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Karlsruhe Institute of Technology

Institute of Engineering in Life Sciences

Section IV: Biomolecular Separation Engineering

Fritz-Haber-Weg 2

76131 Karlsruhe

Tel: +49 721 608 42557
Fax: +49 721 608 46240

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Protein Interaction Engineering

Protein Characterization

The accurate characterization of protein solutions is indispensable for an optimal process design and formulation development. Here, the solubility of the target molecules and the long term stability of biopharmaceuticals are regarded as a bottleneck in the biopharmaceutical development. Therefore, our scientific group focuses on the analysis off protein-protein interactions, the resulting protein phase behavior, the predictability of protein phase behavior and its manipulation. We study model proteins, pharmaceutical relevant molecules like monoclonal antibodies, different classes of enzymes as well as virus like particles under ideal dilute and highly concentrated conditions.


Protein Phase Behavior

Protein Phase Behavior

For the pharmaceutical industry, the stability of biologically active components during processing, formulation and storage is of crucial importance in order to ensure patient safety and product activity. An important stability criterion is the phase behavior of the target molecule. It can be distinguished between the soluble, crystalline and precipitated phase state as well as gel formation. Controlled crystallization and precipitation offer the biopharmaceutical industry a cost effective alternative to conventional separation methods and are acknowledged for formulation purposes. However, undesired aggregation may lead to product loss and changes in the three-dimensional structure of the target molecule. Therefore, knowledge of protein phase behavior is essential for downstream process design.

At our institute, a screening method to generate phase diagrams in high throughput on an automated liquid handling station in microbatch scale was developed. Using a high resolution camera, the generated phase diagrams are evaluated regarding their phase behavior. Using this method, the influence of various process parameters, such as type and concentration of salts or polymers, pH or temperature for various biologically active components can be examined.

 

Literature

N. Rakel, M. Baum, J. Hubbuch, Moving through threedimensional phase diagrams of monoclonal antibodies, Biotechnology progress (2014), DOI: 10.1002/btpr.1947.

K. Baumgartner, L. Galm, J. Nötzold, H. Sigloch, J. Morgenstern, K. Schleining, S. Suhm, S. A. Oelmeier, J. Hubbuch, Determination of protein phase diagrams by microbatch experiments: Exploring the influence of precipitants and pH, International Journal of Pharmaceutics (2014), DOI: 10.1016/j.ijpharm.2014.12.027.


Influence of Additives on Protein Phase Behavior

Additives

Protein therapeutics exhibit their highest biological activity in the native state. Thus, it is essential to ensure that the target protein remains in its native conformation during production, formulation and storage. One possibility for the stabilization of the native state is the addition of additives. Frequently used additives are polymers (e.g. polyethylene glycol) and osmolytes. Osmolytes are low molecular weight additives, which can be grouped in the main categories of free amino acids and derivatives (e.g. glycine), polyols and uncharged sugar (e.g. glycerol), methylamines and urea. At our institute automated screening are carried out to investigate the effect of additives on proteins. Thereby the influence of the additives on the secondary structure of the proteins, on the unfolding behavior of the proteins under thermal stress as well as the protein phase behavior is examined.

 

Literature

L. Galm, J. Morgenstern, J. Hubbuch, Manipulation of lysozyme phase behavior by additives as function of conformational stability, International Journal of Pharmaceutics (2015), DOI: 10.1016/j.ijpharm.2015.08.045.


Protein Conjugation

The term protein conjugation describes the insertion of non-proteinogenic groups into protein molecules, which are not present in the wild type. The aim of this process is the production of molecules with unique properties derived from both the biological component and the synthetic material.
The covalent attachment of synthetic polymers to pharmaceutical proteins is a promising approach to alter physical properties such as thermal stability, solubility, tolerance to organic solvents and proteases and thus to achieve an increase in protein yield during production, formulation and storage. Furthermore, physiological properties like the half-life in circulation and the immune tolerance can be improved. The most common polymer modification in the pharmaceutical industry is the covalent attachment of polyethylene glycol (PEG) to the target molecule (PEGylation). In addition to PEGylation, alternative polymers as modifier for proteins are examined at our institute.

Also in the development of anti-cancer drugs the potential of protein conjugates is made use of. In this case, biologically active cytotoxic drugs are coupled to monoclonal antibodies (mAbs) via a covalent linker. This way the specificity of the mAbs for antigens expressed on the cancer cells enables the targeted delivery of the cytotoxic payload mitigating the systemic effect of the drug. 

