Purification of Influenza A/H1N1 Using Capto Core 700

Extracted from Multimodal Chromatography (PDF), GE Healthcare, 2013

This example shows results from a process stream with the following steps: clarification using ULTA™ Prime GF microfiltration (MF), capture, and polishing using Capto Core 700.

Madin-Darby canine kidney (MDCK) cells (inoculation concentration of 500 000 cells/mL) were grown on Cytodex™ 3 microcarriers for 48 h in an Applikon™ Bioreactor (Applikon Biotechnology). The final cell density was approximately 2 500 000 cells/mL at which point cells were infected with influenza A/Solomon Islands/3/2006 (H1N1) and harvested at 72 h post infection.

After sample clarification by MF, the virus was captured, and eluted fractions from this step were applied to an XK column packed with Capto Core 700 for final purification (Fig 4.60). Because of the robust binding performance of Capto Core 700, equilibration of the medium was achieved using the buffer used for elution in the capture step. The need for buffer exchange or dilution between steps was thereby eliminated, contributing to speeding up the chromatography process. This demonstrates the advantages of the large window of operation that is enabled by Capto Core 700.

Table 4.25 shows the results in terms of hemagglutinin (HA, e.g., total virus titer) recovery, infectious virus titer (Tissue Culture Infectious Dose (TCID50), DNA and protein removal at each step of the process. In this case, good yield of virus HA as well as significant removal of HCP and DNA were observed. In the capture step, DNA was reduced 2.8 log and proteins 5- to 7-fold. Capto Core 700 further reduced protein levels by 3- to 5-fold. The infectivity of the virus was retained throughout the process, as indicated by the titer measured with TCID50 (data not shown).

Flow scheme of the purification process in which steps involving in-process filtration of the sample to reduce bioburden are indicated with asterisks (*). UF/DF = ultrafiltration/diafiltration.

Fig 4.60. Two-step purification of influenza A/H1N1 virus after MF. After capture of the virus, final purification was achieved using Capto Core 700.

Table 4.25. Virus HA yield, TCID50, DNA, total protein, and HCP/HA quotient in a purification scheme incorporating MF, DNA reduction step using Benzonase™ endonuclease, and final chromatography step using Capto Core 700

Step HA yield (%) Titer (TCID50/mL) DNA/HA (ng/μg) Total protein/HA (μg/μg) HCP/HA (μg/μg)
Microfiltration 64 9.7 2672 22.0 32.3
Chromatography – capture 94   4.0 3.1 6.1
Chromatography – polishing (Capto Core 700) 94 9.3 5.0 1.1 1.1

For more information on this example, see application note 29-0003-34, “Purification of influenza A/H1N1 using Capto Core 700.”

Materials

     
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