(194f) Exploiting Bacterial Adaptations for Stable Spray-Dried Enzymes- a Study of a Thermophilic Aldehyde Dehydrogenase

Spray drying is a well-established method of producing protein and peptide powders with controlled particle properties, for a span of applications including dry powder inhalables (DPIs) and continuous process implementation. One of the main drawbacks of the method is the application of heat, shear, and interfacial stresses on protein molecules, often resulting in irreversible denaturation of the product by increasing the likelihood of none-specific protein-protein interactions. Currently, stabilisation of spray dried proteins is achieved by addition of excipients. Excipients are successful in ameliorating protein stability issues, however their use can be limiting due to lung-related toxicity, product-specific interactions and their effect on final powder properties such as morphology and density [1-4]. This presentation will explore an alternative avenue for the stabilisation of proteins in spray drying by primary structure modifications. Structural mutations which enhance protein stability are widely explored in the literature however are largely ignored in their impact on solid-state stabilisation [5]. A thermophilic aldehyde dehydrogenase from Thermus thermophilus (ALDHTt) was used as a model protein to showcase the ability of primary structure features in stabilisation of protein during spray drying. Through evolutionary adaptation, the ALDHTt protein possesses a unique thermostable amino acid extension in its C-terminal [6]. The existence of this intrinsic thermostable structure in a protein, which may be excised using genetic engineering methods, cuts down on introduction of novel mutations and screening of potential thermostable mutants.

Methods

To study the effect of this structural adaptation on spray drying and solid-state stability, the C-terminus of ALDHTt was excised and both proteins (with/without the C-terminal amino acid extension) were recombinantly expressed in E.coli BL21(DE3) cells. The proteins were then spray dried using a design of experiments (DOE) approach to statistically determine the impact of the C-terminus on the rates of enzymatic activity loss and related aggregation. The utilized DOE can be viewed in Figure 2, supplementary image. As spray drying of ALDH has not been reported in the literature, the DOE was also used to optimize the spray drying conditions of ALDHTt, by monitoring the output factors of particle size, moisture content and enzyme activity. Both soluble and insoluble aggregation was assessed using Size-Exclusion High Performance Liquid Chromatography (SE-HPLC) and nephelometric turbidity, respectively. Additionally, the secondary structure conformation was explored using Circular Dichroism (CD) and Fourier Transform Spectroscopy (FTIR), both before and after spray drying. Stability of both proteins at solid-state was monitored at room temperature over a period of two months.

Results

The presence of the thermostable C-terminus extension was found to lower the activity of the native protein in aqueous solution, however it stabilised the oligomeric state of the ALDH tetramer upon solid state formation and during subsequent storage. The behaviour of the genetically engineered protein in solution has given a rationale into the evolutionary exclusion of the C-terminus from ALDH proteins in mesophilic organisms. The removal of the C-terminus extension promoted the presence of insoluble and soluble aggregation after spray drying, by allowing interaction of exposed residues with air/liquid interfaces and with other protein molecules. ALDHTt with the C-terminal extension retained, on average, 24% more activity than without it during spray drying and retained up to 40% more activity during solid-state storage (see Figure 3 and 4 in supplementary image). Loss of activity from the protein lacking a C-terminus was correlated with the prevalence of high-order insoluble aggregates, and a destabilisation of the tetrameric structure by SE-HPLC and CD (see Figure 5 in supplementary image).We propose a mechanism for the protection of oligomeric proteins by the distinct C-terminal extension under shear, interfacial and thermal stresses involved in solid formation. This work is the first report outlining the effect of a structural modification on spray drying and solid-state stability of enzymes and is the first work showcasing the application of the C-terminal bacterial adaptation in the stabilisation of proteins. Although outside the scope of this work , the possibility of replicating the effect of this adaptation in other proteins using modelling and a rational mutagenesis approach may have a high impact in protein stability research. As a secondary goal of this work, the optimum process conditions for spray drying ALDH were found to be 100˚C outlet drying air temperature and a feed flow rate of 1.5 ml/min using a laboratory scale spray drier Büchi B-290. Using these process conditions, dried powders of ALDH were obtained with moisture contents of 7.5%, average of 9.2 ± 2.9 µm and activity retentions of 89%. Specifically, the creation of a stable dried powder of the thermophilic aldehyde dehydrogenase may improve the breadth of this enzyme’s applications in biocatalysis.

References

[1] G. Pilcer, K. Amighi, Formulation strategy and use of excipients in pulmonary drug delivery, International Journal of Pharmaceutics, 392 (2010) 1-19.https://doi.org//10.1016/j.ijpharm.2010.03.017

[2] H.R. Costantino, J.D. Andya, P.A. Nguyen, N. Dasovich, T.D. Sweeney, S.J. Shire, C.C. Hsu, Y.F. Maa, Effect of mannitol crystallization on the stability and aerosol performance of a spray-dried pharmaceutical protein, recombinant humanized anti-IgE monoclonal antibody, J Pharm Sci, 87 (1998) 1406-1411.https://doi.org/10.1021/js9800679

[3] K. Wrzosek, J. Moravčík, M. Antošová, V. Illeová, M. Polakovič, Spray drying of the mixtures of mono-, di-, and oligosaccharides, Acta Chimica Slovaca, 6 (2013) 177-181.https://doi.org/10.2478/acs-2013-0028

[4] F. Emami, A. Vatanara, E.J. Park, D.H. Na, Drying Technologies for the Stability and Bioavailability of Biopharmaceuticals, Pharmaceutics, 10 (2018) 131.https://doi.org/10.3390/pharmaceutics10030131

[5] G. Walsh, Proteins : Biochemistry and Biotechnology, John Wiley & Sons, Incorporated, Hoboken, UNITED KINGDOM.

[6] K. Hayes, M. Noor, A. Djeghader, P. Armshaw, T. Pembroke, S. Tofail, T. Soulimane, The quaternary structure of Thermus thermophilus aldehyde dehydrogenase is stabilized by an evolutionary distinct C-terminal arm extension, Sci Rep, 8 (2018) 13327.https://doi.org/10.1038/s41598-018-31724-8