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Protein aggregation: when proximity becomes dangerous

The aggregation of proteins in the human body might have severe medical consequences, as is the case with Alzheimer’s disease, for example. A less known fact is that the biopharmaceutical industry is also faced with the biological phenomenon of protein aggregation and is working on preventive measures.

A cooperative project by researchers from the Biberach University of Applied Sciences and the Karlsruhe Institute of Technology (KIT) is focusing on the basic mechanisms behind the aggregation of proteins and is attempting to find solutions to the problem. The project is funded by the German Federal Ministry of Education and Research (BMBF) with a total of two million euros, which are equally divided between the Institute of Pharmaceutical Biotechnology at Biberach University of Applied Sciences and three institutes at the KIT. The project will run for three years.

Process of aggregation has not yet been studied in depth

Protein aggregation relates to many different processes of which little is yet known: reversible and irreversible processes are known; processes in which proteins retain their structure and processes associated with the unfolding and refolding of proteins. When a protein is in the process of taking on a different structure, areas that are normally invisible might become exposed to the external side and lead to unwanted immune reactions, Hans Kiefer explains. Kiefer, a biochemist and protein specialist at Biberach University of Applied Sciences, believes that phenomenological solutions are most often used to explain what occurs and few people are interested in what the protein aggregate looks like and how it develops. He is convinced that answers to such fundamental questions are required because “different aggregate forms have different damage potential”.

Prof. Dr. Hans Kiefer. © Biberach University

Some aspects of the aggregation of proteins are well known, for example it is known that the aggregation of proteins turns a transparent solution turbid, and that proteins can shift from a dissolved to a solid state. Proteins can form tiny crystals that are not even visible under the microscope. These proteins maintain their structure and activity. However, they can also form unordered aggregates; in the worst cases, the proteins denature and lose their structure. This situation is normally irreversible and causes the protein to become inactive. Denatured pharmaceutical products become worthless. In the worst cases, aggregates with an autocatalytical effect develop and also contribute to other correctly folded proteins forming aggregates.

In biopharmaceutical manufacturing processes, macromolecules that are used for therapy are exposed to a broad range of mechanical and chemical stress. The cell culture specialist Friedemann Hesse from the Institute of Pharmaceutical Biotechnology explains that an increase in turbidiy is a clear sign of protein aggregates being formed during one of the numerous step involved in downstream processing. However, protein aggregation can also become a problem during upstream processes, i.e. during fermentation. Hesse explains that biopharmaceuticals are mainly produced in fed-batch processes that take around 14 days to complete. During this time, the product is exposed to a broad range of stress factors (e.g. the redox potential as well as bases and acids used to adjust the pH of the culture media) that occur as a result of the culture conditions.

Kiefer goes on to explain that the proteins themselves also affect where and when they aggregate. However, it is generally biomanufacturing processes that promote the aggregation of proteins. For example, monoclonal antibodies can only be stored stably at low temperatures (0ºC, pH 5 rather than pH 7, which increases the charge of the proteins and makes them reject one another). However, cell culture conditions do not allow this, as they need a temperature of 37ºC and a neutral pH (ph 7).

Early detection in the culture broth

Chemist Friedemann Hesse came into contact with cell culture methods for the first time during his doctorate; he worked as post-doctoral student at the GBF (Society of Biotechnological Research) in Braunschweig where he specialised in industrial-scale cell culturing. He also spent eight years at the Austrian Center of Biopharmaceutical Technology in Vienna, where he continued his work on cell culturing. In 2009, Hesse was appointed professor at Biberach University of Applied Sciences. © Pytlik

A group of researchers led by Friedemann Hesse is using fluorescent dyes to identify the factors or process parameters that favour the aggregation of proteins in the fermentation broth. Scientific studies have shown that certain fluorescent dyes attach to or incorporate into certain protein aggregates, thereby changing the signal detected by a fluorescence probe. Hesse’s team is hoping to determine these factors using antibodies as model proteins.

If the researchers manage to identify the factors that lead to the aggregation of proteins, they will try and change the process parameters in order to prevent the aggregation of proteins. This would be the first step towards understanding the mechanisms that underlie protein aggregation. Hesse’s work is of a basic nature as it will provide the researchers with insights into how cells affect the fluorescence signal, how they tolerate the attachment of fluorescent dyes and which fluorescent dyes are actually suitable for protein production processes.

At present, the standard procedure is the removal of the protein aggregates in the purification step. It has been known for quite some time that protein aggregates tend to occur during the downstream process, which is why researchers have mainly focused on finding solutions in this protein manufacturing step. This includes attempting to influence the conditions under which the chromatography step takes place by reducing the protein concentration. Extremely high protein concentrations sometimes also occur in the chromatographic elution step, and flatter gradients are usually chosen to prevent this from occurring. Ultrafiltration is another method that has proven suitable for separating monomeric proteins from larger (e.g. dimeric) protein aggregates.

