evaluation of recombinant prokaryotic lectins (rpls) for ... shukla 15... · evaluation of...

Confidential

Evaluation of Recombinant Prokaryotic Lectins (RPLs)

for Capture of Highly Glycosylated Proteins

Prathima Acharya,Consultant – JSR/KBI,Biosonata Consullting, [email protected]

Abhinav Shukla,SVP, PD & Manuf,KBI Biopharma

HIV Env Vaccine Manufacturing Workshop, Rockville, MD, Sep 15 -2016

Confidential

Ref: Lancet, Volume 5, No. 11, p726–731, November 2005

Surface decorated with glycans Principles of chromatographic

capture - hard to follow(AEX, CEX, Hydrophobic, combination of charge/hydrophobicity,Bind/flow-through modes etc) Regular chromatography capture

development time can be long and expensive

Affinity chromatography can provide relief

HIV Env Proteins – Highly glycosylated

An Attempt to Close the Translational Gap……….. • Academic/• Research lab• Affinity capture using

plant lectins• Develop purification

schemes• Test for activity based on

product profile obtained from PLANT lectin capture step

• Transfer to Industry for GMP production/or produce GMP material in-house

• GMP Production• Current State

• Desired –• Future State for

GMP Production• Replacement of PLANT lectin affinity capture step with commercial resins

• Additional cost and time and duplication of efforts for the capture step

• Replacement can now lead to a different product profile rendering the other steps in the academic process useless

• Expensive process when additional development and manufacturing costs are factored in for redevelopment of capture step and required additional polishing steps

• POC data set may no longer be valid given there is a change in the process of making these proteins

• Enables affinity capture using recombinant lectin resins that can be used both for research and GMP production for human use

• Closing of the “translational gap” expediting potential cures

• Takes away extra cost and time required for redevelopment

• Minimizes possible failures of candidates processed differently than material made from a completely different process

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Advantages of Recombinant Prokaryotic Lectins (RPLs)

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Shake Flask Expression• Shake flask titers of 0.5-2.0 g/L with JM109 for the 7 constructs

• SDS-PAGE and back calculated from chromatography yields• Minimal (≈ 0.04 g/L) expression with BL21

• SDS-PAGE

Confidential

Purification

• Single Nickel Sepharose Fast Flow column• Imidazole gradient elution at pH 7.4 or 8.0 (based on pI of individual RPL)• Pooling based on SDS-PAGE analysis of individual fractions• Identity confirmed by SDS-PAGE (co-elution with GlycoSelect controls) and LCMS

(comparison of intact mass to theoretical is on-going. Waiting for data)• Estimate of pool purities >95% (SDS-PAGE); comparable to GlycoSelect controls

(next slide)

Cost Effective Production of High Quality RPLs in E. coli (high yield & His-tagged)

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Reducing SDS-PAGE (4-12% Bis-Tris/MES)

Target loads of 20 µg/lane

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αGal Gal1 Gal2 Gal3 Gal4 αMan Man2w53 Lectin

(S14) +/- + +/- +/- +/- +/- +

w53 BDS - ++ - - - - +

w78 BDS - +++ - - - - +++

w100 BDS - + - - - - +++

w100 harvest - + - - - - +/-

Goal: Determining the KD of each RPL for the gp120 molecules. Collection of data in triplicate for top hits

Confidential

0.0000

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0 100 200 300 400 500 600 700 800 900

Bind

ing Re

spon

se (n

m)

Concentration (nM)

w53 Dose Response Binding with CH106

HEK Reference

AGN Eluate 1

Man 2 Eluate 1

Gal 1 Eluate

MMC Eluate

Fractogel Eluate

W 53 gp120 Env purified by different columns/lectins {Gal1, Man2, AGN (plant lectin), non-affinity} binding to CH106 antibody –Activity assay

• RPL affinity chromatography has not been optimized (unlike CaptoMMC/Fractogel) and so there is no data to show “inactive” forms exclusion during RPL affinity capture yet. This is listed as future work in the coming months.

Confidential

Capto MMC (RPL) Gal1 (RPL) Man2AGN (plant lectin)

SO3 (Fractogel)

TF<LOQ 1200

PPM<LOQ 900

PPM<LOQ 4000

PPM

W53 112714 12174<LOQ 10000

PPM 26281

W100 14501 1981 8365<LOQ 2000

PPM

W78 26722<LOQ 2800

PPM<LOQ 7500

PPM

Arma 6732 10141 6611

Fluxa 75371 21527 47236 5754

Residual Host Cell Protein (HCP) Analysis of Capture Eluates

Most active product also cleared the most HCP

Residual HCP DataComparison of PoC data from RPLs with optimized non-affinity capture

Can RPLs serve as true affinity resins?

