Advancing purification strategies for large viral modalities using nanofiber adsorbent technology S. Yang, J. Corbiere, C. Daye, B. Sant Mora, J. Fletcher, M. Hummersone, and I. Scanlon Astrea Bioseparations, Horizon Park, Barton Road, Comberton, Cambridge, CB23 7AJ, UK
TU recovery % TU recovery %
Pseudotyped LVV production and purification workflow
155-240nm
360x270x250nm
200
300 nm
• Transient transfection with four plasmids
Centrifugation 1500 x g, 5 min
• DNA 2 µg/VCD
• Reagent: DNA; 2:1 to 3:1
4040
40.8% 40.8%
0.45 µm PES microfiltration
97% 97%
8080
88% 88%
00
6060 4040
LentiHERO® demonstrates very high dynamic binding capacity
110
Loading study
Breakthrough (%)
100 90
Total load 5E+9 TU/mL of adsorbent
80 70 60
LentiHERO®
Optimized Optimized
DNA DNA removal removal(%) (%)
40
Breakthrough CV of load
30 20 10 0
0
100
200
300
Optimized Optimized
400
500
600
Breakthrough TU/mL of adsorbent
3E+9
LH P24
Q membrane P24
LH TU
Q membrane TU
LentiHERO® processed large feed volumes before breakthrough of functional and physical LVV particles
GalV LV
Load volume (mL)
190
300
TU in load
2.1E+07
1.2E+08
1.8E+06
2.01E+06
>2.1E+7*
4.5E+07
Processing with LentiHERO® greatly reduces volumes for formulation at manufacturing scale Estimation of final elution pool volume at manufacturing scales
Clarified LVV harvest volume
Device
Adsorbent volume
Elution volume
Elution volume post-dilution
LentiHERO®
100 mL
1 L (10 CV)
1 L (Nil)
450 mL
2.25 L (5 CV)
Commercial Q membrane
LentiHERO®
1L
10 L (10 CV)
11.25 (5x)
15
25 L (5 CV)
100 80
2.5E+07 2.0E+07
60
36%
1.5E+07
140
0.0E+00
0
4070A
Q Membrane
120
Infectious LVV
100 80
12.5 fold reduction
60 LentiHERO® 0.6 M NaCl elution
40
40 20
5.0E+06
20
LentiHERO® 0.8 M arginine elution
125 L (5x)
0
50
100
150
200
50% 40% 30%
40
9%
10%
3%
2%
0%
0 34
12%
20%
80% 60%
97%
56%
20% 0%
46
Infectious HSV Non-bound
%B
85%
40%
0.3 M NaCl EL
0.6 M NaCl EL
Infectious HSV 1.2 M NaCl EL
dsDNA
Proteins
Strip
Infectious HSV recovery yield was 56%, 9.2E+9 IU/mL adsorbent Most infectious HSV eluted at 0.6 M NaCl
8
80
98.7
100
99.4
87
40 20 0
GalV
HSV purification profile on LentiHERO® using pBACYAC-HSV-1(KOS)
Load volume 20 mL to a screening device measured on average 3E+8 IU/mL adsorbent.
60
4070A
Recovery %
Protocol
Equilibration
Load
Elution
Standard
20 mM Tris, 20 mM MgCl2, pH 7.2
Clarified harvest
20 mM Tris, 20 mM MgCl2, 1.5 M NaCl, pH 7.2
Arginine
20 mM Tris, 20 mM MgCl2, 50 mM arginine, 10% sucrose, pH 7.2
20 mM Tris, 20 mM MgCl2, 800 mM arginine, 10% sucrose, pH 7.2
Clarified harvest + 50 mM arginine, 10% sucrose
GalV HCP removal
dsDNA removal
2.4E+08
• Robust impurity reduction: >98% host cell proteins and >87% residual dsDNA without compromising vector functionality
69%
2.0E+08
100% 80% 60%
1.2E+08
50% 40%
8.0E+07
1.4E+08
7.9E+07
30% 20%
16%
4.0E+07
A: The virion showing characteristic icosahedral morphology; contrast highlights capsid and tegument features.
