Molecular farming in tobacco hairy roots by triggering the secretion of a pharmaceutical antibody

Suvi T. Häkkinen, N. Raven, M. Henquet, Marja-Leena Laukkanen, T. Anderlei, Juha-Pekka Pitkänen, R.M. Twyman, D. Bosch, Kirsi-Marja Oksman-Caldentey, S. Schillberg, Anneli Ritala (Corresponding Author)

Research output: Contribution to journalArticleScientificpeer-review

28 Citations (Scopus)

Abstract

Recombinant pharmaceutical proteins expressed in hairy root cultures can be secreted into the medium to improve product homogeneity and to facilitate purification, although this may result in significant degradation if the protein is inherently unstable or particularly susceptible to proteases. To address these challenges, we used a design of experiments approach to develop an optimized induction protocol for the cultivation of tobacco hairy roots secreting the full-size monoclonal antibody M12. The antibody yield was enhanced 30-fold by the addition of 14g/L KNO3, 19mg/L 1-naphthaleneacetic acid and 1.5g/L of the stabilizing agent polyvinylpyrrolidone. Analysis of hairy root cross sections revealed that the optimized medium induced lateral root formation and morphological changes in the inner cortex and pericycle cells, indicating that the improved productivity was at least partially based on the enhanced efficiency of antibody secretion. We found that 57% of the antibody was secreted, yielding 5.9mg of product per liter of induction medium. Both the secreted and intracellular forms of the antibody could be isolated by protein A affinity chromatography and their functionality was confirmed using vitronectin-binding assays. Glycan analysis revealed three major plant complex-type glycans on both forms of the antibody, although the secreted form was more homogeneous due to the predominance of a specific glycoform. Tobacco hairy root cultures therefore offer a practical solution for the production of homogeneous pharmaceutical antibodies in containment.
Original languageEnglish
Pages (from-to)336-346
Number of pages10
JournalBiotechnology and Bioengineering
Volume111
Issue number2
DOIs
Publication statusPublished - 2014
MoE publication typeA1 Journal article-refereed

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Molecular Farming
Tobacco
Antibodies
Drug products
Pharmaceutical Preparations
Proteins
Polysaccharides
Vitronectin
Affinity chromatography
Povidone
Monoclonal antibodies
Excipients
Staphylococcal Protein A
Affinity Chromatography
Recombinant Proteins
Design of experiments
Proteolysis
Purification
Assays
Peptide Hydrolases

Keywords

  • antibody
  • hairy roots
  • molecular farming
  • secretion
  • tobacco

Cite this

Häkkinen, Suvi T. ; Raven, N. ; Henquet, M. ; Laukkanen, Marja-Leena ; Anderlei, T. ; Pitkänen, Juha-Pekka ; Twyman, R.M. ; Bosch, D. ; Oksman-Caldentey, Kirsi-Marja ; Schillberg, S. ; Ritala, Anneli. / Molecular farming in tobacco hairy roots by triggering the secretion of a pharmaceutical antibody. In: Biotechnology and Bioengineering. 2014 ; Vol. 111, No. 2. pp. 336-346.
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abstract = "Recombinant pharmaceutical proteins expressed in hairy root cultures can be secreted into the medium to improve product homogeneity and to facilitate purification, although this may result in significant degradation if the protein is inherently unstable or particularly susceptible to proteases. To address these challenges, we used a design of experiments approach to develop an optimized induction protocol for the cultivation of tobacco hairy roots secreting the full-size monoclonal antibody M12. The antibody yield was enhanced 30-fold by the addition of 14g/L KNO3, 19mg/L 1-naphthaleneacetic acid and 1.5g/L of the stabilizing agent polyvinylpyrrolidone. Analysis of hairy root cross sections revealed that the optimized medium induced lateral root formation and morphological changes in the inner cortex and pericycle cells, indicating that the improved productivity was at least partially based on the enhanced efficiency of antibody secretion. We found that 57{\%} of the antibody was secreted, yielding 5.9mg of product per liter of induction medium. Both the secreted and intracellular forms of the antibody could be isolated by protein A affinity chromatography and their functionality was confirmed using vitronectin-binding assays. Glycan analysis revealed three major plant complex-type glycans on both forms of the antibody, although the secreted form was more homogeneous due to the predominance of a specific glycoform. Tobacco hairy root cultures therefore offer a practical solution for the production of homogeneous pharmaceutical antibodies in containment.",
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Häkkinen, ST, Raven, N, Henquet, M, Laukkanen, M-L, Anderlei, T, Pitkänen, J-P, Twyman, RM, Bosch, D, Oksman-Caldentey, K-M, Schillberg, S & Ritala, A 2014, 'Molecular farming in tobacco hairy roots by triggering the secretion of a pharmaceutical antibody', Biotechnology and Bioengineering, vol. 111, no. 2, pp. 336-346. https://doi.org/10.1002/bit.25113

