University of Limerick
Witt_2014_study.pdf (4.51 MB)

A study of selected environmental issues related o biopharmaceutical manufacturing using Escherichia coli to produce a recombinant protein

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posted on 2022-12-14, 15:58 authored by Madlen K Witt
Escherichia coli expression systems remain a preferred choice for the production of recombinant proteins for therapeutic, diagnostic and industrial purposes. Low costs and simplicity of culturing as well as straightforward genetic engineering technologies ensure their continued use for laboratory investigations as well as in commercial activities. An E. coli expression system producing a recombinant protein was constructed for this research. The model strain, E. coli MC106 producing recombinant bacterial His6-tagged β-galactosidase, was developed via standard genetic engineering techniques and protein expression was optimised to achieve high concentrations of soluble product. Historically, during upstream processing little consideration was given to the potential environmental impacts of culture media ingredients which were often added in excess to achieve high cell density and hence product yields. The model E. coli strain was utilised to investigate the scope for reducing phosphorus (P) quantities included in a complex (LB and TB) and semi-defined (M9/YE) fermentation media. The findings showed that P reductions of up to 70 % did not adversely affect biomass and product yields attained; however, further P minimisation lead to a drop in dry cell weight as well as protein synthesis, particularly in the case of semi-defined media. Protein functionality, assessed by the kinetic parameters Km and Vmax, was not influenced by the type of media nor the P concentration present. 70 % P reductions would lead to significant P savings in large-scale manufacturing of proteins produced by genetically engineered E. coli strains. The second part of this study entailed purification, at laboratory-scale to electrophoretic homogeneity, of the model protein via a traditional multichromatographic scheme and an affinity-based strategy. Both purification schemes were compared in terms of their environmental impact based on the buffers used. Utilising the engineered affinity-based approach reduced the number of downstream processing steps required to achieve purification from 6 to 4 and increased the final product yield from 11 % to 34 %. Environmental analysis of the chromatographic buffer constituents indicated that, per mg of purified protein, the use of the affinitybased method reduced the total P usage levels by 46 %, total ammonia by 99 %, total water usage by 75 % and total COD by 62 %, although the organic nitrogen levels increased by 75 %. In addition, comparative cost analysis showed a 60 % savings in chemical and chromatography costs per mg of purified product for this purification strategy. Although already widely used at research level, the use of affinity-based purification systems for process-scale protein purification would likely have significant environmental, energy and cost benefits. Furthermore, the study showed additional P savings can be achieved by using alternative buffering systems not containing P compounds during protein purification. Mass balance simulations and environmental modelling was used to highlight the total phosphorus savings that can be achieved when employing the P-reduced fermentation media and the optimised purification strategies.



  • Faculty of Science and Engineering


  • Doctoral

First supervisor

Walsh, Gary

Second supervisor

O'Dwyer, Tom



Other Funding information




Department or School

  • Chemical Sciences

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