HTRF ® HUMAN IGG KIT Direct Human IgG quantification for bioprocess operations CATHY DREXLER June V0 April V0 Sales training

2 Human IgG background Human IgG kit (V0) The immunoglobulin G glycoprotein (IgG) molecule has a four-chain structure, comprising two identical heavy (H) chains and two identical light (L) chains, linked together by inter-chain disulfide bonds (about 150 kDa). Four IgG subclasses have been identified with important amino acid differences in the hinge region. IgGs are major effector molecules of the humoral immune response in humans They account for about 75% of total immunoglobulins in plasma of healthy individuals; whereas IgM, IgA, IgD and IgE, constitute the remaining 25%. IgGs are the only immunoglobulins that can pass through the human placenta, thereby providing protection to the fetus in utero

3 Target Customer Who Cares  High probability users: Biotechs and Pharmas working on biologics Customers manufacturing therapeutic antibodies for preclinical batch production Therapeutic area of interest: all (oncology, inflammation, infectious disease, vaccine, auto-immunity)  Identify customers / Key questions to ask: Do you manufacture therapeutic antibodies? Do you quantify total Human IgG? Which kind of reference technology do you use? (ELISA, AlphaLisa, Fortebio, MSD, Gyros) Human IgG kit (V0)

4 Human IgG kit  Included in the kit: - 1 vial of IgG standard (lyophilized) - 1 vial of anti Fab-d2 - 1 vial of anti human-Fc-K - 1 vial of diluent - 1 vial of conjugate buffer  1000 tests (20 µL final)  Ref 6FHIGPEB Human IgG kit (V0) Store the kit at -20°C upon reception

5 Assay Principle Human IgG kit (V0) This assay is designed for the quantitative determination of human IgGs. Human IgG is measured in a sandwich assay format using 2 different specific antibodies, one labeled with Cryptate (donor) and the second with d2 (acceptor). The intensity of the signal obtained is proportional to the number of antibody-antibody complexes formed and therefore the fluorescence is proportional to the concentration of the antibody. Anti-hFc Cryptate conjugate Anti-FAb d2 conjugate Human IgG

6 Protocol Human IgG kit (V0) Incubate 2h at 18-22°C Read on an HTRF compatible reader 10 µL IgG Sample or calibrator 5 µL anti FAb-d2 conjugate 5 µL anti hFc - K conjugate The reagents are dispensed in the order shown below in a white plate (384 wells small volume):

7 Total IgGs standard curve Human IgG kit (V0) The experiment was done in a 384 LV white plate and read after 2h on a PHERAstar FS Flash lamp.

8 IgG detection with different subclasses The experiment was done in a 384 LV white plate and read after 2h on a PHERAstar FS Flash lamp.

9 IgG detection with different subclasses The experiment was done in a 384 LV white plate and read after 2h on a PHERAstar FS Flash lamp.

10 Assay format and detection on PHERAstar (Laser) Human IgG kit (V0) The kit was run in 384w (20µL) and 1536w (with 8µL, 6µL and 4µL) then read on PHERAstar laser to evaluate the potential loss in 1536 format. There is nealry no loss from 96 to 1536 format when PHERAstar is used

11 Assay format and detection on EnVision (Flash Lamp) Human IgG kit (V0) The kit was run in 384w (20µL) and 1536w (with 8µL, 6µL and 4µL) then read on the EnVision FL to evaluate the potential loss in 1536 format. On our EnVision, the EC50 from 384 (20 µL) to1536 formats was 6% for 8µL, 10% for 6µL and 19% for 4µL.

12 Analytical characteristics Human IgG kit (V0) Diluent Detection limit (dose of mean zero + 3 SD)0.9 ng/ml Range of detection0.9 to 2000 ng/ml Incubation time2H at RT

13 HTRF ® Human IgG offers concrete benefits Human IgG kit (V0)  Cost effective  Easy to run and faster  High robustness HTRFAlphaLISAFortebioMSDSo What? Detection limit 0.9 ng/mL0.24 ng/mL10.9 pg/mL? On request Dynamic range ng/mL ng/mL pg/mL? On request Process 1 incubation step2 incubation steps 96 samples in 30 minutes (low throughput) 2 incubations and 3 washing steps Easy to run and faster Stability YESNO (ligh,T° sensitive, signal ↘ after readout) YESNO (50% signal lost after 1min) Robust Price/well (20µL final) 0.54 € / $ € / $1.25$1 to $3$3.1Cost effective Human IgG assay Confidential

14 Value proposition  Easy to run and faster process: 1 single incubation step cell-based (culture media compatible)  Cost effective: cheaper compared to our competitors  High robustness: signal stability low interferences  Specific for intact antibodies: immunosandwich detection Human IgG kit (V0)

15 Associated HTRF ® products Human IgG kit (V0)  Human Fc kit (62HFCPEB/C)  Human IgG kit (6FHIGPEB/H)  Human IgG Kappa kit (64KAPPEB/C)  Human IgG Lambda kit (64LAMPEB/C)  CHO HCP kit (6FHCPPEG)  CD16a (FcgRIIIa, F158) cellular kit (62C16PAG)  Fc  R variant kits

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