Molecular farming in tobacco hairy roots by triggering the secretion of a pharmaceutical antibody. / Häkkinen, Suvi T.; Raven, N.; Henquet, M.; Laukkanen, Marja-Leena; Anderlei, T.; Pitkänen, Juha-Pekka; Twyman, R.M.; Bosch, D.; Oksman-Caldentey, Kirsi-Marja; Schillberg, S.; Ritala, Anneli (Corresponding Author).

In: Biotechnology and Bioengineering, Vol. 111, No. 2, 2014, p. 336-346.

Research output: Contribution to journalArticleScientificpeer-review

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T1 - Molecular farming in tobacco hairy roots by triggering the secretion of a pharmaceutical antibody

AU - Häkkinen, Suvi T.

AU - Raven, N.

AU - Henquet, M.

AU - Laukkanen, Marja-Leena

AU - Anderlei, T.

AU - Pitkänen, Juha-Pekka

AU - Twyman, R.M.

AU - Bosch, D.

AU - Oksman-Caldentey, Kirsi-Marja

AU - Schillberg, S.

AU - Ritala, Anneli

PY - 2014

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N2 - Recombinant pharmaceutical proteins expressed in hairy root cultures can be secreted into the medium to improve product homogeneity and to facilitate purification, although this may result in significant degradation if the protein is inherently unstable or particularly susceptible to proteases. To address these challenges, we used a design of experiments approach to develop an optimized induction protocol for the cultivation of tobacco hairy roots secreting the full-size monoclonal antibody M12. The antibody yield was enhanced 30-fold by the addition of 14g/L KNO3, 19mg/L 1-naphthaleneacetic acid and 1.5g/L of the stabilizing agent polyvinylpyrrolidone. Analysis of hairy root cross sections revealed that the optimized medium induced lateral root formation and morphological changes in the inner cortex and pericycle cells, indicating that the improved productivity was at least partially based on the enhanced efficiency of antibody secretion. We found that 57% of the antibody was secreted, yielding 5.9mg of product per liter of induction medium. Both the secreted and intracellular forms of the antibody could be isolated by protein A affinity chromatography and their functionality was confirmed using vitronectin-binding assays. Glycan analysis revealed three major plant complex-type glycans on both forms of the antibody, although the secreted form was more homogeneous due to the predominance of a specific glycoform. Tobacco hairy root cultures therefore offer a practical solution for the production of homogeneous pharmaceutical antibodies in containment.

AB - Recombinant pharmaceutical proteins expressed in hairy root cultures can be secreted into the medium to improve product homogeneity and to facilitate purification, although this may result in significant degradation if the protein is inherently unstable or particularly susceptible to proteases. To address these challenges, we used a design of experiments approach to develop an optimized induction protocol for the cultivation of tobacco hairy roots secreting the full-size monoclonal antibody M12. The antibody yield was enhanced 30-fold by the addition of 14g/L KNO3, 19mg/L 1-naphthaleneacetic acid and 1.5g/L of the stabilizing agent polyvinylpyrrolidone. Analysis of hairy root cross sections revealed that the optimized medium induced lateral root formation and morphological changes in the inner cortex and pericycle cells, indicating that the improved productivity was at least partially based on the enhanced efficiency of antibody secretion. We found that 57% of the antibody was secreted, yielding 5.9mg of product per liter of induction medium. Both the secreted and intracellular forms of the antibody could be isolated by protein A affinity chromatography and their functionality was confirmed using vitronectin-binding assays. Glycan analysis revealed three major plant complex-type glycans on both forms of the antibody, although the secreted form was more homogeneous due to the predominance of a specific glycoform. Tobacco hairy root cultures therefore offer a practical solution for the production of homogeneous pharmaceutical antibodies in containment.

KW - antibody

KW - hairy roots

KW - molecular farming

KW - secretion

KW - tobacco

U2 - 10.1002/bit.25113

DO - 10.1002/bit.25113

M3 - Article

VL - 111

SP - 336

EP - 346

JO - Biotechnology and Bioengineering

JF - Biotechnology and Bioengineering

SN - 0006-3592

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