Microtiter miniature shaken bioreactor system as a scale-down model for process development of production of therapeutic alpha-interferon2b by recombinant Escherichia coli

Abstract

BackgroundDemand for high-throughput bioprocessing has dramatically increased especially in the biopharmaceutical industry because the technologies are of vital importance to process optimization and media development. This can be efficiently boosted by using microtiter plate (MTP) cultivation setup embedded into an automated liquid-handling system. The objective of this study was to establish an automated microscale method for upstream and downstream bioprocessing of α-IFN2b production by recombinant Escherichia coli. The extraction performance of α-IFN2b by osmotic shock using two different systems, automated microscale platform and manual extraction in MTP was compared.ResultsThe amount of α-IFN2b extracted using automated microscale platform (49.2 μg/L) was comparable to manual osmotic shock method (48.8 μg/L), but the standard deviation was 2 times lower as compared to manual osmotic shock method. Fermentation parameters in MTP involving inoculum size, agitation speed, working volume and induction profiling revealed that the fermentation conditions for the highest production of α-IFN2b (85.5 μg/L) was attained at inoculum size of 8%, working volume of 40% and agitation speed of 1000 rpm with induction at 4 h after the inoculation.ConclusionAlthough the findings at MTP scale did not show perfect scalable results as compared to shake flask culture, but microscale technique development would serve as a convenient and low-cost solution in process optimization for recombinant protein.

Numerical and experimental assessment of a miniature bioreactor equipped with a mechanical agitator and non‐invasive biosensors

“…The cost of high‐value reagents, the lab intensive repetitive work as well as the complex cell cultivation process make small‐volume vessels attractive, including microplates, assay tubes and Erlenmeyer flasks . However, environmental parameters such as pH, temperature and DO in these vessels during the cell culture are often neglected and unmonitored, which could not always explain the unpredictable, unreproducible or unreliable results reasonably . Moreover, biological parameters or behaviors observed in these systems are not necessarily reproducible at the next larger scale – typically stirred tanks with a volume range of 1–5 L. Therefore parallel small/mini stirred bioreactors with well‐controlled pH, temperature and DO have shown great superiority to address the above scale‐up issues …”

“…With a substantial decrease in working volume, controlling the shear rate to maintain a sufficiently high oxygen transfer becomes quite difficult. Mixing and mass transfer in miniature bioreactors commonly rely on shaking or stirring with magnetic rings, which can provide a mixing time less than 2 s and an oxygen transfer rate higher than 1000 h −1 under certain conditions . Puskeiler et al .…”

“…8 However, environmental parameters such as pH, temperature and DO in these vessels during the cell culture are often neglected and unmonitored, which could not always explain the unpredictable, unreproducible or unreliable results reasonably. [9][10][11][12][13] Moreover, biological parameters or behaviors observed in these systems are not necessarily reproducible at the next larger scale -typically stirred tanks with a volume range of 1-5 L. Therefore parallel small/mini stirred bioreactors with well-controlled pH, temperature and DO have shown great superiority to address the above scale-up issues. 1,3,4,8,[14][15][16] For the past two decades, many miniature bioreactors ranging from milliliter to microliter scale have been developed, such as Ambr15 and Ambr250 from Sartorius (Göttingen, Germany) and bioREACTOR 8 and bioREACTOR 48 from 2Mag (Munich, Germany).…”

“…Mixing and mass transfer in miniature bioreactors commonly rely on shaking or stirring with magnetic rings, which can provide a mixing time less than 2 s and an oxygen transfer rate higher than 1000 h −1 under certain conditions. 9,19,20,8,14,15,[21][22][23] Puskeiler et al 24 developed a gas-inducing impeller to improve the oxygen transfer at milliliter scale. Another challenge for miniature bioreactors is process control, particularly for pH and DO in minimal space.…”