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Cited by 24Protein purification and stabilityViral Infectious Diseases and Gene Expression in InsectsMicrobial Metabolic Engineering and Bioproduction

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

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Kumar, Deepak, Batra, Jyoti, Komives, Claire, Rathore, Anurag S.·2019·Bioengineering
DOI 10.3390/bioengineering6020029PMC PMC6631317
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Abstract

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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  • The 22 amino acids long PelB signal sequence from Erwinia carotovora is the most frequently used for transportation of Fabs and scFvs.

    “The 22 amino acids long PelB (pectate lyase B) signal sequence from Erwinia carotovora [157] is the most frequently used for transportation of the Fabs [88,96] and scFvs [30,101,158,159].”

    From Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments (2020) · cites this paper as [96]

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References 45 · Cited by 24

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  1. [1]Walsh G. Biopharmaceutical benchmarks 2014 Nat. Biotechnol. 2014 32 992 1000 10.1038/nbt.3040 25299917 · DOI · Paper page
  2. [3]Zhang K. Zhang L. Weinreb R.N. Ophthalmic drug discovery: Novel targets and mechanisms for retinal diseases and glaucoma Nat. Rev. Drug Discov. 2012 11 541 559 10.1038/nrd3745 22699774 · DOI
  3. [4]Mullard A. Can next-generation antibodies offset biosimilar competition? Nat. Rev. Drug Discov. 2012 11 426 428 10.1038/nrd3749 22653202 · DOI
  4. [5]Million R.P. Therapeutic area crossroads: Anti-angiogenesis Nat. Rev. Drug Discov. 2008 7 115 116 10.1038/nrd2507 · DOI
  5. [6]Rathore A.S. Follow-on protein products: Scientific issues, developments and challenges Trends Biotechnol. 2009 27 698 705 10.1016/j.tibtech.2009.09.004 19846228 · DOI
  6. [8]Yee L. Blanch H.W. Defined media optimization for growth of recombinant Escherichia coli X90 Biotechnol. Bioeng. 1993 41 221 230 10.1002/bit.260410208 18609541 · DOI
  7. [10]Eiteman M.A. Altman E. Overcoming acetate in Escherichia coli recombinant protein fermentations Trends Biotechnol. 2006 24 530 536 10.1016/j.tibtech.2006.09.001 16971006 · DOI
  8. [11]Kipriyanov S.M. Moldenhauer G. Little M. High level production of soluble single chain antibodies in small-scale Escherichia coli cultures J. Immunol. Methods 1997 200 69 77 10.1016/S0022-1759(96)00188-3 9005945 · DOI
  9. [12]Rathore A.S. Quality by design (QbD)-based process development for purification of a biotherapeutic Trends Biotechnol. 2016 34 358 370 10.1016/j.tibtech.2016.01.003 26879188 · DOI
  10. [13]Rathore A.S. QbD/PAT for bioprocessing: Moving from theory to implementation Curr. Opin. Chem. Eng. 2014 6 1 8 10.1016/j.coche.2014.05.006 · DOI
  11. [14]Rathore A.S. Winkle H. Quality by design for biopharmaceuticals Nat. Biotechnol. 2009 27 26 10.1038/nbt0109-26 19131992 · DOI
  12. [16]Rathore A.S. Roadmap for implementation of quality by design (QbD) for biotechnology products Trends Biotechnol. 2009 27 546 553 10.1016/j.tibtech.2009.06.006 19647883 · DOI
  13. [17]Saida F. Uzan M. Odaert B. Bontems F. Expression of highly toxic genes in E. coli: Special strategies and genetic tools Curr. Protein Pept. Sci. 2006 7 47 56 10.2174/138920306775474095 16472168 · DOI
  14. [18]King D.J. Byron O.D. Mountain A. Weir N. Harvey A. Lawson A.D.G. Proudfoot K.A. Baldock D. Harding S.E. Yarranton G.T. Expression, purification and characterization of B72. 3 Fv fragments Biochem. J. 1993 290 723 729 10.1042/bj2900723 8457200 PMC1132340 · DOI
  15. [19]Cossins A.J. Harrison S. Popplewell A.G. Gore M.G. Recombinant production of a VL single domain antibody in Escherichia coli and analysis of its interaction with peptostreptococcal protein L. Protein Expr. Purif. 2007 51 253 259 10.1016/j.pep.2006.07.013 16949300 · DOI · Paper page
  16. [20]Sletta H. Nedal A. Aune T.E.V. Hellebust H. Hakvåg S. Aune R. Ellingsen T.E. Valla S. Brautaset T. Broad-host-range plasmid pJB658 can be used for industrial-level production of a secreted host-toxic single-chain antibody fragment in Escherichia coli Appl. Environ. Microbiol. 2004 70 7033 7039 10.1128/AEM.70.12.7033-7039.2004 15574897 PMC535149 · DOI
  17. [21]Zhang C. Chen X. Zou R. Zhou K. Stephanopoulos G. Too H.-P. Combining genotype improvement and statistical media optimization for isoprenoid production in E. coli PLoS ONE 2013 8 e75164 10.1371/journal.pone.0075164 24124471 PMC3790805 · DOI
  18. [22]Champion K.M. Nishihara J.C. Joly J.C. Arnott D. Similarity of the Escherichia coli proteome upon completion of different biopharmaceutical fermentation processes Proteomics 2001 1 1133 1148 10.1002/1615-9861(200109)1:9<1133::AID-PROT1133>3.0.CO;2-S 11990508 · DOI
  19. [23]Riesenberg D. Schulz V. Knorre W.A. Pohl H.-D. Korz D. Sanders E.A. Ross A. Deckwer W.-D. High cell density cultivation of Escherichia coli at controlled specific growth rate J. Biotechnol. 1991 20 17 27 10.1016/0168-1656(91)90032-Q 1367313 · DOI
  20. [24]Kumar V. Bhalla A. Rathore A.S. Design of experiments applications in bioprocessing: Concepts and approach Biotechnol. Prog. 2014 30 86 99 10.1002/btpr.1821 24123959 · DOI
