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1.
Vaccine ; 29(41): 7182-7, 2011 Sep 22.
Article in English | MEDLINE | ID: mdl-21651939

ABSTRACT

Foot and mouth disease (FMD) is a highly infectious viral disease that affects cattle, sheep, goats and swine causing severe economic losses worldwide. The efficacy of inactivated vaccines is critically dependent on the integrity of foot and mouth disease virus (FMDV) particles. The recommended method to quantify the active ingredient of vaccines is the 140S quantitative sucrose density gradient analysis. This method has been an immensely valuable tool over the past three decades but it is highly operator dependent and difficult to automate. We developed a method to quantify FMDV particles during the vaccine manufacturing process that is based on separation of components by size-exclusion chromatography and measurement of virus by absorption at 254nm. The method is linear in the 5-70µg/mL range, it is applicable to different FMDV strains, and has a good correlation with the 140S test. The proposed method uses standard chromatographic media and it is amenable to automation. The method has potential as a process analytical technology and for control of final product by manufacturers, international vaccine banks and regulatory agencies.


Subject(s)
Chromatography, Gel/methods , Foot-and-Mouth Disease Virus/isolation & purification , Technology, Pharmaceutical/methods , Viral Load , Viral Vaccines , Virology/methods , Vaccines, Inactivated
2.
Biotechnol Bioeng ; 105(2): 260-75, 2010 Feb 01.
Article in English | MEDLINE | ID: mdl-19739083

ABSTRACT

Experimentation in shaken microplate formats offers a potential platform technology for the rapid evaluation and optimization of cell culture conditions. Provided that cell growth and antibody production kinetics are comparable to those found in currently used shake flask systems then the microwell approach offers the possibility to obtain early process design data more cost effectively and with reduced material requirements. This work describes a detailed engineering characterization of liquid mixing and gas-liquid mass transfer in microwell systems and their impact on suspension cell cultures. For growth of murine hybridoma cells producing IgG1, 24-well plates have been characterized in terms of energy dissipation (P/V) (via Computational Fluid Dynamics, CFD), fluid flow, mixing and oxygen transfer rate as a function of shaking frequency and liquid fill volume. Predicted k(L)a values varied between 1.3 and 29 h(-1); liquid-phase mixing time, quantified using iodine decolorization experiments, varied from 1.7 s to 3.5 h; while the predicted P/V ranged from 5 to 35 W m(-3). CFD simulations of the shear rate predicted hydrodynamic forces will not be detrimental to cells. For hybridoma cultures however, high shaking speeds (>250 rpm) were shown to have a negative impact on cell growth, while a combination of low shaking speed and high well fill volume (120 rpm, 2,000 microL) resulted in oxygen limited conditions. Based on these findings a first engineering comparison of cell culture kinetics in microwell and shake flask formats was made at matched average energy dissipation rates. Cell growth kinetics and antibody titer were found to be similar in 24-well microtiter plates and 250 mL shake flasks. Overall this work has demonstrated that cell culture performed in shaken microwell plates can provide data that is both reproducible and comparable to currently used shake flask systems while offering at least a 30-fold decrease in scale of operation and material requirements. Linked with automation this provides a route towards the high throughput evaluation of robust cell lines under realistic suspension culture conditions.


Subject(s)
Biotechnology/methods , Cell Culture Techniques/methods , Animals , Biotechnology/instrumentation , Cell Culture Techniques/instrumentation , Cell Line , Cell Proliferation , Cell Survival , Hybridomas/metabolism , Immunoglobulin G/analysis , Immunoglobulin G/metabolism , Oxygen/chemistry
3.
Biotechnol Appl Biochem ; 51(Pt 1): 43-51, 2008 Sep.
Article in English | MEDLINE | ID: mdl-18315524

ABSTRACT

The effect of bacterial-cell centrifugation and handling on the initial stages of plasmid processing was investigated. Escherichia coli cells containing either a 6 or 20 kb plasmid were grown in 75- and 450-litre bioreactors, and the process yield of the early recovery stages was characterized in terms of SC pDNA (supercoiled plasmid DNA) recovered. In all cases, the cells were totally recovered using either a continuous-feed, intermittent-solids-discharge, disc-stack centrifuge or a continuous-feed, batch-discharge, solid-bowl centrifuge. The cells were then either processed immediately or stored frozen. The centrifugation method considerably affected the yield of SC pDNA, and there was evidence that the intermittent discharge of cells from a centrifuge operating at high speed led to a sediment containing lysed cells and degraded pDNA. This led to estimated plasmid yield losses of up to 40% as compared with cells recovered from laboratory or solid-bowl centrifuges, where there is evidently no cell stress on discharge. By inference, the cell stress on feed to either of the continuous centrifuges studied was not implicated in product loss. Freezing of the recovered cells gives a convenient hold stage prior to further processing. In all cases, this extra freeze-thaw stage led to loss of SC pDNA, and this was in addition to the loss attributed to cell lysis during centrifugation discharge. Only average yields can be gained from pilot plant-scale studies; separate laboratory-based experiments indicated that this loss of SC pDNA is determined by the time and temperature for which the resuspended cells are held.


