A HIV E A M I T P Q /P - Global HIV Vaccine Enterprise. Gowetski.pdf · Immunoassay 2 +++ V3...
Transcript of A HIV E A M I T P Q /P - Global HIV Vaccine Enterprise. Gowetski.pdf · Immunoassay 2 +++ V3...
ADVANCES IN HIV ENV ANTIGEN MANUFACTURING TOIMPROVE TIMELINES AND PRODUCT QUALITY/PRODUCTIVITY
Daniel Gowetski, Ph.D.Vaccine Production Program/VRC/NIAID
ENV Manufacturing WorkshopJuly 20th, 2017
Development Cycle at the VRCResearch: NIH Main Campus
Clinical Trials:NIH Clinical Center
Process Development:Vaccine Production Program
Manufacturing: Vaccine Clinical Material Production (VCMP)
Q1 Q2 Q3 Q4 Q1 Q2 Q3 Q4 Q1 Q2 Q3 Q4
2016 2017 2018
Process Development
Representative VPP Project Progression
Phase 1 Start
Research
Tech Transfer/Manufacturing
Regulatory
VRCBuilding 40CandidateResearch
VRCVPPLPro cessDevelopm ent
VCMPGMPManufacturing
VRCClinicalT rial
FDAIN D Filing
IndustryL icensee
Q1 Q2 Q3 Q4
2019
TBD
Development: From Research
VRCBuilding 40CandidateResearch
VRCVPPLPro cessDevelopm ent
VCMPGMPManufacturing
VRCClinicalT rial
FDAIN D Filing
IndustryL icensee
Trimer
S200 Gel Filtration – Load: VRC01 elution
Purification Procedure Protein Yield (from 5 L of 293FS cells)
VRC01 18 mg / 5 L
S-200 (Trimer) 11 mg / 5 L
447-52D (Trimer) 8.9 mg / 5 L
V3 cocktail (Trimer) 8.7 mg / 5 L
Protein Yield
Agg1
Agg2
Agg1: Aggregate peak 1Agg2: Aggregate peak 2Trimer: Trimer peak
4 0 5 0 6 0 7 0 8 00
6 0 0
1 2 0 0
1 8 0 0
2 4 0 0
V o lu m e (m L )
A28
0
(Kwon YW, et. al. Nat Struct Mol Biol. 2015 Jul; 22(7): 522–531)
1. Transient expression, VRC01 immuno-affinity capture
2. Concentrate 10-15-fold à Superdex 200 SEC
3. 447-52D (-) selection column
4. V3 cocktail (-) selection
5. Concentrate to 1.25 mg/ml, glycerol 10% (v/v) spike
(Cocktail = 1006-15D, 2219, 2557, 2558, 3074 and 50.1)
Production of pre-clinical product
Production of pre-clinical product
1. Transient expression, VRC01 immuno-affinity capture
2. Concentrate 10-15-fold à Superdex 200 SEC
3. 447-52D (-) selection column
4. V3 cocktail (-) selection
5. Concentrate to 1.25 mg/ml, glycerol 10% (v/v) spike
VRCBuilding 40CandidateResearch
VRCVPPLPro cessDevelopm ent
VCMPGMPManufacturing
VRCClinicalT rial
FDAIN D Filing
IndustryL icensee
Production of clinical product(wish list for ENV?)
1. Productive stable cell line
2. Use conventional/commercial resins
3. Validated viral clearance
4. Robust analytical panel for product release
5. Formulation for long term stability
VRCBuilding 40CandidateResearch
VRCVPPLPro cessDevelopm ent
VCMPGMPManufacturing
VRCClinicalT rial
FDAIN D Filing
IndustryL icensee
Development: From Research to GMP
(Cocktail = 1006-15D, 2219, 2557, 2558, 3074 and 50.1)
(Kwon YW, et. al. Nat Struct Mol Biol. 2015 Jul; 22(7): 522–531)
Acceleration Requires Parallel DevelopmentCell Line/Clone Selection
Upstream Dev
Downstream Dev
Formulation Dev
Analytical Dev
Q1 Q2 Q3 Q4 Q1 Q2 Q3 Q4 Q1 Q2 Q3 Q4
2016 2017 2018
Q1 Q2 Q3 Q4
2019
Acceleration Requires Parallel Development
Robotics, Robotics,& more Robotics
High-throughput Analytics + = Rapid & Parallel
Development
Cell Line/Clone Selection
Upstream Dev
Downstream Dev
Formulation Dev
Analytical Dev
Tech Transfer/Mfg
Q1 Q2 Q3 Q4 Q1 Q2 Q3 Q4 Q1 Q2 Q3 Q4
2016 2017 2018
Q1 Q2 Q3 Q4
2019
What can the VPP leverage to enable this?
How to Define the End of the Process?
Target Attribute Target Meaning Characterization Assay Priority High-Throughput?
