AlphaLISA assays for the development of monoclonal antibody biotherapeutics Margareta Sutija, Ph.D. 18th October 2012
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1 AlphaLISA assays for the development of monoclonal antibody biotherapeutics Margareta Sutija, Ph.D. 18th October PerkinElmer
2 Biotherapeutic solutions 2 2
3 AlphaLISA basic principle Excitation 680 nm AlphaLISA Emission 615 nm Analyte Streptavidin-coated Alpha Donor Bead Antibody Conjugated AlphaLISA Acceptor Bead 3
4 AlphaLISA - Inside the beas Donor beads Excitation: 680nm AlphaLISA TM beads Signal Amplification thioxene S 1D g O 2 S O O S O OSi O O O O SiO Si Eu nm energy transfer Europium chelate O nm 1 D g O 2 Donor: Phthalocyanine Detection: 615 nm Beads Ø : 250 nm 1 O4 2 diffusion: up to 200 nm in solution FRET cascade serves to increase wavelength of light generated
5 AlphaLISA - protocol Add: AALYTE Biotinylated anti-analyte antibody Anti-analyte antibody-conjugated Acceptor beads Incubate for 60 minutes Streptavidin-coated Donor beads Incubate for 30 minutes Excite Donor beads & Read 5 Donor beads release singlet oxygen activating Acceptor bead emission. Technical Data Sheet
6 AlphaLISA technology application in Biotherapeutics Target Identification Protein Development Functional Testing Purification Safety Testing Pre-Clinical Testing Manufacturing & QC Clone Screening Biomarker testing Excitation 680 nm AlphaLISA Emission 615 nm Safety Testing: PD/PK Immunogenicity Analyte Vaccine development Contamination Testing Streptavidin-coated Alpha Donor Bead Antibody Conjugated AlphaLISA Acceptor Bead 6 Flexibility
7 7 Pharmacokinetics
8 Pharmacokinetic Anti-IgG1 and anti-igg4 GEERIC assay Measure the quantity of biotherapeutic monoclonal antibody in serum using a generic approach Antibodies chosen to have very minimal cross-reactivity with monkey serum One antibody is anti-human IgG, heavy and light chain (monkey adsorbed) Other antibody is subclass specific Excitation 680 nm Emission 615nm Monoclonal antibody Biotinylated Anti-IgG Streptavidin-coated Alpha Donor Bead Anti-IgGx-conjugated AlphaLISA Acceptor Bead 8 OT FOR ISOTYPIG
9 Pharmacokinetic Anti-IgG1 GEERIC assay Performance IgG1 LDL in buffer = 85 pg/ml Serum experiments: animal sera spiked with human IgG1 Expected physiological levels during PK studies = 30 ng/ml 600 mg/ml Three sera have been qualified: monkey, mouse, and rat Serum Dilution required LDL (pg/ml) Monkey Rat Mouse 4 74 Specificity 9 Analyte Counts MAX Counts MI cross-reactivity Human IgG % Human IgG % Human IgG % Human IgG % Cyno Monkey % Cross-reactive with: IgG2, IgG3, and IgG4, OT with monkey IgG All IgGs at 0.3 mg/ml
10 Pharmacokinetic Anti-IgG4 GEERIC assay Performance IgG4 LDL in buffer = 35 pg/ml Serum experiments: animal sera spiked with human IgG4 Expected physiological levels during PK studies = 30 ng/ml 600 mg/ml Three sera have been qualified: monkey, mouse, and rat Serum Dilution required LDL (pg/ml) Monkey Rat 2 37 Mouse 4 23 Specificity 10 Analyte Counts MAX Counts MI cross-reactivity Human IgG % Human IgG % Human IgG % Human IgG % Cyno Monkey % Cross-reactive with: IgG2, IgG3, and IgG4, OT with monkey IgG All IgGs at 0.3 mg/ml
