Antigen and antibody development using Octet Biolayer ... · Antigen and antibody development for...

Antigen and antibody

development for IVD

immunoassay using Octet

Biolayer Interferometry

Néstor Santiago Ph.D.

R&D Scientist. Biochemistry section

Biokit R&D

Pall-FortèBio User Meeting - Lyon 04 June 2015

Living Immunoassay Excellence

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Presentation overview


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1. Biokit R&D. Company presentation

2. Introduction

3. Objective

4. Results

5. Conclusions


Biokit Research and Development

Company presentation


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Biokit R&D. Presentation


• Barcelona-based In Vitro Diagnostics (IVD) company created in 1973

• Diagnostics specialization

- infectious diseases & serology

- coagulation

• Focus on Immunoassay

• Global presence

• One of the Werfen companies

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Differential Core Capabilities


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BIO-FLASH ®

Bio-technology

Product & Process

Innovation


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One Core Focus, Three Research Lines


Immunoassay

Excellence Infectious

Serology

Blood Bank

Screening

OEM Contract

Development

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Technologies


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1. Technologies oriented to the achievement of antigens and antibodies:

Viral, bacterial and parasitological culture and purification

Synthetic peptides and recombinant antigen production and purification

Rabbit, goat, guinea pig and lamb polyclonal antibody production and purification

Hybridoma culture and monoclonal antibody production and purification

Recombinant antibody production and purification

2. Technologies oriented to the production of final reagent kits:

Latex particle agglutination (on slide and turbidimetric tests)

Hemagglutination

Enzyme immunoassay (ELISA) & Western blot

Chemiluminescence


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Technologies


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1. Technologies oriented to the achievement of antigens and antibodies:

Viral, bacterial and parasitological culture and purification

Synthetic peptides and recombinant antigen production and purification

Rabbit, goat, guinea pig and lamb polyclonal antibody production and purification

Hybridoma culture and monoclonal antibody production and purification

Recombinant antibody production and purification

2. Technologies oriented to the production of final reagent kits:

Latex particle agglutination (on slide and turbidimetric tests)

Hemagglutination

Enzyme immunoassay (ELISA) & Western blot

Chemiluminescence


Introduction


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Immunoassay


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ANTIBODY

ANTIGEN


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Immunoassay formats


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ELISA

CHEMILUMINESCENCE

TURBIDIMETRY


Objective


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Objective


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Produce recombinant antibody – antigen pair for immunoassay development.


Results


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Recombinant antibody production


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mAb anti-A Hybridoma produced monoclonal

antibody

rIgG anti-A Recombinant IgG produced as two

peptidic chains in HEK293F cells

Fab anti-A Fab fragment generated by papain

digestion of mAb

scFab anti-A Recombinant Fab fragment

produced as a single peptide in

HEK293F cells

scrIgG anti-A Recombinant

IgG produced as a single peptide in

HEK293F cells

scFv-Fc anti-A Recombinant Fc fused scFv

fragment produced as a single

peptide in CHOK1 cells


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Activity assessment. Kinetics assay


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Octet kinetics analysis. 6 ug/mL Antigen A were amine coupled onto amine reactive (AR2G) biosensors at pH6.

anti-A antibodies were analysed in triplicate at five different 3-fold dilutions.

All recombinant antibody formats produced in HEK293F cells bind antigen A.

Sample ID KD (nM) KD Error kon(1/Ms) kon Error kdis(1/s) kdis Error Full X^2 Full R^2

mAb anti-A 0.59 0.008 3.34E+05 1.97E+03 1.97E-04 2.55E-06 1.620 0.995

scrIgG anti-A 2.27 0.039 7.83E+04 5.18E+02 1.78E-04 2.81E-06 1.494 0.994

scFv-Fc anti-A 0.34 0.010 3.27E+05 2.05E+03 1.12E-04 3.06E-06 2.729 0.991

Fab anti-A 5.07 0.042 1.02E+05 5.90E+02 5.17E-04 3.13E-06 0.702 0.992

scFab anti-A 10.43 0.122 8.01E+04 7.90E+02 8.35E-04 5.21E-06 0.567 0.982

Antigen A

AR2G

sensor

mAb anti-A

scrIgG anti-A scFab anti-A scFv-Fc anti-A

Fab anti-A


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Synthetic peptide design


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Antigen A A-P4 peptide Synthetic peptide comprising

mAb anti-A epitope sequence

in antigen A

B

A synthetic peptide (A-P4 peptide) was designed based on the epitope

sequence mapped on Antigen A.