GP41/GP120 Cleavage 100% Cleavage R/NR GXII or PAGE 1 +
Pep Map/In Tact mass Confirm Sequence RP-LC MS 1 –
Glycan Shield N332 = 100%, high Mannose Glycan Occupancy LC MS 1 – –
CD4i Induced Conformational Change
No conformational change in presence of CD4. Immunoassay 2 +++
V3 Negative No/Low available V3 Immunoassay 2 +++
Glycan N332 High Mannose Immunoassay 3 +++
SEC Single Population SEC 4 –
Overall Structure Population Distribution Neg Stain EM 5 – – –
A robust panel of critical quality attributes (CQA) is essential to produce consistent material
High-Throughput Analytics
Immunoassays are good…but which is best?
a) Bio-Layer Interferometry (BLI)
b) Surface Plasmon Resonance
c) Electrochemiluminescence Immunoassay (ECLIA)
d) ELISA
High-Throughput Analytics
Immunoassays are good…but which is best?
a) Bio-Layer Interferometry (BLI)
b) Surface Plasmon Resonance
c) Electrochemiluminescence Immunoassay (ECLIA)
d) ELISA
• In use at the VRC’s Vaccine Immunogenicity Program to assess ENV antigenicity
• Multiplexing and/or 384 well plate capable
• Potential to correlate binding data with other assays (e.g. SEC, glycans)
• High sensitivity and extremely low reagent mass requirements
Back to Research…
Standard:BG505.DS-SOSIP.V3
Lot# 150610 MCRun 170607
4571 Clone 1 lot NLC-670-23-01
4571 Clone 8lot NLC-670-23-02
4571 Clone 10lot NLC-670-23-03
4571 Clone 11lot NLC-670-23-04
0
1×106
2×106
3×106
4×106
5×106
AUC
CR9114
CD
4bs/
CD
4iV1
V2V3
gp12
0/41
Non
-Cog
nate
0
1×106
2×106
3×106
4×106
5×106
AUC
F10517b17b + sCD448d48d+sCD4VRC01VRC13b12
0
1×106
2×106
3×106
4×106
5×106
AUC
PGT145VRC26.25PGT121PGT1282G12
0
1×106
2×106
3×106
4×106
5×106
AUC
447-52D447-52D+sCD430743074+sCD425572557+sCD4
0
1×106
2×106
3×106
4×106
5×106
AUC
PGT151350228ANC195VRC34.01
Sample A
Sample B
Sample C
Sample D
Sample E
• ECLIA-based binding of serial dilutions (from 10 μg/mL) against multiple anti-HIV mAbs
• Area under the curve (AUC) of ECL units are used to compare antigenicity between samples
0
2×105
4×105
6×105
8×105
1×106
EC
L U
nits
0
2×105
4×105
6×105
8×105
1×106
EC
L U
nits
0
2×105
4×105
6×105
8×105
1×106
EC
L U
nits
0
2×105
4×105
6×105
8×105
1×106
EC
L U
nits
0.1 1 10
BLAN
K0
2×105
4×105
6×105
8×105
1×106
Std. BG505.DS-SOSIP.V36/10/15 MCRun 170607
(µg/mL)
EC
L U
nits
0.1 1 10
BLAN
K 4571 Clone 1 lot NLC-670-23-01
(µg/mL)
0.1 1 10
BLAN
K 4571 Clone 8 lot NLC-670-23-02
(µg/mL)
0.1 1 10
BLAN
K 4571 Clone 10 lot NLC-670-23-03
(µg/mL)
F10517b17b+sCD448d48d+sCD4VRC01VRC13b12PGT145VRC26.25PGT121PGT1282G12
447-52D447-52D+sCD430743074+sCD425572557+sCD4
PGT151350228ANC195VRC34.01
0.1 1 10
BLAN
K 4571 Clone 11 lot NLC-670-23-04
(µg/mL)
CR9114
Standard:BG505.DS-SOSIP.V3
Lot# 150610 MCRun 170607
4571 Clone 1 lot NLC-670-23-01
4571 Clone 8lot NLC-670-23-02
4571 Clone 10lot NLC-670-23-03
4571 Clone 11lot NLC-670-23-04
0
1×106
2×106
3×106
4×106
5×106
AUC
CR9114
CD
4bs/
CD
4iV1
V2V3
gp12
0/41
Non
-Cog
nate
0
1×106
2×106
3×106
4×106
5×106
AUC
F10517b17b + sCD448d48d+sCD4VRC01VRC13b12
0
1×106
2×106
3×106
4×106
5×106
AUC
PGT145VRC26.25PGT121PGT1282G12
0
1×106
2×106
3×106
4×106
5×106
AUC
