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Transgenic Research

, Volume 21, Issue 6, pp 1291–1301 | Cite as

Purification of the therapeutic antibody trastuzumab from genetically modified plants using safflower Protein A-oleosin oilbody technology

  • Michael D. McLean
  • Rongji Chen
  • Deqiang Yu
  • Kor-Zheng Mah
  • John Teat
  • Haifeng Wang
  • Steve Zaplachinski
  • Joseph Boothe
  • J. Christopher Hall
Original Paper

Abstract

Production of therapeutic monoclonal antibodies using genetically modified plants may provide low cost, high scalability and product safety; however, antibody purification from plants presents a challenge due to the large quantities of biomass that need to be processed. Protein A column chromatography is widely used in the pharmaceutical industry for antibody purification, but its application is limited by cost, scalability and column fouling problems when purifying plant-derived antibodies. Protein A-oleosin oilbodies (Protein A-OB), expressed in transgenic safflower seeds, are relatively inexpensive to produce and provide a new approach for the capture of monoclonal antibodies from plants. When Protein A-OB is mixed with crude extracts from plants engineered to express therapeutic antibodies, the Protein A-OB captures the antibody in the oilbody phase while impurities remain in the aqueous phase. This is followed by repeated partitioning of oilbody phase against an aqueous phase via centrifugation to remove impurities before purified antibody is eluted from the oilbodies. We have developed this purification process to recover trastuzumab, an anti-HER2 monoclonal antibody used for therapy against specific breast-cancers that over express HER2 (human epidermal growth factor receptor 2), from transiently infected Nicotiana benthamiana. Protein A-OB overcomes the fouling problem associated with traditional Protein A chromatography, allowing for the development of an inexpensive, scalable and novel high-resolution method for the capture of antibodies based on simple mixing and phase separation.

Keywords

Antibody Purification Protein A Oleosin Fusion protein Trastuzumab Nicotiana benthamiana Safflower Transient Transgenic 

Abbreviations

C

Concentration

dpi

Days post-infiltration

E

Elution

FT

Flow-through

HC

Heavy chain

HER2

Human epidermal growth factor receptor 2

hIgG

Human immunoglobulin G

IgG

Immunoglobulin G

kDa

Kilo dalton

LC

Light chain

LE

Leaf extract

OB

Oilbodies

PAGE

Polyacrylamide gel electrophoresis

PBS

Phosphate buffered saline

PVX

Potato virus X

SDS

Sodium dodecyl sulphate

Ti

Tumour inducing

TMV

Tobacco mosaic virus

W

Wash

Notes

Acknowledgments

Funding for this project was provided to JCH from the Ontario Ministry of Agriculture, Food and Rural Affairs (OMAFRA), and the Canada Research Chairs program (CRC). DY received an Industrial Fellowship from MITACS Canada (Mathematics of Information Technology and Complex Systems). Special recognition is given to Linda Veldhuis for help with experiments performed to complete this research.

Supplementary material

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Supplementary material 1 (DOCX 17 kb)
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Supplementary material 2 (TIFF 683 kb)
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Supplementary material 3 (TIFF 4892 kb)
11248_2012_9603_MOESM4_ESM.tif (10.2 mb)
Supplementary material 4 (TIFF 10439 kb)

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Copyright information

© Springer Science+Business Media B.V. 2012

Authors and Affiliations

  • Michael D. McLean
    • 1
    • 2
  • Rongji Chen
    • 3
  • Deqiang Yu
    • 1
    • 2
  • Kor-Zheng Mah
    • 2
  • John Teat
    • 2
  • Haifeng Wang
    • 1
  • Steve Zaplachinski
    • 3
  • Joseph Boothe
    • 3
  • J. Christopher Hall
    • 1
    • 2
  1. 1.School of Environmental SciencesUniversity of GuelphGuelphCanada
  2. 2.PlantForm Corp.GuelphCanada
  3. 3.SemBioSys Genetics, Inc.CalgaryCanada

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