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Keeping as many valuable attachments as possible

The goal of many projects focusing on the purification of recombinant proteins is to rapidly and effectively remove substances and side products that would compromise the quality and quantity of biopharmaceutical compounds. A consortium of industrial and scientific partners is currently focusing on a different step in the biopharmaceutical production process: the goal is to keep as many as possible of the drugs’ ‘attachments’. The project focuses on sialic acids attached to the sugar residues of drugs.

The BMBF-funded cooperative project involves seven partners from research and industry, including Ulm-based Merckle Biotec (biopharmaceuticals producer), Dessau-Roßlau-based IDT Biologika (vaccine producer), the University of Magdeburg (Prof. Udo Reichl from the Department of Bioprocess Engineering and Prof. Andreas Seidel-Morgenstern from the Department of Chemical Process Engineering), Tübingen-based EMC microcollections (managing director: Prof. Karl-Heinz Wiesmüller), Prof. Kai Sundmacher of the Max Planck Institute for Dynamics of Complex Technical Systems in Magdeburg as well as Prof. Stefan Laufer from the Department of Pharmaceutical and Medical Chemistry at the University of Tübingen.

New separation systems, optimal drugs

Purification of biopharmaceutical proteins using a chromatography column. This process is extremely time-consuming and costly. © ratiopharm

The managing director of Merckle Biotec Dr. Hermann Allgaier, who was a co-initiator of the BMBF's downstream funding programme, explains the numerous goals of the cooperative project:

One, the project seeks to develop innovative separation media to improve the purification of biopharmaceutical products.

Second, the biopharmaceutical products will be purified in such a way as to retain their optimal efficiency, explained Dr. Heinz Rotering, leader of the project at Merckle Biotec.

And third, general knowledge about the purification methods is to be communicated to students at Magdeburg University. Downstream processing has not previously played a major role in university research and training.

Special focus on sialic acids

Sialic acids (S) are located at a very exposed site; in this, they resemble aerials. The schematic shows the blood thinner EPO, which has a major influence on the effect and efficacy of a drug. © Merckle Biotec

Chemically speaking, recombinant human proteins are clearly defined polypeptide chains. Second-generation biopharmaceuticals such as antibodies or erythropoietin also have heterogeneous sugar side chains attached to them, which have an effect on a protein's half life and sometimes on its efficacy as well.

The cooperative project focuses on sialic acids, which are natural components of glycoproteins. These sugar residues are known to have a considerable effect on the efficacy of a protein. If the drug's sugar residues contain only a few sialic acids or none at all, this could have a negative effect on the efficacy of a drug, explains Dr. Heinz Rotering.

Contact for other molecules

Sialic acids are carbohydrates with a nine-carbon backbone. More than 40 different naturally occurring sialic acids have been identified. N-acetylneuraminic acid (Neu5Ac or NANA) is the most common member of this group and is the precursor of all other derivatives. Sialic acids are mainly found in vertebrates, but also in some bacteria and higher invertebrates.

Sialic acids are mainly found at the end of the sugar residues of glycoproteins, thereby making a major contribution to the high chemical and biological diversity of glycoconjugates. Their terminal position enables them to easily interact with other molecules. Located on the cell surface, the molecules' negative charge affects the repulsion of cells as well as the interaction between cells and extracellular matrix. 

CHO (Chinese hamster ovary) cells are mainly used for the production of recombinant glycoproteins. However, a significant problem in the formation of glycoproteins in CHO cells is the sialation of the terminal oligosaccharides, which is often incomplete and extremely heterogeneous. Incomplete sialation reduces the half life of such drugs in the blood.

Traditional separation is inefficient

The project partners are aiming to develop separation media that are able to retrieve all of these highly active molecules at any purification step. Traditional separation media often lead to molecule losses, said project coordinator Dr. Heinz Rotering.

Ion exchange chromatography separates the proteins according to their charge. However, the charge of recombinant proteins is not only determined by the terminal negatively charged sialic acids, but also by the charge of the polypeptide chain and potentially also by other residues. Therefore, ion exchange chromatography only indirectly detects the valuable sialic acids.