447-52D447-52D+sCD430743074+sCD425572557+sCD4
0
1×106
2×106
3×106
4×106
5×106
AUC
PGT151350228ANC195VRC34.01
Sample A Sample B Sample C Sample D Sample E
Standard:BG505.DS-SOSIP.V3
Lot# 150610 MCRun 170607
4571 Clone 1 lot NLC-670-23-01
4571 Clone 8lot NLC-670-23-02
4571 Clone 10lot NLC-670-23-03
4571 Clone 11lot NLC-670-23-04
0
1×106
2×106
3×106
4×106
5×106
AUC
CR9114
CD
4bs/
CD
4iV1
V2V3
gp12
0/41
Non
-Cog
nate
0
1×106
2×106
3×106
4×106
5×106
AUC
F10517b17b + sCD448d48d+sCD4VRC01VRC13b12
0
1×106
2×106
3×106
4×106
5×106
AUC
PGT145VRC26.25PGT121PGT1282G12
0
1×106
2×106
3×106
4×106
5×106
AUC
447-52D447-52D+sCD430743074+sCD425572557+sCD4
0
1×106
2×106
3×106
4×106
5×106
AUC
PGT151350228ANC195VRC34.01
Standard:BG505.DS-SOSIP.V3
Lot# 150610 MCRun 170607
4571 Clone 1 lot NLC-670-23-01
4571 Clone 8lot NLC-670-23-02
4571 Clone 10lot NLC-670-23-03
4571 Clone 11lot NLC-670-23-04
0
1×106
2×106
3×106
4×106
5×106
AUC
CR9114
CD
4bs/
CD
4iV1
V2V3
gp12
0/41
Non
-Cog
nate
0
1×106
2×106
3×106
4×106
5×106
AUC
F10517b17b + sCD448d48d+sCD4VRC01VRC13b12
0
1×106
2×106
3×106
4×106
5×106
AUC
PGT145VRC26.25PGT121PGT1282G12
0
1×106
2×106
3×106
4×106
5×106
AUC
447-52D447-52D+sCD430743074+sCD425572557+sCD4
0
1×106
2×106
3×106
4×106
5×106
AUC
PGT151350228ANC195VRC34.01
Standard:BG505.DS-SOSIP.V3
Lot# 150610 MCRun 170607
4571 Clone 1 lot NLC-670-23-01
4571 Clone 8lot NLC-670-23-02
4571 Clone 10lot NLC-670-23-03
4571 Clone 11lot NLC-670-23-04
0
1×106
2×106
3×106
4×106
5×106
AUC
CR9114
CD
4bs/
CD
4iV1
V2V3
gp12
0/41
Non
-Cog
nate
0
1×106
2×106
3×106
4×106
5×106
AUC
F10517b17b + sCD448d48d+sCD4VRC01VRC13b12
0
1×106
2×106
3×106
4×106
5×106
AUC
PGT145VRC26.25PGT121PGT1282G12
0
1×106
2×106
3×106
4×106
5×106
AUC
447-52D447-52D+sCD430743074+sCD425572557+sCD4
0
1×106
2×106
3×106
4×106
5×106
AUC
PGT151350228ANC195VRC34.01
Standard:BG505.DS-SOSIP.V3
Lot# 150610 MCRun 170607
4571 Clone 1 lot NLC-670-23-01
4571 Clone 8lot NLC-670-23-02
4571 Clone 10lot NLC-670-23-03
4571 Clone 11lot NLC-670-23-04
0
1×106
2×106
3×106
4×106
5×106
AUC
CR9114
CD
4bs/
CD
4iV1
V2V3
gp12
0/41
Non
-Cog
nate
0
1×106
2×106
3×106
4×106
5×106
AUC
F10517b17b + sCD448d48d+sCD4VRC01VRC13b12
0
1×106
2×106
3×106
4×106
5×106
AUC
PGT145VRC26.25PGT121PGT1282G12
0
1×106
2×106
3×106
4×106
5×106
AUC
447-52D447-52D+sCD430743074+sCD425572557+sCD4
0
1×106
2×106
3×106
4×106
5×106
AUC
PGT151350228ANC195VRC34.01
Adaption to Suit Process Development
• In place of AUC of ECL units, single points from sample dilutions are backfit against the standard curve to calculate a concentration
• ECLIA-based binding of serial dilutions (from 10 μg/mL) of a reference standard
Backfit Conc in µg/mLSample Singleplex Multiplex Ratio S/M
1 173.9 206.3 84%2 354.4 373.6 95%3 473.1 526.9 90%4 327.4 309.2 106%5 155.1 200.0 78%6 396.4 389.5 102%7 393.8 448.9 88%8 445.9 419.5 106%9 1083.2 904.0 120%10 821.7 899.1 91%
Averageforsampleswith%CV<30% 96%
n(includedsamples) 70
1 1 0 1 0 0 1 0 0 0 1 0 0 0 0 1 0 0 0 0 00
5 0 0 0 0 0
1 0 0 0 0 0 0
1 5 0 0 0 0 0
V R C 0 1 C u rv e s
C o n c e n tra tio n o f tr im e r in n g /m L
EC
L R
es
po
ns
e U
nit
s S in g le
M u lti
