96a956f19c60eaa233bc4c9c9c6dbacf pmc_34696262.pdf 58fbf1ba1d6afc47e22799b7878c6ab8824348cf pmc_34696262.pdf 84474fbfefc4c7840a59fd5ae477af802b1cb66b20cfe731c119d02281630bf3 pmc_34696262.pdf Title: A Four-Step Purification Process for Gag VLPs: From Culture Supernatant to High-Purity Lyophilized Particles Subject: Gag-based virus-like particles (VLPs) have high potential as scaffolds for the development of chimeric vaccines and delivery strategies. The production of purified preparations that can be preserved independently from cold chains is highly desirable to facilitate distribution and access worldwide. In this work, a nimble purification has been developed, facilitating the production of Gag VLPs. Suspension-adapted HEK 293 cells cultured in chemically defined cell culture media were used to produce the VLPs. A four-step downstream process (DSP) consisting of membrane filtration, ion-exchange chromatography, polishing, and lyophilization was developed. The purification of VLPs from other contaminants such as host cell proteins (HCP), double-stranded DNA, or extracellular vesicles (EVs) was confirmed after their DSP. A concentration of 2.2 0.8 109 VLPs/mL in the lyophilized samples was obtained after its storage at room temperature for two months. Morphology and structural integrity of purified VLPs was assessed by cryo-TEM and NTA. Likewise, the purification methodologies proposed here could be easily scaled up and applied to purify similar enveloped viruses and vesicles. Keywords: Gag VLPs; purification; clarification; chromatography; lyophilization; EVs; nanoparticle quantification Author: Irene González-Domínguez, Elianet Lorenzo, Alice Bernier, Laura Cervera, Francesc Gòdia and Amine Kamen Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Wed Oct 20 09:47:33 2021 CEST ModDate: Wed Oct 20 09:50:22 2021 CEST Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 19 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 4414344 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- NNKDOL+VnURWPalladioL Type 1 Custom yes yes no 10 0 YJPSFM+URWPalladioL-Ital Type 1 Custom yes yes no 15 0 STXKAQ+URWPalladioL-Bold Type 1 Custom yes yes no 20 0 JGXUGX+URWPalladioL-Roma Type 1 Custom yes yes no 24 0 KVEXVN+CMSY10 Type 1 Builtin yes yes no 28 0 LPJXBF+VnURWPalladioL-Bold Type 1 Custom 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Double-Stranded DNA (dsDNA) Quantification Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@262b2c86 Item: Title: Dynamic Light Scattering (DLS) Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@371a67ec Item: Title: Nanoparticle Tracking Analysis (NTA) Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@5ed828d Item: Title: Cryogenic Transmission Electron Microscopy (Cryo-TEM) Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@50d0686 Item: Title: Sodium Dodecyl Sulfate-Polyacrylamide Gel (SDS-PAGE) Electrophoresis and Western Blot Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7a3d45bd Item: Title: Thermostability Studies of Gag-eGFP VLPs Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1e7c7811 Item: Title: Preliminary Serial Filtration Experiments Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77ec78b9 Item: Title: Preparative Clarification Experiments Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1a3869f4 Item: Title: Ion-Exchange Chromatography (IEX) Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@a38d7a3 Item: Title: SEC Chromatography Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77f99a05 Item: Title: Lyophilization Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@63440df3 Item: Title: Results Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@3aeaafa6 Children: Item: Title: Stability Studies on Gag VLPs Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@76a3e297 Item: Title: Development of Four Steps Downstream Process of Gag VLPs Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4d3167f4 Children: Item: Title: Clarification Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@ed9d034 Item: Title: IEX Chromatography Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@6121c9d6 Item: Title: SEC Chromatography Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@87f383f Item: Title: Lyophilization of Gag-eGFP VLPs Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4eb7f003 Item: Title: Discussion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@eafc191 Children: Item: Title: Purification Train and Unit Capacity Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@612fc6eb Item: Title: VLP Quality and Purification Yield Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1060b431 Item: Title: Conclusions Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@612679d6 Item: Title: References Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@11758f2a Info: Title: A Four-Step Purification Process for Gag VLPs: From Culture Supernatant to High-Purity Lyophilized Particles Author: Irene González-Domínguez, Elianet Lorenzo, Alice Bernier, Laura Cervera, Francesc Gòdia and Amine Kamen Subject: Gag-based virus-like particles (VLPs) have high potential as scaffolds for the development of chimeric vaccines and delivery strategies. The production of purified preparations that can be preserved independently from cold chains is highly desirable to facilitate distribution and access worldwide. In this work, a nimble purification has been developed, facilitating the production of Gag VLPs. Suspension-adapted HEK 293 cells cultured in chemically defined cell culture media were used to produce the VLPs. A four-step downstream process (DSP) consisting of membrane filtration, ion-exchange chromatography, polishing, and lyophilization was developed. The purification of VLPs from other contaminants such as host cell proteins (HCP), double-stranded DNA, or extracellular vesicles (EVs) was confirmed after their DSP. A concentration of 2.2 0.8 109 VLPs/mL in the lyophilized samples was obtained after its storage at room temperature for two months. Morphology and structural integrity of purified VLPs was assessed by cryo-TEM and NTA. Likewise, the purification methodologies proposed here could be easily scaled up and applied to purify similar enveloped viruses and vesicles. 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Checksum: 59b84abd Type: CRC32 Checksum: 96a956f19c60eaa233bc4c9c9c6dbacf Type: MD5 Checksum: 58fbf1ba1d6afc47e22799b7878c6ab8824348cf Type: SHA-1