Novel approach of high cell density recombinant bioprocess development: Optimisation and scale-up from microlitre to pilot scales while maintaining the fed-batch cultivation mode of E. coli cultures

Abstract

BackgroundBioprocess development of recombinant proteins is time consuming and laborious as many factors influence the accumulation of the product in the soluble and active form. Currently, in most cases the developmental line is characterised by a screening stage which is performed under batch conditions followed by the development of the fed-batch process. Performing the screening already under fed-batch conditions would limit the amount of work and guarantee that the selected favoured conditions also work in the production scale.ResultsHere, for the first time, high throughput multifactorial screening of a cloning library is combined with the fed-batch technique in 96-well plates, and a strategy is directly derived for scaling to bioreactor scale. At the example of a difficult to express protein, an RNase inhibitor, it is demonstrated that screening of various vector constructs and growth conditions can be performed in a coherent line by (i) applying a vector library with promoters and ribosome binding sites of different strength and various fusion partners together with (ii) an early stage use of the fed-batch technology. It is shown that the EnBase® technology provides an easy solution for controlled cultivation conditions in the microwell scale. Additionally the high cell densities obtained provide material for various analyses from the small culture volumes. Crucial factors for a high yield of the target protein in the actual case were (i) the fusion partner, (ii) the use of of a mineral salt medium together with the fed-batch technique, and (iii) the preinduction growth rate. Finally, it is shown that the favorable conditions selected in the microwell plate and shake flask scales also work in the bioreactor.ConclusionsCultivation media and culture conditions have a major impact on the success of a screening procedure. Therefore the application of controlled cultivation conditions is pivotal. The consequent use of fed-batch conditons from the first screening phase not only shortens the developmental line by guarantying that the selected conditions are relevant for the scale up, but in our case also standard batch cultures failed to select the right clone or conditions at all.

Reducing conditions are the key for efficient production of active ribonuclease inhibitor in Escherichia coli

“…As the previous results [8] indicated aggregation in the cytoplasm as a major problem, the following work was focussed on the control of the redox conditions. This was parallel approached by using a dsbA - mutant to remove of the strong oxidising activity of DsbA, and in parallel applying reducing agents.…”

Detection of growth rate‐dependent product formation in miniaturized parallel fed‐batch cultivations

“…This may cause problems during industrialization Correspondence: Prof. Dr. Peter Neubauer (peter.neubauer@tu-berlin.de), Chair of Bioprocess Engineering, Institute of Biotechnology, Technische Universität Berlin, Ackerstraße 76, ACK24, 13355 Berlin, Germany Abbreviations: EPG, endo-polygalacturonase; OD 600 , optical density at 600 nm of production processes [2]. Ideally, the cultivation conditions should remain as similar as possible during scale-up [3].…”

Constitutive overexpression of Pseudoalteromonas carrageenovora arylsulfatase in E. coli fed-batch culture

“…Most of the arylsulfatase activity (68.4%) in the fed-batch culture was produced in the extracellular medium. This high level secretion at high cell density could suggest that the change in permeability of the outer membrane may result from a disturbance of cell envelope biogenesis caused by competition between the overproduced protein and envelope components for the sites of translocation across the cytoplasmic and outer membranes, or for common metabolic precursors [22][23][24][25]. Nesmeyanova's studies indicate that translocation across the outer membrane may have resulted in a change of alkaline phosphatase conformation due to transmembrane movement, or alteration of the enzyme environment, and probably depends on the mechanism of protein translocation.…”