Link to the University of Pittsburgh Homepage
Link to the University Library System Homepage Link to the Contact Us Form

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. (Unpublished)

Primary Text

Download (1MB) | Preview


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.


Social Networking:
Share |


Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairAtaai, Mohammad
Committee MemberMorsi, Badie
Committee MemberGlorioso, Joseph C.glorioso@pitt.eduGLORIOSO
Committee MemberKoepsel, Richard
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; Release the ETD for access worldwide immediately.
Institution: University of Pittsburgh
Schools and Programs: Swanson School of Engineering > Chemical Engineering
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: Viral Vector Production; Cell Culture; Gene Therapy; GLP Assay Development; Herpes Simplex Virus Type 1; Optimization of Culture Conditions
Other ID:, etd-02112002-130710
Date Deposited: 10 Nov 2011 19:31
Last Modified: 15 Nov 2016 13:36


Monthly Views for the past 3 years

Plum Analytics

Actions (login required)

View Item View Item