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Production of Therapeutic Single-Chain Variable Fragments (ScFv) in Pichia pastoris

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Therapeutic Antibodies

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2313))

Abstract

The interest in the use of monoclonal antibodies as therapeutic molecules has raised in the recent years. Due to their high affinity and specificity towards other biological molecules, antibodies are being widely used to treat a broad range of human diseases such as cancer, rheumatism, and cardiovascular diseases. Currently, the production of IgG-like antibodies is mainly obtained from stable or transient mammalian expression systems that allow proper folding and posttranslational modifications. Despite the technological advances of the last decade, the use of these systems still has a rather high production cost and long processing times. For these reasons, researchers are increasingly interested in alternative antibody production methods as well as alternative antibody formats. Bacterial systems, such as Escherichia coli, are extensively being used for recombinant protein production because their easy manipulation and cheap costs. However, the presence of lipopolysaccharides (LPS) traces in the already fractionated recombinant protein makes these systems not good candidates for the preparation of therapeutic molecules. Yeast systems, such as Pichia pastoris, present the convenient easy manipulation of microbial systems but show some key advantages of eukaryotic expression systems, like improved folding machinery and absence of LPS. They are especially suitable for the production of antibody fragments, which do not need human-like glycosylation, avoiding the high costs of mammalian systems. Here, the protocol for the expression and purification of a single-chain antibody fragment (scFv) in P. pastoris is provided, in deep detail for lab manipulation and briefly for a 5L-bioreactor production.

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Authors and Affiliations

  1. Department of Psychiatry and Neurochemistry, Institute of Neuroscience and Physiology, The Sahlgrenska Academy at the University of Gothenburg, Mölndal, Sweden

    Laia Montoliu-Gaya PhD & Sandra Villegas PhD

  2. Protein Design and Immunotherapy Group, Departament de Bioquímica i Biologia Molecular. Facultat de Biociències, Universitat Autònoma de Barcelona, Barcelona, Spain

    Sandra Villegas PhD

Authors
  1. Laia Montoliu-Gaya PhD
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  2. Sandra Villegas PhD
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Correspondence to Sandra Villegas PhD .

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Editors and Affiliations

  1. Department of Neurology, Rigshospitalet, Glostrup, Denmark

    Gunnar Houen

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Montoliu-Gaya, L., Villegas, S. (2022). Production of Therapeutic Single-Chain Variable Fragments (ScFv) in Pichia pastoris. In: Houen, G. (eds) Therapeutic Antibodies. Methods in Molecular Biology, vol 2313. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1450-1_8

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  • DOI: https://doi.org/10.1007/978-1-0716-1450-1_8

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