Clinical-grade oncolytic adenovirus purification using polysorbate 20 as an alternative for cell lysis

Mafalda G. Moleirinho, Sara Rosa, Manuel J.T. Carrondo, Ricardo J. S. Silva, Åsa Hagner-McWhirter, Gustaf Ahlén, Mats Lundgren, Paula M. Alves, Cristina Peixoto

Research output: Contribution to journalArticle

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Abstract

Introduction: Oncolytic virus therapy is currently considered as a promising therapeutic approach for cancer treatment. Adenovirus is well-known and extensively characterized as an oncolytic agent. The increasing number of clinical trials using this virus generates the demand for the development of a well-established purification approach. Triton X-100 is commonly used in cell lysis buffer preparations. The addition of this surfactant in the list of substances with the very high concern of the Registration, Evaluation, Authorization and Restriction of Chemicals (REACH) regulation promoted the research for effective alternatives. Methods: In this work, a purification strategy for oncolytic adenovirus compatible with phase I clinical trials, using an approved surfactant – Polysorbate 20 was developed. The proposed downstream train, composed by clarification, concentration using tangential flow filtration, intermediate purification with anion exchange chromatography, followed by a second concentration and a final polishing step was evaluated for both Triton X-100 and Polysorbate 20 processes. The impact of cell lysis with Polysorbate20 and Triton X-100 for each downstream step was evaluated in terms of product recovery and impurities removal. Overall, 61 ± 4% of infectious viral particles were recovered. Depletion of host cell proteins and ds-DNA was 99.9% and 97.1%, respectively. Results & Conclusion: The results indicated that Polysorbate 20 can be used as a replacement for Triton X-100 during cell lysis with no impact on product recovery, potency, and purity. Moreover, the developed process is scalable and able to provide a highly purified product to be used in phase I and II clinical trials.

Original languageEnglish
Pages (from-to)366-374
Number of pages9
JournalCurrent Gene Therapy
Volume18
Issue number6
DOIs
Publication statusPublished - 1 Jan 2018

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Polysorbates
Octoxynol
Adenoviridae
Clinical Trials, Phase I
Surface-Active Agents
Oncolytic Virotherapy
Phase II Clinical Trials
Virion
Anions
Chromatography
Buffers
Clinical Trials
Viruses
DNA
Therapeutics
Research
Neoplasms
Proteins

Keywords

  • Adenovirus
  • Cell lysis
  • Downstream processing
  • GMP process development
  • Oncolytic virus
  • Polysorbate 20

Cite this

Moleirinho, Mafalda G. ; Rosa, Sara ; Carrondo, Manuel J.T. ; Silva, Ricardo J. S. ; Hagner-McWhirter, Åsa ; Ahlén, Gustaf ; Lundgren, Mats ; Alves, Paula M. ; Peixoto, Cristina. / Clinical-grade oncolytic adenovirus purification using polysorbate 20 as an alternative for cell lysis. In: Current Gene Therapy. 2018 ; Vol. 18, No. 6. pp. 366-374.
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Clinical-grade oncolytic adenovirus purification using polysorbate 20 as an alternative for cell lysis. / Moleirinho, Mafalda G.; Rosa, Sara; Carrondo, Manuel J.T.; Silva, Ricardo J. S.; Hagner-McWhirter, Åsa; Ahlén, Gustaf; Lundgren, Mats; Alves, Paula M.; Peixoto, Cristina.

In: Current Gene Therapy, Vol. 18, No. 6, 01.01.2018, p. 366-374.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Clinical-grade oncolytic adenovirus purification using polysorbate 20 as an alternative for cell lysis

AU - Moleirinho, Mafalda G.

AU - Rosa, Sara

AU - Carrondo, Manuel J.T.

AU - Silva, Ricardo J. S.

AU - Hagner-McWhirter, Åsa

AU - Ahlén, Gustaf

AU - Lundgren, Mats

AU - Alves, Paula M.

AU - Peixoto, Cristina

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PY - 2018/1/1

Y1 - 2018/1/1

N2 - Introduction: Oncolytic virus therapy is currently considered as a promising therapeutic approach for cancer treatment. Adenovirus is well-known and extensively characterized as an oncolytic agent. The increasing number of clinical trials using this virus generates the demand for the development of a well-established purification approach. Triton X-100 is commonly used in cell lysis buffer preparations. The addition of this surfactant in the list of substances with the very high concern of the Registration, Evaluation, Authorization and Restriction of Chemicals (REACH) regulation promoted the research for effective alternatives. Methods: In this work, a purification strategy for oncolytic adenovirus compatible with phase I clinical trials, using an approved surfactant – Polysorbate 20 was developed. The proposed downstream train, composed by clarification, concentration using tangential flow filtration, intermediate purification with anion exchange chromatography, followed by a second concentration and a final polishing step was evaluated for both Triton X-100 and Polysorbate 20 processes. The impact of cell lysis with Polysorbate20 and Triton X-100 for each downstream step was evaluated in terms of product recovery and impurities removal. Overall, 61 ± 4% of infectious viral particles were recovered. Depletion of host cell proteins and ds-DNA was 99.9% and 97.1%, respectively. Results & Conclusion: The results indicated that Polysorbate 20 can be used as a replacement for Triton X-100 during cell lysis with no impact on product recovery, potency, and purity. Moreover, the developed process is scalable and able to provide a highly purified product to be used in phase I and II clinical trials.

AB - Introduction: Oncolytic virus therapy is currently considered as a promising therapeutic approach for cancer treatment. Adenovirus is well-known and extensively characterized as an oncolytic agent. The increasing number of clinical trials using this virus generates the demand for the development of a well-established purification approach. Triton X-100 is commonly used in cell lysis buffer preparations. The addition of this surfactant in the list of substances with the very high concern of the Registration, Evaluation, Authorization and Restriction of Chemicals (REACH) regulation promoted the research for effective alternatives. Methods: In this work, a purification strategy for oncolytic adenovirus compatible with phase I clinical trials, using an approved surfactant – Polysorbate 20 was developed. The proposed downstream train, composed by clarification, concentration using tangential flow filtration, intermediate purification with anion exchange chromatography, followed by a second concentration and a final polishing step was evaluated for both Triton X-100 and Polysorbate 20 processes. The impact of cell lysis with Polysorbate20 and Triton X-100 for each downstream step was evaluated in terms of product recovery and impurities removal. Overall, 61 ± 4% of infectious viral particles were recovered. Depletion of host cell proteins and ds-DNA was 99.9% and 97.1%, respectively. Results & Conclusion: The results indicated that Polysorbate 20 can be used as a replacement for Triton X-100 during cell lysis with no impact on product recovery, potency, and purity. Moreover, the developed process is scalable and able to provide a highly purified product to be used in phase I and II clinical trials.

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KW - Cell lysis

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KW - GMP process development

KW - Oncolytic virus

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