Subject(s)
Cell Separation/methods , Centrifugation/methods , DNA, Superhelical , Escherichia coli , Plasmids , DNA Damage , DNA, Superhelical/analysis , DNA, Superhelical/chemistry , DNA, Superhelical/metabolism , Electrophoresis, Polyacrylamide Gel , Escherichia coli/cytology , Escherichia coli/genetics , Escherichia coli/growth & development , Fermentation , Freezing , Molecular Weight , Plasmids/analysis , Plasmids/chemistry , Plasmids/metabolism , Temperature
4.
Biotechnol Prog ; 23(4): 858-65, 2007.
Article in English | MEDLINE | ID: mdl-17672520

ABSTRACT

Supercoiled circular (SC) plasmid DNA is often subjected to fluid stress in large-scale manufacturing processes. It is thus important to characterize the engineering environment within a particular unit operation as well as within the associated ancillary equipment during process design for plasmid DNA manufacture so as to avoid shear-induced degradation of the SC isoform, which would compromise product efficacy in therapeutic applications. In the past few years, ultra scale-down (USD) tools were developed within our laboratory to mimic the engineering environments experienced by biomolecules within a range of manufacturing-scale ancillary, primary recovery, and purification operations, using milliliter quantities of material. Through the use of a USD shear device, the effect of elongational strain rate on SC plasmid DNA degradation was studied in this paper, and from that, the impact of a centrifugal pump, a Mono pump, and a disk-stack centrifuge feed zone on SC plasmid DNA degradation was predicted and experimentally verified at scale. Model predictions, over the range of conditions studied, were in good agreement with experimental values, demonstrating the potential of the USD approach as a decisional tool during bioprocess design.


Subject(s)
Biotechnology/methods , DNA/chemistry , Industrial Microbiology/instrumentation , Plasmids/metabolism , Alcohols/chemistry , DNA, Superhelical/chemistry , Electrophoresis, Agar Gel , Equipment Design , Fermentation , Industrial Microbiology/methods , Models, Statistical , Models, Theoretical , Stress, Mechanical , Time Factors
5.
J Virol Methods ; 137(2): 213-8, 2006 Nov.
Article in English | MEDLINE | ID: mdl-16854472

ABSTRACT

The traditional plaque forming and TCID(50) methods to determine replication competent virus titres rely on several cycles of replication and infection to generate a plaque with an incubation period of 24-72 h post-infection typically required. We developed a method to quantify infective viral particles based on early detection of cellular events by capillary cytometry. The method uses a capillary cytometer as a precise cell counter that can discriminate infected from non-infected cells. The general protocol was developed using a Guava PCA, genetically modified HSV-1 virus and polyclonal antibodies against antigens expressed on the cell membrane. Infection was detected after 1 h incubation and a plateau in the number of infected cells was observed between 7 and 9 h. A good correlation between titres obtained by the plaque forming method and the proposed method was observed for a ratio of infected to total cells between 0.5 and 0.05. The rapid and automated analysis (10 s/1000 events acquired per sample) makes the method particularly useful for high-throughput applications. The proposed method can be extended easily to determine the titre of other viruses providing a powerful tool for virology and antiviral screening.


Subject(s)
Antigens, Viral/analysis , Flow Cytometry/methods , Herpesvirus 1, Human/growth & development , Viruses/growth & development , Animals , Antibodies, Viral , Cell Line , Cricetinae , Herpesvirus 1, Human/immunology , Herpesvirus 1, Human/isolation & purification , Statistics as Topic , Viral Plaque Assay , Viruses/isolation & purification
6.
Biotechnol Prog ; 21(6): 1577-92, 2005.
Article in English | MEDLINE | ID: mdl-16321039

ABSTRACT

The risk of a pandemic with a virulent form of influenza is acknowledged by the World Health Organization (WHO) and other agencies. Current vaccine production facilities would be unable to meet the global requirement for vaccine. As a possible supplement a DNA vaccine may be appropriate, and bioprocess engineering factors bearing on the use of existing biopharmaceutical and antibiotics plants to produce it are described. This approach addresses the uncertainty of timing of a pandemic that precludes purpose-built facilities. The strengths and weaknesses of alternative downstream processing routes are analyzed, and several gaps in public domain information are addressed. The conclusion is that such processing would be challenging but feasible.


