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Bioengineering 2019, 6(2), 29;

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

Department of Chemical Engineering, Indian Institute of Technology, Hauz Khas 110016, India
Department of Biomedical, Chemical and Materials Engineering, San Jose State University, San Jose, CA 95192, USA
Author to whom correspondence should be addressed.
Received: 26 January 2019 / Revised: 20 March 2019 / Accepted: 23 March 2019 / Published: 28 March 2019
PDF [2966 KB, uploaded 28 March 2019]


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. View Full-Text
Keywords: quality by design; Ranibizumab; design of experiments; media development quality by design; Ranibizumab; design of experiments; media development

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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited (CC BY 4.0).

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Kumar, D.; Batra, J.; Komives, C.; Rathore, A.S. QbD Based Media Development for the Production of Fab Fragments in E. coli. Bioengineering 2019, 6, 29.

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