Impact of Process Conditions on the Centrifugal Recovery of a Disabled Herpes Simplex Virus

Lotfian, Pantea; Levy, M. Susana; Coffin, Robert S.; Fearn, Tom; Ayazi-Shamlou, Parviz

doi:10.1021/bp025599g

Cited by 8 publications

(6 citation statements)

References 32 publications

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“…However, these studies were carried out with vaccine purified using centrifugation-based methods which are not readily scaled for commercial production. Indeed, a number of groups have defined laboratory-scale procedures for purification of herpes viruses based upon centrifugation [28], [29], gradients [30], [31], [32], [33], filtration [34] and affinity chromatography [35], [36].…”

Section: Introductionmentioning

confidence: 99%

High-Purity Preparation of HSV-2 Vaccine Candidate ACAM529 Is Immunogenic and Efficacious In Vivo

et al. 2013

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Genital herpes is a sexually transmitted infection (STI) caused by herpes simplex virus 2 (HSV-2) and to a lesser extent herpes simplex virus 1 (HSV-1). Infection by HSV-2 is life-long and is associated with significant cost to healthcare systems and social stigma despite the highly prevalent nature of the disease. For instance, the proportion of HSV-2 seropositive to seronegative adults is approximately 1 in 5 in the US and greater than 4 in 5 in some areas of sub-Saharan Africa. The replication-defective vaccine strain virus dl5-29 was re-derived using cells appropriate for GMP manufacturing and renamed ACAM529. Immunization with dl5-29 was previously reported to be protective both in mice and in guinea pigs, however these studies were performed with vaccine that was purified using methods that cannot be scaled for manufacturing of clinical material. Here we describe methods which serve as a major step towards preparation of ACAM529 which may be suitable for testing in humans. ACAM529 can be harvested from infected cell culture of the trans-complementing cell line AV529 clone 19 (AV529-19) without mechanical cell disruption. ACAM529 may then be purified with respect to host cell DNA and proteins by a novel purification scheme, which includes a combination of endonuclease treatment, depth filtration, anion-exchange chromatography and ultrafiltration/diafiltration (UF/DF). The resultant virus retains infectivity and is ∼ 200-fold more pure with respect to host cell DNA and proteins than is ACAM529 purified by ultracentrifugation. Additionally, we describe a side-by-side comparison of chromatography-purified ACAM529 with sucrose cushion-purified ACAM529, which shows that both preparations are equally immunogenic and protective when tested in vivo.

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Section: Introductionmentioning

confidence: 99%

High-Purity Preparation of HSV-2 Vaccine Candidate ACAM529 Is Immunogenic and Efficacious In Vivo

et al. 2013

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“…A fresh stock of material was prepared for each test run. The infected cells were separated from the culture medium by using a Beckman model J2‐21 centrifuge equipped with a 25° fixed‐angle rotor and six 250 ml (200‐ml‐working‐volume buckets) operating at 1600 g for 15 min, as described previously [22]. The cells in the pellet fraction were re‐suspended in HBSS (Hanks balanced salt solution; Invitrogen; catalogue no.…”

Section: Methodsmentioning

confidence: 99%

Shear‐induced release of disabled herpes simplex virus from baby‐hamster kidney cells

Lotfian

Levy

et al. 2003

Biotech and App Biochem

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A new process route is proposed to increase the production yield of disabled herpes simplex virus type 1 (HSV-1 DIS). Infected baby-hamster kidney (BHK) cells were subjected to a range of shear rates between 3.69 x 10(3) s(-1) and 51.3 x 10(3) s(-1) in the gap between a pair of co-axial cylinders. Analysis of the supernatant fractions of sheared material established that optimal virus release was achieved by exposing the infected cells to a shear rate of 42.7 x 10(3) s(-1) for a period of 1 min. Compared with the current laboratory process, the titre of HSV-1 DIS was increased over 30-fold, from about 1 x 10(6) to 30 x 10(6) pfu (plaque-forming units)/ml. Evaluation of the supernatant fractions by flow cytometry, total protein assay, PAGE and dot-blot assays showed no evidence of cell disruption, supporting the hypothesis that shear-induced release of the cell-membrane-bound virus was achieved without compromising downstream purification. The proposed method is scalable, and since no additional chemicals are required, it provides an attractive option for enhanced recovery of virus particles for therapeutic applications.

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“…A major challenge is the need for better delivery systems. Viral vectors are popular, but there are safety concerns regarding their use [3], and it is proving difficult to make them in large quantities and sufficient purity [4,5]. DNA complexes pose fewer problems than viral vectors, but suffer from unacceptably low transfection efficiencies.…”

Section: Introductionmentioning

confidence: 99%

The fractal structure of polycation–DNA complexes

Sarkar¹,

Lee²,

Hart³

et al. 2005

Biotech and App Biochem

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We used static light scattering to obtain new measurements on the internal structure of aggregated non-viral gene-delivery particles in colloidal suspension. The vector particles are prepared by charge neutralization of plasmid DNA either by poly-L-lysine or by a Lipofectin/integrin-targeting peptide. We use established theories of the stability of colloidal particles and fractal concepts to explain the aggregation processes and demonstrate the existence of a new property (fractal dimension) of the aggregated vector particles. Aggregation is shown to produce particles with fractal dimensions in the range between 1.8 and 2.4; the former suggests a loose three-dimensional structure and the latter characterizes an aggregation process that leads to the formation of particles with tightly packed structures. We show that the fractal dimension of the vector particles is sensitive to changes in physicochemical conditions (ionic strength) of the buffer solution and propose that fractal dimension may provide a useful means of monitoring the physical state of non-viral delivery-vector particles during preparation and storage.

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Impact of Process Conditions on the Centrifugal Recovery of a Disabled Herpes Simplex Virus

Cited by 8 publications

References 32 publications

High-Purity Preparation of HSV-2 Vaccine Candidate ACAM529 Is Immunogenic and Efficacious In Vivo

High-Purity Preparation of HSV-2 Vaccine Candidate ACAM529 Is Immunogenic and Efficacious In Vivo

Shear‐induced release of disabled herpes simplex virus from baby‐hamster kidney cells

The fractal structure of polycation–DNA complexes

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