Affinity capture step (RPLs as resins)

• RPL resins were made at KBI for PoCstudies

• Most active product also cleared the most HCP

• Generic protocols provided by GlycoSeLect used (no development or optimization)

• Produces equivalent or better results than non-affinity capture

• Almost Protein-A like affinity clearance of HCPs with no effort during these PoC studies

• Can clear HCPs better upon optimization

• May have the potential to reduce the number of polishing steps that may be required following the capture step

Non-affinity capture step(capto MMC/Fractogel)

• Capto MMC and Fractogel are commercially available resins

• Most active product also cleared the most HCP

• Process devpt was performed on wash and elution buffers to optimize the capture step for each Env

• Had to be tailored for each Env; even replaced by Fractogel for one Env -w53

• Requires development and most likely re-optimization for each envelope

• May require multiple polishing steps following the capture step

Confidential

SampleName: W53 Capto MMC ph 6.5 eluate

SampleName: W53 Gal1 eluate

SampleName: W53 Ref, Lot 2925p147

SampleName: W53 Man2 eluate

Minutes8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00 44.00 46.00 48.00 50.00 52.00

Goal: Downstream w53 material was submitted and evaluated for CH106 binding by dose response. Samples were subsequently

analyzed by released glycan to evaluate differences

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• Creation of a library of resins for initial scouting using Forte Bio in a HT fashion• Based on this data and other glycan data, select one or two RPL resins• Pick RPL resins based on activity data• If possible a mix of resin can also be generated to increase yields (if two or more

RPL resins show good activity upon affinity capture)• Protein A resins typically come with customized residual protein A kits (ELISA)• Develop residual RPL kit to detect (ELISA) based on conserved sequence regions

of the lectin analogous to protein A kits• Research grade resin will be produced for research labs (analogous to protein A

affinity capture for antibodies)• GMP grade resin will be produced for GMP production (analogous to protein A

affinity capture for antibodies)• Thereby closing the Translational Gap ……….

Next steps…. Closing the Translational Gap

Confidential

Gal-3 Reducing SDS-PAGE (4-12% Bis-Tris/MES)

Frac

tion

A5

Frac

tion

A6

Frac

tion

B2

RP

L-G

al-3

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Std

Frac

tion

B4

Frac

tion

B6

Frac

tion

C1

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tion

C2

Frac

tion

B1

UFDF Pool

Protein Concentration: 5.0 mg/mL (UV280)

Pooled Fractions: A3 – C1

Pool Volume: 205 mL

Purified RPL-Gal-1: 1.0 g (shipped)

Frac

tion

C3

Frac

tion

A4

Frac

tion

A3

(Approximately 10 µg protein loaded/lane)

PG9

PGT128

2F5

4E10

VRC01

0.002

Co-Evolution of Transmitted/Founder Broad Neutralizing Antibodies From African Individual CH505

(CH505 bnAb- HIV-1producer)

Antibody (the CH103HCDR3-

binder bnAbB Cell

lineage)Transmitted/Founder Virus

41 weeks

92 weeks

14 weeks

Week 4 Week 14Week 20Week 22Week 30Week 53Week 78Week 100Week 136Week 160

Onset breadth Tier 2

Virus Neutralization

Autologous Neutralization

Onset Broad Virus Neutralization

CH103-CH106Isolated

CH

106

CH

103

CH

104

CH

105

H.X. Liao et al. Nature 496: 469; 2013

CH505 Envelopes selected as vaccine immunogens

CH505 transmitted-founder (TF) and Env variants generated during viral evolution drove affinity maturation

of CH103 bnAb lineage

Antibody: UCAT/F gp120 Kd = ~200 nM

Env:

CH103

CH505 wk53

CH505 wk78

CH505 wk100

CH103 lineage intermediateantibodies

CH505 TF

CH505 wk136

18H.X. Liao et al. Nature 496: 469;

2013

CH505 Envelopes selected as vaccine immunogens

CH505 transmitted-founder (TF) and Env variants generated during viral evolution drove affinity maturation

of CH103 bnAb lineage

Antibody: UCAT/F gp120 Kd = ~200 nM

Env:

CH103

CH505 wk53

CH505 wk78

CH505 wk100

CH103 lineage intermediateantibodies

CH505 TF

CH505 wk136

19H.X. Liao et al. Nature 496: 469;

2013

The key to patient access is creating processesthat can take these vaccine candidates intoclinical trials rapidly

A process platform for Env proteins• Need for a Platform process for Env proteins

• Creating a process platform can help save time in development and enable human trials to proceed

• Clinical trials in this space are proof-of-concept studies and likely even more antigens will be needed

• Key aspects of the platform• CHO DG44 cell line, cell culture process development,

downstream process development, analytical methods development

• Use of antigenic binding to human antibodies as a key tool for confirming process decisions

• Process issues encountered & solved for multiple antigens

Platform for Cell Culture Process• Parameters shaded in gray are

defined across molecules. Parameters shaded in yellow require molecule specific optimization

• For all Env molecules the operating parameters, basal medium, feed type and some of the supplement additions have defined

• The need for additional supplements is molecule specific• Reasons for supplement

addition:• Biocompatability in SU

bioreactors• Increase in productivity

ScaleTemperature Set point 37.0 ± 0.5°C Temperature Shift 33.0 ± 0.5°C on Day 6DO Set point 30%pH Set point 6.90 ± 0.1

