A Parallel Purification Strategy for Rapid Processing of Lentiviral Vector Suspension Feedstock Samples Sujeong Yang, Emma Burman, Joseph Fletcher, Beatriz Sant Mora, Daniella Steel, Ian Scanlon, Marc Hummersone Astrea Bioseparations Ltd, Horizon Park, Barton Road, Comberton, Cambridge, CB23 7AJ, UK
4
Figure 1 B
Minimal Affect of Optimized Clarification Step on Lentiviral Recovery
Figure 5 A
1
Adherent
Centrifugation at 300 x g, followed by filtration by 0.45um PES, no buffer exchange step
Suspension
Centrifugation at 1500 x g, followed by filtration by 0.45um PES then buffer exchanged with equilibrium buffer using 100kDa ultrafiltration (Condition 3).
Adherent and suspension feedstocks were processed according to the experimental plan in Figure 2. Purification was performed following the Nereus LentiHERO® NL100100 technical user guideline.
Figure 2
Harvest/Nuclease treatment
Adherent LV
A
Removal of impurities
HEK HCP
10
dsDNA µg
HCP µg
15 10
Figure 3
1
TU/ML (Crude Harvest 2.55E+07)
300
100
2
300
300
1.88E+07
3
1500
100
2.47E+07
4
1500
300
1.75E+07
5
4400
100
2.16E+07
6
4400
300
1.61E+07
Load
1.52E+07
Clarification was optimised by testing 6 conditions (Figure 3). Feed clarified by centrifugation at 1500 x g, followed by 100 kDa MWCO filtration (Condition #3) showed the highest TU titer, indicating minimal loss of functional LVV.
6 A
B
Total Time
Elution
Washing
Loading
20
25
Time (min) Elution
Equilibration
C
800
Load
0
Elution
M
1
2
3
98 62 49 38
4 2
3.2
kDA
28 0.67
Load
0.11
17
Elution
Total Time
6.0E+07
0.0E+00
2
3
B Transmission Electron Microscopy Images of LVV purified by LentiHERO®.
100 nm
28
LV-specific p24 around 24kDa molecular weight were identified in the eluted fractions (Figure 7A). TEM images revealed the typical healthy morphologies of LVVs from the eluates following LentiHERO® purification (Figure 7B).
p24
High Dynamic Binding Capacity of Nereus LentiHERO®
Figure 8 1
2
3
4
5
• Nereus LentiHERO® enables parallel purification of samples to scale out screening studies. • The high dynamic binding capacity of Nereus LentiHERO® demonstrates the significant potential of the composite nanofiber adsorbent at processing scale.
Dynamic binding capacity
60%
6
Condition
• Nereus LentiHERO® purification, used with an optimised clarification process, forms an efficient sample preparation workflow for suspension feedstock.
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LV specific p24 Gag protein was detected using rabbit polyclonal anti-p24 IgG by Western blot analysis.
38
7
Key Findings
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M:Marker, Lane 1: LV load Lane 2: Flow-through, Lane 3: LV Eluate
9.0E+07
Samples of suspension feedstock clarified from each of the above 6 conditions were purified using the Nereus LentiHERO® spin columns in a bench top centrifuge. This enabled 6 conditions to be prepared in less than 25 minutes (Figure 4A). Conditions 3 and 5 resulted in the highest TU recovery (Figure 4B).
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6
0
14
3.0E+07 15
8
1200
Elution
17
Total TU
Loading Washing
10
Load
Silver stained SDS-PAGE gel
LV p24 detection
1
49
1.2E+08
5
TU
Lentiviral Structure is Well Preserved
kDA
1.5E+08
Equilibration
0
Suspension LV
dsDNA
10
1388.6
1600
400
0
62
% Breakthrough (C/C0)
A
Adherent LV
HEK HCP
0.67
Elution
Figure 7
Infectious LV recovery Post clarification & LentiHERO® purification
Parallel operation time
0
Suspension feedstock post clarification contained high levels of HCP. Nereus LentiHERO® purification significantly reduced HCP and process related dsDNA contamination from both adherent and suspension feed (Figure 6A & B). HCP was significantly reduced, with barely detectable level of HCP in the eluate (Figure 6C).