Representative Standard Curves
Structural Assessment Through Antigenicity
Target Attribute Target Meaning Characterization ECLIA Assay
ENV (BG505) PDB: 5FYL
Structural Assessment Through Antigenicity
Target Attribute Target Meaning Characterization ECLIA Assay
CD4bs Accessible CD4bs VRC01, VRC07, N6
ENV (BG505) PDB: 5FYL Fab PDB: 5FYK
Structural Assessment Through Antigenicity
Target Attribute Target Meaning Characterization ECLIA Assay
CD4bs Accessible CD4bs VRC01, VRC07, N6
ENV (BG505) PDB: 5FYL Fab PDB: 4OLU
Structural Assessment Through Antigenicity
Target Attribute Target Meaning Characterization ECLIA Assay
CD4bs Accessible CD4bs VRC01, VRC07, N6
ENV (BG505) PDB: 5FYL Fab PDB: 5TE7
Structural Assessment Through Antigenicity
Target Attribute Target Meaning Characterization ECLIA Assay
CD4bs Accessible CD4bs VRC01, VRC07, N6
V1/V2 100% Proper fold PGT145, CAP256, PGDM1400, PG9
ENV (BG505) PDB: 5FYL Fab PDB: 5V8L
Structural Assessment Through Antigenicity
Target Attribute Target Meaning Characterization ECLIA Assay
CD4bs Accessible CD4bs VRC01, VRC07, N6
V1/V2 100% Proper fold PGT145, CAP256, PGDM1400, PG9
ENV (BG505) PDB: 5FYL Fab PDB: 5DT1
Structural Assessment Through Antigenicity
Target Attribute Target Meaning Characterization ECLIA Assay
CD4bs Accessible CD4bs VRC01, VRC07, N6
V1/V2 100% Proper fold PGT145, CAP256, PGDM1400, PG9
ENV (BG505) PDB: 5FYL Fab PDB: 4RQQ
Structural Assessment Through Antigenicity
Target Attribute Target Meaning Characterization ECLIA Assay
CD4bs Accessible CD4bs VRC01, VRC07, N6
V1/V2 100% Proper fold PGT145, CAP256, PGDM1400, PG9
ENV (BG505) PDB: 5FYL Fab PDB: 5VJ6
Structural Assessment Through Antigenicity
Target Attribute Target Meaning Characterization ECLIA Assay
CD4bs Accessible CD4bs VRC01, VRC07, N6
V1/V2 100% Proper fold PGT145, CAP256, PGDM1400, PG9
V3 Glycan Positive High Mannose N332, glycan consistency PGT121-PGT128
ENV (BG505) PDB: 5FYL Fab PDB: 4TVP
Structural Assessment Through Antigenicity
Target Attribute Target Meaning Characterization ECLIA Assay
CD4bs Accessible CD4bs VRC01, VRC07, N6
V1/V2 100% Proper fold PGT145, CAP256, PGDM1400, PG9
V3 Glycan Positive High Mannose N332, glycan consistency PGT121-PGT129
ENV (BG505) PDB: 5FYL Fab PDB: 5C7K
Structural Assessment Through Antigenicity
Target Attribute Target Meaning Characterization ECLIA Assay
CD4bs Accessible CD4bs VRC01, VRC07, N6
V1/V2 100% Proper fold PGT145, CAP256, PGDM1400, PG9
V3 Glycan Positive High Mannose N332, glycan consistency PGT121-PGT129
GP120/41 Fold 100% Proper fold, gp120/41 cleavage
35O22, PGT151, VRC34
ENV (BG505) PDB: 5FYL Fab PDB: 4TVP
Structural Assessment Through Antigenicity
Target Attribute Target Meaning Characterization ECLIA Assay
CD4bs Accessible CD4bs VRC01, VRC07, N6
V1/V2 100% Proper fold PGT145, CAP256, PGDM1400, PG9
V3 Glycan Positive High Mannose N332, glycan consistency PGT121-PGT129
GP120/41 Fold 100% Proper fold, gp120/41 cleavage
35O22, PGT151, VRC34
ENV (BG505) PDB: 5FYL Fab PDB: 4NUG
Structural Assessment Through Antigenicity
Target Attribute Target Meaning Characterization ECLIA Assay