Agitation (1-impeller) 50 rpm → 55 rpm

Air overlay 1.6 SLPMAir Sparge 0.5 SLPM Max. Oxygen Sparge 5 SLPMMax. CO2 Sparge 5 SLPM

Medium CD OptiCHO + 8 mM Glutamine

Target VCD 0.50 x 106 cells/mLBase 1M Sodium carbonate

Feed Type: LTI Feed A+B (1:1) 15% on Day 0, 10% current wv each on Days 3, 6, and 9

Supplement 1 addition: HT Supplement 1X current wv each on Days 0 and 4

Supplement 2 addition: CystineSupplement 3 addition: TyrosineSupplement 4 addition: Soy:Yeastolate Hydrolysate (2:3) 5g/L current wv each on Days 4 and 8

Supplement 5 addition: C1615Harvest Add 10g/L Hydrolysate on harvest

High throughput process development• Accelerated development approaches critical for non-

platform molecules• Rapid experimentation made possible by integrated use

of high through cell culture and high throughput analytics

Rameez, S.; Mostafa, S. S.; Miller, C.; Shukla, A. A. High-throughput miniaturized bioreactors for cell culture process development: Reproducibility, scalability and control. Biotechnology Progress 2014, (30): 718-727.

Platform for Downstream Processing• Parameters shaded in gray are

defined across molecules. Parameters shaded in yellow require molecule specific optimization

• Load and elution conditions for three of the unit operations require molecule specific definition given the heterogeneity of this class of molecules

• Env antigens structurally sensitive to hydrophobic surfaces, hence HIC not employed

Downstream process performance for various Envproteins

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Log HCP

(pp

m)

Downstream Process

Platform HCP Clearance

TF Demo

TF ENG

TF GMP

w100 Demo

w100 ENG

w100 GMP

w78 Demo

w78 GMP

SEC‐HPLC % Main Peak

Sample ID TF Demo

TF ENG

TF GMP

w100 Demo

w100 ENG

w100GMP

w78Demo

w78 GMP

BDS 99.3% 98.9% 98.8% 98.9% 99.3% 99.2% 99.5% 99.6%

gp120 Env Portfolio

CH505TF

CH505w100

CH505w78

CH505w53

B63521

Clone SelectionUpstream/Downstream Process Assessment &

confirmation of SPR activity

Verification of Compatibility with

Single Use Bioreactor

Pilot scaleRun

Engineering Run cGMP Run

Developing an affinity purification step for gp120 Envs

• Platform process is highly effective for this family of proteins but will need modification for a new class of proteins

• An affinity capture step based on lectin can be used broadly for all gp120 or gp140 proteins since they are heavily glycosylated

• Commercial lectin sources (from Aganthus nivalis) are heterogeneous and have significant lot-to-lot variability

• Recombinant prokaryotic lectins as chromatographic ligands offer the possibility of being a unique capture step for highly glycosylated proteins

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Commercially Available Glycoproteins

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norm

alized

kob

s(1/(M*s)

AlphaGal

Gal1

Gal2

Gal3

Gal4

AlphaMan

Man2

KD Screening for gp120 Variants

Gal1 and Man2 Data collected in Triplicate

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w53 Lectin (S14) w53 BDS w78 BDS w100 BDS Fluxa BDS ARMA BDS w100 harvest

KD

(nM

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αGalGal1Gal2Gal3Gal4αManMan2

Gal1 and Man2 show the highest bindingto gp120 proteins

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Product quality attributes• Yield• HCP• Potency

gp120 panel

RPL-Gal1 RPL-Man2 Optimized non-affinity

capture

Commercial broad spectrum

lectin

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UV 1_280 Cond Conc B pH Fraction

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Chromatogram obtained for TF binding onto Capto MMC (0.46cm IDx10cm BH)


68666462605856545250484644424038

680660640620600580560540520500480460440420400380360340320300280260240220200180160140120100806040200

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Step Buffer CV Flowrate(cm/h)

Sanitization 0.5M NaOH 5 300

Equilibration 25mM Tris, pH 7.2 8 300

Load clarified harvest, 2.5x diluted, pH 7.2 NA 150

EQ Wash 25mM Tris, pH 7.2 3 150Intermediate

Wash25mM Tris, 0.5M urea, pH

8.8 5 300

pH Wash 25mM Tris, pH 8.8 5 300

Elution 25mM Tris, 300mM NaCl, pH 8.8 10 300

Strip 25mM Tris, 2M NaCl, pH 9.0 5 300


Storage 0.1M NaOH 5 300

Zoomed Chromatogram (elution to salt strip)

31


80706050403020100-10

32003100300029002800270026002500240023002200210020001900180017001600150014001300120011001000900800700600500400300200100

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Chromatogram obtained for TF binding onto RPL-AGN immobilized onto Agarose (0.46cm IDx10cm BH)


605550454035302520

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1.C

.6

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.1



Equilibration 20 mM MES, 130 mM NaCl, 10 mM CaCl2, pH 7.0 10 300

Loading Unadjusted Harvest loaded to 1.5 mg/mLResin

NA 35 (17 min RT)

