Composite nanofiber chromatography membrane for fast LV purification: Comparison with contemporary methods Daniella Steel, Emma Burman, Ian Scanlon, Joseph Fletcher, Adam Pinnock, Ben Wallis, Marc Hummersone, Sujeong Yang. Astrea Bioseparations Ltd, Horizon Park, Barton Road, Comberton, Cambridge, CB23 7AJ, UK
3
1
A
Method
LentiHERO® is a weak anion exchange composite nanofiber membrane housed in a spin column device. Wash, load and elution steps were performed using a benchtop centrifuge at 1000 x g for 2 minutes. Product A is a bead-based adsorbent, where the process steps were gravity fed. Product B is a strong anion exchange membrane adsorbent, where process steps were performed with a syringe. Each device was loaded with unconcentrated, GFP-packaged lentiviral feedstock (LV-sffv-GFP). The feedstock was generated in adherent HEK293T cells in DMEM with 2% FBS, and had been clarified through 0.45 µm PES filtration prior to loading. The final concentration and buffer exchange of the eluates from each product was performed with an ultrafiltration device, 100kDa MWCO using OptiMem™ reduced serum medium. These concentrated LV samples were analysed for LV titers (physical titer, infectivity titer, viral genome copies titer) and contaminant levels of host cell proteins and total residual DNA.
Centrifugation
16
2
Product A
Bead-based resin
Syringe/Gravity
42
Product B
AEX membrane
Syringe/Gravity
45
Washing Elution *Volume(mL) 2
15
10
12
5
20
5
2
NaCl
2
0.6M
5
3
1.2M
15
4
1.2M
* Not including diluents or buffer exchange volume
Table 1. The operation time of LentiHERO® and Product A were measured by the same operator while Product B operation time was referenced as written in the manufacture’s protocol. All 3 methods had recommendations in common to further process the eluate with buffer exchange and concentration, therefore these additional steps were excluded from the operation time. Furthermore, the LentiHERO® eluate volume was 50% smaller and the eluate salt concentrations were lower compared to the alternative methods. Figure 1.
Operation Time
Product A
0%
Product B
LentiHERO®
Product A
Product B
60% 40% 20% 0%
LentiHERO®
Product A
Product B
Total Infectious LV recovery (TU) Product A
Product B
2.16E+09
*1.46E+09
2.16E+09
Elution
1.3E+09
5.55E+08
1.1E+09 * Reached the maximum load
Table 2. Total Infectious LV recovery (TU/mL) of the load and eluate after purification with LentiHERO®, Product A and Product B. Product A load was restricted by the capacity advised by the product manual.
LentiHERO® process removed host cell proteins and Total Residual DNA contaminants LentiHERO® showed higher than 95% removal of HCP and total residual DNA from the load challenge Contaminant
Product
80%
Host Cell Protein
Total Residual DNA
LentiHERO
97.6 ± 0.12%
95.0 ± 1.99%
Product A
99.8 ± 0.01%
98.7 ± 0.9%
Product B
97.1 ± 0.2%
95.5 ± 2.42%
®
60%
40%
20%
0%
Host cell proteins LV input
LentiHERO®
dsDNA Product A
Product B
Figure 3. HEK293 host cell protein (HCP) levels were measured by HEK HCP ELISA, and total dsDNA quantified by PicoGreen assay.
Summary
50 45
Total LV particle recovery, vector genome recovery and infectious recovery were significantly improved using the LentiHERO® spin columns without compromising on the purity afforded by the resin or the membrane adsorbents, as measured by reduction in host cell proteins and total residual DNA. Using the LentiHERO® spin columns resulted in approximately 60% reduction in processing time.
40 35
Time (min)
LentiHERO®
HCP and total DNA in the eluates
Elution
10
0%
20%
80%
Load
HCP and dsDNA recovery (%)
AEX membrane
20%
40%
Table. 3
Operation time (min)
LentiHERO®
40%
60%
100%
Equipment Loading
80%
60%
Vector genome recovery
LentiHERO®
LentiHERO® LV purification system is 60% faster than alternative methods
Total Time Equilibration
80%
C 100%
Table. 2
Operational process time can be significantly reduced when using the LentiHERO® protocol
Platform
LV particle recovery 100%
Product
4
Table 1.
B
Figure 2. LV recovery yield of the concentrated eluates was measured by infectious LV recovery (A), LV particle recovery (B) and vector genome recovery (C). Infectious LV (TU/mL) was tested by transduction assay in Jurkat cells measured by flow cytometry. Optimal infection range was 1-20% GFP positive cells, and only those data sets were used for calculation. LV particle recovery (VP/mL) was measured by P24 ELISA (ZeptoMetrix kit). LV viral vector genome was quantified by qPCR targeting Psi sequence. The graph represented the relative percentage (%) of recovery against LV load following a mass balance. N=3/group.
Each purification was performed using either LentiHERO®, Product A or Product B, following the manufacturer’s instructions.
2
Infectious LV recovery 100%
Viral genome recovery (%)
Here, we demonstrate the LV recovery and purification of a novel spin column, Nereus LentiHERO®. This is an electrospun composite nanofiber membrane, functionalized with a weak anion exchange chemistry. The performance of Nereus LentiHERO® was compared to two commonly used lab-scale chromatography methods; a gravity fed bead-based adsorbent, and an anion exchange membrane adsorber operated in accordance with the supplier’s instructions. As processing speed can also be a concern, operation times were also examined.
Increased LV recovery yield purified by LentiHERO®
Figure 2.
P24 recovery (%)
The development of therapeutic assets that use lentiviral vectors (LV), including ex-vivo and in vivo gene therapy, is growing rapidly. To keep pace with this increasing demand for LV, the bottlenecks regarding low LV recovery and poor contamination removal must be addressed. Purification steps with chromatographybased methods often have poor step yield recovery, and so during lab-scale development purity is often compromised to reduce LV loss during feedstock processing. As a consequence, therapeutic candidates may not be representative of the therapeutic assets during downstream processing stages where chromatography is used at larger scale bioprocessing.
Transduction Unit recovery (%)
Introduction
30
In summary, the composite nanofiber membrane LentiHERO® spin column provides increased yield and faster LV processing, without compromising on purity at lab-scale. The use of chromatographical tools during lab-scale vector development will enable candidates that are more representative of largescale manufacturing to progress through the development pipeline faster.
25 20 15 10 5 0
Product A
LentiHERO® Equilibration
Loading
Washing
Product B Elution
Figure 1. represents the cumulative operation time of the three methods, as outlined in Table 1. The LentiHERO® process is 64% faster than Product B, and 62% faster that Product A.
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