High-throughput SAXS analysis of protein-based drugs

Novo Nordisk researches, produces and markets protein-based drug formulations, such as insulins or blot clotting factors. Many of Novo Nordisk’s protein and peptide based drugs are modified by chemically attaching sugar or fatty acid molecules, thus significantly changing the properties of the molecules and improve on properties such as duration of action or stability.  In this focus project between Novo Nordisk and the University of Copenhagen small-angle scattering was applied to increase the amount of information that can be obtained from scattering data and the speed at which it can be determined.

Read the interesting one pager from this project: SAXS Analysis of Protein-Based drugs.

Techniques and Methods

First the LINX team at the University of Copenhagen (KU) developed a pipeline software that can automate processing and analysis of a large number of small-angle X-ray scattering (SAXS) datasets which are measured on a daily base with Novo Nordisk’s in-house SAXS instruments. Using statistical tests, the software automatically selects good frames from all recorded frames, processes them and determines the radius of gyration and the pair distance distribution function (PDDF) p(r) for each sample. These parameters describe the size, shape and compactness of protein structures without the need for further input.

In a next step, the LINX team at KU developed a model-based analysis software for the simultaneous analysis of X-ray and neutron scattering of proteins with attached modifications. Based on the previously developed WillItFit?-framework, the new software works with molecules which are decorated chemically. The modifications can have a significant influence on the interactions between the individual proteins and their ability to self-assemble into larger oligomeric structures. With the help of the new software an improved description and understanding  of the properties of drug molecules in formulations can be obtained, which aids in the selection of lead molecules and in  optimisation of drug formulations.

Finally, the LINX team at KU demonstrated in a proof-of-concept study how the powerful beam intensity of a synchrotron can be utilized for time-resolved SAXS measurements, where the frames are recorded with a temporal resolution of only a few milliseconds with the help of a stopped flow device. Different insulin hexamer formulations were mixed in the stop-flow cell with different additives and the course of the SAXS patterns recorded over a few hundreds of milliseconds. Model-free analyses (radius of gyration, PDDF) were applied to study the temporal course of the dissociation or oligomerization of the insulin hexamers at these time scales.

Project Information

Participants: Novo Nordisk, University of Copenhagen.
Start date, end date:
 May 2016 – June 2018.
High-throughput SAXS analysis of protein-based drugs (FP04.001, Colloid materials).