Wash 20 mM Tris, 130 mM NaCl, 10 mM CaCl2, pH 7.0 3 35

Elution 0.5M Mannose, 20 mM Tris, 130 mM NaCl, 10 mM CaCl2,

pH 7.05 150

Sugar Strip1M Mannose, 20 mM Tris, 130

mM NaCl, 10 mM CaCl2, pH 7.0

10 300

Strip 2M NaCl 10 300


Step Buffer 10 300

32


706050403020100-10

32003100300029002800270026002500240023002200210020001900180017001600150014001300120011001000900800700600500400300200100

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5250484644424038363432302826242220

600580560540520500480460440420400380360340320300280260240220200180160140120100806040200

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333231302928272625242322212019181716151413121110987654321

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Chromatogram obtained for TF binding onto RPL-Gal1 immobilized onto CNBrSepharose (0.46cm IDx10cm BH)

Zoomed Chromatogram (elution to salt strip) Step Buffer CV Flowrate

(cm/h)

Equilibration20 mM Tris, 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, pH

7.510 300

Loading Unadjusted Harvest to 1.5 mg/mLResin

NA 35 (17 min RT)

Wash20 mM Tris, 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, pH

7.5

3 35

5 150

Elution 0.5M Galactose, 20 mM Tris, 150 mM NaCl, 1 mM CaCl2, 1

mM MgCl2, pH 7.510 300

Strip 2M NaCl 5 300


7.510 300

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Chromatogram obtained for TF binding onto RPL-Man2 immobilized onto CNBr Sepahrose (0.46cm IDx10cm BH)


5452504846444240383634323028262422

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(elution to salt strip)



7.510 300


NA 35 (17min RT)

Wash 120 mM Tris, 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, pH

7.5

3 35

5 150

Elution 0.5M Mannose, 20 mM Tris,

150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, pH 7.5

10 300

Sugar Strip1M Mannose, 20 mM Tris,


5 300

Strip 2M NaCl 5 300


7.510 300

ForteBio™ Octet®

34

%RSD ≤10% %RSD ≤20%

Typical Run Time (1 sample)

Maximum Samples per day (8 hr run time)

Binding Potency Precision

≤$5 ~$150

384‐well capacity X ✓ X

Maintenance Minimal Minimal (15 minutes/month or after heavy use)

Moderate (20 minutes/week + 90 minutes/month)

Cost per sample

BLI/Octet SPR/BiacoreOvernight Plate Coating +

6.5 hours

~$10

Kinetics Precision N/A

ELISAParameter*Potency ≤ 30 mins. Kinetics ~35 min.

~100 minutes

***6**Potency = 176 Kinetics = 26

≤5

%RSD ≤30%? %RSD ≤10% N/A

Comparing Octet Apparent KD and Biacore® KD

Cameron, C.A. et al Development of BLI-Based Potency Assays as an Increased Throughput Alternative to SPR for the Analysis of In-Process and Drug Substance Samples; 2016 KBI Poster

TF CH106

35

0

0.1

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0 100 200 300 400 500 600 700 800 900

nm binding

shift (respo

nse)

Concentration (nM)

TF Dose Response Curve

TF HEK Reference

TF AGN

TF Gal1

TF Man2

TF MMC

TF IA2

36

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Bind

ing Re

spon

se (n

m)

Concentration (µg/mL)

TF Dose Response Binding with CH103_IA2

AGN Eluate 1

Gal 1 Eluate

Man 2 Eluate

MMC Eluate

TF VRC01

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ing Re

spon

se (n

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TF Dose Response Binding with VRC01

AGN Eluate 1

Gal 1 Eluate

Man 2 Eluate

MMC Eluate

TF IA3.2

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ing Re

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m)


TF Dose Response Binding with CH103_IA3.2

AGN Eluate 1

Gal 1 Eluate

Man 2 Eluate

MMC Eluate

TF

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TF Dose Response Curve

TF HEK Reference

TF AGN

TF Gal1

TF Man2

TF MMC

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TF Dose Response Binding with CH103_IA2

AGN Eluate 1

Gal 1 Eluate

Man 2 Eluate

MMC Eluate

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se (n

m)


TF Dose Response Binding with VRC01

AGN Eluate 1Gal 1 EluateMan 2 EluateMMC Eluate

0.0000

0.0500

0.1000

0.1500

0.2000

0.2500

0 50 100 150 200

Bind

ing Re

spon

se (n

m)


TF Dose Response Binding with CH103_IA3.2

AGN Eluate 1Gal 1 EluateMan 2 EluateMMC Eluate

Normalized Glycan TF

SampleName: Fluxa AGN elu SampleName: Fluxa Gal1 elu SampleName: Fluxa Man2 elu SampleName: Fluxa MMC elu

Minutes10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00 44.00 46.00 48.00 50.00 52.00

41

Loading (mg/mL

Resin)RP Yield% HCP (ppm)

