(M1330-01-01) Utility of Small Angle X-Ray Scattering (SAXS) in Process Development, Characterization and Comparability of Lipid Nanoparticles, Liposomes and Fusion Proteins

Purpose: Large molecule drug modalities like lipid nanoparticles (LNPs), liposomes and engineered fusion proteins have extensively expanded the delivery of a variety of active pharmaceutical ingredients (APIs). LNPs are made of different types of lipids encapsulating a drug cargo, typically a therapeutic nucleic acid. Liposomes are made of one or more phospholipid bilayers with an aqueous core and can hold and release drugs in a controlled manner. Engineered fusion proteins are modified proteins made to treat diseases or function as vaccine antigens. These three types of large molecule modalities are largely administered in solution and their efficacy depends on their specific morphology. To fully optimize the process development and control the physical attributes of these large molecules, we need analytical tools that can monitor size and molecular conformation under different solution conditions. Small Angle X-ray Scattering (SAXS) is a powerful technique to probe the electron density distributions of biological samples and has been commonly used for structural characterization of proteins and nucleic acids in solution [1, 2]. The purpose of this investigation is to explore the utility of routine in-laboratory SAXS measurements for biopharmaceutical development with line of sight towards process optimization, structural characterization, and comparability studies of LNPs, liposomes and engineered proteins.

Methods: We use SAXS to characterize LNP morphology, liposome lamellarity and folding/unfolding of engineered fusion proteins under different solution conditions. LNPs are made using small-scale mixing with or without RNA load. Liposome lamellarity is controlled by undergoing different extrusion parameters. The quality of the folded state of an engineered fusion protein is tested under different excipient conditions. SAXS data are collected using a commercial in-laboratory instrument with a CuK-alpha x-ray source. In SAXS, x-ray scattering from LNP, liposome and engineered proteins in solution are collected in an area detector positioned at a certain distance from the sample. The scattering image is condensed to a scattering profile given by scattering intensity, I, and a wave vector, q, which is a function of the scattering angle. The scattering profile provides conformational information about the samples to aid process development. Replicate measurements and time course studies using SAXS in conjunction with orthogonal analytical methods provides a way to monitor process robustness and sample stability.

Results: For LNPs, we compare changes in SAXS profiles under different mixing and formulation conditions and benchmark results with what is currently available in the literature [3, 4]. Characterization with SAXS, in conjunction with CryoEM imaging and modeling approaches, can be used to elucidate the full structure of LNPs [5]. In Figure 1, we show comparisons of SAXS profiles of LNPs made with or without RNA cargo. This suggests a notable change in internal structure of LNPs after encapsulation of RNA. For liposomes, it is important to assess lamellarity or the number of lipid bilayers, N, because liposome structure and integrity can affect drug properties. We find that SAXS of liposomes provides a quick way to assess lamellarity by noting peak profiles and determining N from modeling [6]. In Figure 2, we show changes in SAXS profiles of liposomes after undergoing different extrusion conditions. As noted in the figure, liposomes with a high lamellarity (number of lipid bilayers of 5 or higher) have a sharper first peak which corresponds to the bilayer spacing distance. As the number of extrusions increase the intensity of the first peak decreases indicating a decreased number of lipid bilayers. Therefore, SAXS, together with particle size measurements and benchmarked with CryoEM imaging, allows fast process development turnaround for liposomes. Lastly, for engineered proteins, we noted that SAXS is a great tool for characterization of protein conformation under different excipient conditions [7]. We can measure the radius of gyration, pair distribution function and show Kratky plots, I(q)·q² vs. q, a qualitative way to assess protein flexibility or degree of unfolding in protein samples. In Figure 3, Kratky plots of the same engineered fusion protein in 2 different excipient conditions highlight the importance of choosing the correct excipient for maintaining protein stability.

Conclusion: We showed the utility of routine in-laboratory SAXS measurements for accelerated biopharmaceutical development of different types of large molecule samples: LNPs, liposomes and engineered proteins.

References: [1] Broset & Tainer, Curr. Opin. Struct. Biol. 2019: 58, 197; [2] Tokuda, et al. Biophys. Rev. 2016: 8, 139; [3] Yanez Arteta et al., PNAS 2018: 115, E3351; [4] Cornebise et al., Adv. Funct. Mat. 2022: 32, 2106727; [5] Cardenas et al., Curr. Opin. Coll. Interf. Sci. 2023: 66, 101705; [6] Konarev et al., J. Appl. Cryst. 2020: 53, 236; [7] Zhang et al. Pharmaceutics 2022: 14, 69


Figure 1: SAXS profiles of LNP with and without cargo


Figure 2: SAXS profiles of liposomes showing decreasing lamellarity from N = 5 to N = 2


Figure 3: SAXS Kratky plot of the same engineered fusion protein incubated in 2 different excipients. Protein in excipient 1 is in the correct fold while protein in excipient 2 is showing signs of unfolding.