Rotering reports that attempts to optimise ion exchange chromatography have often not been successful as hoped, and additional purification steps have been necessary in order to obtain highly sialated molecules. This is an expensive and time-consuming process, and severely impedes economic business operations.

Since a protein molecule can have more than ten sialic acid residues, the project partners hope that a specific selection method will lead to a less heterogenous mixture of protein subforms, i.e. to a “more defined” molecule.

Method also suitable for vaccine antigens

The new method will not only be suitable for the purification of recombinant glycoproteins, but will also improve the isolation of active vaccine components, i.e. vaccine antigens. A surface protein of the influenza virus could be used as a vaccine antigen. It is known that sialic acid also plays an important role in human influenza infections. Specific influenza virus surface glycoproteins bind to the sialic acids on the surface of human epithelial cells when the virus enters the human body, thereby enabling the virus to enter the cells.

Wanted: sialic acid catchers

The goal of the cooperative project is to be able to purify proteins with valuable sialic acids, and if possible also based on the concentration of sialic acids. © Merckle Biotec

Affinity chromatography of the aforementioned proteins requires ligands that "capture" the salic acids (and hence the recombinant proteins or the viral antigen). This part of the project is done by Tübingen-based EMC microcollections whose peptide libraries are screened for potential sialic acid-binding ligands. The "hits" will then be produced in large enough quantities to produce larger chromatography columns.

Merckle Biotec provides purified recombinant proteins and their precursors required for the screening process, for the subsequent validation of the capture proteins and for the downstream experiments carried out at Magdeburg University. The valuable test material will also be used to determine the purification steps where the separation method can be best used. IDT Biologika GmbH will provide cell cultures of inactivated influenza viruses, swine flu viruses, human influenza viruses and modified bird flu virus strains.

The three-year cooperative project, funded with a total of 1.2 million euros, began in early 2009. Initial success has already been achieved, ligand screening has already led to the first hits and further analyses are being carried out to determine whether these proteins are effective sialic acid catchers.

Pragmatic ligand search leads to initial success

The search for ligands is an extremely pragmatic process: the researchers are testing already known proteins, with a special focus on a very large number of potential candidates. Among the most promising candidates are molecules that are able to recognise all sialic acid binding sites. Ideally, the researchers would like to be able to separate the substances according to the number of sialic acid residues.

Candidate screening focuses on synthetic peptides provided by Tübingen-based EMC microcollections. Using a random peptide library consisting of about two million individual components, EMC isolates peptides that are able to recognise bound sialic acids. In addition, the researchers are also screening the large number of publications dealing with sialic acid-binding proteins. According to Rotering, EMC microcollections has already been able to synthesise and supply the University of Magdeburg with 30 promising peptides.

Structural analogues produced with molecular modelling

At the Department of Bioprocess Engineering at the University of Magdeburg, scientists in Prof. Udo Reichl’s team are investigating whether these peptides specifically bind to sialic acid-containing proteins. The validation of the ligands and the availability of structural analogues developed with molecular modelling will then enable the scientists to develop innovative separation materials. These are the beginning of the scientists’ attempts to optimise the separation methods, which will conclude with the integration of the new purification method into the entire process chain.

Attempts to obviate the subsequent optimisation of purivation when producing ideal biotech drugs

If the project partners are successful, then their work would render unnecessary the purification steps that have so far been needed to improve the yield of highly active sialic acids. The researchers hope to achieve the same positive effect in the production of vaccines, i.e. the targeted isolation of the influenza virus from a culture broth.

In addition to the obvious economic success, i.e. cost- and time reduction, this development would bring about another positive effect. According to Heinz Rotering, a less heterogeneous compound would bring the scientists another step closer on the road towards the development of an “ideal biotechnological drug”.

Another idea would be to use the new separation method for investigating and subsequently specifically influencing the sialation of a drug in the cell. This seems feasible because the new ligands can also be used for quick analytical separations. Knowledge relating to the influence of cultivation parameters, media or cell clones on the sialation of proteins would help to specifically optimise biotechnological production methods very early on in the development process.

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