AGN

1.77

25 NARPL-Gal1 57 <900RPL-Man2 40 <4000

Capto MMC 44 <1200

• Similar HCP levels after affinity and optimized non-affinity capture steps• Roughly comparable yields• Very similar binding activity to commercial lectin or non-affinity capture• Inactive/less active forms of the product are cleared on non-affinity capture

step on Capto MMC

42

Chromatogram obtained for w78 binding onto Capto MMC (0.46cm IDx10cm BH)


1101009080706050403020100-10

2100

2000

1900

1800

1700

1600

1500

1400

1300

1200

1100

1000

900

800

700

600

500

400

300

200

100

0

1601551501451401351301251201151101051009590858075706560555045403530252015105

ml

mAU mS/cm

Out

-Was

te

Out

-Was

te

^̂̂^

1.G

.12^

1.G

.10^

1.G

.8^

1.G

.6^

1.G

.4^^

1.G

.1^

Out

-Was

te

Out

-Was

te

Out

-Was

te


8886848280787674727068666462

480

460

440

420

400

380

360

340

320

300

280

260

240

220

200

180

160

140

120

100

80

60

40

20

0

-20

-40

38

36

34

32

30

28

26

24

22

20

18

16

14

12

10

8

6

4

2

Zoomed mode

ml

mAU mS/cm

Out

-Was

teFr

ac

^̂

1.H

.1

1.G

.12

1.G

.11

1.G

.10

1.G

.9

1.G

.8

1.G

.7

1.G

.6

1.G

.5

1.G

.4^

1.G

.2

1.G

.1^



Sanitization 0.5 N NaOH 3 300

Equilibration 50 mM MES, pH 6.3 8 300

Load Harvest, 3x diluted with WFI, pH 6.3 NA 150

Wash 1 50 mM MES, pH 6.3 3 150

Wash 2 50 mM HEPES, pH 6.9 5 300

Step Elution 50 mM Tris, 150 mM NaCl, pH 8.2 10 300

Strip 25 mM Tris, 2 M NaCl, pH 9.0 5 300

Sanitization 0.5 N NaOH 5 300

Storage 0.1 N NaOH 5 300

43

Chromatogram obtained for w78 binding onto RPL-AGN immobilized onto Agarose (0.46cm IDx10cm BH)




NA 35 (17 min RT)



pH 7.05 150



10 300



Step Buffer 10 300

UV 1_280 UV 2_320 Cond Conc B pH Fraction

9080706050403020100-10

330032003100300029002800270026002500240023002200210020001900180017001600150014001300120011001000900800700600500400300200100

0-100

1551501451401351301251201151101051009590858075706560555045403530252015105

ml

mAU mS/cm

Out

-Was

te

Out

-Was

te

^̂

1.C

.6^

1.C

.4^

1.C

.2^^

1.B

.11^

1.B

.9^

1.B

.7^

1.B

.5^

1.B

.3^^

1.A

.12^

1.A

.10^

1.A

.8^

1.A

.6^

1.A

.4^^

1.A

.1

Out

-Was

te

Out

-Was

te


7065605550454035

380

360

340

320

300

280

260

240

220

200

180

160

140

120

100

80

60

40

20

0

-20

-40

-60

-80

-100

-120

-140

-160

24

23

22

21

20

19

18

17

16

15

14

13

12

11

10

9

8

7

6

5

4

3

2

1

Zoomed mode

ml

mAU mS/cm

1.C

.5

1.C

.4

1.C

.3

1.C

.2^

1.B

.12

1.B

.11

1.B

.10

1.B

.9

1.B

.8

1.B

.7

1.B

.6

1.B

.5

1.B

.4

1.B

.3^

1.B

.1

1.A

.12

1.A

.11

1.A

.10

1.A

.9

1.A

.8

1.A

.7

1.A

.6

1.A

.5

Elution


Heterogeneous elution observed for AGN

44

Chromatogram obtained for w78 binding onto RPL-Gal1 immobilized onto CNBrSepahrose (0.46cm IDx10cm BH)



7.510 300


NA 35 (17 min RT)


7.5

3 35

5 150


mM MgCl2, pH 7.510 300

Strip 2M NaCl 5 300


7.510 300


706050403020100-10

330032003100300029002800270026002500240023002200210020001900180017001600150014001300120011001000900800700600500400300200100

0-100

1551501451401351301251201151101051009590858075706560555045403530252015105

ml

mAU mS/cm

Out

-Was

te

Out

-Was

te

^̂

1.E

.2^

1.D

.12^

1.D

.10^^

1.D

.7^

1.D

.5^

1.D

.3^

1.D

.1^

1.C

.11^^

1.C

.8

Out

-Was

te

Out

-Was

te


5452504846444240383634323028

500480460440420400380360340320300280260240220200180160140120100

80604020

0-20-40-60-80

-100-120-140-160

29

28

27

26

25

24

23

22

21

20

19

18

17

16

15

14

13

12

11

10

9

8

7

6

5

4

3

2

1

Zoomed mode

ml

mAU mS/cm

1.E

.2

1.E

.1

1.D

.12

1.D

.11

1.D

.10^

1.D

.8

1.D

.7

1.D

.6

1.D

.5

1.D

.4

1.D

.3

1.D

.2

1.D

.1

1.C

.12

1.C

.11^

1.C

.9

1.C

.8Zoomed Chromatogram (elution to salt strip)

