Never diffuse alone: understanding antibody teamwork to design tomorrow's therapeutics
Monoclonal antibodies (mAbs) are used to treat a large variety of diseases. In order to allow patients to inject these therapeutic molecules autonomously, it is crucial to understand the origin of and to control their viscosity. An international team of researchers used an interdisciplinary approach to understand how molecular interactions and motions influence the viscosity of different mAbs. The study has now been published in the journal Molecular Pharmaceutics.
Monoclonal antibodies (mAb) are therapeutic molecules with a highly diverse spectrum of activity. They are used to treat a large variety of cancers as well as some infectious and autoimmune conditions, for example airway infections with the respiratory syncytial virus (RSV), Crohn's disease and rheumatoid arthritis.
An important limitation of mAbs is their rapid degradation by the digestive system. This implies that they need to be administered intravenously, which required hospitalisation of the patients. To facilitate treatment, the pharmaceutical industry is therefore interested in producing mAb formulations that patients can self-administer via injections. To this end, it is essential to control the viscosity of these formulations since injectability decreases rapidly above a certain viscosity threshold.
The types of interactions between the mAbs (i.e. whether they tend to repel each other or rather come close together into so-called clusters) are major determinants of their viscosity. So far this knowledge had been limited to a narrow selection of model mAbs.
Discover Ilaria Mosca's InnovaXN PhD project in this short video
Teaming up with a partner from the pharmaceutical industry (company Lonza AG, from Basel, Switzerland) a team of researchers from the University of Tübingen (Germany) and the ILL designed a study of five different therapeutic mAbs. "Our goal was to perform a detailed, multi-method study of how these mAbs behave in solution - starting from their interactions up to their movement in solution", explains Ilaria Mosca, first author of the study and PhD student at the ILL in the framework of InnovaXN, a EU-funded doctoral training programme bringing together large-scale research infrastructures and industry.
The authors performed a complex set of experiments ranging from laboratory-based rheology (measuring the flow properties of fluids and soft materials under stress)to neutron experiments at the ILL. In a first step, rheology revealed that, despite their similar molecular structures, the viscosities of the mAbs studied differ drastically. This result shows how important subtle protein-protein interactions are in determining their macroscopic properties.
To further characterise these molecular interactions, the team used small-angle neutron scattering (SANS) on the D11 instrument at the ILL. At body temperature (37°C), the mAbs appeared to be less attracted to each other than at lower temperature. This may indicate that the mAbs group together into so-called clusters when transported or stored at ambient or cold temperatures, which then dissolve upon injection.
Apparent diffusion coefficients D obtained from fitting QENS data for different IgG antibodies vs their crowding in solution, at ~7°C - storage temperature in a standard fridge (right). Lines represent the theoretical monomeric and dimeric diffusion of charged spheres. Symbols below the monomer lines corroborate the presence of small clusters of a few monomers. The diffusion varies a lot among the antibodies despite their very similar sequences (left). They in fact show almost identical sequences in the Fc region (blue - 100% identity), while they differ in the extreme parts of the Fab regions (red - 0% identity),
The presence of mAb clusters was indeed confirmed with the help of neutron backscattering on ILL instrument IN16B, a technique which allows to track molecular dynamics and diffusion on very small time scales. "We observed lifetimes on the nanosecond time scale (1 nanosecond = 10-9 seconds)", says Tilo Seydel, instrument co-responsible of IN16B. "The clusters dissolve and re-form rapidly in solution. On average, each cluster consists of 2-3 mAb molecules, but with a strong dependence on the type of mAb. This links back to their highly different viscosities." The experimental data were further supported by molecular dynamics simulations.
"On the European Photon and Neutron (EPN) campus in Grenoble, the proximity of the labs to the ILL experimental halls and the availability of such a large selection of neutron instrumentation really makes it an ideal environment for experiments of such complexity", says Ilaria Mosca.
The study now published in the journal Molecular Pharmaceutics underlines the strength of interdisciplinary approaches, including both lab bench methods and large scale facilities. In particular, neutron backscattering provides access to molecular dynamics on time and energy scales that are not accessible otherwise. This information is invaluable for a detailed understanding of therapeutic molecules and for the optimisation of future administration routes.
ILL Instruments : D11 - Lowest momentum transfer & lowest background small-angle neutron scattering instrument and IN16B - Versatile high flux backscattering spectrometer
Reference : Mosca, I., Pounot, K., Beck, C., Colin, L., Matsarskaia, O., Grapentin, C., Seydel, T. & Schreiber, F.
Biophysical determinants for the viscosity of concentrated monoclonal antibody solutions.
Molecular Pharmaceutics (2023), 20(9), 4698-4713
doi.org/10.1021/acs.molpharmaceut.3c00440
ILL Contacts : Olga Matsarskaia, Tilo Seydel
Industrial contact: Christoph Grapentin, Lonza AG, Basel, Switzerland