Multiplexing Immunogenicity Assays with the MSD … · Multiplexing Immunogenicity Assays with the MSD U-PLEX

Slide 1© 2015 Meso Scale Diagnostics, LLC. All rights reserved.

Multiplexing Immunogenicity Assays with the MSD U-PLEX® Platform

Dr. Mark Dürr

Senior Application Scientist – Central Europe

Meso Scale Discovery

[email protected]

Biologics & Biosimilars Congress 2016, Berlin


Once upon a time…

• Biotherapeutics are increasing in complexity

• Testing domain specificity of ADAs elicited by multi-domain

therapeutics may be required depending on risk factors

• Multiple PK assays may be required

Then

MAb

Now…

Pegylated ADCBi-specific BiosimilarFusion


Multiple Bioanalytical Assays for the Same Samples

Multiple drugs

• Combination therapies

• Originator and biosimilar

Epitope specificity

• Drug conjugates

• Pegylated drugs

• Multi-domain therapeutics

Identification interference

• Drug Interference

• False bridging

Immunogenicity

• ADAs to one or more

therapeutics?

• Do the ADAs recognize a

biosimilar or an originator drug?

• Is one domain of a multi-domain

therapeutic more immunogenic?

• Which functional domain is

impacted by a NAb?

PK

• Multiple drugs

• Drug domains or

fragments


Multiplexing

Sample conservation (1-25 µL)

Increased throughput

The MSD Advantage

Large dynamic range

Ease of use

High sensitivity

vs.

0.01 0.1 1 10 100 1000 10000100

1000

10000

100000

1000000

IFN

IL-1

IL-2

IL-4

IL-6

IL-8

IL-10

IL-12p70

IL-13

TNF

Concentration (pg/mL)

Sig

nal

MULTI-SPOT 96-well 7-Spot Plate


384 well plate:

1 or 4 assays/well

Multiplex Assays

Working electrode

“spot”

Counter electrode

Dielectric

Support

1 2

A

B

Working electrode

“spot”

Counter electrode

Dielectric

1 2

A

B

4

7

10

96 well plate

1, 4, 7 or 10 assays/well

1

Manufactured using deposition of conducting and insulating layers

Spatially independent (rigorously defined)

Specific reagents are deposited on each “spot”

Automated image analysis

96 and 384 well options

90 seconds or less to read a whole plate

Positional Multiplexing, not

based upon different

wavelengths of fluorophores.

MULTI-SPOT 96-well 7-Spot Plate


Multiplexed Antibody Detection Assays

Anti-species detection

®

No chemical modification of materials coated on plate

Reduction in amount of material required to coat plates

Increased throughput

Reduced sample requirements


MSD Applications for Therapeutic Proteins

• Immunogenicity• High sensitivity

• Large dynamic range

• Drug tolerance

• Neutralizing Ab response• Binding assays

• Cell binding assays

• Receptor phosphorylation assays

• Intracellular signaling markers

• Cytokine Assays

• PK/DM• High sensitivity

• Large dynamic range

• Mechanism of Action• Intracellular signaling

• Secreted markers

• Cell binding

• PD• Intracellular signaling

• Secreted markers

• Product development

and release testing• Quantitation

• Isotyping

• Potency

• Host cell Protein

• Protein A


The U-PLEX® Technology Platform

Design and build a personalized multiplex quickly and easily with the assays that are right for the your study.

Create custom combinations from a selection of MSD®

assays, your own in-house antibodies, or both.

Easy workflow and simple protocol similar to ELISA; no spotting required.

Compatible with all MSD instruments and software.

Open platform: compatible with antibodies, proteins, peptides, carbohydrates, nucleic acids, and oligonucleotides.

Flexibility that empowers you to make personalized multiplex assay combinations quickly

and easily.


• U-PLEX plates

• Up to 10 spots, each spot activated to

capture a specific U-PLEX linker

(represented by color)

• U-PLEX linkers

• 10 linkers, each designed to bind to a

specific spot on the U-PLEX plate

• U-PLEX reagents

• Assay reagents (e.g., MSD GOLD SULFO-TAG™ NHS-Ester)

• Antibodies, Calibrators

• Diluents

• Read Buffer T (4X)

