Why is protein aggregation bad
If proteins clump together in the human body, this has serious medical consequences, as in Alzheimer's disease, for example. What is less well known is that the biopharmaceutical industry is also plagued by the biological phenomenon of protein aggregation and tries to avoid it at all costs.
In a research project, researchers from Biberach University and the Karlsruhe Institute of Technology (KIT) are fundamentally addressing the problem of protein aggregation and developing solutions from it. The project, which is fully funded with two million euros by the Federal Ministry of Research, is shared equally between the Institute for Pharmaceutical Biotechnology and three KIT institutes. It has a term of three years.
The process of clumping has been little studied
Protein aggregation includes many different, little-known and studied processes. Reversible and irreversible processes are known; those in which proteins retain their structure and those that are associated with unfolding or refolding. If a protein folds over, areas are expressed outwards that are normally invisible and lead to dreaded immune reactions, explains Hans Kiefer. The Biberach biochemist and protein specialist has found that phenomenological solutions are usually required. Usually nobody was interested in the shape of the unit and how it came about. He is convinced that answers to these fundamental questions make sense because the “respective aggregate forms have different damage potential”.
What is certain is that when a clear solution aggregates, it becomes cloudy, and proteins go from the dissolved to the solid state. Proteins either form tiny crystals that cannot be seen even under a microscope; this means that structure and activity are retained. Or a disordered aggregate results, in the worst case the proteins denature and lose their structure. Usually this condition is irreversible and associated with loss of activity. If it is a pharmaceutical product, it becomes worthless. In the worst case, aggregates are formed that act autocatalytically and clump other correctly folded proteins together.
In the biopharmaceutical manufacturing process, the therapeutically used macromolecules are exposed to various forms of mechanical and chemical stress. This becomes evident in downstream processing when the protein solution becomes cloudy during one of the numerous separation steps, says cell culture specialist Friedemann Hesse from the Biberach Institute for Pharmaceutical Biotechnology. Protein aggregation can, however, already become a problem in the upstream, during fermentation. As a rule, explains Hesse, these are fed-batch processes that run for 14 days. Meanwhile, the product is exposed to the culture conditions with all possible stress factors: Dangers arise from the redox potential, from alkalis and acids to regulate the pH value.
Where and when aggregation can occur also depends on the proteins, says Kiefer. In general, however, biomanufacturing tends to promote clumping. For example, if you want to store monoclonal antibodies in a stable solution as a solution, you would choose low temperatures (zero degrees Celsius, pH 5 instead of pH 7, because the proteins are more strongly charged and repel each other), if the cell culture would allow it. Because there the manufacturer is forced to work at 37 degrees Celsius and a neutral pH value of 7.
Early detection in the culture broth
The group around cell culture specialist Friedemann Hesse wants to clarify which factors or process control parameters lead to protein aggregation in the culture broth. She wants to measure this with the help of fluorescent dyes. It is known from the literature that certain fluorescent dyes attach to or are stored in certain aggregates and thus change their signal, which a fluorescent probe could detect. Implementing this on the antibody model protein in the course of the culture is the aim of Hesse AG.
If the specific clumping detection is successful with the fluorescence spectrometer, an attempt should be made to influence the aggregation with modified process parameters. These would be the first steps towards understanding the mechanisms underlying clumping. Hesse's work has a fundamental character because it has to clarify how the cells influence the fluorescence signal, are compatible with these dyes and which fluorescent dyes are suitable for the process.
Usually an attempt is made to separate these protein clumps only during the purification. The problem has been known for a long time in the downstream and has been better investigated. Here the manufacturers try to influence the conditions of the chromatography step, for example by lowering the protein concentration. Extremely high protein concentrations also occur selectively in chromatographic elution steps, which one tries to avoid with flatter gradients. It is also possible to separate a monomeric protein from larger (e.g. already dimeric) aggregates by means of ultrafiltration.
Osmolytes as possible aggregation inhibitors
Manufacturers try to prevent their valuable molecules from clumping together with optimized pH values, salt conditions and the addition of stabilizing substances. These substances (osmolytes), which were already used to store the active ingredients, were found by chance in organisms that protect themselves from protein aggregation in the cell by producing certain protein-stabilizing substances, reports Hans Kiefer.
His working group has two goals. On the one hand, controlled aggregation is to be created in separate model systems - half with common proteins and half with pharmaceutically important proteins - and the necessary analytics are to be developed. The targeted formation of fibrils has already been achieved with two proteins. Kiefers AG also wants to bring about other mechanisms such as amorphous precipitation (aggregating solid particles without a specific structure) in a controlled manner. The large range of selected proteins is intended to ensure that the aggregation mechanisms are understood in principle. If this succeeds, osmolytes should be added in order to clarify their aggregation-preventing influence on the molecular level. Initial work on these natural substances has already been carried out, and the protein specialist from Biberach is currently preparing a publication. His group works with 30 different osmolytes, which belong to the substance classes sugar molecules (polyalcohols), amino acids and methylamines.
Osmolytes occur in bacteria, but also in higher organisms such as the tardigrade strain. These survivors are protected from dehydration, high and low temperatures and even cosmic rays. They are known to produce a sugar called trehalose, which acts like a natural antifreeze. However, the eight-legged organisms also produce other substances: “Maybe it depends on the right mixture,” Kiefer formulates a kind of hypothesis.
First, Kiefer's group wants to investigate the mode of action of these protective substances and their respective anti-clumping potential, before a chemo-informatic analysis should attribute properties to the inhibition of aggregation. If successful, Hans Kiefer thinks of new molecules with improved effects, of new additives for industry.
These new aggregation inhibitors could already be added to the culture medium and tested, says Friedemann Hesse. So far, however, their use has been difficult because they only show their inhibitory potential at high concentrations (up to 20 percent of the solvent), which in turn influences the cell culture. On top of that, you need large quantities of it, which causes high costs. It would therefore be attractive to have more effective substances that are already effective in lower concentrations.
Little is known about the molecular sequence of protein aggregation in the biopharmaceutical process. Presumably, the mere collision of the molecules is not enough, but they have to lose their three-dimensional structure, at least for a short time, and partially open up. According to Kiefer, this process is more or less common to all soluble proteins. But even the folded state is not a static state; Experiments and computer simulations have shown that protein molecules move very strongly. If proteins show amino acids that are normally hidden inside for a short time, this can trigger irreversible aggregation, permanent refolding. If these hydrophobic areas of the macromolecule that have come out to the outside come up against an equally "extroverted" neighboring protein, these proteins can very easily attach to one another, Kiefer tries to explain.
The situation is different with (micro) crystals or amorphous aggregates, in which the proteins do not unfold, but only come into contact with one another via their normal surface without losing their structure. In this case, it is a question of reversible aggregation, which usually occurs when the solvent conditions are changed in such a way that the proteins are no longer soluble. This process is relatively well understood.
Biberach analyzes are being further developed at three KIT institutes
The analysis methods developed in Biberach are to be further developed at three KIT institutes. Jürgen Hubbuch from the Institute for Molecular Processing of Bioproducts examines two steps in purification where protein aggregation is particularly important: in concentration via ultrafiltration and in hydrophobic chromatography. His working group wants to clarify when the clumps form and how they can be reduced as best as possible.
KIT working groups around the crystallization expert Matthias Kind (Institute for Thermal Process Engineering) and the specialist for solid-liquid separation Hermann Nirschl from the Institute for Mechanical Process Engineering want to bring about the targeted precipitation / aggregation in their projects, without loss of activity, and this as a cleaning step establish, similar to what happens in a joint project on crystallization, in which the Biberach-born Hans Kiefer is also involved.
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