Osmolytes as potential aggregation inhibitors

Biopharmaceutical manufacturers tend to prevent the aggregation of their valuable molecules by optimizing the pH and salt conditions of the culture media and by adding stabilizing substances. “These osmolytes, which are used for storing the pharmaceutical substances, have been discovered by pure chance in organisms that protect themselves against cellular protein aggregates by producing protein stabilizing substances,” said Hans Kiefer.

Kiefer’s team of researchers has two goals: 1) the use of two model systems (involving 50% common proteins and 50% pharmaceutically important proteins) to provoke the controlled aggregation of the proteins and 2) development of the required analytical methods. The team has already shown that two proteins are able to form fibrils. Kiefer’s team is also focused on provoking other mechanisms (e.g. amorphous precipitation, which refers to the aggregation of solid particles without a particular structure). The researchers will use a broad range of different proteins in order to gain an in-depth understanding of the aggregation mechanisms. Once this has been achieved, they will add osmolytes to the protein solutions in order to test whether they are able to prevent the formation of aggregates on the molecular level. Initial work has been done on these natural substances and Hesse is currently preparing one of their studies for publication. Hesse’s research group works with 30 different osmolytes that belong to the following substance classes: carbohydrate molecules (polyalcohols), amino acids and methylamines.

Osmolytes are found in bacteria as well as in higher organisms such as water bears (tardigrades). Tardigrades, which are around 1.5 mm in size and have four pairs of stubby legs, can protect themselves against dehydration, high and low temperatures and even cosmic radiation. Tardigrades produce trehalose, a natural disaccharide that acts like a natural anti-freeze agent as well as other substances: “Maybe, it is the mixture that is key in protecting the tardigrades against adverse environmental effects,” said Kiefer, giving a possible explanation for the protective effect. At first, Kiefer’s team will investigate the mechanism of action of the protective substances and their anti-aggregation potential. Chemoinformatic analyses will then be carried out to find out how protein aggregation can be prevented. These analyses will also provide further information about the substances’ properties. If the researchers are successful, Hans Kiefer foresees the development of new molecules with an improved effect as well as new substances that can be used for industrial purposes.

Crystallisation (here: of insulin) is also a type of aggregation, albeit a reversible one. © Lilly Pharma

What about adding aggregation inhibitors to the culture medium and testing them? This is still quite difficult, as these substances only exhibit an inhibitory effect at rather high concentrations (up to 20 per cent of the solvent). It goes without saying that such high concentrations also have an effect on the cell culture. The use of such large quantities is of course also costly. It would therefore be far more attractive to have substances available that can already exert their effect at much lower concentrations.

Little is yet known about the molecular mechanisms that underlie the aggregation of proteins in biopharmaceutical processes. It is generally assumed that slightly more than just touching each other is required. The proteins need to lose their three-dimensional structure and open up, at least briefly. According to Kiefer, this is what the majority of soluble proteins do. It is also worth noting that the folded state is not a static one; experiments and computer simulations have shown that protein molecules move around a lot. If proteins display amino acids that are normally hidden inside, this can lead to an irreversible aggregation, i.e. a permanent change in the three-dimensional protein structure. “When a protein in which the hydrophobic areas have been turned to the outside then meets a similarly “extrovert” molecule, the two proteins bind easily to each other,” said Kiefer in an attempt to explain the phenomenon.

The situation is different with (micro)crystals or amorphous aggregations where rather than unfolding, the proteins only come into contact with each other on their normal surface, with no structure alteration. Such reversible aggregations occur when the solvent conditions are being changed to a degree that the proteins become insoluble. This process has been extensively studied.

The analyses performed in Biberach will be further developed at three KIT institutes

Three KIT institutes will develop the Biberach analysis methods further. Jürgen Hubbuch from the KIT Institute of the Molecular Processing of Bioproducts is focusing specifically on two purification steps in which the aggregation of proteins can do a lot of damage; i.e. protein concentration by way of ultrafiltration and hydrophobic chromatography. His team hopes to clarify when aggregations form and how they can be prevented.

 KIT research groups led by Matthias Kind (Institute of Thermal Process Engineering), an expert in crystallization, and Hermann Nirschl (Institute of Mechanical Process Engineering), an expert in solid-liquid separation, will carry out projects in which they will provoke the precipitation/aggregation of proteins without this leading to a loss of activity in the proteins and subsequently use these methods for purifying proteins. This project is similar to a cooperative project on crystallization in which Hans Kiefer is also participating.

Website address: https://www.gesundheitsindustrie-bw.de/en/article/news/protein-aggregation-when-proximity-becomes-dangerous