U-PLEX Fundamentals


U-PLEX Linker Coupling

Biotin-binding domain

Matched linker

and spot

One spot of a plate


• Select your analytes and choose the

appropriate antibodies

• Combine the antibodies to make one solution

• Up to 10 U-PLEX-coupled antibodies can

be pooled

• Do not combine U-PLEX-coupled

antibody solutions that share the same

linker

• Final solution is used to coat plates

• One step, not 10

• Antibodies bind to their spots on the plate from

solution

Combine U-PLEX-Coupled Antibodies


Multiplexing with U-PLEXTM Products

• U-PLEX plates

• 10-spot plates activated to capture specific

U-PLEX linkers (represented by color)

• Linker-Coupled Capture

• Biotinylated molecules coupled to U-PLEX

linkers

• Add 50 mL of solution and the coupled

antibodies bind to the correct spot

U-PLEX PlateU-PLEX Linker

Coupled Antibodies


Antibody Assays on the MSD Platform

ADA

SULFO-TAG™

drug/antigen

Biotin-drug/antigen

Bridging format

Used for more than 95% of Immunogenicity

assays for monoclonal antibody therapeutics

• Not species specific

• Detects multiple isotypes

• High tolerance to free drug

• Detect low affinity antibodies

• High matrix tolerance

• Rapid

• Sensitive

ADA

SULFO-TAG™ Anti-

species antibody

Drug/ Antigen

Anti-species / Direct format

Used for more than 90% of serology/vaccine

assays

Useful for drugs that are challenging to

conjugate, drugs that can’t be modified or do

not bridge

Species and isoptype specificity governed by

detection reagents

Streptavidin plate Bare plate


Bridging Immunogenicity Assays with U-PLEX

• Same reagents as used for single assays with MSD Streptavidin plates

• Requirements: 1 – 2 µg biotinylated / SULFO-TAG drug per plate

SULFO-TAG

drug 1SULFO-TAG

drug 2

Biotin drug 1 Biotin drug 2

Mastermix 1

Couple to Linker 1

Mastermix 2

Couple to Linker 10

Linker 1 Linker 10

Couple each Master mix with a U-PLEX linker



SULFO-TAG

drug 1 SULFO-TAG

drug 2


Mastermix 1

Coupled to Linker 1

Mastermix 2

Coupled to Linker 10

Linker 1 Linker 10

Combine U-PLEX Coupled Mastermixes Incubate with Samples

SULFO-TAG

drug 1 SULFO-TAG

drug 2


Mastermix 1

Coupled to Linker 1

Mastermix 2


Linker 1 Linker 10

ADA 1 ADA 2



Incubate with Samples

SULFO-TAG

drug 1 SULFO-TAG

drug 2


Mastermix 1

Coupled to Linker 1

Mastermix 2


Linker 1 Linker 10

ADA 1 ADA 2

Transfer to U-PLEX plate

• Incubate 1h

• Wash

• Add Read Buffer

• Read plate (90 seconds)

1

10

SULFO-TAG

drug 1

Biotin drug 1

Linker 1

ADA 1

SULFO-TAG

drug 2

Biotin drug 2

Linker 10

ADA 2

SPOT 1 SPOT 10


Example

0

2000

4000

6000

8000

10000

12000

14000

16000

1000 500 250 125 1000 500 250 125

Spot 1 Drug A Spot 10 Drug B

SIG

NA

L Dual Sp.

A Specific

B Specific

Neg. C.

ng/mL ADA

• U-PLEX Immunogenicity assay for Drug A and Drug B.

• Titration of four control ADAs tested

SPOT 1 DRUG A SPOT 10 DRUG B

Control

ADA


AAPS 2015 Posters

MSD U-PLEX® Technology Differentially Measures Domain-Specific Anti-Drug Antibodies

J. Postelnek 1, H. Myler 1, R. Santockyte 1, D. Georlette 2, P. Oberoi 2, S. P. Piccoli 1

1 Bristol-Myers Squibb Company, 2 Meso Scale Discovery

• U-PLEX to differentially measure domain specific anti-drug antibodies (ADA) against polyethylene

glycol (PEG; 20 kDa linear and 40 kDa branched), interferon lambda 1a, or interferon alfa-2a using

positive controls and clinical samples.

• High degree of comparability with singleplex assays

Evaluation of a Multiplexed Anti Drug Antibody (ADA) Assay for Immunogenicity Testing in

Support of Combination Biologic Therapies

P. R. Conliffe 1, M. Ourmanova 1, I. DuBey 1, A. Chow 2, L. Luo 3, P. Oberoi 2, R. Pillutla 3

1 Smithers Avanza, 2 Meso Scale Discovery, LLC, 3 Bristol-Myers Squibb Company

• Comparable performance with ADA specific detection to each biologics at levels similar to that of

the single assay. Drug tolerance assessment also shows comparable assay performance across

the two assays suggesting a potential savings in sample volume, analysis time and cost.