45

Chromatogram obtained for w78 binding onto RPL-Man2 immobilized onto CNBr Sepahrose (0.46cm IDx10cm BH)



7.510 300


NA 35 (17min RT)


7.5

3 35

5 150



10 300



5 300

Strip 2M NaCl 5 300


7.510 300


80706050403020100-10

3400330032003100300029002800270026002500240023002200210020001900180017001600150014001300120011001000

900800700600500400300200100

0

1551501451401351301251201151101051009590858075706560555045403530252015105

ml

mAU mS/cm

Out

-Was

te

Out

-Was

te

^̂

1.C

.1^

1.B

.11^

1.B

.9^^

1.B

.6^

1.B

.4^̂

1.B

.1^

1.A

.11^

1.A

.9^

1.A

.7^

1.A

.5^̂

1.A

.2^

Out

-Was

te

Out

-Was

te


6560555045403530

380

360

340

320

300

280

260

240

220

200

180

160

140

120

100

80

60

40

20

0

-20

-40

-60

21

20

19

18

17

16

15

14

13

12

11

10

9

8

7

6

5

4

3

2

1

Zoomed mode

ml

mAU mS/cm

Out

-Was

te

^̂

1.C

.1

1.B.

12

1.B.

11

1.B.

10

1.B.

9^

1.B.

7

1.B.

6

1.B.

5

1.B.

4

1.B.

3^

1.B.

1

1.A.

12

1.A.

11

1.A.

10

1.A.

9

1.A.

8

1.A.

7

1.A.

6

1.A.

5

1.A.

4^

1.A.

2


W78 CH106

46

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0 100 200 300 400 500 600 700 800 900

nm binding

shift (respo

nse)

Concentration (nM)

w78 Dose Response Curve

w78 HEK Reference

w78 AGN

w78 Gal1

w78 Man2

w78 MMC

W78 IA2

47

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0 10 20 30 40 50 60 70 80 90 100

Bind

ing Re

spon

se (n

m)


w78 Dose Response Binding with CH103_IA2

AGN Eluate 1

Gal 1 Eluate

Man 2 Eluate

MMC Eluate

48

Loading (mg/mL


AGN

1.45

29 NARPL-Gal1 23 <2800RPL-Man2 36 <7500

Capto MMC 49 26722

• Improved HCP levels after affinity capture compared to Capto MMC process• Roughly comparable yields• Very similar binding activity to commercial lectin or non-affinity capture• Inactive/less active forms of the product are cleared on non-affinity capture

step on Capto MMC

9/20/201649

- Confidential



9080706050403020100-10

2100

2000

1900

1800

1700

1600

1500

1400

1300

1200

1100

1000

900

800

700

600

500

400

300

200

100

0

1601551501451401351301251201151101051009590858075706560555045403530252015105

ml

mAU mS/cm

Out

-Was

te

Out

-Was

te

^̂̂

1.C

.1^

1.B

.11^

1.B

.9^

1.B

.7^

1.B

.5^̂

1.B

.2^̂

Out

-Was

te

Out

-Was

te

Out

-Was

te


747270686664626058565452504846

580560540520500480460440420400380360340320300280260240220200180160140120100

806040200

-20-40

46

44

42

40

38

36

34

32

30

28

26

24

22

20

18

16

14

12

10

8

6

4

2

Zoomed mode

ml

mAU mS/cm

Out

-Was

teFr

ac

^̂

1.C

.1

1.B

.12

1.B

.11

1.B

.10

1.B

.9

1.B

.8

1.B

.7

1.B

.6

1.B

.5

1.B

.4^

1.B

.2

1.B

.1^

Zoomed Chromatogram (elution to salt strip) Step Buffer CV Flowrate

(cm/h)


