Yes

No / Bulk crystallisation of protein therapeutic molecules towards their controlled drug delivery is of interest to the biopharmaceutical industry. The complexity of biotherapeutic molecules is likely to lead to complex material properties of crystals in the solid state and to complex transitions. This complexity is explored using batch crystallised lysozyme as a model. The effects of drying and milling on the solid-state transformations of lysozyme crystals were monitored using differential scanning calorimetry (DSC), X-ray powder diffraction (XRPD), FT-Raman, and enzymatic assay. XRPD was used to characterise crystallinity and these data supported those of crystalline lysozyme which gave a distinctive DSC thermogram. The apparent denaturation temperature (Tm) of the amorphous lysozyme was ∼201 °C, while the Tm of the crystalline form was ∼187 °C. Raman spectra supported a more α-helix rich structure of crystalline lysozyme. This structure is consistent with reduced cooperative unit sizes compared to the amorphous lysozyme and is consistent with a reduction in the Tm of the crystalline form. Evidence was obtained that milling also induced denaturation in the solid-state, with the denatured lysozyme showing no thermal transition. The denaturation of the crystalline lysozyme occurred mainly through its amorphous form. Interestingly, the mechanical denaturation of lysozyme did not affect its biological activity on dissolution. Lysozyme crystals on drying did not become amorphous, while milling-time played a crucial role in the crystalline-amorphous-denatured transformations of lysozyme crystals. DSC is shown to be a key tool to monitor quantitatively these transformations.

Acoustic focusing of microscale protein crystals with acoustophoresis technology could reduce clogs during experiments with the scientific technique serial femtosecond x-ray crystallography (SFX). SFX determines molecular structures of proteins, these structures are valuable in drug discovery and fundamental biomedical research. Lysozyme crystals were focused in their own mother liquor and dilutions with PBS buffer. The aim of these tests were to study how the acoustic contrast factor Φ changes with the medium. Recorded experiments were analyzed using the particle tracking software Trackmate to extract velocities and radii. The lysozyme crystals changed morphologies in large dilutions of PBS buffert, they either became rounder or broke into fragments. The changed forms are likely caused by dissolution behaviors; some dilutions were unstable, but not unstable enough to dissolve the crystals completely.  Measured velocities during focusing of the crystals had large variance. Sinusoidal fits of the velocities had significant increases in amplitudes for larger dilutions of PBS. A change in acoustic contrast factor Φ could be the cause for the increased amplitudes, but the results do not rule out other causes. There are currently major knowledge gaps about using protein crystals as particles with acoustophoresis technologies, hence many ideas for future works have been proposed in this master thesis report. / Akustisk fokusering av mikrometers-stora proteinkristaller med hjälp av ultraljudsteknik skulle kunna reducera proppar under experiment med tekniken seriell femtosekundskristallografi (SFX). SFX kan avgöra vilken struktur proteinmolekyler har, dessa strukturer är värdefulla för industriell utveckling av nya läkemedel och fundamental biomedicinsk forskning. Proteinkristaller av lysozym har fokuserats i sin egen kristalliseringslösning och utspädningar av PBS. Målet med de här experimenten var att se om den akustiska flödeskontrasten Φ kunde bli påverkad. Fokuseringar spelades in och partiklarnas hastigheter vid fokuseringarna mättes med hjälp av Trackmate (en mjukvara för partikelspårning). I de större utspädningarna förändrades lysozymkristallernas former, de blev antingen mer runda eller så blev de små bitar. Förändringen skedde förmodligen på grund av upplösningsmekanismer i instabila utspädningar.  Uppmätta hastigheter vid testerna hade stor varians, men kurvanpassningar av data tyder på att hastigheterna ökar signifikant med utspädningar av PBS. Den akustiska flödeskontrasten Φ kan vara orsaken för ökade hastigheter, men andra hypoteser gick inte att utesluta med resultaten. Stora kunskapshål finns om akustisk fokusering av proteinkristaller, därför har många idéer för framtida experiment och arbeten föreslagits i rapporten.