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Influenza virus purification

IP.com Disclosure Number: IPCOM000244184D
Publication Date: 2015-Nov-20
Document File: 2 page(s) / 18K

Publishing Venue

The IP.com Prior Art Database

Abstract

During cell cultivation of virus large amounts of DNA and HCP Host Cell Proteins are present in the harvest and need to be removed The most common way of chromatographic removal of DNA is by using Capto Q and protein may be removed with Capto Core 700 In this idea the purifications are done simultaneously Instead of an inactive lid Capto Core 700 is provided with Q ligands on the outer surface

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Title: Influenza virus purification

Summary and Background

During cell cultivation of virus large amounts of DNA and HCP (Host Cell Proteins) are present in the harvest and need to be removed. The most common way of chromatographic removal of DNA is by using Capto Q, and protein may be removed with Capto Core 700. In this idea the purifications are done simultaneously. Instead of an inactive lid, Capto Core 700 is provided with Q-ligands on the outer surface.

Materials

Hollow fiber 500C 4X2MA

Microfilter, 2 µm och 0.6 µm (capsules)

Capto Core 700

Capto Q

Capto Core 700 with Q-ligands in the lid, two different levels of Q (LS-007959A (high) and B (low))

Equipment

Peristaltisk pump for filtration

ÄKTA10, PB_E10 for chromatography

Experimental

Influenza virus is cultivated in Vero cells in a Wave 25 bioreactor which yields about 6 L.

The microfilters are wet with water and then the harvest is run through. The sticky bottom residue is left and 5.3 L is filtrated. The hollow fiber is also rinsed with water (to rinse away the base in which it was stored). When pH is fine we concentrate to about 200 mL + death volume. 2 L 50 mM Tris, pH 7.5 + 0.5 M NaCl is used for diafiltration. When the DF is ready we have about 150 mL in the beaker and after pumping the filter dry we have 256 mL which gives a concentration factor of 20.6x.

Chromatografic method according to table 1:

Step

Volume

(CV)

Flow

(cm/h)

Buffer

Equilibration

7

300

50 mM Tris, pH 7.5 + 0.5 M NaCl

Sample load

7

100

Influenza

Wash

 

300

50 mM Tris, pH 7.5 + 0.5 M NaCl

CIP

5 + 15 min pause + 5

100...