Integrated process development: The key to improve Fab production in E. coli.

Bioprocess development and optimization is a challenging, costly, and time-consuming effort. In this multidisciplinary task, upstream processing (USP) and downstream processing (DSP) are conventionally considered distinct disciplines. This consideration fosters "one-way" optimization disregarding interdependencies between unit operations; thus, the full potential of the process chain cannot be achieved. Therefore, it is necessary to fully integrate USP and DSP process development to provide balanced biotechnological production processes. The aim of the present study was to investigate how different host/secretory signal/antigen binding fragment (Fab) combinations in E. coli expression systems influence USP, primary recovery performance and the final product quality. We ran identical fed-batch cultivations with 16 different expression clones to study growth and product formation kinetics, as well as centrifugation efficiency, viscosity, extracellular DNA, and endotoxin content, important parameters in DSP. We observed a severe influence on cell growth, product titer, extracellular product, and cell lysis, accompanied by a significant impact on the analyzed parameters of DSP performance. Our results provide the basis for future research on integrated process development considering interdependencies between USP and DSP; however, individual products need to be considered specifically. These interdependencies need to be understood for rational decision-making and efficient process development in research and industry.

Three-dimensional chromatography for purification and characterization of antibody fragments and related impurities from Escherichia coli crude extracts.

Antibody fragments (Fab) are often produced by recombinant methods in Escherichia coli as no glycosylation is needed. Besides the correctly expressed Fab molecule, a multitude of host cell impurities and product related impurities are present in the crude sample. The identification and characterization of the product-related impurities, such as modified Fab-molecules or free light chain, are of utmost importance. The objective of this work was to design a purification strategy to isolate and characterize Fab and related impurities. A three-dimensional chromatography method was established, consisting of two affinity steps (Protein G and Protein L) and subsequent cation exchange chromatography, followed by mass spectrometry analysis of the purified samples. The procedure was automated by collecting the eluted target species in loops and directly loading the samples onto the high-resolution cation exchange chromatography column. As an example, four different Fab molecules are characterized. All four samples contained mainly the correct Fab, while only one showed extensive N-terminal pyroglutamate formation of the Fab. In another case, we found a light chain variant with uncleaved amino acids from the lead molecule, which was not used for the formation of whole Fab as only correct Fab was found in that sample. Impurities with lower molecular weights, which were bound on the Protein L column, were observed in all samples, and identified as fragments of the light chain. In conclusion, we have devised a platform for characterizing Fab and Fab-related impurities, which significantly facilitated strain selection and optimization of cultivation conditions.

Extraction of recombinant periplasmic proteins under industrially relevant process conditions: Selectivity and yield strongly depend on protein titer and methodology.

In this work, we attempted to identify a method for the selective extraction of periplasmic endogenously expressed proteins, which is applicable at an industrial scale. For this purpose, we used an expression model that allows coexpression of two fluorescent proteins, each of which is specifically targeted to either the cytoplasm or periplasm. We assessed a number of scalable lysis methods (high-pressure homogenization, osmotic shock procedures, extraction with ethylenediaminetetraacetic acid, and extraction with deoxycholate) for the ability to selectively extract periplasmic proteins rather than cytoplasmic proteins. Our main conclusion was that although we identified industrially scalable lysis conditions that significantly increased the starting purity for further purification, none of the tested conditions were selective for periplasmic protein over cytoplasmic protein. Furthermore, we demonstrated that efficient extraction of the expressed recombinant proteins was largely dependent on the overall protein concentration in the cell.

Tunable recombinant protein expression with E. coli in a mixed-feed environment.

Controlling the recombinant protein production rate in Escherichia coli is of utmost importance to ensure product quality and quantity. Up to now, only the genetic construct, introduced into E. coli, and the specific growth rate of the culture were used to influence and stir the productivity. However, bioprocess technological means to control or even tune the productivity of E. coli are scarce. Here, we present a novel method for the process-technological control over the recombinant protein expression rate in E. coli. A mixed-feed fed-batch bioprocess based on the araBAD promoter expression system using both D-glucose and L-arabinose as assimilable C-sources was designed. Using the model product green fluorescent protein, we show that the specific product formation rate can be efficiently tuned even on the cellular level only via the uptake rate of L-arabinose. This novel approach introduces an additional degree of freedom for the design of recombinant bioprocesses with E. coli. We anticipate that the presented method will result in significant quality and robustness improvement as well as cost and process time reduction for recombinant bacterial bioprocesses in the future.