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Scaling-up The Production and Purification of HSV-based Gene Therapy Vectors and Development of GLP Assay System to Analyze Quantity and Quality of Clinical-grade HSV Vector Stocks

Ozuer, Ali (2002) Scaling-up The Production and Purification of HSV-based Gene Therapy Vectors and Development of GLP Assay System to Analyze Quantity and Quality of Clinical-grade HSV Vector Stocks. Doctoral Dissertation, University of Pittsburgh.

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    Abstract

    Herpes simplex virus type-1 (HSV-1) represents an ideal vector for use in many gene therapy applications. HSV-based vectors have recently been employed in human clinical trials to treat malignant brain tumors. The production of HSV-1 vectors must now be scaled-up to meet the demand for large volumes of high-titer clinical-grade vector stocks. However, in order to develop efficient large-scale production and purification methods, not only the factors affecting the efficiency of HSV-1 vector production have to be systematically investigated but also new Good Laboratory Practices (GLP) vector assay systems needs to developed. We evaluated the dependence of viral yield on genetic background, culture pH, glucose and lactate concentration as well as serum content. High-level glucose consumption, with the corresponding increase in lactate synthesis, observed within the first 24 hours post infection period. Media replacement at various times following infection resulted in a 2-fold overall increase. The influence of culture pH on viral yield and virus stability was also investigated. Lower pH (6.8) not only enhanced the viral yield but also increased the half-life of virus compare to higher pH values (7.4 and 8.0). The use of serum-free media did not result in high viral yield. However, our results show that serum content of 5% provided similar viral yields as that of 10%, suggesting a potential cost reduction and ease in the purification of vector. Then, we successfully scaled-up the vector production by employing CellCube bioreactor. Finally, we developed and a very sensitive assay system relevant for meeting and surpassing current FDA requirements for clinical grade viral vector stocks. Our real time PCR assay is linear from 10 to 10⁷ copies of HSV and 1 to 10⁵ copies of host cell genomic DNA. Our PicoGreen micro-plate assay is fast and accurate, with a detection limit as low as 0.5 ng of HSV DNA corresponding to ∼3X10⁶ HSV particles. The resultant combination of real-time PCR and PicoGreen micro-plate DNA quantitation assays represents a standard in the field of HSV gene therapy vector quality assessment.


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    Item Type: University of Pittsburgh ETD
    ETD Committee:
    ETD Committee TypeCommittee MemberEmail
    Committee ChairAtaai, Mohammad M.ataai@engrng.pitt.edu
    Committee MemberMorsi, Badie I.morsi@engrng.pitt.edu
    Committee MemberGlorioso, Joseph C.glorioso@pitt.edu
    Committee MemberKoepsel, Richard
    Title: Scaling-up The Production and Purification of HSV-based Gene Therapy Vectors and Development of GLP Assay System to Analyze Quantity and Quality of Clinical-grade HSV Vector Stocks
    Status: Unpublished
    Abstract: Herpes simplex virus type-1 (HSV-1) represents an ideal vector for use in many gene therapy applications. HSV-based vectors have recently been employed in human clinical trials to treat malignant brain tumors. The production of HSV-1 vectors must now be scaled-up to meet the demand for large volumes of high-titer clinical-grade vector stocks. However, in order to develop efficient large-scale production and purification methods, not only the factors affecting the efficiency of HSV-1 vector production have to be systematically investigated but also new Good Laboratory Practices (GLP) vector assay systems needs to developed. We evaluated the dependence of viral yield on genetic background, culture pH, glucose and lactate concentration as well as serum content. High-level glucose consumption, with the corresponding increase in lactate synthesis, observed within the first 24 hours post infection period. Media replacement at various times following infection resulted in a 2-fold overall increase. The influence of culture pH on viral yield and virus stability was also investigated. Lower pH (6.8) not only enhanced the viral yield but also increased the half-life of virus compare to higher pH values (7.4 and 8.0). The use of serum-free media did not result in high viral yield. However, our results show that serum content of 5% provided similar viral yields as that of 10%, suggesting a potential cost reduction and ease in the purification of vector. Then, we successfully scaled-up the vector production by employing CellCube bioreactor. Finally, we developed and a very sensitive assay system relevant for meeting and surpassing current FDA requirements for clinical grade viral vector stocks. Our real time PCR assay is linear from 10 to 10⁷ copies of HSV and 1 to 10⁵ copies of host cell genomic DNA. Our PicoGreen micro-plate assay is fast and accurate, with a detection limit as low as 0.5 ng of HSV DNA corresponding to ∼3X10⁶ HSV particles. The resultant combination of real-time PCR and PicoGreen micro-plate DNA quantitation assays represents a standard in the field of HSV gene therapy vector quality assessment.
    Date: 15 February 2002
    Date Type: Completion
    Defense Date: 28 November 2001
    Approval Date: 15 February 2002
    Submission Date: 11 February 2002
    Access Restriction: No restriction; The work is available for access worldwide immediately.
    Patent pending: No
    Institution: University of Pittsburgh
    Thesis Type: Doctoral Dissertation
    Refereed: Yes
    Degree: PhD - Doctor of Philosophy
    URN: etd-02112002-130710
    Uncontrolled Keywords: Viral Vector Production; Cell Culture; Gene Therapy; GLP Assay Development; Herpes Simplex Virus Type 1; Optimization of Culture Conditions
    Schools and Programs: Swanson School of Engineering > Chemical Engineering
    Date Deposited: 10 Nov 2011 14:31
    Last Modified: 22 Feb 2012 12:25
    Other ID: http://etd.library.pitt.edu/ETD/available/etd-02112002-130710/, etd-02112002-130710

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