80% 60%
99% 40%
76%
98% 20 nm
73% B
20%
10%
0.0E+00
0%
Total infectious HSV
A
100%
90% 70%
1.6E+08
TEM images of HSV-1 post LentiHERO®
Proteins and dsDNA clearance
HSV recovery yields, n=2
• Efficient purification of non-VSV-G lentiviral vectors (4070A: 100%, GalV: 36%) achieved
0%
Arginine
Standard
% Recovery
dsDNA removal
Arginine
B: Intact, spherical HSV-1 particles with envelope and underlying capsid visible. The outer lipid envelope appears as a dark peripheral ring due to uranyl acetate staining. Scale bar = 20 nm.
HCP removal 20 nm
6
Upstream-downstream workflow of oncolytic HSV-1
• Infectious HSV recovery achieved 69%, ~1.4E+8 IU/mL adsorbent by 0.8 M arginine elution with high impurity clearance
Bioreactor volume of LVV (L) assuming 1E+7 TU/mL
1
HSV-1 (KOS) infection to Vero cells and monitor cytopathic effect (CPE)
2
Harvest the virus after 3 days (supernatant + cell lysate)
4
Virus purification via LentiHERO® nanofiber adsorbent
5
Virus titration: TCID50 assay dsDNA: PicoGreen assay HCP: microBradford assay
3
Clarification and 0.45 µm PES filtration post-nuclease treatment
Sample 2 replicates
Increasing virus dilution
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30
44%
60%
Volume (mL)
Infectious unit , IU/mL ≅ 0.69 x 1/ TCID50
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45
4.51E+07
Sample 1 replicates
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60
300
0
TCID50 assay on 96-well plate
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75 450
100
120
100%
0.3 M + 0.6 M NaCl elution fractions 100%
70%
Total proteins and dsDNA clearance 140
1.0E+07
Elution pool volume post-dilution
0
5L
3.0E+07
Infectious HSV recovery, n=4 80% 90
Standard
10 L (Nil)
200 L Commercial Q membrane
Total TU yield
4
• Arginine elution eliminates the need for post-elution dilution, simplifying the process • Low salt or arginine elution minimizes buffer consumption and reduces processing volume
Any data or results provided are only examples and do not provide any guarantee of similar results in future.
Chromatogram with stepwise NaCl elution
150
TU in non-bound
4.0E+07
9E+8
*Normalized for the initial TU breakthrough
4070A LV
Infectious LVV recovery
82% lentiviral recovery with yields > 2E+9 TU/mL of adsorbent and > 90% dsDNA/HCP clearance
175*
CV
Strip 20 mM Tris, 20 mM MgCl2, 2 M NaCl, pH 7.2
*The final LVV yield surpassed the loaded LVV quantity, a phenomenon verified through two independent measurements
50 L 500*
Stepwise NaCl elution 20 mM Tris, 20 mM MgCl2, 0.3 M, 0.6 M, 1.2 M NaCl, pH 7.2
HCP HCPremoval removal(%) (%) Standard Standard
Commercial Q membrane
50
Run
TU in eluate
Elution pool (L)
Breakthrough of TU and P24 on LentiHERO® (LH) and Q membrane devices 120
Load Clarified harvest
28
00
Standard Standard
• Physical LVV: P24 ELISA • TU assay: Jurkat cells, GFP+ Flow cytometry • HCPs: HEK HCP ELISA • dsDNA: PicoGreen
Higher binding capacity of VSV-G LVV particles using LentiHERO® nanofiber adsorbent
HSCs, T cells, neurons
B. Infectious LVVs (TU) recovery yields by LentiHERO®1
3.5E+07
2
HSCs, T cells, neurons
Equilibration 20 mM Tris, 20 mM MgCl2, pH 7.2
mAU
Analytics
*Note: This schematic representation highlighting the approximate size relationships between viral structures. Fine structural details are omitted for clarity regarding size.