Rheology

Auswirkung auf das Fließverhalten durch steigende Viskosität
Effects of increasing viscosity on liquid flow.
Rheology
Illustration of complex rheology.

With rheological measurements we study the flow characteristics of biopharmaceutical solutions. Rheological measurements provide information on solution inner structure as well as implicit flow characteristics. A great advantage of these measurements is that their measurement accuracy is independent of the target molecule concentration and varying solution conditions. So that this analytical method is also suitable for highly concentrated dosage forms.

 

Viscosity

Increasing Upstream Processing and formulation titers result in a significant increase of solution viscosity. Downstream and formulation scientists have to face the challenge to evaluate and minimize the resulting alterations in processability, production and administration. Our group is working on the better characterization of parameters which influence the viscosity of protein solutions and based on this knowledge the specific manipulation of solution viscosity e.g. by the addition of additives or protein conjugation. We are able to determine the dynamic viscosity with the help of dynamic light scattering (Zetasizer Nano, Malvern) and with a mechanical rheometer (MCR 301, Anton Paar).

 

Complex Rheology

Protein solutions are complex in structure, form macromolecular networks in crowded solutions and show viscoelastic behavior. That implies that the material exhibits both viscous and elastic characteristics when undergoing deformation. The characteristics are represented by the measurable parameters G’ (storage modulus) and G’’ (loss modulus). G’ describes the elastic component of the measured sample, G’’ the viscous component of the probe. G’ and G’’ are measured over the radial frequency ω for the characterization of given protein samples. The parameters show a characteristic curve shape that is shifted along the x-axes with changing molecular weight or the formation of intermolecular scaffolds of the tested material. We could demonstrate that the crossover-point-frequency (ωCO) of G’ and G’’ is sensitive concerning solution conditions which influence the protein long term stability. Furthermore we could directly correlate the ωCO values of given samples with the studied protein phase behavior. The frequency sweep measurements are conducted on a High Frequency Rheometer. Currently we are also working on the establishment of the microrheological technique in our lab.

 

Literature

M.-T. Schermeyer, H. Sigloch, K. Bauer, J. Hubbuch, Squeeze flow rheometry as a novel tool for the characterization of highly concentrated protein solutions, Biotechnology and Bioengineering (2015), DOI: 10.1002/bit.25834.

K. C. Bauer, M.-T. Schermeyer, J. Seidel, J. Hubbuch, Impact of polymer surface characteristics on the microrheological measurement quality of protein solutions - a tracer particle screening, International Journal of Pharmaceutics (2016), accepted.


Analytics of Molecular Properties

Analytik molekularer Eigenschaften

One aim of our scientific group is to characterize protein solutions, so that one can predict the solution stability and thereby be able to directly manipulate the protein solution towards a better long term stability or targeted phase transition. For the specific characterization of these complex solutions the knowledge about the molecular properties is indispensable. One important component is the study of protein mobility hence the determination of the protein diffusion coefficient. The diffusion of proteins reflects their interactions in solution. This approach allows the correlation with the stability and viscosity of a protein sample by dynamic light scattering measurements. It is suitable for the investigation of dilute as well as concentrated protein solutions.

Protein-protein interactions strongly depend on the protein surface characteristics, like surface charge and surface hydrophobicity. In comparison to the extensively studied impact of the surface charge on the colloidal and conformational stability, the impact of the surface hydrophobicity is studied unsatisfactorily. Our group has developed a method to determine a hydrophobicity index of the protein surface by the measurement of the surface tension in an automated microscale format. To define the influence of the given hydrophobicity and to correlate the diffusion coefficient with the conformational and colloidal stability of a protein solution, we perform temperature dependent stability tests besides to long term stability experiments (see protein phase behavior). With the help of fluorescence and static light scattering measurements protein unfolding and agglomeration can be determined. The high throughput measurements are realized with temperature ramps on the UnIt system (Unchained Labs). The parameters of interest are the agglomeration temperature (Tagg) and the melting temperature (Tm).

 

Literature

 

N. Rakel, K.C. Bauer, L. Galm, J. Hubbuch, From Osmotic Second Virial Coefficient (B22) to Phase Behavior of a Monoclonal Antibody, Biotechnology Process (2015), DOI: 10.1002/btpr.2065.

L. Galm, S. Amrhein, J. Hubbuch, Predictive approach for protein aggregation: Correlation of protein surface characteristics and conformational flexibility to protein aggregation propensity, Biotechnology and Bioengineering (2016), DOI: 10.1002/bit.25949.