Effective Process Development to Reduce Host Cell Proteins and Maximize Lentiviral Vector Yield with Innovative Nanofiber Adsorbent Sujeong Yang, Hanna Dreja, Caroline Daye, Emma Burman, Joe Fletcher, Adam Pinnock, Ben Wallis, Marc Hummersone, Ian Scanlon Astrea Bioseparations, Horizon Park, Barton Road, Comberton, Cambridge, CB23 7AJ, UK
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To perform bench-scale purification with innovative nanofiber adsorbent as a rapid screening tool to optimize clarification of suspension LV feed prior to LVV capture. To establish the LV purification workflow from benchscale to process development scale, incorporating nanofiber adsorbents that maximize the lentiviral recovery and impurities removal.
Novel nanofiber adsorbent with low shear flow path and high binding capacity for maximising LVV recovery Average mean flowpath diameter of 860 nm giving excellent flow characteristics for ATMP targets
Comparison of Infectious LVV titer post clarification Condition #1-3 LVV TU titer post clarification
Run detail
100%
1
Centrifugation, 0.45µm PES filtration
1.47E+08
1.5E+08
80%
2
Centrifugation, 0.45µm PES filtration, Diafiltration 100kDa, Buffer Ex 1xVOL
1.67E+08
3
Depth filtration CA
1.68E+08
1.2E+08
60%
9.0E+07 40%
6.0E+07
0.0E+00
The clarification steps overall gave less than 20% loss of infectious LVVs compared to the crude harvest. Condition 2 and 3 resulted in the most infectious LVVs (n=3).
0% Motherfeed Condition 1 Condition 2 Condition 3
TU/mL
5 Bed Volume 10 mL
Nereus LentiHERO® Spin column screening outcome from Condition #1-3 Infectious LVV recovery
3.0E+08
1
• Centrifugation and 0.45 µm PES filtration • Depth filtration (CA)
30%
20% 15% 1.0E+08
Centrifugation, 0.45µm PES filtration
Centrifugation, 0.45µm PES filtration
Depth filtration
• Condition 3: Depthfiltration (CA)
0.0E+00
0% Condition1
Condition2
0 HCP removal
Condition3 Recovery
Condition1
Nereus LentiHERO® bench scale purification
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Infectious LVV recovery 3.00E+07
100%
2.50E+07
80% 70%
2.00E+07
60% 40%
45%
50%
41%
1.50E+07
40% 30%
1.00E+07
Recovery (%)
80% Total TU
Recovery (%)
60%
20%
20%
5.00E+06
0% TU recovery
HCP
dsDNA
10%
0.00E+00
3x VOL
TU
Buffer to the feed volume
1x VOL 2x VOL 3x VOL
80
1500
60
1000
40
500 0
LV production: HEK 293T, Suspension cells TU assay: Jurkat cell, Flow cytometry GFP+ HCP: HEK HCP ELISA dsDNA: PicoGreen assay
0%
20 0
50
100
150
Chrom.1 UV mAU
METHOD
70%
120 100
2000
Post Nereus LentiHERO® purification
120%
2x VOL
140
% Recovery from the load
Infectious LVV and impurities level in the feed post buffer exchange
160
3000 2500
UV (mAU)
Optimization of buffer exchange volume for ultrafiltration
Viral genome recovery
3500
Conductivity (ms/cm)
Suspension LV load
1x VOL
Condition3
Process Development Scale Purification: Processing clarified suspension LV feedstock with LentiHERO® 1 capsule
LentiHERO® 1 purification
Before Buffer Ex
dsDNA removal Condition2
Buffer exchange* TFF or Ultrafiltration *buffer exchanged with EQ buffer, 0.02M Tris, 0.02M MgCl2, 0.1 M NaCl, pH7
2
Most HCPs were removed by the purification step post clarification. Condition 2 gave a high recovery yield with an efficient removal of dsDNA.