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Synthetic peptide binding study


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mAb

scrIgG

rIgG

Fab

scFab Lot.A

scFab Lot.B

A-P4

None of the different recombinant antibody formats produced

in HEK293 cells binds A-P4 peptide.

Streptavidin

(SA)

biosensor

BA-P4 peptide

A-P4 peptide binding assessment in Octet® system. Biotinylated A-P4 peptide was immobilized onto SA (streptavidin coated) biosensors at 15µg/mL.

100nM (IgG formats) or 300nM (Fab formats) antibody solution was tested for binding.

mAb Fab rIgG scrIgG scFab


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What is going on?


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mAb rIgG

?

rIgG mAb

HEK293F Hybridoma

SDS-PAGE analysis

Differences in Light Chain M.W. are observed between mAb and rIgG anti-A.


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Mass spectrometry analyses were carried out in the Proteomics facility from UAB.

Heavy Chain Mass Spectra

Peptide Mass Fingerprinting (PMF)

mAb and rIgG share same aminoacid sequence.

mAb

rIgG


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VL CL

rIgG LC NH2 COOH

Peptide Mass Fingerprinting (PMF)

… …

Glycosylation site

LC1 peptides LC2 peptides

CDR1 CDR2 CDR3

A theoretical glycosylation site has been identified in CDR1 region of the

antibody light chain.


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Antibody structure


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http://www.novimmune.com/science/antibodies.html TM & © Novimmune SA.

http://www.accessexcellence.org/RC/VL/GG/ecb/ecb_images/04_32_antibody.jpg

Addition of sugars in CDR1 region of the antibody light chain might be

affecting antigen binding.

http://www.novimmune.com/science/antibodies.html

http://www.novimmune.com/science/antibodies.html




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Sugars make the difference

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97

64

39

51

28

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M.W.

kDa PNGase F

Reaction mix

- - + - - +

- + + - + +

rIgG mAb

Deglycosylation reaction.

SDS-PAGE analysis of rIgG and mAb anti-A. Samples were

non-treated; underwent incubation in presence of reaction mix

with or without PNGase F (N-glycosydase).

rIgG light chain is highly glycosylated.


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Hybridoma

mAb anti-A

HEK293F

rIgG anti-A

CHOK1

scFv-Fc anti-A

Binding studies in Octet® system. A. 10ug/mL mAb , rIgG or scFv-Fc anti-A were amine coupled onto AR2G biosensors at pH4. 100nM Antigen A solution was tested for binding.

B. Biotinylated A-P4 peptide was immobilized onto SA (streptavidin coated) biosensors at 15µg/mL. 100nM mAb, rIgG or 1µM scFv-Fc anti-A

solution was tested for binding.

Antibody production system comparison

Antigen A

A A-P4 peptide

B

CHOK1 produced recombinant anti-A antibody binds synthetic A-P4 peptide.

AR2G

biosensor

Antigen A

A

B SA

biosensor

B

A-P4

peptide


Conclusions


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Conclusions


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• A set of different anti-antigen A recombinant antibody formats have been

produced in HEK293F cells

• Synthetic peptides based on natural antigen A bind to antibodies produced in

hybridoma cells but not to recombinant antibodies produced in HEK293 cells.

• Natural and recombinant anti-A antibodies share the same amino acid

sequence.

• Glycosilation of N50 in light chain CDR1 region of recombinant antibodies

produced in HEK293 cells could explain differences in specificity.

• A recombinant antibody format produced in CHOK1 cells binds antigen A

derived peptides.

• Inconvenient recombinant antibody glycosylation could be overcome using

CHOK1 cells as production system instead of HEK293F cells.

Thank you!


Date post:	27-Jul-2019
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