings.
Trenzyme has been providing services in the fields of molecular biology, biochemistry and cell line development for the past decade. The company's international client base includes mainly pharmaceutical and biotechnology companies. The Department of Downstream Processing at Trenzyme is run by Dr. Christoph Glanemann, who obtained his doctorate at the University of Cologne and spent research periods at the Massachusetts Institute of Technology and the Harvard Medical School.
Dr. Glanemann, what does Trenzyme's Downstream Processing Unit do?
We do everything from the processing of biomass to the analysis of the purified proteins. With respect to the target protein, we usually work in the microgram and small gram scale. But we are constantly searching to increase the yield and quality of the products by optimally selecting and combining different chromatography methods and media, for example those with higher binding capacities.
Which processes do you normally use?
We have state-of-the-art FPLC and HPLC systems and of course we also use all the relevant chromatography methods (e.g., affinity, ion exchange, reversed phase, size exclusion). Following the classical procedure of “capture, purify, polish”, we can combine several methods depending on the degree of protein purity we want to achieve. Protein analysis also involves a broad range of methods, from SDS-PAGE to ELISA to complex mass spectrometry methods. In addition, if requested by our clients, we also carry out functional protein analyses, for example by using in vitro enzyme activity assays or cell-based assays in cell culture. The majority of our projects originate directly from the research and development activities of our clients, which means that only limited information is available about the protein we are working on. Typical projects we carry out on behalf of clients include the expression (upstream process development) as well as the complete process development required for purifying the protein.
What is your strategy for purifying the proteins?
In general, the protein purification process is developed and optimised in small-scale analytical experiments before it is transferred to a larger scale. During this development phase, we determine the optimal selection as well as the best order of different chromatography methods. An important criterion for the selection of a specific order of different chromatography steps is, for example, the suitability of certain methods for reducing the sample volume from several litres to a smaller volume that is more practical. This also involves the selection and optimisation of a specific chromatography method according to the properties of the protein under investigation (e.g., theoretical isoelectric point or molecular weight), for example ion exchange chromatography or gel filtration. When we use ion exchange chromatography, we need to determine the pH at which the sought-after protein can be best separated from all other proteins in the sample. In addition, the entire process development phase also requires us to document all steps involved in order to be able to reproduce the processes with the same high quality at a later stage.
How do you handle process-associated contaminations?
Depending on the issue in hand, certain contaminations can either be prevented right from the start by choosing a specific processing procedure or can be reduced during the process. For example, the contamination with endotoxins can be prevented by expressing the protein in systems other than bacteria. In the case of certain contaminations that cannot be prevented right from the start by careful selection of the procedure used, they can be reduced to a specific threshold value by combining several chromatography steps.
It is known that the cost of purification constitutes a considerable proportion of the total cost of protein production: how do you try to reduce these costs?
The interlinkage of upstream and downstream processing, the selection of special chromatography methods and the well thought-out transfer from small-scale to large-scale processes help us to save valuable time and minimise costs. Typical client projects take between a few days and several weeks, depending on the complexity of the project. Our company structure is also a decisive factor in the effort to reduce costs and save time. Our compact management and communication structure, which generates very little red tape, saves us valuable time. The ability to develop and optimise both upstream as well as downstream processing helps us prevent downtimes as well as communication problems between the two areas. There is no need to send samples back and forth between one area and the other and the employees of different departments can contact each other 24 hours a day, something that considerably speeds up the procedure. And the fact that this all works so smoothly is the result of how we deal with the upstream processing.
What conditions are created during upstream processing that enable the smooth transfer to downstream processing and hence the ability to save time?
Firstly, we can chose between all relevant protein expression systems, from E. coli, to yeasts, to insect and mammalian cells. This saves time and hence costs. Instead of testing one expression system after the other, we are able to develop several approaches in parallel and test them for optimal protein expression. The best system can then be used directly in the downstream process. Let me give you an example: a client who commissions us to carry out the expression and purification of a protein. This client not only wishes to achieve high protein quantities, but also wants to use the protein in a functional assay. However, it is still unknown whether posttranslational modifications have an effect on the function of the protein. Or putting it succinctly, the client wants guaranteed protein functionality at the same time as a high quality protein. The ability to express the protein simultaneously in bacteria, yeasts, insect and mammalian cells also enables the small-scale analysis of the proteins produced in all expression systems simultaneously. Imagine the following scenario: the expression of the protein in bacteria and yeasts leads to a high protein yield, but the protein does not pass the critical functional test. On the other hand, the expression of the protein in insect or mammalian cells might lead to smaller protein quantities, but the protein produced with these systems passes the functional test. Such an approach leads to the quickest possible selection of the optimal expression system and the development of the upstream process, at the same time as allowing us to establish downstream protocols.