LentiHERO® - A Highly Efficient Screening Tool
Figure 4
4 2
98
3
10
Host cell proteins and dsDNA were measured by HEK HCP ELISA or PicoGreen assay, respectively. n=4 /feed
Conditions for Clarification
Condition
20
HCP 99.7 ± 0.3% dsDNA 83.6 ± 0.8%
2000
6.82
6
1.3
0
Ultrafiltration MWCO
dsDNA
8
5
Centrifugation speed (x g)
30
Removal of impurities
24.6
25 20
2
40
Suspension LV
B
HCP 94.8 ± 0.7% dsDNA 90.1 ± 0.7%
30
LentiHERO® Purification
50.07
50
High HCP in Clarified Suspension Feed Removed by LentiHERO® Purification
Figure 6
Clarification Centrifuge/0.45 µm PES filtration Buffer exchange TFF or Ultrafiltration MWCO
Clarification Centrifuge/0.45 µm PES filtration
59.07
59.20
P24
Suspension Feedstock
Adherent Feedstock
63.33
60
Suspension feedstock processed with Condition 3 and processed adherent feedstock were purified with LentiHERO®. LVV recoveries were similar for both physical and infectious titers (Figure 5A & B). Range of values for both feedstocks: P24 recovery, 3-4E+10; TU recovery, 1-3E+8. n=4/feed.
5
Experimental Plan
LVV recovery
80 70
Clarification Conditions
While suspension culture systems are often used for large scale processing due to ease of scaling up and reduced costs, sample preparations can be lengthy due to the high levels of contaminants, such as host cell proteins (HCP). Therefore, we developed a process for LVV purification from suspension feedstock using a novel spin column, the Nereus LentiHERO® (Figure 1A). This device contains a functionalized high-flow, high-capacity composite nanofiber adsorbent, AstreAdept® (Figure 1B), previously shown to rapidly deliver high LVV yields from adherent production systems. After optimization of the suspension feedstock clarification step, we compared the performance of the Nereus LentiHERO® purification step from both types of LVV feedstocks.
B
Feedstock System
% recovery to Load
A
dsDNA µg
Bottlenecks caused by sample preparation during the development of lentiviral vector (LVV) therapeutic assets are common, both in upstream and downstream process development, and negatively impact pipeline milestones.
HCP µg
Abstract
FT 5
50% 40%
FT 3
FT 2
FT 4
30%
Fraction
Accumulated Volume (mL)
PP/mL
FT1
4
1.34E+08
6%
FT2
8
5.23E+08
23%
FT3
12
6.41E+08
28%
FT4
16
6.53E+08
28%
FT5
20
8.02E+08
35%
C/C0
20%
* Load concentration is 2.30E+09 PP/mL
FT 1
10% 0% 0
5
10
15
20
Table 1
Accumulated Volume (mL) DBC15%BT/Device is 1.5E+10 Single Nereus LentiHERO® unit loaded at 20mV/min, analysed by P24 ELISA to calculate % of physical LVVs break through. C0: titer of Load, C: titer of flow-through (FT).
P24 recovery
TU recovery
n=4
Load
Elution
Load
Adherent LV
6.52E+10
4.10E+10
4.84E+08
2.88E+08
Suspension LV
5.08E+10
2.56E+10
1.75E+08
9.73E+07
Elution
Nereus LentiHERO® purification of suspension feedstock processed with the optimised clarification conditions demonstrated high dynamic binding capacity (DBC, Figure 8), with recovery of physical particles (PP) and infectious particles (Table 1). When extrapolated to bioprocessing size this interpolates to a DBC of 1.25E+11 PP/mL, indicating Nereus LentiHERO® is a powerful purification tool for the development of downstream processing conditions.