A comparison of statistical approaches used for the optimization of soluble protein expression in Escherichia coli

Papaneophytou, Christos; Kontopidis, George

doi:10.1016/j.pep.2015.12.014

Cited by 18 publications

(16 citation statements)

References 35 publications

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“…Besides the identification of the optimal medium, the significant role of process parameters (temperature, pH, agitation) on cell growth and product quality has also been demonstrated in this study, complementing already published research [45]. QbD-based media development was performed to achieve greater process understanding so as to achieve optimal product titer and quality.…”

Section: Discussionmentioning

confidence: 52%

QbD Based Media Development for the Production of Fab Fragments in E. coli

Kumar¹,

Batra²,

Komives

et al. 2019

Bioengineering

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Ranibizumab is a biotherapeutic Fab fragment used for the treatment of age-related macular degeneration and macular oedema. It is currently expressed in the gram-negative bacterium, Escherichia coli. However, low expression levels result in a high manufacturing cost. The protein expression can be increased by manipulating nutritional requirements (carbon source, nitrogen source, buffering agent), process parameters (pH, inducer concentration, agitation, temperature), and the genetic make-up of the producing strain. Further, understanding the impact of these factors on product quality is a requirement as per the principles of Quality by Design (QbD). In this paper, we examine the effect of various media components and process parameters on the expression level and quality of the biotherapeutic. First, risk analysis was performed to shortlist different media components based on the literature. Next, experiments were performed to screen these components. Eight components were identified for further investigation and were examined for their effect and interactions using a Fractional Factorial experimental design. Sucrose, biotin, and pantothenate were found to have the maximum effect during Fab production. Furthermore, cyanocobalamin glutathione and biotin-glutathione were the most significant interactions observed. Product identification was performed with Liquid Chromatography–Mass Spectrometry (LC-MS), the expression level was quantified using Bio-layer Interferometry, Reverse Phase-HPLC, and SDS-PAGE, and product quality were measured by RP-HPLC. Overall, a five-fold enhancement of the target protein titer was obtained (from 5 mg/L to 25 mg/L) using the screened medium components vis-a-vis the basal medium, thereby demonstrating the efficacy of the systematic approach purported by QbD.

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Section: Discussionmentioning

confidence: 52%

QbD Based Media Development for the Production of Fab Fragments in E. coli

Kumar¹,

Batra²,

Komives

et al. 2019

Bioengineering

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“…Papaneophytou et al 20 in similar investigation selected four effective parameters (cell density, inducer amount, temperature, and induction duration) to be optimized using the RSM. They reported the effectiveness of this method to enhance the soluble protein production from 1.8 to 9.2 mg/L.…”

Section: Discussionmentioning

confidence: 99%

“…The adequacy of the regression was checked by the analysis of variance (ANOVA) and the plots related to the residual analysis. 20 …”

Section: Methodsmentioning

confidence: 99%

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Osmotic conditions could promote scFv antibody production in the Escherichia coli HB2151

Mesgari-Shadi¹,

Sarrafzadeh²

2017

Bioimpacts

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Introduction: Single chain variable fragment (scFv) antibodies are reduced forms of the whole antibodies that could be regarded as an alternative tool for diagnostic and therapeutic purposes. The optimization of processes and environmental conditions is necessary to increase the production yields and enhance the productivity. This can result in a cost-effective process and respond to the high demand for these antibodies. Methods: In this research, physical and chemical factors influencing the batch fermentation was investigated in 50 mL batch tubes using minimum media to find the optimum conditions for production of a single chain variable fragment antibody in the Escherichia coli HB2151. Experimental designs were used to screen the effective parameters and to optimize the main factors. Results: Arabinose was used instead of IPTG as a cheaper and nontoxic inducer and its optimum concentration was determined 0.1% (w/w). Induction duration time and filling volume fraction were set on the relatively better states 24 hours and 1/10 respectively. Regarding our previous study, stationary phase of the cell growth was selected as induction start time that showed higher specific scFv production yields (YP/X) in the minimum media. Finally, a statistical experimental design was extended to a central composite design (CCD) and analysis was performed based on sucrose and sorbitol concentrations producing osmotic condition for induction. The optimum region in the contour plot for the periplasmic scFv production was an osmotic circle area with total sugar molarity 0.8 to 0.9. Conclusion: Sugars such as sucrose and sorbitol producing osmotic conditions could lead to periplasmic scFv concentrations up to 2.85 mg/L of culture media improving scFv concentration near to five times of the average of the screening step (0.59 mg/L).

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“…30 In our laboratory, we have successfully used IF approaches to determine the optimum conditions for the expression and purification of various recombinant proteins. 41,42 Thus, in this work, we subsequently examined whether we can develop an IF approach to determine the best conditions to measure the activity of LDH-B. Our proposed method to determine LDH-B activity is composed of three different pH values (9, 9.5, and 10), three NaCl concentrations (50, 150, and 250 mM), and two incubation times (5 and 10 min), and we called the method IF-BPST (incomplete factorial−buffer/pH/salt/time).…”

Section: Response Surface Methodologymentioning

confidence: 99%