Multiplexing Possibilities – Epitope Specificity

SULFO-TAG

Domain 1

Biotin

Domain 1

ADA 1

SULFO-TAG

Domain 2

Biotin Domain

2

ADA 2

SULFO-TAG

Domain 3

Biotin

Domain 3

ADA 3

Multi-Domain

Therapeutic

ADA Epitope specificity:

• Drug conjugates

• Pegylated drugs



Multiplexing Possibilities – Drug Interference

1

10

SULFO-TAG

drug

Biotin Target

(Receptor)

Competitive format

SULFO-TAG drug

Biotin drug

ADA

Bridging format

Receptor spot

100

1000

10000

100000

0.01 0.1 1 10 100 1000

ADA conc., ng/ml

Ave. counts

High signal on the

interference spot

throughout the range of

ADA response with no Free

Drug

Streptavidin spot

100

1000

10000

100000

1000000

0.01 0.1 1 10 100 1000

ADA conc., ng/ml

Ave. counts

SPOT 1

SPOT 10

Linker 1

Linker 10


Multiplexing Possibilities – Drug Interference

1

10 SULFO-TAG

drug

Biotin Target

(Receptor)

drug

Competitive format

SULFO-TAG drug

Biotin drug

ADA

Bridging format

SPOT 1

SPOT 10

Receptor spot

0

10000

20000

30000

40000

50000

60000

70000

80000

90000

0.01 0.1 1 10 100

Free Drug conc., ug/ml

Ave. counts

0 ng/ml

0.32 ng/ml

8 ng/ml

40 ng/ml

200 ng/ml

Streptavidin spot

100

1000

10000

100000

0.01 0.1 1 10 100

Free Drug conc., ug/ml

Ave. counts

0 ng/ml

0.32 ng/ml

8 ng/ml

40 ng/ml

200 ng/ml

ADA Conc.

Linker 1

Linker 10

ADA Conc.

drug


Multiplexing Possibilities – Identification of False Bridging

1

10

SULFO-TAG

drug

Biotinylated

control Ab

SULFO-TAG drug

Biotin drug

ADA

Drug Specific ADA will not bridge with negative control antibody – no

significant signal on Spot 10

Signal on Spot 1 from Drug

Specific ADA

Linker 1

Linker 10


Multiplexing Possibilities – Identification of False Bridging

1

10

SULFO-TAG

drug

Biotinylated

control Ab

SULFO-TAG drug

Biotin drug

RF

RF

Linker 1

Linker 10

Rheumatoid Factor (RF) can bridge both drug and negative control antibody – signals on Spot 1

AND Spot 10


Multiple Bioanalytical Assays for the Same Samples

Multiple drugs

• Combination therapies

• Originator and biosimilar

Epitope specificity

• Drug conjugates

• Pegylated drugs


Identification interference

• Drug Interference

• False bridging

Immunogenicity

• ADAs to one or more

therapeutics?

• Do the ADAs recognize a

biosimilar or an originator drug

• Is one domain of a multi-domain

therapeutic more immunogenic?

• Which functional domain is

impacted by a NAb?

PK

• Multiple drugs

• Drug domains or

fragments


MSD for PK Assays

• Electrochemiluminescence provides a large dynamic range: allows for quantitation of

high and low drug concentrations with fewer sample dilutions

• Less sample required

• Flexible assay formats

• Matrix tolerance

Range of ELISA

Same capture and detection antibodies used in MSD and ELISA Assays


Validation and Quality Control of U-PLEX Linkers and Plates

• Specificity of Linkers and Plate

• U-PLEX Components (plate and linkers)

• Reproducibility (intra-lot, inter-lot, inter-linker)

• Linker to Linker

• Linker reproducibility (inter-vial, inter-lot, multiple operator, robustness)

• Plate reproducibility

• %CVs, binding capacity

• Quality control release specifications

• Real time stability of plates and linkers (room temperature and 2-8°C)

• Robustness to common additives and samples in assays


Plate and Linker Specificity

Specificity Testing

• Biotinylated monoclonal antibody coupled to

linker.

• 9 uncoupled linkers are combined with the

coupled linker in Stop Solution and coated on a

10-assay plate.

• SULFO-TAG Protein A/G is used to detect the

coated antibody.

• Signals from the uncoated spots are measured

relative to the coated spot.

• Procedure repeated for each spot - 90

combinations of non-specific interactions

measured in multiple replicates.


