Effect of culture medium, host strain and oxygen transfer on recombinant Fab antibody fragment yield and leakage to medium in shaken E. coli cultures

dc.contributor.authorUkkonen, Kaisaen
dc.contributor.authorVeijola, Johannaen
dc.contributor.authorVasala, Anttien
dc.contributor.authorNeubauer, Peteren
dc.date.accessioned2015-11-21T00:48:20Z
dc.date.available2015-07-17T12:00:00Z
dc.date.issued2013
dc.date.submitted2015-07-16
dc.description.abstractBackground: Fab antibody fragments in E. coli are usually directed to the oxidizing periplasmic space for correct folding. From periplasm Fab fragments may further leak into extracellular medium. Information on the cultivation parameters affecting this leakage is scarce, and the unpredictable nature of Fab leakage is problematic regarding consistent product recovery. To elucidate the effects of cultivation conditions, we investigated Fab expression and accumulation into either periplasm or medium in E. coli K-12 and E. coli BL21 when grown in different types of media and under different aeration conditions. Results: Small-scale Fab expression demonstrated significant differences in yield and ratio of periplasmic to extracellular Fab between different culture media and host strains. Expression in a medium with fed-batch-like glucose feeding provided highest total and extracellular yields in both strains. Unexpectedly, cultivation in baffled shake flasks at 150 rpm shaking speed resulted in higher yield and accumulation of Fabs into culture medium as compared to cultivation at 250 rpm. In the fed-batch medium, extracellular fraction in E. coli K-12 increased from 2-17% of total Fab at 250 rpm up to 75% at 150 rpm. This was partly due to increased lysis, but also leakage from intact cells increased at the lower shaking speed. Total Fab yield in E. coli BL21 in glycerol-based autoinduction medium was 5 to 9-fold higher at the lower shaking speed, and the extracellular fraction increased from ≤ 10% to 20-90%. The effect of aeration on Fab localization was reproduced in multiwell plate by variation of culture volume. Conclusions: Yield and leakage of Fab fragments are dependent on expression strain, culture medium, aeration rate, and the combination of these parameters. Maximum productivity in fed-batch-like conditions and in autoinduction medium is achieved under sufficiently oxygen-limited conditions, and lower aeration also promotes increased Fab accumulation into extracellular medium. These findings have practical implications for screening applications and small-scale Fab production, and highlight the importance of maintaining consistent aeration conditions during scale-up to avoid changes in product yield and localization. On the other hand, the dependency of Fab leakage on cultivation conditions provides a practical way to manipulate Fab localization.en
dc.identifier.eissn1475-2859
dc.identifier.uriurn:nbn:de:kobv:83-opus4-68757
dc.identifier.urihttp://depositonce.tu-berlin.de/handle/11303/4848
dc.identifier.urihttp://dx.doi.org/10.14279/depositonce-4551
dc.languageEnglishen
dc.language.isoenen
dc.rights.urihttps://creativecommons.org/licenses/by/2.0/en
dc.subject.ddc570 Biowissenschaften; Biologieen
dc.subject.ddc610 Medizin und Gesundheiten
dc.subject.otherAutoinduktionde
dc.subject.otherFab-Fragmentde
dc.subject.otherFed-batch-Verfahrende
dc.subject.otherPeriplasmatische Expressionde
dc.subject.otherSauerstoffzufuhrde
dc.subject.otherautoinductionen
dc.subject.otherfab fragmenten
dc.subject.otherfed-batchen
dc.subject.otheroxygen transferen
dc.subject.otherperiplasmic expressionen
dc.titleEffect of culture medium, host strain and oxygen transfer on recombinant Fab antibody fragment yield and leakage to medium in shaken E. coli culturesen
dc.typeArticleen
dc.type.versionpublishedVersionen
dcterms.bibliographicCitation.articlenumber73en
dcterms.bibliographicCitation.doi10.1186/1475-2859-12-73en
dcterms.bibliographicCitation.journaltitleMicrobial Cell Factoriesen
dcterms.bibliographicCitation.originalpublishernameBioMed Centralen
dcterms.bibliographicCitation.originalpublisherplaceLondonen
dcterms.bibliographicCitation.volume12en
tub.accessrights.dnbfree*
tub.affiliationFak. 3 Prozesswissenschaften>Inst. Biotechnologiede
tub.affiliation.facultyFak. 3 Prozesswissenschaftende
tub.affiliation.instituteInst. Biotechnologiede
tub.identifier.opus46875
tub.publisher.universityorinstitutionTechnische Universität Berlinen
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