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

Ukkonen, Kaisa; Veijola, Johanna; Vasala, Antti; Neubauer, Peter

doi:10.1186/1475-2859-12-73

Cited by 65Protein purification and stabilityMonoclonal and Polyclonal Antibodies ResearchBacterial Genetics and Biotechnology

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

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Ukkonen, Kaisa, Veijola, Johanna, Vasala, Antti, Neubauer, Peter·2013·Microbial Cell Factories

DOI 10.1186/1475-2859-12-73PMC PMC3733871

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Abstract

BACKGROUND: 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.

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Metabolic stress leads to high accumulation of antibody fragments in the periplasm, saturating the secretory machinery and generating a more permeable membrane structure that allows higher diffusive leakage of periplasm proteins outside the cells.
“The metabolic stress leads to a high accumulation of antibody fragments in the periplasm saturating the secretory machinery. This event generates a more permeable membrane structure that, after sufficient product accumulation in the periplasm, allows a higher diffusive leakage of the periplasm proteins outside the cells [86,144].”
From Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments (2020) · cites this paper as [86]
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Authors: Ukkonen, K., Veijola, J., Vasala, A., & Neubauer, P.
Affiliations: University of Oulu (FI)
Technische Universität Berlin (DE)
Corresponding author: Ukkonen, Kaisa
ORCID: Neubauer, Peter
Venue: Microbial Cell Factories
Publisher: Springer Science and Business Media LLC
Published: 2013
Volume: 12
Issue: 1
Pages: 73
ISSN: 1475-2859 (ISSN-L)
DOI: 10.1186/1475-2859-12-73
PMC: PMC3733871
OpenAlex: W2154955381
Cited by: 65
References: 46
Citations by year: 2026: 6 · 2025: 3 · 2024: 2 · 2023: 4 · 2022: 4 · 2021: 8 · 2020: 6 · 2019: 6 · 2018: 6 · 2017: 6 · 2016: 3 · 2015: 4 · 2014: 4 · 2013: 3
FWCI: 2.09
Type: journal article
Language: en
Open access: gold
doi.org
Licence: cc-by
Locations: Microbial Cell Factories (publishedVersion, cc-by)
Europe PMC (PubMed Central) (publishedVersion, cc-by)
PubMed Central (publishedVersion, cc-by)
PubMed (publishedVersion)
DepositOnce (submittedVersion, cc-by)
Technische Universität Berlin – Universitätsbibliothek (cc-by)
Funders: Tekes (1695/31/2010)
Topics: Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein purification and stability
Health Sciences › Medicine › Radiology, Nuclear Medicine and Imaging › Monoclonal and Polyclonal Antibodies Research
Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
Keywords: antibody Fab fragment, recombinant antibody production, Escherichia coli expression, culture medium effects, shaking speed, air flow rate, oxygen transfer, cell lysis
MeSH terms: Culture Media, Escherichia coli, Hydrogen-Ion Concentration, Immunoglobulin Fab Fragments, Oxygen, Recombinant Proteins, Periplasm, Batch Cell Culture Techniques

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

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