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Rapid isolation of lentivirus particles from cell culture media via a hydrophobic interaction chromatography method on a polyester, capillary-channeled polymer fiber stationary phase

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Abstract

Lentiviruses are increasingly used as gene delivery vehicles for vaccines and immunotherapies. However, the purification of clinical-grade lentivirus vectors for therapeutic use is still troublesome and limits preclinical and clinical experiments. Current purification methods such as ultracentrifugation and ultrafiltration are time consuming and do not remove all of the impurities such as cellular debris, membrane fragments, and denatured proteins from the lentiviruses. The same challenges exist in terms of their analytical characterization. Presented here is the novel demonstration of the chromatographic isolation of virus particles from culture media based on the hydrophobicity characteristics of the vesicles. A method was developed to isolate lentivirus from media using a hydrophobic interaction chromatography (HIC) method performed on a polyester, capillary-channeled polymer (PET C-CP) stationary phase and a standard liquid chromatography apparatus. The method is an extension of the approach developed in this laboratory for the isolation of extracellular vesicles (EVs). Quantitative polymerase chain reaction (qPCR) was used to verify and quantify lentiviruses in elution fractions. Load and elution mobile phase compositions were optimized to affect high efficiency and throughput. The process has been visualized via scanning electron microscopy (SEM) of the fiber surfaces following media injection, the elution of proteinaceous material, and the elution of lentiviruses. This effort has yielded a rapid (<10 min), low-cost (< $15 per column, providing multiple separations), and efficient method for the isolation/purification of lentivirus particles from cell culture media at the analytical scale.

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Funding

Financial support for the chromatography development efforts from the National Science Foundation, Division of Chemistry, under grant CHE-1608663 is gratefully acknowledged.

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Authors and Affiliations

  1. Department of Chemistry, Biosystems Research Complex, Clemson University, Clemson, SC, 29634, USA

    Sisi Huang & R. Kenneth Marcus

  2. Department of Bioengineering, Life Sciences Facility, Clemson University, Clemson, SC, 29634, USA

    Terri F. Bruce

  3. Department of Biological Sciences, Life Sciences Facility, Clemson University, Clemson, SC, 29634, USA

    Hui Ding & Yanzhang Wei

Authors
  1. Sisi Huang
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  2. Terri F. Bruce
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  3. Hui Ding
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  4. Yanzhang Wei
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  5. R. Kenneth Marcus
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Contributions

S. Huang developed and performed the chromatographic separations and wrote the manuscript, H. Ding and Y. Wei provided the cell cultures and guidance in performing qPCR analyses, and T. Bruce and R. K. Marcus provided guidance, supervised the project, and reviewed and edited the manuscript.

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Correspondence to R. Kenneth Marcus.

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Huang, S., Bruce, T.F., Ding, H. et al. Rapid isolation of lentivirus particles from cell culture media via a hydrophobic interaction chromatography method on a polyester, capillary-channeled polymer fiber stationary phase. Anal Bioanal Chem 413, 2985–2994 (2021). https://doi.org/10.1007/s00216-021-03232-8

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  • DOI: https://doi.org/10.1007/s00216-021-03232-8

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