Subject(s)
Biotechnology/methods , Disease Outbreaks/prevention & control , Influenza Vaccines/isolation & purification , Influenza, Human/epidemiology , Influenza, Human/prevention & control , Adsorption , Biomedical Engineering/methods , Bioreactors , Chemistry, Pharmaceutical , Chromatography , Escherichia coli/genetics , Fermentation , Fractional Precipitation , Hot Temperature , Humans , Hydrogen-Ion Concentration , Influenza Vaccines/genetics , Plasmids/genetics , Plasmids/isolation & purification , Ultrafiltration , Vaccines, DNA/genetics , Vaccines, DNA/isolation & purification
7.
Biotechnol Appl Biochem ; 38(Pt 3): 271-81, 2003 Dec.
Article in English | MEDLINE | ID: mdl-12901722

ABSTRACT

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.


Subject(s)
Bioreactors , Cell Culture Techniques/methods , Kidney/virology , Mechanotransduction, Cellular/physiology , Physical Stimulation/methods , Simplexvirus/growth & development , Simplexvirus/isolation & purification , Animals , Cell Culture Techniques/instrumentation , Cells, Cultured , Cricetinae , Motion , Physical Stimulation/instrumentation , Pilot Projects , Shear Strength , Stress, Mechanical , Virus Inactivation
8.
Nucleic Acids Res ; 31(11): e65, 2003 Jun 01.
Article in English | MEDLINE | ID: mdl-12771229

ABSTRACT

A method is described for high-throughput monitoring of DNA backbone integrity in plasmids and artificial chromosomes in solution. The method is based on the denaturation properties of double-stranded DNA in alkaline conditions and uses PicoGreen fluorochrome to monitor denaturation. In the present method, fluorescence enhancement of PicoGreen at pH 12.4 is normalised by its value at pH 8 to give a ratio that is proportional to the average backbone integrity of the DNA molecules in the sample. A good regression fit (r2 > 0.98) was obtained when results derived from the present method and those derived from agarose gel electrophoresis were compared. Spiking experiments indicated that the method is sensitive enough to detect a proportion of 6% (v/v) molecules with an average of less than two breaks per molecule. Under manual operation, validation parameters such as inter-assay and intra-assay variation gave values of <5% coefficient of variation. Automation of the method showed equivalence to the manual procedure with high reproducibility and low variability within wells. The method described requires as little as 0.5 ng of DNA per well and a 96-well microplate can be analysed in 12 min providing an attractive option for analysis of high molecular weight vectors. A preparation of a 116 kb bacterial artificial chromosome was subjected to chemical and shear degradation and DNA integrity was tested using the method. Good correlation was obtained between time of chemical degradation and shear rate with fluorescence response. Results obtained from pulsed- field electrophoresis of sheared samples were in agreement with those obtained using the microplate-based method.


Subject(s)
Chromosomes, Artificial, Bacterial , DNA/analysis , Fluorometry/methods , Plasmids , Automation , DNA/chemistry , Electrophoresis, Agar Gel , Fluorescent Dyes/chemistry , Hydrogen-Ion Concentration , Kinetics , Nucleic Acid Denaturation , Organic Chemicals , Reproducibility of Results
9.
Biotechnol Prog ; 19(1): 209-15, 2003.
Article in English | MEDLINE | ID: mdl-12573027

ABSTRACT

Despite continuous improvements in culturing and recovery techniques, high-titer stocks of purified disabled herpes simplex virus type-1 (HSV-1 DIS) vector for drug discovery and use in preclinical and clinical trials are currently difficult to achieve. Efforts to improve their centrifugal recovery have been addressed in this paper. The operation of a swing-out centrifuge rotor was assessed, and its operational conditions were defined for the recovery of viable HSV-1 DIS. 80% virus recovery was achieved after 90 min at 26000g. The 20% loss of virus was attributed to damage to the viral envelope by overcompaction of the pellet and impaction with the base of the centrifuge tube. Virus recovery was increased by a further 10% by using a fixed-angle centrifuge rotor operating at 26000g. Plaque assays of recovered HSV-1 DIS gave values on the order of 10(6) pfu/mL, compared to values typically above 10(9) pfu/mL obtained for the replication-competent HSV-1 viron.


Subject(s)
Centrifugation/instrumentation , Herpesvirus 1, Human/isolation & purification , Kidney/virology , Animals , Cell Separation/instrumentation , Cell Separation/methods , Cells, Cultured , Centrifugation/methods , Cricetinae , Equipment Design , Equipment Failure Analysis , Herpesvirus 1, Human/physiology , Humans , Kidney/physiology , Pilot Projects , Sensitivity and Specificity , Virus Cultivation/instrumentation , Virus Cultivation/methods , Virus Inactivation
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