Labor Dr. Merk & Kollegen brings its expertise to a BMBF-funded cooperative project, which also involves two other partners from the Ulm region. The partners are working on the efficient detection of even minute traces of proteases and their effective separation from therapeutic proteins.

Werner Dangel, the company's managing partner, says that it has specific know-how in biotechnology, virology, cytotoxicology and microbiology, which is offered to biopharmaceutical and medical product manufacturers. The company founder, Dr. Walter Merk, who is still an active member of the company, spent many years in a leading position in the Biotechnological Research and Production division of Boehringer Ingelheim. The laboratory knows very well that its clients have very high safety standards regarding the purification of biotechnologically produced substances.

Biopharmaceutical companies using living microorganisms, for example mammalian cells or E. coli bacteria, for the production of pharmaceutical substances, must always take into account the potential risk arising from viruses, endotoxins, prions or certain proteases that might be present in cell cultures. In-process and final controls involving sensitive and specific bioassays are used to check whether these undesired contaminants have been reliably and successfully removed during the purification process.

In our GMP testing laboratory, explains biologist Dr. Werner Dangel, we can simulate the manufacturer's purification process (filtration, chromatography, etc.) on a laboratory scale. The active addition of viruses to the substance that is being purified and the use of validated virus detection methods enable the scientists to quantitatively assess the efficiency of virus removal in each individual purification step.

To analyse laboratory-scale purification processes, the Ochsenhausen-based company has a collection of reference strains consisting of more than 100 viruses, bacteria and cell systems at its disposal. In addition, it has all the necessary authorisations required by the infection, zoonosis and genetic engineering laws in place.

The aim of the aforementioned collaborative project is to develop a resin with selective affinity ligands to remove proteolytic activity from fermented cell culture solutions. The goal of biopharmaceutical purification systems is to completely remove such proteases, as even trace amounts can result in the proteolytic degradation of larger proteins such as therapeutic proteins.
Proteases occur naturally in the tissues and cells of all organisms. Many proteases have different pH optimums and some of them are still active at -20◦C. The presence of trace amounts of proteases in the final product has a decisive impact on the formulation, quality and stability of the biopharmaceutical product. Therefore, biopharmaceutical manufacturers are forced to stabilise or freeze-dry their therapeutic proteins in a time-consuming and costly way.

In close cooperation with Dr. Joachim Walter, who has over 25 years experience in the development and production of biopharmaceutical proteins with mammalian cell cultures, the Ochsenhausen-based company is working on effective ways to analyse and remove proteases.

In principle, explains Werner Dangel, these ‘troublemakers’ can play a big role in any purification process, depending on the type of biopharmaceutical product manufactured and the purification process used. When the bacterial cells are disrupted in order to release the pharmaceutical substance produced, the proteases will come into contact with the substance, degrade it and lead to a reduction in protein yield.

Commercial tests that enable the detection of proteases are often unspecific and differ considerably in their detection rates. In addition, these tests do not differentiate between proteases such as pepsin, trypsin or chymotrypsin that are still present in the medium. In general, explains Dangel, it is possible to reduce the proteolytic activity using protease inhibitors. However, this is impossible when purifying biopharmaceutical proteins due to the inhibitors’ toxicity, which can be quite high.

Currently, the removal of proteases needs to be carried out in a time-consuming process for each individual purification step. The collaborative project wants to change this situation. The plan is to use protease scavengers (scavenger = chromatography column) prior to purifying the proteins in chromatographic columns as well as at the end of the purification process. These scavengers bind to the proteases and allow the target protein to pass through.

The success of the project depends on whether the researchers succeed in selecting suitable protease catchers and retain them in the scavenger. Boris Mizaikoff, a chemist from Ulm, is working on this particular project and hopes to find highly specific ligands that bind to proteases and couple them to the chromatography materials to prevent them from being eluted. It is still too early in the three-year project to provide details on the composition of these scavengers, explains the coordinator of the project, Werner Dangel.