Equilibration 50mM AcOH, pH 5 8 300

Load Clarified Harvest, 3x diluted with EQ buffer NA 150

EQ Wash 50mM AcOH, pH 5 3 150

Wash 50mM MES, pH 6 5 300

Elution 50mM MES, 230mM NaCl, pH 6.5 10 300

Strip 25mM Tris, 2M NaCl, pH 9.0 5 300


Store 0.1M NaOH 5 300

9/20/201650

- Confidential




Equilibration 50mM acetate, pH 5.0 8 300

Load Clarified Harvest 2x diluted with WFI and adjusted to pH 5.0 NA 150

EQ Wash 50mM acetate, pH 5.0 5 150

Wash 50mM acetate, 130mM NaCl, pH 5.0 5 300

Elution 50mM acetate, 250mM NaCl, pH 5.0 10 300

Strip 2M NaCl 5 300


Store 0.1M NaOH 5 300


1009080706050403020100-10-20

2100

2000

1900

1800

1700

1600

1500

1400

1300

1200

1100

1000

900

800

700

600

500

400

300

200

100

0

1551501451401351301251201151101051009590858075706560555045403530252015105

ml

mAU mS/cm

Out

-Was

te

Out

-Was

te

^̂̂^

1.C

.2^

1.B

.12^

1.B

.10^

1.B

.8^

1.B

.6^

1.B

.4

Out

-Was

te

Out

-Was

te

Out

-Was

te


7068666462605856545250

500

480

460

440

420

400

380

360

340

320

300

280

260

240

220

200

180

160

140

120

100

80

60

40

20

0

-20

38

36

34

32

30

28

26

24

22

20

18

16

14

12

10

8

6

4

Zoomed mode

ml

mAU mS/cm

Frac

^̂

1.C

.3

1.C

.2

1.C

.1

1.B

.12

1.B

.11

1.B

.10

1.B

.9

1.B

.8

1.B

.7

1.B

.6

1.B

.5

1.B

.4


9/20/201651

- Confidential

Chromatogram obtained for w53 binding onto RPL-AGN immobilized onto Agarose (0.46cm IDx10cm BH)




NA 35 (17 min RT)



pH 7.05 150



10 300



Step Buffer 10 300

Heterogeneous elution observed for AGN


80706050403020100-10

330032003100300029002800270026002500240023002200210020001900180017001600150014001300120011001000

900800700600500400300200100

0-100

1551501451401351301251201151101051009590858075706560555045403530252015105

ml

mAU mS/cm

Out

-Was

te

Out

-Was

te

^̂

1.C

.6^

1.C

.4^

1.C

.2^^

1.B

.11^

1.B

.9^

1.B

.7^

1.B

.5^

1.B

.3^^

1.A

.12^

1.A

.10^

1.A

.8^

1.A

.6^

1.A

.4^^

1.A

.1

Out

-Was

te

Out

-Was

te


6055504540353025

46

44

42

40

38

36

34

32

30

28

26

24

22

20

18

16

14

12

10

8

6

4

2

29

28

27

26

25

24

23

22

21

20

19

18

17

16

15

14

13

12

11

10

9

8

7

6

5

4

3

2

1

0

Zoomed mode

ml

% mS/cm

1.C

.5

1.C

.4

1.C

.3

1.C

.2^

1.B.

12

1.B.

11

1.B.

10

1.B.

9

1.B.

8

1.B.

7

1.B.

6

1.B.

5

1.B.

4

1.B.

3^

1.B.

1

1.A.

12

1.A.

11

1.A.

10

1.A.

9

1.A.

8

1.A.

7

1.A.

6

1.A.

5

1.A.

4^


Elution

9/20/201652

- Confidential

Chromatogram obtained for w53 binding onto RPL-Gal1 immobilized onto CNBrSepahrose (0.46cm IDx10cm BH)



7.510 300


NA 35 (17 min RT)


7.5

3 35

5 150


mM MgCl2, pH 7.510 300

Strip 2M NaCl 5 300


7.510 300


706050403020100-10

330032003100300029002800270026002500240023002200210020001900180017001600150014001300120011001000

900800700600500400300200100

0

1551501451401351301251201151101051009590858075706560555045403530252015105

ml

mAU mS/cm

Out

-Was

te

Out

-Was

te

^̂

1.G

.4^

1.G

.2^

1.F.

12^^

1.F.

9^

1.F.

7^

1.F.

5^

1.F.

3^

1.F.

1^^

1.E

.10

Out

-Was

te

Out

-Was

te


4846444240383634323028262422

480

460

440

420

400

380

360

340

320

300

280

260

240

220

200

180

160

140

120

100

80

60

40

20

0

-20

-40

-60

25

24

23

22

21

20

19

18

17

16

15

14

13

12

11

10

9

8

7

6

5

4

3

2

Zoomed mode

ml

mAU mS/cm

1.G

.4

1.G

.3

1.G

.2

1.G

.1

1.F.

12^

1.F.

10

1.F.

9

1.F.

8

1.F.

7

1.F.

6

1.F.

5

1.F.

4

1.F.

3

1.F.

2

1.F.

1^

1.E

.11

1.E

.10


9/20/201653

- Confidential

Chromatogram obtained for w53 binding onto RPL-Man2 immobilized onto CNBr Sepahrose (0.46cm IDx10cm BH)



7.510 300


NA 35 (17min RT)


7.5

3 35

5 150



10 300



5 300

Strip 2M NaCl 5 300


7.510 300


80706050403020100-10

330032003100300029002800270026002500240023002200210020001900180017001600150014001300120011001000

900800700600500400300200100

0-100

1551501451401351301251201151101051009590858075706560555045403530252015105

ml

mAU mS/cm

Out

-Was

te

Out

-Was

te

^̂

1.E

.8^

1.E

.6^

1.E

.4^^

1.E

.1^

1.D

.11^̂

1.D

.8^

1.D

.6^

1.D

.4^

1.D

.2^

1.C

.12^̂

1.C

.9^

Out

-Was

te

Out

-Was

te


6055504540353025

360

340

320

300

280

260

240

220

200

180

160

140

120

100

80

60

40

20

0

-20

-40

-60

-80

-100

-120

22

21

20

19

18

17

16

15

14

13

12

11

10

9

8

7

6

5

4

3

2

1

Zoomed mode

ml

mAU mS/cm

Out

-Was

te

^̂

1.E.

