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Purification of clinical-grade disulfide stabilized antibody fragment variable—Pseudomonas exotoxin conjugate (dsFv-PE38) expressed in Escherichia coli

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Abstract

Immunotoxins are rationally designed cancer targeting and killing agents. Disulfide stabilized antibody Fv portion—toxin conjugates (dsFv-toxin) are third generation immunotoxins containing only the antibody fragment variable portions and a toxin fused to the VH or VL. Pseudomonas exotoxin fragment (PE-38) is a commonly used toxin in immunotoxin clinical trials. dsFv-toxin purification was previously published, but the recovery was not satisfactory. This report describes the development of a cGMP production process of the dsFv-toxin that incorporated a novel purification method. The method has been successfully applied to the clinical manufacturing of two dsFv-PE38 immunotoxins, MR1-1 targeting EGFRvIII and HA22 targeting CD22. The two subunits, VL and VH PE-38 were expressed separately in Escherichia coli using recombinant technology. Following cell lysis, inclusion bodies were isolated from the biomass harvested from fermentation in animal source component-free media. The dsFv-toxin was formed after denaturation and refolding, and subsequently purified to homogeneity through ammonium sulfate precipitation, hydrophobic interaction and ion-exchange chromatography steps. It was shown, in a direct comparison experiment using MR1-1 as model protein, that the recovery from the new purification method was improved three times over that from previously published method. The improved recovery was also demonstrated during the clinical production of two dsFv-PE38 immunotoxins—MR1-1 and HA22.

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Acknowledgment

The authors would like to thank Dr. Ira Pastan for the expression vectors and assay protocols. Mr. Joe Newland, Ray Rose, and Dr. Edward Wang contributed to the materials preparation for process development and cGMP production. Dr. Xiaoyi Yang and Gopalan Soman performed the bioactivity assay.

This project has been funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under contracts N01-CO-12400. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does the mentioned of trade names, commercial products, or organizations imply endorsement by the US government.

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Author notes

  1. Hua Jiang

    Present address: Novavax Inc., Rockville, MD, USA

  2. Andrew Burnette

    Present address: Zyngenia Inc., Gaithersburg, MD, USA

  3. Steven L. Giardina

    Present address: Science Applications International Corporation, Frederick, MD, USA

  4. Toby T. Hecht

    Present address: Translational Research Program, DCTD, National Cancer Institute, Bethesda, MD, USA

Authors and Affiliations

  1. Biopharmaceutical Development Program, Frederick National Laboratory for Cancer Research, SAIC-Frederick Inc., Frederick, MD, 21702, USA

    Hua Jiang, Yueqing Xie, Andrew Burnette, John Roach, Steven L. Giardina, Gautam Mitra & Jianwei Zhu

  2. Biological Resources Branch, DTP, DCTD, Frederick National Laboratory for Cancer Research, Frederick, MD, 21702, USA

    Toby T. Hecht & Stephen P. Creekmore

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  1. Hua Jiang
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Correspondence to Jianwei Zhu.

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Hua Jiang and Yueqing Xie contributed equally to this work.

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Jiang, H., Xie, Y., Burnette, A. et al. Purification of clinical-grade disulfide stabilized antibody fragment variable—Pseudomonas exotoxin conjugate (dsFv-PE38) expressed in Escherichia coli . Appl Microbiol Biotechnol 97, 621–632 (2013). https://doi.org/10.1007/s00253-012-4319-2

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