Nucleation Studies of Lactobacillus brevis Alcohol Dehydrogenases in a Stirred Crystallizer Monitored by In Situ Multi-Angle Dynamic Light Scattering (MADLS)

Nucleation remains one of the least understood steps during protein crystallization, although it strongly impacts product quality attributes, including total crystal numbers, final crystal size distributions, and thus downstream processing. In this work, the nucleation behavior of Lactobacillus brevis alcohol dehydrogenase (LbADH) wild type (WT) and five mutants (Q207D, Q126H, K32A, D54F, and T102E) is investigated in a stirred 7 mL crystallizer monitored by in situ multi-angle dynamic light scattering (MADLS). Nucleation was studied with highly pure homotetrameric LbADHs by establishing a crystallization, lyophilization, and re-solubilization protocol combined with size exclusion chromatography (SEC) and size exclusion high-performance liquid chromatography (SE-HPLC), yielding tetramer purities above 94% and removing low molecular weight impurities. During stirred batch crystallizations initiated by the addition of polyethyleneglycol 550 monomethyl ether (PEG 550 MME), SEC and SE-HPLC revealed decreasing tetramer peak areas but essentially constant peak apex positions, indicating that no long-lasting oligomeric intermediates accumulate at detectable levels. Time-resolved MADLS measurements using a custom-made flow-through cuvette in a bypass to the stirred crystallizer uncovered transient cluster populations. All protein variants exhibited an initial tetramer peak, followed by the formation of larger aggregates and a rapid rise in signal above a hydrodynamic diameter of 1000 nm, coinciding with the onset of macroscopic turbidity. A simple mesoscale nucleation model was formulated, yielding end-of-nucleation times, crystallized fractions, critical soluble concentrations, and apparent nucleation rate constants. The crystal contact mutations modulate both the timing and magnitude of the nucleation burst (rapid build-up of nuclei/cluster populations). The mutant Q207D showed strongly attenuated nucleation compared to the WT, whereas the other mutants (K32A, D54F, and particularly T102E) display markedly accelerated nucleation at nearly invariant critical concentrations. The combined workflow demonstrates how in situ MADLS, together with a tailored kinetic description, can provide mechanistic insight into protein nucleation in stirred batch crystallizers.