purification of viruses

PurificationPurification

To obtain the viral particles in a single form out of cell debris i.e. removal of all impurities.

Purification outlinePurification outline

1.Preparation of harvest.2.Methods of purification.3.Criteria of purity.4.Maintenance of purified

virus.

Preparation of harvest

Bacteria

Plant

Animal Insect

Methods of purificationMethods of purification

1.Filtration.2.Centrifugation.3.Adsorption.4.Precipitation.5.Solvent extraction.6.Other methods.

Methods of purificationMethods of purification

1.Filtration.2.Centrifugation.3.Adsorption.4.Precipitation.5.Solvent extraction.6.Other methods.

Filtration: :

1.1. UltrafiltrationUltrafiltration 2. Gel filtration2. Gel filtration

Ultra-filtrationUltra-filtration

Gel filtrationGel filtration

Methods of purificationMethods of purification

1.Filtration.2.Centrifugation.3.Adsorption.4.Precipitation.5.Solvent extraction.6.Other methods.

centrifugation

Differential

Gradient

Isopycnic Cushion

Methods of purificationMethods of purification

1.Filtration.2.Centrifugation.3.Adsorption.4.Precipitation.5.Solvent extraction.6.Other methods.

Adsorption: :

1.1. Ion exchangeIon exchange 2. Heamadsroption2. Heamadsroption

Ion exchange chromatography

HeamadsorptionHeamadsorption

Methods of purificationMethods of purification

1.Filtration.2.Centrifugation.3.Adsorption.4.Precipitation.5.Solvent extraction.6.Other methods.

Precipitation: :

1. Chemical1. Chemical• AmmoniumsufateAmmoniumsufate• PEGPEG• Zinc acetaeZinc acetae

2. Physical2. Physical• Isoelectric piont

(pH change)

Methods of purificationMethods of purification

1.Filtration.2.Centrifugation.3.Adsorption.4.Precipitation.5.Solvent extraction.6.Other methods.

Methods of purificationMethods of purification

1.Filtration.2.Centrifugation.3.Adsorption.4.Precipitation.5.Solvent extraction.6.Other methods.

Other methodsOther methods

• Electrophoresis.• Serological methods.• Enzymatic reactions.• HPLC

Electrophoresis

HPLCHPLC

Criteria of purityCriteria of purity

Chemical purityChemical purity

Physical purityPhysical purity

Biological purityBiological purity

Criteria of purityCriteria of purity

SpectrophotomeSpectrophotometerter

IsoradiationIsoradiationInfectivity Infectivity

serologicalserological

E.M.E.M.

Criteria of purityCriteria of purity

SpectrophotomeSpectrophotometerter

IsoradiationIsoradiationInfectivity Infectivity

serologicalserological

E.M.E.M.

MarkingMarking

Criteria of purityCriteria of purity

SpectrophotomeSpectrophotometerter

IsoradiationIsoradiationInfectivity Infectivity

serologicalserological

E.M.E.M.

Infectivity::

1. Local lesion assay1. Local lesion assay 2. Plaque assay2. Plaque assay

y = 1.9608x

020406080

100120140160180

0 2 4 6 8 10Conc.(mg/ml)

No. o

f loc

al le

sion

s

Virus preparationVirus preparation

y = 1.9608x

020406080

100120140160180

0 2 4 6 8 10Conc.(mg/ml)

No. o

f Pla

quesث

Criteria of purityCriteria of purity

SpectrophotomeSpectrophotometerter

IsoradiationIsoradiationInfectivity Infectivity

serologicalserological

E.M.E.M.

Criteria of purityCriteria of purity

SpectrophotomeSpectrophotometerter

IsoradiationIsoradiationInfectivity Infectivity

serologicalserological

E.M.E.M.

44 - -MaintenanceMaintenance::

Lyophlization

The half life of infectivity can be measured in seconds at The half life of infectivity can be measured in seconds at 60 60 ooC; minutes at 37 C; minutes at 37 ooC, hours at 20C, hours at 20ooC , days at 4 C , days at 4 ooC and C and years at -70 years at -70 ooC or lower,C or lower, (Fenner (Fenner et alet al, 1987), 1987)