CD4bs Accessible CD4bs VRC01, VRC07, N6
V1/V2 100% Proper fold PGT145, CAP256, PGDM1400, PG9
V3 Glycan Positive High Mannose N332, glycan consistency PGT121-PGT129
GP120/41 Fold 100% Proper fold, gp120/41 cleavage
35O22, PGT151, VRC34
ENV (BG505) PDB: 5FYL Fab PDB: 5I8H
Structural Assessment Through Antigenicity
Target Attribute Target Meaning Characterization ECLIA Assay
CD4bs Accessible CD4bs VRC01, VRC07, N6
V1/V2 100% Proper fold PGT145, CAP256, PGDM1400, PG9
V3 Glycan Positive High Mannose N332, glycan consistency PGT121-PGT129
GP120/41 Fold 100% Proper fold, gp120/41 cleavage
35O22, PGT151, VRC34
V3 Negative No available V3 1006-15D, 3074, 447-52D
ENV (BG505) PDB: 5FYL Fab PDB: 3MLW
Structural Assessment Through Antigenicity
Target Attribute Target Meaning Characterization ECLIA Assay
CD4bs Accessible CD4bs VRC01, VRC07, N6
V1/V2 100% Proper fold PGT145, CAP256, PGDM1400, PG9
V3 Glycan Positive High Mannose N332, glycan consistency PGT121-PGT129
GP120/41 Fold 100% Proper fold, gp120/41 cleavage
35O22, PGT151, VRC34
V3 Negative No available V3 1006-15D, 3074, 447-52D
ENV (BG505) PDB: 5FYL Fab PDB: 3MLZ
Structural Assessment Through Antigenicity
Target Attribute Target Meaning Characterization ECLIA Assay
CD4bs Accessible CD4bs VRC01, VRC07, N6
V1/V2 100% Proper fold PGT145, CAP256, PGDM1400, PG9
V3 Glycan Positive High Mannose N332, glycan consistency PGT121-PGT129
GP120/41 Fold 100% Proper fold, gp120/41 cleavage
35O22, PGT151, VRC34
V3 Negative No available V3 1006-15D, 3074, 447-52D
ENV (BG505) PDB: 5FYL Fab PDB: 3C2A
KSIRIGPGQAFY305 318
Structural Assessment Through Antigenicity
Target Attribute Target Meaning Characterization ECLIA Assay
CD4bs Accessible CD4bs VRC01, VRC07, N6
V1/V2 100% Proper fold PGT145, CAP256, PGDM1400, PG9
V3 Glycan Positive High Mannose N332, glycan consistency PGT121-PGT129
GP120/41 Fold 100% Proper fold, gp120/41 cleavage
35O22, PGT151, VRC34
V3 Negative No available V3 1006-15D, 3074, 447-52D
ENV (BG505) PDB: 5FYL
UPLC SEC Profile: Immuno-affinity (+/- sel)/SEC Product
Structural Assessment Through Antigenicity
Target Attribute Target Meaning Characterization ECLIA Assay
CD4bs Accessible CD4bs VRC01, VRC07, N6
V1/V2 100% Proper fold PGT145, CAP256, PGDM1400, PG9
V3 Glycan Positive High Mannose N332, glycan consistency PGT121-PGT129
GP120/41 Fold 100% Proper fold, gp120/41 cleavage
35O22, PGT151, VRC34
V3 Negative No available V3 1006-15D, 3074, 447-52D
ENV (BG505) PDB: 5FYL Fab PDB: 5FYK
UPLC SEC Profile: Immuno-affinity (+ sel) Product
Structural Assessment Through Antigenicity
Target Attribute Target Meaning Characterization ECLIA Assay
CD4bs Accessible CD4bs VRC01, VRC07, N6
V1/V2 100% Proper fold PGT145, CAP256, PGDM1400, PG9
V3 Glycan Positive High Mannose N332, glycan consistency PGT121-PGT129
GP120/41 Fold 100% Proper fold, gp120/41 cleavage
35O22, PGT151, VRC34
V3 Negative No available V3 1006-15D, 3074, 447-52D
ENV (BG505) PDB: 5FYL Fab PDB: 3C2A
UPLC SEC Profile: Immuno-affinity (V3- sel) Strip
High-Throughput In-Process Analytics
Target Attribute Target Meaning In-Process ECLIA Assay Priority High-Throughput?
