Figure 1.
Flow-chart for improved NOMV production.
Process performance is evaluated with the NonaMen concept, a nonavalent PorA vaccine comprised of three trivalent RL production strains (ΔrmpM–ΔlpxL1 mutant strains; Supplementary Table S2). Production of trivalent bulk NOMV is depicted in phases A to H. The transition from one phase to the next requires a specific unit operation (1 to 10). The seedlot (A) is first expanded in shake flask and bioreactor pre-cultures, then used to inoculate the production bioreactor. Harvested cells (B) are concentrated with microfiltration (C), and vesicle release is stimulated with a detergent-free buffer containing the chelating agent EDTA (D). Cells are discarded, and the crude NOMV are concentrated with ultrafiltration (E). Any residual DNA is digested with nuclease (F), and the extract is purified with gel filtration chromatography. The non-sterile bulk (G) is then sterilized by filtration to obtain the bulk NOMV (H). The trivalent product can be stored for at least one year before mixing as nonavalent vaccine and diluting to dose concentration (I).
Table 1.
Specifications for improved NOMV production.
Figure 2.
Upstream process characteristics.
A) The upstream process is performed at 40 L scale, which is representative for large-scale (800 L [34]). Biomass growth (black line) on chemically defined production medium is highly reproducible (regression R2 = 0.977). Cultivations from RL production strain 1, 2, and 3 are indicated by squares, circles, and triangles (respectively). Duplicates for each strain are indicated with open and closed symbols, giving a total of 6 cultivations. B) Oxygen consumption is monitored continuously. Cultivations are aligned at the time-point of maximal oxygen consumption (t = 0), which represents onset of the stationary phase. Harvest point of the cultivations has previously been optimized at t = 3 hours [35].
Figure 3.
Downstream process characteristics.
A) PorA content and recovery are shown for the consecutive product phases of the downstream process, from crude NOMV to bulk NOMV (Figure 1, phase D to H). PorA content (black bars) reaches final values from phase G onwards, indicating that the gel filtration chromatography effectively removes impurities. PorA recovery relative to phase D (grey bars) shows that losses are spread equally across the process, but notably not during sterile filtration (phase G to H), resulting in a reproducible and good overall recovery. Error bars indicate standard deviation of combined data from the three RL production strains (n = 9). B) Total protein and PorA yield of RL bulk NOMV (phase H) and large-scale reference processes. Reference 1 [31] is most comparable the current process since both are detergent-free (NOMV product). References 2 [20] and 3 [21] use detergent-extraction (DOMV product). All RL strains have a higher PorA yield than the references. RL strain 3 has a lower yield than the other two RL strains, resulting in a total protein yield that is comparable to reference 2 but still higher than the other references. The results of individual RL strains are reprodicible, therefore this yield difference is not caused by the process. Error bars indicate standard deviation of replicate batches (n = 3).
Table 2.
Quality Control testing of bulk NOMV.
Figure 4.
Protein composition and appearance of NOMV.
A) Protein composition of trivalent bulk NOMV is shown in lanes 1 to 3 for RL strains 1, 2 and 3, respectively (phase H), and composition of the nonavalent vaccine (phase I) in lane 4. Bulk NOMV from the three RL production strains are comparable, therefore the nonavalent vaccine has an equally comparable protein composition. B) Vesicle size of trivalent bulk NOMV is similar for RL strain 1 (black line), RL strain 2 (grey line), and RL strain 3 (dashed line). All bulk NOMV are non-aggregated and have comparable, homogeneous size distributions, with averages between 74 and 87 nm. C) Electron micrograph of nonavalent vaccine (non-adjuvated) after dilution to dose concentration shows that vaccine contains fully intact, non-aggregated OMV, an important benefit of the detergent-free production process.