Antibody production in Bacillus megaterium: Strategies and physiological implications of scaling from microtiter plates to industrial bioreactors

Abstract

Bacillus megaterium was used as an alternative high potential microbial production system for the production of antibody fragment D1.3 scFv. The aim of the study was to follow a holistic optimization approach from medium screening in small scale microtiter platforms, gaining deeper process understanding in the bioreactor scale and implementing advanced process strategies at larger scales (5-100 L). Screening and optimization procedures were supported by statistical design of experiments and a genetic algorithm approach. The process control relied on a soft-sensor for biomass estimation to establish a μ-oscillating time-dependent fed-batch strategy. Several cycles of growth phases and production phases, equal to starving phases, were performed in one production. Flow cytometry was used to monitor and characterize the dynamics of secretion and cell viability. Besides the biosynthesis of the product, secretion was optimized by an appropriate medium design considering different carbon sources, metal ions, (NH(4))(2)SO(4), and inductor concentrations. For bioprocess design, an adapted oscillating fed-batch strategy was conceived and successfully implemented at an industrially relevant scale of 100 L. In comparison to common methods for controlling fed-batch profiles, the developed process delivered increased overall productivities. Thereby measured process parameters such as growth stagnation or productivity fluctuations were directly linked to single cell or population behavior leading to a more detailed process understanding. Above all, the importance of single cell analysis as key scale-free tool to characterize and optimize recombinant protein production is highlighted, since this can be applied to all development stages independently of the cultivation platform.

Expression of Recombinant Antibodies

“…The Gram-positive bacteria Bacillus brevis (76, 77), Bacillus subtilis (78, 79), and Bacillus megaterium (80–85) have already been successfully used for the production of different antibody fragments. In addition, B. megaterium does not produce alkaline proteases and provides high stability of plasmid vectors during growth allowing stable transgene expression during long term cultivation in bioreactors (86).…”

Recombinant production of the antibody fragment D1.3 scFv with different Bacillus strains

“…Little consumption of xylose by B. megaterium MS941 after depletion of the main carbon source was described earlier [30] and only slightly influences the amount of recombinant protein [39]. Due to the used optimized xylose-inducible promoter P xylA the D1.3 scFv concentration was found to be around tenfold enhanced compared to data published by David et al [40]. …”

“…Lower oxygen tension slowed the energy generation of the respiratory chain and the present fructose concentration caused overflow metabolism [44], indicated by the secretion of acetate during growth (Fig. 2 and Additional file 1: Figure S1) which has been described previously [40, 45]. In this study, maximal acetate concentrations of 0.45–0.55 g L −1 were attained in shake flask and bioreactor cultivations, but only 0.25 g L −1 in microtiter plate experiments.…”

“…With this system, 390 µg L −1 of anti-CRP scFv [64], 410 µg L −1 of D1.3 scFv [33] and 3.5 µg L −1 of the more complex D1.3 scFab were produced and secreted in their active forms by B. megaterium strain MS941 [64] deficient in the main extracellular protease NprM lacking around 98% of the overall extracellular protease activity [36]. When compared to the production and secretion by a B. megaterium strain additionally deficient in xylose metabolization [30], D1.3 scFv secretion was enhanced to 700 µg L −1 in shake flasks and even 3 mg L −1 in 3 L bioreactors [40, 65]. With E. coli , about 1–2 mg L −1 of D.13 scFv were recovered as soluble protein in the culture supernatant and as periplasmic fraction [66], showing the inefficient production in Gram-negative bacteria.…”

Cultivo de <i>Escherichia coli</i> BL21 (DE3) para produção de L-asparaginase II

“…De acordo com o esquema de bioprocessos de David et al (2011) crescimento neste sistema permitiu obter até 4,5 g L -1 , enquanto que para agitador orbital foi 4,2 g L -1 , isto devido, provavelmente, ao aumento da eficiência do consumo de glicose para formação de biomassa e energia; ou então pelo aumento do oxigênio no meio de cultivo. A Tabela 22 mostra os valores de Ys/x e os intervalos da fase logarítmica de crescimento.…”