40
20
5%
Total TU
• Ultrafiltration with 100kDa or 300kDa MWCO • TFF (*cross flow filtration 100kDa)
60
10%
5.0E+07
Clarification with a buffer exchange
• Condition 2: CF at 1500 x g, TFF 100kDa with EQ buffer change
80 25%
1.5E+08
Fresh LV harvest
100
29%
24% 2.0E+08 Total TU
Primary clarification
Conditions 2 and 3 gave the highest recovery of TU titer after Nereus LentiHERO purification. n=2
Post clarification & Nereus LentiHERO® purification
35%
31%
2.5E+08
Study Overview: LVV purification workflow using nanofiber adsorbents at bench and PD scales
RESULT
Impurities removal
Post clarification & Nereus LentiHERO® purification
Removal %
Bed Volume 1 mL
Recovery (%)
Recovery %
Bench-scale
(Crude Harvest 1.75E+08)
RESULT
20%
3.0E+07
Nanofiber adsorbents in spin column and capsule formats for bench-scale and PD scale processing
TU/ML
Condition
1.8E+08
Recovery (%)
Aim
The significant presence of host cell proteins in suspension lentiviral (LV) feedstock highlights the need to optimize clarification in order to maximize downstream processing lentiviral vector (LVV) recovery and reduction of impurities. Effective process development tools are required to rapidly purify LVV while minimising LVV loss and damage.
TU/mL
Challenge
0
200
Chrom.1 Cond mS/cm
100
METHOD
100
• HEK 293 T, Suspension cells
69.38
80
LentiHERO® 1 capsule
60
• 200 mL of harvest clarified centrifugation and 0.45 µm filtration
40 20
• Diafiltration through 100 kDa filter
0
Load
Elution
• Concentration to <150 mL to enable loading via superloop
Result: Functional LVV recovery was 53% (measured by GFP integration into Jurkat cells).
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High recovery and high purity with optimized workflow using LentiHERO® 1 Process Development scaled Capsule
Recovery
Result: 1× volume (VOL) buffer exchange to feed gave the highest infectious titer, with >40% removal of HCPs and
LOAD Physical Particles*/mL adsorbent
LOAD TU** /mL adsorbent
RECOVERY TU** (600 mM NaCl)
Adherent LV 242 mL load
4.5E+11
4.7E+09
2.6E+09 (54%)
Suspension LV 200 mL harvest load
1.75E+12
2.5E+09
LV
dsDNA. These feeds were submitted for the purification step, performed with Nereus LentiHERO®. 1× VOL to the feed resulted in the highest infectious titer, with 70% TU recovery to the load and was adopted for the UF/DF step.
1.3E+09 (53%)
Impurities level from LVV post LentiHERO® 1 purification Adherent LV feed
3
100%
Optimization of Centrifugation and Ultrafiltration
The effects of centrifugation speeds and MWCO UF filter on fresh harvest were examined. Conditions for clarification Condition 1
TU/ML
RESULT
(Crude Harvest 2.55E+07)
300
100
2
300
300
1.88E+07
3
1500
100
2.47E+07
1.52E+07
4
1500
300
1.75E+07
5
4400
100
2.16E+07
6
4400
300
1.61E+07
Infectious LVV recovery
Post clarification & Nereus LentiHERO® purification
1.5E+08 1.2E+08
Total TU
Equilibration Loading Washing Elution
9.0E+07 6.0E+07 3.0E+07
0
5
10
15
20
25
Time (min) Elution
Washing
Loading
Equilibration
0.0E+00
1
Total time
2
3
Any data or results provided are only examples and do not provide any guarantee of similar results in future. The products of Astrea Bioseparations may be covered by or for use under one or more patents: astreabioseparations.com/patents All trademarks, trade names, trade dress, product names and logos are the property of Astrea UK Services Ltd.
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Condition
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100%
HEK HCP
hcDNA
100% 77%
80% 70% 60% 50%
39%
30%
Ultrafiltration MWCO
Parallel operation time
100%
90%
40%
Centrifugation speed (x g)
Total time
100%
Suspension LV feed
5
6
• Minimizing processing time is essential to maintain infectivity potential, and Nereus LentiHERO® enabled six conditions to be screened within 25 minutes. • UF gave optimal conditions when considering the final recovery under low shear conditions. • Condition 3 was adopted as the clarification step in subsequent workflow with a LentiHERO® 1 chromatography capsule.
20%
18%
15%
10%
3%
2%
0%
Harvest
Elution
Harvest
Post TFF
Elution
* Zeptometrix P24 ELISA ** Jurkat transduction
Key Findings • Nereus LentiHERO® purification forms an efficient sample preparation workflow for suspension feedstock including clarification methods. • Minimizing processing time is essential to maintain infectivity potential of LVVs, and Nereus LentiHERO® enabled parallel purification of samples within 25 minutes. • The high DBC of LentiHERO® 1 PD scaled capsule demonstrates the significant potential of this innovative adsorbent for bioprocessing at scale. • LentiHERO® 1 purification achieved the high purity LVVs with >98% removal of HCPs and >85% removal of dsDNA from the harvest.