The big picture
From immune disorders to certain types of cancers and infections, therapeutic proteins have revolutionised the treatment of a number of diseases. Such proteins are often unstable in solution and freeze-drying (lyophilisation) is commonly used to extend product shelf lifetime. During lyophilisation, a protector is used to avoid instability that could result in protein aggregation, often associated with a loss of potency. Understanding the mechanisms involved is key in the development of stable freeze-dried protein biopharmaceuticals. For this, neutron and X-ray scattering can be excellent tools, allowing researcher to see the inner structure of the materials and obtain essential information about protein–protein interactions.
In a new study using neutron and X-ray scattering measurements on bovine serum albumin (BSA), a protein relevant for pharmaceutical applications, researchers observed for the first time two populations of protein molecules, characterised by two different inter-molecular distances. This observation, now published in the scientific journal Soft Matter, is in contrast with the results previously obtained for lysozyme, an enzyme existing in many animal species and often used in studies of this kind. The results obtained highlight the importance of protein selection: lysozyme may not be a suitable model for studying stability-related aspects of larger pharmaceutical proteins.
In the words of Viviana Cristiglio, researcher at ILL and first author of the study, “The use of small-angle scattering techniques is invaluable in this field, providing detailed insights that are essential for advancing protein stabilisation methods. This research lays the groundwork for developing more stable and effective therapeutic proteins for pharmaceutical use.”
Taking a closer look
But what do we know about the stabilisation process and what are scientists trying to find out? We know that, for stabilisation to be effective, the protein and the protector must be present in a single glassy phase (a state of matter that combines the rigidity of crystals with the random molecular structure of liquids), with bonds formed between the protein and sugar molecules. However, this is not the full story. Several additional mechanisms have been suggested and may actually be at work simultaneously. One of them is a ‘‘dilution’’ hypothesis, according to which the protector molecules may serve as a physical barrier to reduce protein–protein contacts that could lead to instability and aggregation.
Using small-angle neutron and X-ray scattering techniques (SANS and SAXS), scientists can measure the typical protein–protein distances in the sample. In previous studies, a protein interaction peak with a larger spacing (corresponding to longer protein–protein distances) has been detected in formulations with a higher sugar content, supporting the “dilution” hypothesis. Up to now, lysozyme has been widely used as model protein in such studies. There have, however, been concerns that lysozyme behaviour during freezing may not necessarily be representative of the larger protein molecules commonly used in biopharmaceuticals.
For the first time
In the newly published study, freeze-dried samples of bovine serum albumin were investigated using neutrons and X-rays in Grenoble (France): at the Institut Laue Langevin (ILL) using instrument D16 (a cold neutron diffractometer), and at the ID02 beamline of the European Synchrotron Radiation Facility (ESRF). The study was performed within a collaboration joining experts from the pharmaceutical company AbbVie, ILL and ESRF.
Using small-angle neutron and X-ray scattering techniques (SANS and SAXS), researchers observed for the first time two protein interaction peaks in the SAXS and SANS patterns. The presence of two peaks indicates that there are two populations of protein molecules characterised by different inter-molecular distances. This contrasts with the results obtained for freeze-dried lysozyme samples, in which a single protein interaction peak was observed.
In addition, this study provides direct experimental support for the “dilution” hypothesis as a stabilisation mechanism: in samples with a higher sugar content, the protein–protein distances were longer for both populations, while the fraction of the protein population with a shorter protein–protein distance was lower. Both factors would favour better stability (better protection against aggregation) in sugar-rich mixtures.
What next?
Taking into account that a single protein interaction peak is observed before freeze-drying, as well as the different behaviour of lysozyme and BSA in frozen solutions (observed in high-resolution X-ray diffraction), researchers have put forward a new hypothesis that explains their observations and has implications on the stability of freeze-dried products (see figure below). These results are therefore certainly not the end of the story. The next steps will include testing this hypothesis further, investigating the SANS and SAXS results in detail, and further exploring the potential of these techniques.
Additionally, future studies will focus on proteins that are currently in use in pharmaceutical applications. This will enhance both the understanding and the application of stabilisation processes, directly benefiting the development of real-world therapeutic proteins.
"The collaboration with pharmaceutical experts such as AbbVie inc. is crucial," says Viviana Cristiglio, adding: "Their expertise can drive the fundamental science behind our research towards real-world applications, ensuring that our findings have a tangible impact on the development of effective biopharmaceuticals”.
Schematic representation of the hypothesis that the two populations of protein molecules are formed during the freezing step of the lyophilisation process, when a fraction of the BSA molecules and the majority of sugar molecules are expelled from the growing ice crystals (d1 population) with the second (d2) population remaining near the surface of the ice. These d2-population molecules wiil be present at the air/solid interface in the freeze-dried materials.
ILL instrument:D16
ILL contact person: Viviana Cristiglio
Publication: V. Cristiglio, S. Feng, M. Sztucki, X. Yuan, E. Shalaev, Two populations of protein molecules detected by small-angle neutron and X-ray scattering (SANS and SAXS) in lyophilized protein:lyoprotector (disaccharide) systems, Soft Matter 20 (2024) 19. http://dx.doi.org/10.1039/D4SM00028E.