“We want to be able to capture the most important three or four proteases that play a key role in biopharmaceutically relevant mammalian cells such as CHO, NSO and BHK. We hope that the new test will provide us with quantitative and qualitative information about the protease concentration and the protease spectrum,” said Dangel outlining the goal of the protease project.

If the partners are successful, then the biopharmaceutical manufacturers will be able to obtain detailed information on the proteases found in the fermentation broth. With this new method, the manufacturers will be able to tell as to which proteases occur at which purification step. This will save the manufacturers a complete process step and make the costly downstream processing much simpler. Highly sensitive protease tests will also be able to identify traces of these “substance killers” in the final product and remove them by way of scavengers. In addition, in terms of product stabilisation, the freeze-drying of the substance might become obsolete and subsequently be replaced by more user-friendly fluid formulations.

The three project partners will divide the tasks as follows: Labor Dr. Merk & Kollegen will ferment the mammalian cell cultures, provide protease-containing fermentation solutions as test material and develop sensitive protease assays for the identification of trace proteases and the assessment of protease removal. In addition, the protease assay will be used to identify potential ligands. Labor Dr. Merk & Kollegen will also focus on the cytotoxic testing of suitable ligands and assess the user friendliness, cost-effectiveness and the efficiency of the newly developed methods in the fermentation and purification on a laboratory scale.

The company Atoll GmbH is a chromatography specialist from Weingarten (district of Ravensburg) which sells materials required for the preparation and purification of proteins, in particular for use in biopharmaceutical production. Atoll GmbH will bring its skills in method development and screening of all kinds of chromatography materials to the cooperative project.
Atoll GmbH will provide screening tools with packed columns in 96-microtitre plate format, ready packed, and characterised disposable columns that can be used with the chromatography materials of the majority of manufacturers. These can be used for processes in which the validation of sanitation procedures proves problematic (e.g. for products such as the planned protease scavenger). This saves time in packaging the columns and there is also the advantage of working with a perfectly packed column. Dr. Jürgen Friedle of Atoll brings over 20 years of experience with chromatographic separation media to the project.

Another important part of the project is to be dealt with by Professor Boris Mizaikoff from the Institute of Analytical and Bioanalytical Chemistry at Ulm University, who works together with researchers of many disciplines and has great experience in project work. His team will investigate and develop biomimetic receptor materials for proteases which will then be used as synthetic ligands in the molecular-selective protease scavenger or in the planned protease assay.

If the project is successful, then the Ochsenhausen-based Labor Dr. Merk & Kollegen will be able to offer biopharmaceutical manufacturers a test kit that allows them to perform tests at the various purification steps and determine where, which and how many proteases occur at those steps. This would then be a protease test that is not only more sensitive than currently used tests, but one that also provides information about the spectrum of proteases present in the fermentation broth. In addition, a chromatography column (scavenger) would be available that is able to efficiently remove proteases both at the smallest scale as well as at the technical production scale, while allowing all biopharmaceutically relevant substances to pass through.

The design of the planned products will also take into account that the future users will be able to comply with GMP guidelines. Since pharmaceutical manufacturers are, increasingly, outsourcing downstream processing steps, the Ochsenhausen-based company has also plans to expand the portfolio of safety tests and maybe offer protease analytics or protease removal as services.
These are still plans for the future, but Werner Dangel can well envisage the possibility of developing new methods based on these findings in order to identify foreign bodies that have unwanted side effects as well as removing them from the fermentation broth or from the final product, in order to make pharmaceutical products even safer.

In principle, the avenue chosen by the project partners has the advantage that the substance is “freed” from substances which may have a negative impact in a more gentle way than currently used purification methods, which require the binding of the substance to chromatographic material surfaces and the subsequent removal by physical means. This might prove quite problematic as the substance may suffer from this kind of treatment; it might refold or, in the worst case, be destroyed. Therefore, the market needs more gentle purification methods that can easily be scaled up.