  21. [25]Erdag B. Koray Balcioglu B. Ozdemir Bahadir A. Serhatli M. Kacar O. Bahar A. Seker U.O.S. Akgun E. Ozkan A. Kilic T. Identification of novel neutralizing single-chain antibodies against vascular endothelial growth factor receptor 2 Biotechnol. Appl. Biochem. 2011 58 412 422 10.1002/bab.61 22172104 · DOI
  22. [26]Bradford M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding Anal. Biochem. 1976 72 248 254 10.1016/0003-2697(76)90527-3 942051 · DOI · Paper page
  23. [27]Nilson B.H.K. Lögdberg L. Kastern W. Björck L. Åkerström B. Purification of antibodies using protein L-binding framework structures in the light chain variable domain J. Immunol. Methods 1993 164 33 40 10.1016/0022-1759(93)90273-A 8360508 · DOI
  24. [29]Shokri A. Sandén A. Larsson G. Cell and process design for targeting of recombinant protein into the culture medium of Escherichia coli Appl. Microbiol. Biotechnol. 2003 60 654 664 10.1007/s00253-002-1156-8 12664143 · DOI
  25. [30]Sørensen H.P. Mortensen K.K. Advanced genetic strategies for recombinant protein expression in Escherichia coli J. Biotechnol. 2005 115 113 128 10.1016/j.jbiotec.2004.08.004 15607230 · DOI
  26. [31]Huang C.-J. Lin H. Yang X. Industrial production of recombinant therapeutics in Escherichia coli and its recent advancements J. Ind. Microbiol. Biotechnol. 2012 39 383 399 10.1007/s10295-011-1082-9 22252444 · DOI
  27. [32]Carter P. Kelley R.F. Rodrigues M.L. Snedecor B. Covarrubias M. Velligan M.D. Wong W.L.T. Rowland A.M. Kotts C.E. Carver M.E. High level Escherichia coli expression and production of a bivalent humanized antibody fragment Nat. Biotechnol. 1992 10 163 167 10.1038/nbt0292-163 1368228 · DOI
  28. [33]Dragosits M. Frascotti G. Bernard-Granger L. Vázquez F. Giuliani M. Baumann K. Rodríguez-Carmona E. Tokkanen J. Parrilli E. Wiebe M.G. Influence of growth temperature on the production of antibody Fab fragments in different microbes: A host comparative analysis Biotechnol. Prog. 2011 27 38 46 10.1002/btpr.524 21312353 · DOI · Paper page
  29. [34]Cabilly S. Growth at sub-optimal temperatures allows the production of functional, antigen-binding Fab fragments in Escherichia coli Gene 1989 85 553 557 10.1016/0378-1119(89)90451-4 2697647 · DOI
  30. [35]Gasser B. Saloheimo M. Rinas U. Dragosits M. Rodríguez-Carmona E. Baumann K. Giuliani M. Parrilli E. Branduardi P. Lang C. Protein folding and conformational stress in microbial cells producing recombinant proteins: A host comparative overview Microb. Cell Fact. 2008 7 11 10.1186/1475-2859-7-11 18394160 PMC2322954 · DOI
  31. [36]Frenzel A. Hust M. Schirrmann T. Expression of recombinant antibodies Front. Immunol. 2013 4 217 10.3389/fimmu.2013.00217 23908655 PMC3725456 · DOI
  32. [37]Wilms B. Hauck A. Reuss M. Syldatk C. Mattes R. Siemann M. Altenbuchner J. High-cell-density fermentation for production of L-N-carbamoylase using an expression system based on the Escherichia coli rhaBAD promoter Biotechnol. Bioeng. 2001 73 95 103 10.1002/bit.1041 11255157 · DOI
  33. [38]Jeske M. Altenbuchner J. The Escherichia coli rhamnose promoter rhaPBAD is in Pseudomonas putida KT2440 independent of Crp–cAMP activation Appl. Microbiol. Biotechnol. 2010 85 1923 1933 10.1007/s00253-009-2245-8 19789867 · DOI
  34. [39]Wegerer A. Sun T. Altenbuchner J. Optimization of an E. coli L-rhamnose-inducible expression vector: Test of various genetic module combinations BMC Biotechnol. 2008 8 2 10.1186/1472-6750-8-2 18194555 PMC2254391 · DOI
  35. [40]Singh S. Du Preez J.C. Pillay B. Prior B.A. The production of hemicellulases by Thermomyces lanuginosus strain SSBP: Influence of agitation and dissolved oxygen tension Appl. Microbiol. Biotechnol. 2000 54 698 704 10.1007/s002530000440 11131398 · DOI
  36. [41]Carlson H.K. Stoeva M.K. Justice N.B. Sczesnak A. Mullan M.R. Mosqueda L.A. Kuehl J.V. Deutschbauer A.M. Arkin A.P. Coates J.D. Monofluorophosphate is a selective inhibitor of respiratory sulfate-reducing microorganisms Environ. Sci. Technol. 2015 49 3727 3736 10.1021/es505843z 25698072 · DOI
  37. [42]Rock C.O. Calder R.B. Karim M.A. Jackowski S. Pantothenate kinase regulation of the intracellular concentration of coenzyme A J. Biol. Chem. 2000 275 1377 1383 10.1074/jbc.275.2.1377 10625688 · DOI
  38. [43]Gande R. Dover L.G. Krumbach K. Besra G.S. Sahm H. Oikawa T. Eggeling L. The two carboxylases of Corynebacterium glutamicum essential for fatty acid and mycolic acid synthesis J. Bacteriol. 2007 189 5257 5264 10.1128/JB.00254-07 17483212 PMC1951862 · DOI
  39. [44]Schäffner J. Winter J. Rudolph R. Schwarz E. Cosecretion of chaperones and low-molecular-size medium additives increases the yield of recombinant disulfide-bridged proteins Appl. Environ. Microbiol. 2001 67 3994 4000 10.1128/AEM.67.9.3994-4000.2001 11525996 PMC93120 · DOI
  40. [45]Papaneophytou C. Kontopidis G. A Comparison of Statistical Approaches Used for the Optimization of Soluble Protein Expression in Escherichia coli Elsevier Amsterdam, The Netherlands 2016 26721705
  41. [46]Rosano G.L. Ceccarelli E.A. Recombinant protein expression in Escherichia coli: Advances and challenges Front. Microbiol. 2014 5 172 10.3389/fmicb.2014.00172 24860555 PMC4029002 · DOI · Paper page

