Spray Drying Is a Viable Technology for the Preservation of Recombinant Proteins in Microalgae

Abstract

Microalgae are promising host organisms for the production of encapsulated recombinant proteins such as vaccines. However, bottlenecks in bioprocess development, such as the drying stage, need to be addressed to ensure feasibility at scale. In this study, we investigated the potential of spray drying to produce a recombinant vaccine in microalgae. A transformant line of Chlamydomonas reinhardtii carrying a subunit vaccine against salmonid alphavirus was created via chloroplast engineering. The integrity of the recombinant protein after spray drying and its stability after 27 months storage at -80 °C, +4 °C and room temperature were assessed by immunoblotting. The protein withstood spray drying without significant losses. Long-term storage at +4 °C and room temperature resulted in 50% and 92% degradation, respectively. Optimizing spray drying and storage conditions should minimize degradation and favour short-term storage at positive temperatures. Using data on yield and productivity, the economics of spray drying- and freeze drying-based bioprocesses were compared. The drying stage corresponded to 41% of the total production cost. Process optimization, genetic engineering and new market strategies were identified as potential targets for cost reduction. Overall, this study successfully demonstrates the suitability of spray drying as a process option for recombinant protein production in microalgae at the industrial scale.

Keywords: Chlamydomonas reinhardtii; microalgae; oral vaccination; recombinant protein; scale-up; spray drying; techno-economic analysis.

Figure A1

Process flow diagram of the production of a microalgae-based edible vaccine with a freeze-drying strategy.

Figure A2

Process flow diagram of the production of a microalgae-based edible vaccine with a spray-drying strategy.

Figure 1

Illustration of the E2-ecto transgene inserted in the C. reinhardtii plastome. The four level 0 parts (promoter, 5′UTR, CDS and 3′UTR) were assembled to form the level 1 transcription unit, which was targeted to a neutral site between psbH and trnE2 in the plastome of the psbH-deletion mutant TN72, with transformants selected based on restoration of PsbH function, as described by Wannathong et al. [22]. The coding sequence encodes a 511-residue chimeric protein comprising the cholera toxin beta (CTB) subunit, three copies of a GGGGS linker, the SAV E2 ectodomain (E2), a single copy of GGGGS and the HA epitope tag.

Figure 2

Process steps for the large-scale production of a microalgae-based edible vaccine against SAV, from the preparation of the inoculum for large-scale cultivation in photobioreactors (PBR) to the oral delivery of the vaccine. The scope of the present techno-economic analysis (TEA) is delimited by the red dotted-line rectangle. (Figure created with BioRender.com.)

Figure 3

Evaluation of E2-ecto vaccine integrity throughout the manufacturing process, with a focus on the effects of spray drying as compared to freeze drying. (a) Immunoblot analysis of E2-ecto vaccine in TN72:E2-ecto cells after cultivation (“culture”, biological triplicates), centrifugation (“slurry”, technical triplicates), spray drying (“SD”, technical triplicates) or freeze drying (“FD”, technical triplicates). Samples were normalized at 30 µg of biomass loaded per well. Positive control (+): TN72:ptxD, negative control (–): TN72:pSRSapI. (b) Densitometry analysis of the culture (biological triplicates), slurry, spray-dried (SD) and freeze-dried (FD) samples (technical triplicates) in the 800 nm channel. Data are expressed as the average signal intensity ± one standard error. Individual data points are shown as white circles. Student’s t-tests were applied, with statistically significant differences identified with * (p-value < 0.05).

Figure 4

Evaluation of E2-ecto vaccine stability in spray-dried microalgal powder after long-term storage at different temperatures. (a) Immunoblot analysis of E2-ecto vaccine in spray-dried (SD) microalgal powder stored at –80 °C, +4 °C and ambient temperature (RT) for 27 months. The freeze-dried (FD) microalgal powder stored at –80 °C in similar conditions was used as reference. Each sample was analysed in technical triplicates, except for the FD powder, for which one of the replicates was lost during storage. Samples were normalized at 30 µg of biomass loaded per well. Positive control (+): TN72:ptxD, negative control (–): TN72:pSRSapI. (b) Densitometry analysis in the 800 nm channel of the spray-dried (SD) samples stored at –80 °C, +4 °C and ambient temperature for 27 months. Data are expressed as the average signal intensity relative to the average signal intensity of the FD powder stored at –80 °C ± one standard error. Individual data points are shown as white circles. Student’s t-tests were applied, with statistically significant and highly significant differences identified with * (p-value < 0.05) and ** (p-value < 0.01), respectively.

Figure 5

Details of the (a) capital expenditures (CAPEX) and (b) operating expenditures (OPEX) for the production of a microalgae-based edible vaccine with a freeze-drying or spray-drying strategy. An annual production of 286 kg_DCW of microalgae is considered with a vaccine yield of 3.15 g_vaccine/kg_DCW (base case scenario).