Downstream Processing of Lentiviral Vectors: Releasing Bottlenecks

Bandeira, Vanessa Sofia; Peixoto, Cristina; Rodrigues, Ana Paula Leite; Cruz, Pedro; Alves, Paula M.; Coroadinha, Ana S.; Carrondo, Manuel J.T.

doi:10.1089/hgtb.2012.059

Cited by 76 publications

(82 citation statements)

References 34 publications

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“…23,24 In these reports, residual plasmids in vector stocks were not transfected into target cells during in vitro exposure to lentiviral vectors. Treatment with benzonase is not, however, routine in most preclinical laboratory studies.…”

Section: Discussionmentioning

confidence: 99%

Integration-specific In Vitro Evaluation of Lentivirally Transduced Rhesus CD34+ Cells Correlates With In Vivo Vector Copy Number

Uchida

Evans

Hsieh

et al. 2013

Molecular Therapy - Nucleic Acids

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Hematopoietic stem cell (HSC) gene therapy using integrating vectors has a potential leukemogenic risk due to insertional mutagenesis. To reduce this risk, a limitation of ≤2 average vector copy number (VCN) per cell is generally accepted. We developed an assay for VCN among transduced CD34+ cells that reliably predicts in vivo VCN in 16 rhesus recipients of CD34+ cells transduced with a green fluorescent protein (GFP) (or yellow fluorescent protein (YFP))-encoding lentiviral vector. Using GFP (or YFP)-specific probe/primers by real-time PCR, VCN among transduced CD34+ cells had no correlation with VCN among granulocytes or lymphocytes in vivo assayed 6 months post-transplantation. This was a likely result of residual plasmids present in the vector preparation. We then designed self-inactivating long terminal repeat (SIN-LTR)-specific probe/primers, which detect only integrated provirus. Evaluation with SIN-LTR probe/primers resulted in a positive correlation of VCN among transduced CD34+ cells with granulocytes and lymphocytes in vivo. The transduced CD34+ cells had higher VCN (25.1 ± 5.6) as compared with granulocytes (2.8 ± 1) and lymphocytes (2.4 ± 0.7). In summary, an integrated provirus-specific real-time PCR system demonstrated nine- to tenfold higher VCN in transduced CD34+ cells in vitro, as compared with VCN in vivo. Therefore, the restriction of ≤2 VCN before infusion might unnecessarily limit gene transfer efficacy.

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Section: Discussionmentioning

confidence: 99%

Integration-specific In Vitro Evaluation of Lentivirally Transduced Rhesus CD34+ Cells Correlates With In Vivo Vector Copy Number

Uchida

Evans

Hsieh

et al. 2013

Molecular Therapy - Nucleic Acids

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“…between any viruses or VLP variants [7,8]. Monoliths have been frequently used for separation of enveloped VLPs [9][10][11] and enveloped viruses [12][13][14], such as lentivirus [15], baculovirus [16], and influenza virus [17]. Recently, we have shown that by using ion-exchange monoliths, the separation of VLPs from extra cellular particles is possible [9].…”

Section: Introductionmentioning

confidence: 99%

Separation of HIV‐1 gag virus‐like particles from vesicular particles impurities by hydroxyl‐functionalized monoliths

Steppert

Burgstaller

Klausberger

et al. 2017

J of Separation Science

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The downstream processing of enveloped virus-like particles is very challenging because of the biophysical and structural similarity between correctly assembled particles and contaminating vesicular particles present in the feedstock. We used hydroxyl-functionalized polymethacrylate monoliths, providing hydrophobic and electrostatic binding contributions, for the purification of HIV-1 gag virus-like particles. The clarified culture supernatant was conditioned with ammonium sulfate and after membrane filtration loaded onto a 1 mL monolith. The binding capacity was 2 × 10 /mL monolith and was only limited by the pressure drop. By applying either a linear or a step gradient elution, to decrease the ammonium sulfate concentration, the majority of double-stranded DNA (88-90%) and host cell protein impurities (39-61%) could be removed while the particles could be separated into two fractions. Proteomic analysis and evaluation of the p24 concentration showed that one fraction contained majority of the HIV-1 gag and the other fraction was less contaminated with proteins originated from intracellular compartments. We were able to process up to 92 bed volumes of conditioned loading material within 3 h and eluted in average 7.3 × 10 particles per particle fraction, which is equivalent to 730 vaccination doses of 1 × 10 particles.

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“…The method developed from a disk/tube monolithic column can be easily scaled-up to litres for bulk purification of biomolecules [29]. Recently, CIM DEAE monolithic columns have been used for the purification of many viruses and bacteriophages including Tomato mosaic virus [16,30], filamentous potato virus [17], Pseudomonas phage LUZ19 [31], Rubella virus [32], Staphylococcus phage ISP [33], viral vectors such as Canine Adenoviral Vectors [14], and Lentiviral Vectors [34]. …”

Section: Discussionmentioning

confidence: 99%

Concentration and purification of enterovirus 71 using a weak anion-exchange monolithic column

Venkatachalam

Szyporta

Kiener

et al. 2014

Virol J

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BackgroundEnterovirus 71 (EV-71) is a neurotropic virus causing Hand, Foot and Mouth Disease (HFMD) in infants and children under the age of five. It is a major concern for public health issues across Asia-Pacific region. The most effective way to control the disease caused by EV-71 is by vaccination thus a novel vaccine is urgently needed. Inactivated EV-71 induces a strong, virus-neutralizing antibody response in animal models, protecting them against a lethal EV-71 challenge and it has been shown to elicit cross-neutralizing antibodies in human trials. Hence, the large-scale production of purified EV-71 is required for vaccine development, diagnosis and clinical trials.MethodsCIM® Monolith columns are single-piece columns made up of poly(glycidyl methacrylate co-ethylene dimethacrylate) as support matrix. They are designed as porous channels rather than beads with different chemistries for different requirements. As monolithic columns have a high binding capacity, flow rate and resolution, a CIM® DEAE-8f tube monolithic column was selected for purification in this study. The EV-71 infected Rhabdomyosarcoma (RD) cell supernatant was concentrated using 8% PEG 8000 in the presence of 400 mM sodium chloride. The concentrated virus was purified by weak anion exchange column using 50 mM HEPES + 1 M sodium chloride as elution buffer.ResultsHighly pure viral particles were obtained at a concentration of 350 mM sodium chloride as confirmed by SDS-PAGE and electron microscopy. Presence of viral proteins VP1, VP2 and VP3 was validated by western blotting. The overall process achieved a recovery of 55%.ConclusionsEV-71 viral particles of up to 95% purity can be recovered by a single step ion-exchange chromatography using CIM-DEAE monolithic columns and 1 M sodium chloride as elution buffer. Moreover, this method is scalable to purify several litres of virus-containing supernatant, using industrial monolithic columns with a capacity of up to 8 L such as CIM® cGMP tube monolithic columns.

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Downstream Processing of Lentiviral Vectors: Releasing Bottlenecks

Cited by 76 publications

References 34 publications

Integration-specific In Vitro Evaluation of Lentivirally Transduced Rhesus CD34+ Cells Correlates With In Vivo Vector Copy Number

Integration-specific In Vitro Evaluation of Lentivirally Transduced Rhesus CD34+ Cells Correlates With In Vivo Vector Copy Number

Separation of HIV‐1 gag virus‐like particles from vesicular particles impurities by hydroxyl‐functionalized monoliths

Concentration and purification of enterovirus 71 using a weak anion-exchange monolithic column

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