E. coli fermenting

Figure 8.7 Overview of the manufacture of Betaferon, a recombinant human IFN-(3 produced in E. coli. The product differs from native human IFN-(3 in that it is unglycosylated and cysteine residue 17 had been replaced by a serine residue. E. coli fermentation is achieved using minimal salts/glucose media and product accumulates intracellularly in inclusion body (IB) form. During downstream processing, the lbs are solubilized in butanol, with subseguent removal of this denaturant to facilitate product refolding. After two consecutive gel-filtration steps, excipients are added, the product is filled into glass vials and freeze-dried. It exhibits a shelf life of 18 months when stored at 2-8 °C...

Some of the earliest work on NIR of bioprocesses was performed on the nutrients and metabolites in a fermentation broth. A classic paper (if 1996 is antiquity) was written by Hall et al.30 on the determination of acetate, ammonia, biomass, and glycerol in E. coli fermentations. This early paper used NIR to simultaneously monitor all the above-mentioned parameters. The correlation coefficients were all better than 0.985 with variable SEPs acetate, 0.7 g/1 ammonia, 7 mM glycerol, 0.7 g/1 and biomass, 1.4 g/1. While later work with more modem equipment has attained better results, this remains as one of the first. The work was performed at line in a cuvette, but rapidly enough to be considered a process measurement. 

Panitch et al. (1997) produced impressive yields of 700 mg/L by E. coli fermentation of a simple small artificial protein comprising 60 repeats of an (AG)4 motif with 23 and 33 amino acid N- and C-terminal fusions, respectively. The (AG)240 repeat units were not separated after expression, and the fusion sequences comprised just 10% of the whole fusion protein. This meant that loss of polypeptide material due to inefficient cleavage by... 

The other isoforms, Iso-2 and Iso-3, expressed in the E. coli fermentation of IFN-a-2b, were characterized using a similar approach. The isolated Iso-2 and Iso-3 were enzymatically digested with trypsin, and the resulted peptide mixtures were mass-mapped using RP-HPLC/ESI-MS. The results indicated that Iso-2 was a correctly folded IFN-a-2b acetylated on the amino group of the N-terminal cysteine. Iso-3 was similarly determined to be a glutathionated form (Cys-98) of the partially reduced IFN-a-2b that was pyruvated on the N-terminal cysteine. The complete structures for IFN-a-2b, Iso-2, Iso-3, and Iso-4 are shown in Figure 19-11. 

Another fermentation study of note was reported by Hall et al. in 1996 [135]. A simultaneous NIR assay for acetate, ammonia, biomass, and glycerol was developed for an industrial Escherichia coli (E. coli) fermentation broth. The PLS equation produced was capable of predicting with standard errors of, respectively, 0.7 g/L, 1.4 g/L, 0.7 g/L, and 7 mmol/L for the listed constituents. Standard wet chemistry methods were used to calibrate the NIR equation. 

Panahi M, Alii Z, Cheng X, et al. Recombinant protein expression plasmids optimized for industrial E. coli fermentation and plant systems produce biologically active human insulin-like growth factor-1 in transgenic rice and tobacco plants. Transgenic Res., 2004 13(3) 245-259. 

The production of fuU-sized antibodies is restricted to mammahan cell systems that possess the correct production and glycosylation machinery (see Part IV, Chapter 1). Antibody fragments, on the other hand, can also be expressed at much lower cost in microbial expression systems, such as bacterial, yeast, or fungal fermentation [49, 50]. For the high-level expression of antibody fragments, E. coli fermentation provides a well-established tech-... 

Sav (which afforded low levels of pure protein), all mutants could be produced in high yield and purified from 10 litre E. coli fermentations. 

Gnerrero-German et al. [288] developed a new bioprocess for the purification of plasmid DNA from E. coli ferments nsing a hollow-fiber tangential filtration (as a pretreatment of the feed solution) and tandem anion-exchange membrane chromatography as a second step. 

Figure 10.11 shows the effect of rotation speed for filtration of baker s yeast suspension and E. coli fermentation broth [25]. 

The environmental burden of small- and large-scale processes has to be reduced as much as possible. Waste reduction (mass and energy) of course has an ethical component, but also economic competitiveness dictates that cleaner solutions are found. Auxiliary materials, including their regeneration or disposal costs, may contribute significantly to the cost price of the product. An example is the production of recombinant insulin by E. coli fermentation as is described by Datar and Rosen [1]. The auxiliary materials in the downstream processing were estimated to contribute approximately 12% of the production costs, and waste treatment to approximately 5%. 

A method was described for the RPLC determination of recombinant methionylaspartyl-human growth hormone (MD-HGH) in Escherichia coli (E. coli) fermentation broth [7], which utilizes mobile phases containing the anionic surfactant SDS and 1-propanol, under micellar conditions. A C4 column was used at 60°C for the separation. The methodology is directly applicable to the analysis of samples solubilized via sulfitolysis in the presence of SDS, and offers superior resolution in comparison with chromatography in the absence of the surfactant. 

Alternatively, spectrophotometric techniques can be used for the determination of acetic acid based on the UV absorbance at 205 nm (Tippkotter et al., 2008). The system was applied for the determination of acetic acid in E. coli fermentation broth. 

They have noted that anaerobically grown E. coli ferment thymidine to thymine, formate (or H2 and CO2), acetate, and ethanol. The process is inhibited by phosphate or sulfate—an inhibition attributed to inhibition... 

Foundation Lockheed Martin energy US 5869301 1999 E. coli Fermentation... 

Application of bipolar electrodialysis for the simultaneous removal of inhibitory acetate and pH control during E. coli fermentation was investigated by Wong and co-workers (2010). The final biomass and recombinant protein concentrations obtained iuCTeasedby up... 

Wong M, Woodley JM, Lye GJ 2010. Application of bipolar electrodialysis to E. Coli fermentation for simultaneous acetate removal and pH control. Biotechnol Lett 32(8) 1053-1057. 

Figure 1. Flow sheet for the production of / -galoctosidase from E. coli. Fermentation and enzyme release (F), followed by a number of purification stages including centrifugation (C), precipitation (P), extraction (ET), ultrafiltration (UF), and chromatography (GC and AC). The nmnbers refer to the volumetric flow rate (above the lines) and enzyme yield (below the lines).

Hileman, R., G. Holleschak, L. Turner, et al. 2001. Characterization and Equivalence of the Cry3Bbl Protein Produced by E. coli Fermentation and Com Event MON 863 Lab Project Number 17274 01-01-39-30. Unpublished study prepared by Monsanto Company. 75 p. 

PDO from glucose. The researAers initially used a recombinant E. coli strain that produces glycerol from o-glucose followed by K. pneumonia that finally produces 1,3-PEX ) up to 60-70g/L using the glycerol formed after E. coli fermentation. 

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