GP41/GP120 Cleavage 100% Cleavage PGT151/VRC34/35O22 1 +++
Quaternary Structure Intact 4º Epitopes PGT145/PGDM1400/PG9PGT151/CAP256/ 1 +++
Glycan Shield N332 = 100% Occcupancy PGT121-PGT129 1 +++
CD4i Induced Conformational Change
No conformational change in presence of CD4. 17b/48d + sCD4 2 +++
V3 Negative No/Low available V3 447-52D/1006-15D/3074 2 +++
Glycan N332 High Mannose PGT121-PGT129 3 +++
SEC Single Population 447-52D/1006-15D/3074 4 +++
Overall Structure Population Distribution Congruence of ELCIA 5 +++
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From Antigenicity to Commercial Resin Chemistry
GP120/GP41 (–) Asp+Glu (+) Arg+Lys Ala+Val+Leu+Ile+Phe+Pro+Tyr+Trp
90º
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Vendor ResinTOSOH Toyopearl® MX-Trp-650MPall HEA HyperCel™Pall MEP HyperCel™Pall PPA HyperCel™Millipore Eshmuno® HCXGE Capto™ adhereGE Capto™ Core 700GE Capto™ MMCBio-Rad Macro-Prep® CFT™ Type I 40 µmBio-Rad Macro-Prep® CHT™ Type I 40 µmBio-Rad Macro-Prep® CHT™ Type I 80 µmBio-Rad Nuvia™ cPrime™
Vendor ResinTOSOH Toyopearl® Butyl-650MTOSOH Toyopearl® Ether-650MTOSOH Toyopearl® Hexyl-650CTOSOH Toyopearl® Phenyl-650MTOSOH Toyopearl® PPG-600MTOSOH Toyopearl® SuperButyl-550CGE Butyl Sepharose™ 4 FFGE Butyl-S Sepharose™ 6 FFGE Capto™ ButylGE Capto™ Phenyl (high sub)GE Octyl Sepharose™ 4 FFGE Phenyl Sepharose™ 6 FF (high sub)Bio-Rad Macro-Prep® MethylBio-Rad Macro-Prep® t-Butyl
Vendor ResinTOSOH Toyopearl® GigaCap® CM-650MTOSOH Toyopearl® GigaCap® S-650MTOSOH Toyopearl® MegaCap® II SP-550 ECTOSOH Toyopearl® SP-650MThermoFisher POROS® 50 HSThermoFisher POROS® XSPall CM Ceramic HyperD® FPall S Ceramic HyperD® 20Pall S Ceramic HyperD® FPall S HyperCel™Millipore Eshmuno® CPXMillipore Eshmuno™ SMillipore Fractogel® EMD COO- (M)Millipore Fractogel® EMD SE Hicap (M)Millipore Fractogel® EMD SO3- (M)GE Capto™ SGE CM Sepharose™ FFGE CM Sepharose™ HPGE SOURCE™ 30SGE SP Sepharose™ FFGE SP Sepharose™ XLBio-Rad Macro-Prep® CMBio-Rad Macro-Prep® High SBio-Rad MacroCap SPBio-Rad Nuvia™ HR-SBio-Rad Nuvia™ SBio-Rad UNOsphere™ S
Anion Exchange Cation Exchange Hydrophobic Interaction
Multimodal
Overview: 32 resins (in triplicate), 12 step elution pseudo-gradient, ~1400 samples for ECLIA
1 2 3 4 5 6 7 8 9 10 11 12A R01 R09 R17 R25 R01 R09 R17 R25 R01 R09 R17 R25B R02 R10 R18 R26 R02 R10 R18 R26 R02 R10 R18 R26C R03 R11 R19 R27 R03 R11 R19 R27 R03 R11 R19 R27D R04 R12 R20 R28 R04 R12 R20 R28 R04 R12 R20 R28E R05 R13 R21 R29 R05 R13 R21 R29 R05 R13 R21 R29F R06 R14 R22 R30 R06 R14 R22 R30 R06 R14 R22 R30G R07 R15 R23 R31 R07 R15 R23 R31 R07 R15 R23 R31H R08 R16 R24 R32 R08 R16 R24 R32 R08 R16 R24 R32
Elution Steps Chart:Buffer: 10mM Sodium Citrate
pH 5Fraction Number [NaCl] pH
1 2.5 mM 5
2 25 mM 5
3 50 mM 5
4 75 mM 5
5 100 mM 5
6 125 mM 5
7 150 mM 5
8 200 mM 5
9 250 mM 5
10 300 mM 5
11 350 mM 5
12 2 M 5
Equilibration: 10 mM Sodium Citrate, 2.5 mM NaCl pH 5
Load: Clarified Harvest TFF’dinto equilibration buffer
Wash: Equilibration Buffer
Elution: Programmed steps from 2.5 mM NaCl to 250 mM
NaCl
Strip: 2 M NaCl
CIP: 0.1 N NaOH, 1 hr
Storage: 20% Ethanol
ENV 4571 IEX Capture Conventional Resin Screen
A280 Output (Yellow [low] à Blue [high])
[low] à [high]
2000 mM
350 mM300 mM250 mM
200 mM150 mM
100 mM
50 mM
Programmable Step Elutions [NaCl]
25 mM2.5 mM
ENV 4571 IEX Capture Conventional Resin Screen
Load à
ENV 4571 IEX Capture Conventional Resin Screen A280 output, using pathlength correction and volume calculation to report total mass/well (mg)
[0.001] à [0.738]
Output: ECLIA CD4bs Concentration (μg/mL)