Plate and Linker Specificity Results

• Tested 20 batches of plates and 3 lots of

linkers. Non-specific interactions were

less than 0.06%.

• 90 non-specific conditions per 60 different

combinations of plates and linkers = 5400

combinations

Each color shows the histogram for 90

conditions for all three linkers (270 conditions).

The MSD release specification for spot-linker specificity is less than 0.12% for every

combination.


Plate Reproducibility: Intra-Plate and Inter-Plate

• A total of 22 batches of plates were produced and tested during validation.

• 6 plates were tested from each batch.

• Intra-plate and inter-plate reproducibility from all batches: % CV all below 5%. All batches

met specifications.


U-PLEX Assays and Assay Development

Human

• 40 Assays

• 9 popular assay combinations*

Non Human Primate

• 30 Assays

• 4 popular assay combinations

Mouse

• 14 Assays

• 2 popular assay combinations

Single analyte detection with U-PLEX Antibody Sets on MSD

GOLD™ Small Spot Streptavidin

Open Spots

• Develop in-house assays

• Combine with MSD assays for ultimate flexibility

Assays Assay Development

* Popular combinations of analytes grouped into separate Cat. Nos. for ordering convenience


U-PLEX Human, NHP and Mouse Groups


Detailed Information on U-PLEX and Custom Assay Design

Visit www.mesoscale.com/u-plex

http://www.mesoscale.com/u-plex


MESO SCALE DISCOVERY, MESO SCALE DIAGNOSTICS, MSD, MSD GOLD, DISCOVERY WORKBENCH, MULTI-ARRAY, MULTI-SPOT,

QUICKPLEX, SECTOR, SECTOR PR, SECTOR HTS, SULFO-TAG, U PLEX, S-PLEX, V PLEX, STREPTAVIDIN GOLD, MESO, Parsec,

www.mesoscale.com, SMALL SPOT (design), 96 WELL 1, 4, 7, & 10-SPOT (designs), 384 WELL 1 & 4-SPOT (designs), MSD (design), U-PLEX

(design), S-PLEX (design), V PLEX (design), and SPOT THE DIFFERENCE are trademarks and/or service marks of Meso Scale Diagnostics, LLC.

©2015 Meso Scale Diagnostics, LLC. All rights reserved.


Supplemental Slides


Linker Reproducibility: Inter-Vial

• Inter-vial linker reproducibility was

assessed by testing 22 vials of each

linker from the beginning, middle and

end of the linker vialing run.

• All 10 linkers passed specifications

with 22 vials showing < 10% CV.

• Typical vials are within 5% of the

mean signals.

• Representative data are shown on

the right.

Linker 9

100%

100%

99%

100%

101%

99%

97%

102%

102%

60%

70%

80%

90%

100%

110%

120%

130%

140%

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

No

rma

lize

d S

ign

al

Vial number

Pass Marginal Pass Test anomaly, CV > 0.0% Out of range

Linker 10

101%

96%

99%

101%

99%

103%

102%

97%

98%

60%

70%

80%

90%

100%

110%

120%

130%

140%

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

No

rma

lize

d S

ign

al

Vial number

Pass Marginal Pass Test anomaly, CV > 0.0% Out of range


• Couple:

• Combine antibodies and linkers

• No purification of reagents

• No complex washing

• Combine coupled antibodies

• Coat:

• Simple 50 mL addition to plates

• Incubate for 1 hour to overnight

• Plates are ready to use or store for 7 days

• Complete (Run Assay):

• Add 25–50 mL of samples for 1 hour

• Add 25 mL of detection antibody for 1 hour

• Read

Build Personalized Multiplexes in 3 Simple Steps


U-PLEX Development Packs

• U-PLEX Development Packs are open platforms

for assay development• No MSD Assays

• From 2 to 10 activated spots depending on your need

• Each Development Pack comes with the following

reagents:• U-PLEX Plate with up to 10 activated spots

• Up to 10 U-PLEX Linkers

• U-PLEX Stop Solution

• Read Buffer

• Additional reagents required: • Biotinylated capture antibody (or other suitable reagents)

• A detection reagent labeled with MSD GOLD SULFO-TAG

NHS-Ester

Stop Solution and Read Buffer

10-U-PLEX Linkers


Plates and Linkers Designed for Optimal Multiplexing

• Plates have activated spots in an optimal pattern, not linear pattern

• Assay packs are configured with the appropriate plate and linkers

Documents

Multiplexing Immunogenicity Assays with the MSD … · Multiplexing Immunogenicity Assays with the MSD U-PLEX