Abstract
Tremendous advancements in cell and protein engineering methodologies and bioinformatics have led to a vast increase in bacterial production clones and recombinant protein variants to be screened and evaluated. Consequently, an urgent need exists for efficient high-throughput (HTP) screening approaches to improve the efficiency in early process development as a basis to speed-up all subsequent steps in the course of process design and engineering. In this study, we selected the BioLector micro-bioreactor (µ-bioreactor) system as an HTP cultivation platform to screen E. coli expression clones producing representative protein candidates for biopharmaceutical applications. We evaluated the extent to which generated clones and condition screening results were transferable and comparable to results from fully controlled bioreactor systems operated in fed-batch mode at moderate or high cell densities. Direct comparison of 22 different production clones showed great transferability. We observed the same growth and expression characteristics, and identical clone rankings except one host-Fab-leader combination. This outcome demonstrates the explanatory power of HTP µ-bioreactor data and the suitability of this platform as a screening tool in upstream development of microbial systems. Fast, reliable, and transferable screening data significantly reduce experiments in fully controlled bioreactor systems and accelerate process development at lower cost.
Highlights
Tremendous advancements in cell and protein engineering methodologies and bioinformatics have led to a vast increase in bacterial production clones and recombinant protein variants to be screened and evaluated
We evaluated the μ-bioreactor system BioLector operated in an enzymatic substrate release fed-batch mode as a screening cultivation platform with a set of 22 different E. coli host/protein/leader combinations
Transferability of μ‐bioreactor Fab clone screenings to the benchtop bioreactor. In this first part of the study, the Fab production clone characterizations in fed-batch-like HTP μ-bioreactor cultivations were evaluated with respect to their transferability to fed-batch data from benchtop bioreactor cultivations
Summary
Tremendous advancements in cell and protein engineering methodologies and bioinformatics have led to a vast increase in bacterial production clones and recombinant protein variants to be screened and evaluated. Abbreviations CDM Cell dry mass DO Dissolved oxygen Fab Fragment antigen binding GI Genome integrated HCD High cell density HTP High-throughput μ-bioreactor Micro-bioreactor MTP Micro titer plate scFv Single-chain variable fragment SS Signal sequence STR Stirred tank reactor WCB Working cell bank In recent years, both the development of host strains and the identification/design of promising protein candidates have accelerated significantly with steady progress in the fields of molecular biology, genetic engineering, synthetic biology, protein engineering, and bioinformatics. The number of potential protein candidates was boosted by protein engineering approaches utilizing the large number of native protein sequences, designed protein scaffolds, phage displays, and the option to combine functional protein units, such as constant scaffolds with randomized variable r egions[8,9,10] This is further supported by the use of directed evolution employing machine learning[11]. Potential combinations is increased further by the broad range of genetic elements supporting the transcription, translation, and processing of respective proteins of interest[13]
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