<0.6 μg/mL ECLIA result reported as 0.0 μg/mL[0.0] à [236.1]
ENV 4571 IEX Capture Conventional Resin Screen
SDS PAGE Analysis of CEX “Hits”
Lane # Sample Description Conc. by A280 (mg/mL)
1 Benchmark Protein Ladder
2 pos. ctrl (PGDM1400 & 447-52D purified) 0.107
3 Plate 2 Load neg. ctrl well 3.18
4 CEX R15 - Fraction 5 triplicate pool 0.606
5 CEX R15 - Fraction 9 duplicate pool 0.651
6 CEX R16 - Fraction 5 triplicate pool 0.780
7 CEX R16 - Fraction 9 triplicate pool 1.218
8 CEX R04 - Fraction 5 triplicate pool 0.283
9 CEX R04 - Fraction 9 triplicate pool 0.364
10 Blank
1 2 3 4 5 6 7 8 9 10
R15 R16 R04Peak Strip Peak Strip Peak Strip+Ctrl Load
HIV Trimer Conventional Resin Screen of AEX CaptureOverview: 15 resins (in duplicate), 3 pH, 12 step elution pseudo-gradient, ~1400 samples
Equilibration: Appropriate buffer
Load: Clarified Harvest TFF’dinto EQ buffer
Wash: EQ Buffer
Elution: Programmed steps from 50 mM NaCl to 550 mM
NaCl
Strip: 2 M NaCl
CIP: 0.1 N NaOH, 1 hr
Storage: 20% Ethanol
1 2 3 4 5 6 7 8 9 10 11 12A R01 R09 R01 R09 R01 R09 R01 R09 R01 R09 R01 R09B R02 R10 R02 R10 R02 R10 R02 R10 R02 R10 R02 R10C R03 R11 R03 R11 R03 R11 R03 R11 R03 R11 R03 R11D R04 R12 R04 R12 R04 R12 R04 R12 R04 R12 R04 R12E R05 R13 R05 R13 R05 R13 R05 R13 R05 R13 R05 R13F R06 R14 R06 R14 R06 R14 R06 R14 R06 R14 R06 R14G R07 R15 R07 R15 R07 R15 R07 R15 R07 R15 R07 R15H R08 R16 R08 R16 R08 R16 R08 R16 R08 R16 R08 R16
Low pH pH=6
Mid pH pH=8
High pH pH=10
Equil, wash, and elution buffers will be at these pH's for these samples
Elution Steps Chart:
Buffer: 20mM MES pH 6, Tris-HClpH 8, or Glycine-OH pH 10
Fraction Number [NaCl]
1 50 mM
2 100 mM
3 150 mM
4 200 mM
5 250 mM
6 300 mM
7 350 mM
8 400 mM
9 450 mM
10 500 mM
11 550 mM
12 2 M
HIV Trimer Conventional Resin Screen of AEX CaptureA280 output, using pathlength correction and volume calculation to report total mass/well
[0.001] à [0.783]
HIV Trimer Conventional Resin Screen of AEX CaptureOutput: ECLIA (CD4bs) Concentration (μg/mL)
<0.6 μg/mL ECLIA result reported as 0.0 μg/mL[0.0] à [159.1]
HIV Trimer Conventional Resin Screen of AEX CaptureOutput: ECLIA (CD4bs) Concentration (μg/mL)
<0.6 μg/mL ECLIA result reported as 0.0 μg/mL[0.0] à [159.1]
BG505 Conventional Resin Screen of HIC PolishOverview: 15 resins (in duplicate), 1 pH, 12 step elution pseudo-gradient, ~350 samples
Equilibration: 15 mM Sodium Citrate, 1.8 M Ammonium
Sulfate pH 6
Load: CEX Capture Material
Wash: Equilibration Buffer
Elution: Programmed steps from 1.6 M [(NH4)2SO4] to 0.1 M
Strip: 15 mM Sodium Citrate pH 6
CIP: 0.1 N NaOH, 1 hr
Storage: 20% Ethanol
1 2 3 4A R01 R09 R01 R09B R02 R10 R02 R10C R03 R11 R03 R11D R04 R12 R04 R12E R05 R13 R05 R13F R06 R14 R06 R14G R07 R15 R07 R15H R08 R16 R08 R16
Elution Buffers Chart:
Buffer: 15mM Sodium Citrate pH 6Fraction Number [(NH4)2SO4]
1 1600 mM
2 1400 mM
3 1200 mM
4 1000 mM
5 800 mM
6 600 mM
7 400 mM
8 200 mM
9 100 mM
10 0 mM
BG505 Conventional Resin Screen of HIC PolishTop: A280, total protein (mg)
Bottom: ECLIA CD4bs Concentration (μg/mL)
[0.000] à [0.124]
[0.9] à [1309.3]
ENV Trimer 4571 Conventional Resin Scale-up Runs1. Clarified Harvest was UF/DF processed to low conductivity2. Capture Step: AEX, bind/elute3. Polishing Step 1: AEX, bind/elute (lane 2)4. Polishing Step 2: HIC bind/elute (lane 5)
Gel samples were run under non-reducing conditions
Lane Sample Load Volume(μL)
1 Ladder 7
2 AEX capture à polish 1 Pool 20
3 HIC load (high [(NH4)2SO4)] 20
4 HIC polish wash 20
5 HIC polish elution 20
6 HIC polish Strip 40
7 Blank
1 2 3 4 5 6 7
Platform-y-ness?