Authors
Kumar, D., Batra, J., Komives, C., & Rathore, A. S.
Affiliations
  • Department of Chemical Engineering, Indian Institute of Technology, Hauz Khas 110016, India
  • San Jose State University (US)
Corresponding author
Rathore, Anurag S.
ORCID
Venue
Bioengineering
Publisher
MDPI AG
Published
2019-03-28
Volume
6
Issue
2
Pages
29
ISSN
2306-5354 (ISSN-L)
DOI
10.3390/bioengineering6020029
PMC
PMC6631317
OpenAlex
W2924184670
Cited by
24
References
45
Citations by year
2026: 1 · 2025: 4 · 2024: 2 · 2023: 5 · 2022: 4 · 2021: 3 · 2020: 3 · 2019: 2
FWCI
0.99
Type
journal article
Language
en
Open access
Licence
Locations
  • Bioengineering (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Informationszentrum Lebenswissenschaften (ZB MED) (submittedVersion, cc-by)
Funders
Department of Biotechnology , Ministry of Science and Technology (BT/COE/34/SP15097/2015)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein purification and stability
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Viral Infectious Diseases and Gene Expression in Insects
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Microbial Metabolic Engineering and Bioproduction
Keywords
quality by design, fermentation medium optimization, Fab fragment, recombinant protein expression, Escherichia coli, biologic therapy, sucrose, biotin, pantothenic acid, vitamin B12, glutathione, fractional factorial design, risk analysis, protein abundance, product quality

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