97% 97%
90% 90%
• FLOW RATE: 5 mv/min
• Nuclease treatment 50 U/mL, 1 hr, 37°C • Harvest post 48 hrs transfection
Early retroviral trials
GalV
Protocol Standard
600
2020
• ELUTION BUFFER: 0.02 M Tris, 0.6 M NaCl, 0.02 M MgCl2, pH 7
• Transfection: PEIpro LV transfection kit
Herpes simplex virus Vaccinia virus
Broad (many cell types) Broad (human/primate)
Infectious HSV/mL adsorbent
100
80-120nm
CAR-T, iPSC, in vivo delivery
VSV-G 4070A
Recovery %
Lentivirus
Clinical Use
100 100
82% 82%
6060
2020
• EQ BUFFER: 0.02 M Tris, 0.02 M MgCl2, pH 7.2
• HEK293, suspension cells
0
Impurity removal after arginine elution
Tropism
120 120
Downstream process
• VCD 2 or 3E+6 cells/mL
Pseudotype
HSV purification profile on LentiHERO® using wt HSV-1(KOS)
Load volume 15 mL to a screening device measured 1.5E+10 IU/mL adsorbent.
A. Lentiviral pseudotypes in cell and gene therapy
Impurities removal %
Clarification
LVV Production
20-25nm
20 mM Tris, 20 mM MgCl2, 800 mM arginine, pH 7.2
8080
Upstream process
AAV
20 mM Tris, 20 mM MgCl2, 0.6 M NaCl, pH 7.2
Clarified harvest + 50 mM arginine
Impurities removal % Impurities removal %
• Investigate scalability potential for industrial implementation of nanofiber-based purification
Larger viral vectors for gene therapy: LVV, HSV, and VACV
Clarified harvest
20 mM Tris, 20 mM MgCl2, 50 mM arginine, pH 7.2
100 100
• Assess host cell driven proteins and dsDNA removal to determine impurity clearance capability
1
20 mM Tris, 20 mM MgCl2, pH 7.2
LentiHERO® functional LVV recovery
• Evaluate purification performance of a novel nanofiber adsorbent for LVV with different pseudotyped envelopes (VSV-G, 4070A, GalV) and oncolytic virus such as HSV
7
Non-VSV-G pseudotyped LVV purification by LentiHERO®1
% Recovery or removal
Aims of study
Elution
% Recovery
Standard Optimized
5
Lentiviruses can be pseudotyped with different envelope glycoproteins to enhance transduction efficiency and specific tropism. This impacts downstream purification strategies.
Load
Impurities removal %
Equilibration
% Buffer B (2 M NaCl)
Traditional resin-based chromatography approaches face limitations in scalability, yield, and host cell protein (HCP) clearance. To address these bottlenecks, this study investigates a novel nanofiber-based adsorbent as an alternative purification strategy. This innovative platform offers promising advantages in binding capacity, impurity clearance, and process throughput, potentially transforming the downstream processing of large viral vectors (Box 1).
Protocol
% Recovery
Maximizing the LVV recovery yield with high purity via optimized protocol
Oncolytic viruses, like herpes simplex virus (HSV) and vaccinia virus (VACV), are gaining traction in cancer therapy due to their selective replication in tumor cells, capacity to induce cell lysis, and ability to trigger anti-tumor immune responses. Similarly, pseudotyped lentiviral vectors (LVV), such as GalV and VSV-G, enhance gene therapy by enabling cell-type-specific transduction. This can can impact downstream purification strategies.
mAU (UV280)
3
Introduction
CPE
Conclusions • LentiHERO® weak AEX nanofiber effectively purifies a range of viral vectors, including: - Pseudotyped lentiviruses (4070A, VSV-G, GalV) - Oncolytic virus HSV-1 • High recovery yields achieved: - 4070A: 100%, VSV-G: 82%, GalV: 36%, HSV-1: 69%
• Superior host cell proteins clearance: >97% host cell protein removal • Scalable and versatile platform suitable for industrial applications • Potential expansion to other viral vectors like Ad5, Vaccinia, and VLPs • Supports processing intensification for gene therapy and oncolytic virus manufacturing