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




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




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




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


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


0

1×106

2×106

3×106

4×106

5×106

AUC


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


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


0

1×106

2×106

3×106

4×106

5×106

AUC


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


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


0

1×106

2×106

3×106

4×106

5×106

AUC


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


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


0

1×106

2×106

3×106

4×106

5×106

AUC


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


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


0

1×106

2×106

3×106

4×106

5×106

AUC


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



CD4bs Accessible CD4bs VRC01, VRC07, N6

ENV (BG505) PDB: 5FYL Fab PDB: 5FYK




ENV (BG505) PDB: 5FYL Fab PDB: 4OLU




ENV (BG505) PDB: 5FYL Fab PDB: 5TE7




V1/V2 100% Proper fold PGT145, CAP256, PGDM1400, PG9

ENV (BG505) PDB: 5FYL Fab PDB: 5V8L





ENV (BG505) PDB: 5FYL Fab PDB: 5DT1





ENV (BG505) PDB: 5FYL Fab PDB: 4RQQ





ENV (BG505) PDB: 5FYL Fab PDB: 5VJ6





V3 Glycan Positive High Mannose N332, glycan consistency PGT121-PGT128

ENV (BG505) PDB: 5FYL Fab PDB: 4TVP






ENV (BG505) PDB: 5FYL Fab PDB: 5C7K






GP120/41 Fold 100% Proper fold, gp120/41 cleavage

35O22, PGT151, VRC34

ENV (BG505) PDB: 5FYL Fab PDB: 4TVP







35O22, PGT151, VRC34

ENV (BG505) PDB: 5FYL Fab PDB: 4NUG







35O22, PGT151, VRC34

ENV (BG505) PDB: 5FYL Fab PDB: 5I8H







35O22, PGT151, VRC34

V3 Negative No available V3 1006-15D, 3074, 447-52D

ENV (BG505) PDB: 5FYL Fab PDB: 3MLW







35O22, PGT151, VRC34


ENV (BG505) PDB: 5FYL Fab PDB: 3MLZ







35O22, PGT151, VRC34


ENV (BG505) PDB: 5FYL Fab PDB: 3C2A

KSIRIGPGQAFY305 318







35O22, PGT151, VRC34


ENV (BG505) PDB: 5FYL

UPLC SEC Profile: Immuno-affinity (+/- sel)/SEC Product







35O22, PGT151, VRC34


ENV (BG505) PDB: 5FYL Fab PDB: 5FYK

UPLC SEC Profile: Immuno-affinity (+ sel) Product







35O22, PGT151, VRC34


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 +++

Robots,Cell Line Development robotic automation for two major processes:

Clone Selection Clone Expansion& Downselection

Robots, Robots,Upstream uses robotic cell culture for optimization and final clone selection

Robots, Robots, & More Robots

Integrated Absorbance on Ron Added Integrated Fluorescence on Max

Downstream uses robotic liquid handlers for resin screening, process optimization, column cleaning studies, clone selection support, upstream process support, and…

Advancing Development

A case study in pairing high-throughput analytics with downstream automation

From Antigenicity to Commercial Resin Chemistry

GP120/GP41 (–) Asp+Glu (+) Arg+Lys Ala+Val+Leu+Ile+Phe+Pro+Tyr+Trp

90º

High Throughput Resin Screening

Vendor ResinTOSOH Toyopearl® CM-650MTOSOH Toyopearl® DEAE-650MTOSOH Toyopearl® DEAE-650STOSOH Toyopearl® GigaCap® DEAE-650MTOSOH Toyopearl® GigaCap® Q-650MTOSOH Toyopearl® GigaCap® Q-650STOSOH Toyopearl® NH2-750FTOSOH Toyopearl® Q-600C ARTOSOH Toyopearl® SuperQ-650MTOSOH Toyopearl® SuperQ-650SThermoFisher POROS® 50 DThermoFisher POROS® 50 HQThermoFisher POROS® 50 PIThermoFisher POROS® XQPall DEAE Ceramic HyperD® FPall HyperCel™ STAR AXPall Q Ceramic HyperD® 20Pall Q Ceramic HyperD® FPall Q HyperCel™Millipore Eshmuno™ QMillipore Fractogel® EMD DEAE (M)Millipore Fractogel® EMD DMAE (M)Millipore Fractogel® EMD TMAE (M)Millipore Fractogel® EMD TMAE Hicap (M)Millipore Fractogel® EMD TMAE Medcap (M)GE ANX Sepharose™ 4FF high subGE Capto™ DEAEGE Capto™ QGE DEAE Sepharose™ FFGE Q Sepharose™ FFGE SOURCE™ 30QGE Q Sepharose™ XLBio-Rad Macro-Prep® DEAEBio-Rad Macro-Prep® High QBio-Rad Nuvia™ QBio-Rad UNOsphere™ Q

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


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]


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





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




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]

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



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




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

a hiv e a m i t p q /p - global hiv vaccine enterprise. gowetski.pdf · immunoassay 2 +++ v3...

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