8

1.E.

7

1.E.

6

1.E.

5

1.E.

4^

1.E.

2

1.E.

1

1.D

.12

1.D

.11

1.D

.10^

1.D

.8

1.D

.7

1.D

.6

1.D

.5

1.D

.4

1.D

.3

1.D

.2

1.D

.1

1.C

.12

1.C

.11^

1.C

.9

1.C

.8


w53 CH106

54

0.0000

0.1000

0.2000

0.3000

0.4000

0.5000

0.6000

0.7000

0 100 200 300 400 500 600 700 800 900

Bind

ing Re

spon

se (n

m)

Concentration (nM)


HEK Reference

AGN Eluate 1

Man 2 Eluate 1

Gal 1 Eluate

MMC Eluate

Fractogel Eluate

w53 IA2

55

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0 10 20 30 40 50 60 70 80

Bind

ing Re

spon

se (n

m)



AGN Eluate 1

Gal 1 Eluate

Man 2 Eluate

Fractogel Eluate

w53 VRC01

0.0000

0.1000

0.2000

0.3000

0.4000

0.5000

0.6000

0 10 20 30 40 50 60 70 80

Bind

ing Re

spon

se (n

m)


w53 Dose Response Binding with VRC01

AGN Eluate 1

Gal 1 Eluate

Man 2 Eluate

Fractogel Eluate

w53 IA3.2

0.0000

0.0500

0.1000

0.1500

0.2000

0.2500

0 10 20 30 40 50 60 70 80

Bind

ing Re

spon

se (n

m)


w53 Dose Response Binding with CH103_IA3.2

AGN Eluate 1

Gal 1 Eluate

Man 2 Eluate

Fractogel Eluate

w53

58

0.0000

0.1000

0.2000

0.3000

0.4000

0.5000

0.6000

0.7000

0 100 200 300 400 500 600 700 800 900

Bind

ing Re

spon

se (n

m)

Concentration (nM)


HEK Reference

AGN Eluate 1

Man 2 Eluate 1

Gal 1 Eluate

Fractogel Eluate

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0 20 40 60 80

Bind

ing Re

spon

se (n

m)



AGN Eluate 1

Gal 1 Eluate

Man 2 Eluate

Fractogel Eluate

0.0000

0.1000

0.2000

0.3000

0.4000

0.5000

0.6000

0 10 20 30 40 50 60 70 80

Bind

ing Re

spon

se (n

m)


w53 Dose Response Binding with VRC01

AGN Eluate 1Gal 1 EluateMan 2 EluateFractogel Eluate

0.0000

0.0500

0.1000

0.1500

0.2000

0.2500

0 20 40 60 80

Bind

ing Re

spon

se (n

m)


w53 Dose Response Binding with CH103_IA3.2

AGN Eluate 1Gal 1 EluateMan 2 EluateFractogel Eluate

Normalized Glycan w53

SampleName: w53 Man2 elu SampleName: w53 Gal1 elu SampleName: w53 fractogel elu

Minutes10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00 44.00 46.00 48.00 50.00 52.00

60

Loading (mg/mL


AGN

1.01

21 NARPL-Gal1 42 12174RPL-Man2 29 <10000

Capto MMC 97 112714Fractogel SO3- 29 26281

• Similar HCP levels after affinity and optimized non-affinity capture steps• Roughly comparable yields• Very similar binding activity to commercial lectin or non-affinity capture• Inactive/less active forms of the product are cleared on non-affinity capture

step on Fractogel SO3

Summary of binding activity for Env proteins

UCA IA3.2 1A2 CH106TF + + NT +W53 - + NT +W78 - +

(weak)+ +

W100 - NT + +ARMA - + NT +

61

Summary• Non-affinity platform process has enabled progression of

gp120s into the clinic• RPLs can serve as a generic affinity capture step for

highly glycosylated proteins• Very comparable binding activity and high purity obtained

without any optimization on RPL affinity chromatography• RPLs are shown to be less heterogeneous in binding and

will have greater lot-to-lot reproducibility given recombinant production

• Useful tool for lab-bench and larger-scale purification of gp120 and gp140 based proteins

Acknowledgments

Duke Human Vaccine Institute: Dr. Barton Haynes, Prof. Thomas Denny, Dr. Munir Alam and team

Dr. Gerry Kovacs and team

Dr. Michael Pensiero and team

Cell line development, Upstream & Downstream PDAnalytical development, Formulation Development, cGMP Manufacturing, QA/QC

Dr. Paul ClarkeDr. Roisin Thompson

evaluation of recombinant prokaryotic lectins (rpls) for ... shukla 15... · evaluation of...

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