Downstream Processing of Chlamydomonas reinhardtii TN72 for Recombinant Protein Recovery

Abstract

The green microalga Chlamydomonas reinhardtii is under development as a production host for recombinant proteins and whole-cell therapeutics. In particular, the cell wall-reduced strain TN72 is used as a model organism for protein expression and algal synthetic biology. However, the bioprocessing characteristics of TN72 and other C. reinhardtii strains have yet to be examined. Here we use a TN72 strain expressing a protein-based antibiotic (Pal) to study the scale-up of cell harvest and product recovery. Cell harvest was examined with 100L cultures in two intermittent-discharge continuous-flow disc-stack centrifuges at flow rates of 150-250 L.h^-1, as well as with an ultra scale-down (USD) mimic of the centrifuges. Solids recovery exceeded 99.5% and the loss of product to the supernatant was below 2-3%. TN72 is intact following the high shear conditions of the feed zone, however discharge from both disc-stack centrifuges resulted in full cell breakage and in the case of Pal, partial degradation in the subsequent hours. We demonstrated that shake flask cultivation and the USD centrifuge technique can be used to predict the pilot-scale clarification efficiency and product release at the centrifuge inlet for TN72, but not the cell breakage on discharge. This study outlines a number of challenges for scale-up of recombinant protein production in the microalgal host in particular for whole cell therapeutics, but also opportunities for the bioprocessing of intracellular products from TN72.

Keywords: algae; disc-stack centrifugation; downstream processing; endolysin; scale-up; ultra scale-down.

Figure 1

Flow sheet outlining the processing steps involved in both pilot-scale and USD studies. Pilot-scale cultures of Chlamydomonas reinhardtii TN72_pal were grown in a 4 × 25 L modular hanging bag photobioreactor system before harvest with Gea Westfalia continuous-flow, intermittent-discharge disc-stack centrifuges. Disruption of the cellular component at pilot-scale was performed using a SPX 1000 homogenizer. In the USD study, cultures were grown in 1 L shake flasks or the hanging bag system and then processed using the USD rotating shear device to mimic the shear cells are subject to upon entrance into the Gea Westfalia disc-stack centrifuge. Subsequently, a bench top centrifuge was used to mimic the separation of cells from culture media that occurs within the pilot-scale centrifuge.

Figure 2

Comparison of the solids remaining (%) (A) and product loss (%) to the supernatant (B) after ultra scale-down (USD) centrifugation of a lab-scale and a pilot-scale Chlamydomonas reinhardtii TN72_pal culture. Both types of culture were exposed to two levels of shear in the rotating shear device followed by bench-scale centrifugation at equivalent settling area. Solids remaining (%) were determined by optical density measurements and the loss of the product Pal (%) to the supernatant by quantitative western blot analysis. The same pilot-scale culture was used for the 200 L.h⁻¹ GEA Westfalia CSA-1 run (see Figure 3 and Table 1). The linear regression lines correspond to the lab-scale culture data for high shear (red) and no shear (blue). Error bars represent ± 1 SD (n ≥ 3).

Figure 3

Solids remaining (%) (A) and product loss (%) (B) to the supernatant during harvest of Chlamydomonas reinhardtii TN72_pal by continuous-flow disc-stack centrifugation. Three 100 L pilot-scale cultures were harvested in a GEA Westfalia CSA-1 at flow rates of 150 L.h⁻¹ (Q/Σ 65 × 10⁻⁹ m.s⁻¹), 200 L.h⁻¹ (Q/Σ 87 × 10⁻⁹ m.s⁻¹), and 250 L.h⁻¹ [(Q/Σ 109 × 10⁻⁹ m.s⁻¹], respectively. Solids remaining (%) in the supernatant was determined by optical density measurements and the loss of the product Pal (%) to the supernatant by quantitative western blot analysis. Error bars represent ± 1 SD (n ≥ 3).

Figure 4

Integrity of Chlamydomonas reinhardtii TN72_pal cells during harvest by continuous-flow intermittent-discharge disc-stack centrifugation. A 100 L pilot-scale culture was harvested in a GEA Westfalia CSA-1 at a flow rate of 150 L.h⁻¹ (Q/Σ 65 × 10⁻⁹ m.s⁻¹). Samples were taken before harvest, from the centrifuge bowl during the run and the discharge after the harvest. The cell integrity was determined by particle size measurements (A1–A3) and light microscopy (B1–B3). Error bars represent ± 1 SD (n ≥ 5).

Figure 5

Integrity of Chlamydomonas reinhardtii TN72_pal cells before and after ultra scale-down (USD) centrifugation. For the USD centrifugation studies, cells from two pilot-scale bag cultures were exposed to two levels of shear in the rotating shear device followed by bench-scale centrifugation at equivalent settling area. The cell integrity was determined by particle size measurements (A1–A3) and light microscopy (B1–B3). Error bars represent ± 1 SD (n≥5).

Figure 6

Recovery (%) of the recombinant protein Pal during harvest by continuous-flow intermittent-discharge disc-stack centrifugation (A) and comparison to the recovery of endogenous proteins (B). Chlamydomonas reinhardtii TN72_pal pilot-scale cultures were harvested in a GEA Westfalia CSA-1 at a flow rate of 150 L.h⁻¹ (Q/Σ 65 × 10⁻⁹ m.s⁻¹) (A) and 250 L.h⁻¹ [(Q/Σ 109 × 10⁻⁹ m.s⁻¹] (B), respectively. The amount of Pal, D1 and RbcL in the cell material was determined by quantitative western blot analysis, and the total protein by Coomassie-stained SDS-PAGE. Error bars represent ± 1 SD (n ≥ 3).

Figure 7

Integrity of Chlamydomonas reinhardtii TN72 cells (A1–3) and product release (B) during harvest with a GEA Westfalia Pathfinder PSC-1 and subsequent homogenization. A 100 L pilot-scale culture was harvested at a flow rate of 120 L·h⁻¹ (Q/ 95 × 10⁻⁹ m·s⁻¹). Subsequently, the cells were treated with a SPX 1000 homogenizer. The cell integrity was determined by particle size measurements and the amount of the product Pal by western blot analyses. Error bars represent ± 1 SD (n ≥ 4).