4571 (BG505-isolate ENV)
Versus
5177 (CH505-isolate ENV)
4571 vs 5177 ENV Sequences
Construct 4571 5177
pI 8.80 8.82
Peptide length 634 623
MW (Da) 71138 69950
Ext. Coef.(OD = 1 mg/mL)
1.57 1.58
AliphaticIndex 81.1 83.1
Asp+Glu 54 54
Arg+Lys 67 68
5177 AEX and CEX Resin Screen
ElutionBufferSteps:
FractionNumber [NaCl]
1 50mM
2 100mM
3 150mM
4 200mM
5 250mM
6 300mM
7 350mM
8 400mM
9 450mM
10 500mM
11 550mM
12 2M
Equilibration: 20 mMAppropriate Buffer, 10 mM
NaCl
Load: Clarified Harvest buffer exchanged into EQ buffer
Wash: EQ Buffer
Elution: Programmed steps from 50 mM NaCl to 550 mM
NaCl
Strip: 2 M NaCl
CIP: 0.1 N NaOH, 1 hr
Storage: 20% Ethanol
1 2 3 4 5 6 7 8 9 10 11 12A R01 R01 R01 R01 R01 R01 R09 R09 R09 R09 R09 R09B R02 R02 R02 R02 R02 R02 R10 R10 R10 R10 R10 R10C R03 R03 R03 R03 R03 R03 R11 R11 R11 R11 R11 R11D R04 R04 R04 R04 R04 R04 R12 R12 R12 R12 R12 R12E R05 R05 R05 R05 R05 R05 R13 R13 R13 R13 R13 R13F R06 R06 R06 R06 R06 R06 R14 R14 R14 R14 R14 R14G R07 R07 R07 R07 R07 R07 R15 R15 R15 R15 R15 R15H R08 R08 R08 R08 R08 R08 R16 R16 R16 R16 R16 R16
AEX CEX/MM
LowpH pH=6 LowpH pH=5
MidpH pH=8 MidpH pH=6
HighpH pH=10 HighpH pH=7Equil,wash,andelutionbufferswillbeatthesepH'sforthesesamples
Buffers: 20 mM Sodium Citrate pH 5, MES pH 6, MES pH 7, Tris-HCl pH 8, Glycine-NaOH pH 10
5177 AEX and CEX Resin Screen – (mg) by A280
[0.006] à [0.849]
5177 AEX and CEX Screen – CD4bs ECLIA (μg/mL)
[<1.1] à [604.6]
5177 AEX R04: A280 with CD4bs/V3(-) ECLIA
[<1.1] à [604.6]
5177 AEX R04: A280 with CD4bs/V3(-) ECLIA
Selected for SDS PAGE
1
CD4bs ECLIATotal Protein
V3(–):CD4bs Ratio CD4bs ECLIATotal Protein
V3(–):CD4bs Ratio
5177 AEX and CEX Resin Screen – Top Hits
Lane Resin pH [NaCl] (mM) Mass loaded (ug)
1 Ladder
2 R02 8 50 5
3 R02 10 50 4
4 R04 6 100 5
5 R04 8 50 4
6 R10 5 350 5
7 R13 5 350 5
8 R13 5 400 5
9 R14 5 400 4
10 R16 5 450 3
1 2 3 4 5 6 7 8 9 10
ECLIA 2 3 4 5 6 7 8 9 10
CD4bs 177 339 248 235 590 465 414 604 445
V3(-):CD4bs 4.7 3.6 4.3 2.3 0.5 0.8 1.0 4.4 3.0
Conclusions
1. Advancing high-throughput, low material consuming analytical assays upfront can return maximal dividends in development.
2. Robotics at every development step will: • enhance throughput• minimize errors• make more efficient use of limited materials/resources
3. Downstream high-throughput resin screening can allow quick selection of conventional resins to advance the purification development process.
4. To deliver the project turn-around-time needed for experimental medicine strategies, the approach to developing a manufacturing process should itself become a platform.
Acknowledgements!Cell Line and Upstream• Joe Horwitz• Peifeng Chen• Stephanie Barclay• Lizz Carey• Isaac Godfroy• Julia Frederick• Isaac Godfroy• Alvenne Goh• Stephanie Golub• Abasha Williams• Dan Schankel• Liz Scheideman• Naga Chalamalasetty• Venkat Mangalampalli
Analytical• Jon Cooper• Sarah O’Connell• Brad Tippett• Tina Khin• Nadji Lambert• Philip Yang• Chris Barry• Paula Lei• Vera Ivleva• Aakash Patel• Yile Li• Nathan Barefoot
Technical Services• Janel Holland-Linn• Troy Chase
Project Management• Kevin Carlton• Gretchen Schieber
Chief• Frank Arnold
VRC Research• Peter Kwong• Anita Shah• Hui Geng• Adrian McDermott• Sandeep Narpala• Guillaume Stewart Jones
Downstream• Adam Charlton• Zach Schneiderman• Nicole Cibelli• Haley Fuller• Sara Witter• Krishna Gulla• Jordan Ficca• Daniel Ragheb• Nga Tran• Lena Wang
Formulation• KC Cheng• Lisa Kueltzo• Dina Manceva• Rajoshi Chaudhuri