11 Pharmacokinetic Functional ligand binding assay Measure Ab levels in serum (at different time points) using a generic IgG assay A) Conjugation of IgG1 or IgG4 subclass to Acceptor bead B) Target should be biotinylable Excitation 680 nm Emission 615nm Biotinylated target Streptavidin-coated Alpha Donor Bead Sample (antibody) -Cell lysate -Cell supernatant Anti-IgG-conjugated AlphaLISA Acceptor Bead 11
12 Pharmacokinetic Functional ligand binding assay Experiment: detection of human anti-tfalpha monoclonal antibody (IgG1 subclass) in monkey serum Antibody against TF was diluted in 10% monkey serum and detected using anti-igg1- conjugated Acceptor beads with biotinylated TF as ligand Calibration curve of monoclonal antibody in 10-fold diluted monkey serum The lower detection limit 0.3 ng/ml of monoclonal antibody Dynamic range 5 fold Excitation 680 nm Emission 615nm Biotinylated TFa Anti- TFa antibody Streptavidin-coated Alpha Donor Bead Anti-IgGx-conjugated AlphaLISA Acceptor Bead 12 Counts Counts EC50 LDL MAX MI S/B (ng/ml) (pg/ml)
13 Pharmacokinetic Functional ligand binding assay Ab-bAb Pairs Counts MAX Counts MI S/B EC50 (ng/ml) LDL (pg/ml) Hook HB E-06 MS E-06 Comparison of standard curve in buffer (HB) or 10% monkey serum (1/10 MS) 13
14 Ab Characterisation: Fucosylated IgG Assay Based on the detection of core fucose by lens culinaris agglutinin, which has a high specificity for 1,6-linked fucose Detection of human IgG4 IgG4 (purified from myeloma plasma) in hybridoma serum-free medium (Hybridoma-SFM Gibco) Typical concentration of monoclonal antibody in cell culture supernatant is mg/ml. Here 0.1 mg/ml is easily detected. Excitation 680 nm Emission 615 nm Monoclonal Biotinylated antibody Anti-human IgG Streptavidin-coated Alpha Donor Bead LCA lectin-conjugated AlphaLISA Acceptor Bead ote: hybridoma serum-free media contain biotin, which interferes in AlphaLISA 14
15 AlphaLISA Signal (counts) Ab Characterisation: Fucosylated IgG Assay Specificity for different subclasses Invivogen anti-tfalpha human monoclonals 1,000, ,000 10,000 IgG1 mab IgG2 mab IgG3 mab IgG4 mab IgG4 from kit 1, Log [Analyte] (g/ml) IgG type MAX counts MI counts S/B EC50 (g/ml) LDL (g/ml) LQL (g/ml) HOOK IgG1 mab ,6 8,38E-07 1,35E-08 4,71E-08 1,00E-05 IgG2 mab ,1 8,38E-07 1,10E-08 3,89E-08 1,00E-05 IgG3 mab ,3 9,06E-07 1,08E-08 3,60E-08 1,00E-05 IgG4 mab ,1 7,64E-07 1,89E-08 6,00E-08 1,00E-05 IgG4 from kit (from human myeloma plasma) ,3 4,57E-06 1,04E-08 3,95E-08 3,00E-05 15
16 16 Contamination Testing
17 Contamination Testing process- and product-related impurities compared to ELISA, indicates better inter- and intra-assay precision and improved CVs Media Components: Albumin Insulin Cell Components: CHO HCP E. coli HCP S0 HCP Leachables: Protein A 17 Simplicity in design
18 AlphaLISA Host Cell Protein assays assays measure the presence of contaminating proteins in purification samples Immunosandwich assay HCP Biotinylated antibody -1 Antibody -2 conjugated Acceptor Bead Assay Lower detection limit * (ng/ml) Upper limit ** ( g/ml) CHO HCP CHO HCP (broad reactivity) PER.C6 HCP E. coli HCP S0 HCP * Calculated by interpolating the average background counts (12 wells without analyte) + 3 x standard deviation value on the standard curve. 18 ** Corresponds to analyte concentration on the standard curve giving maximum signal.
19 AlphaLISA Signal (counts) AlphaLISA Signal (counts) AlphaLISA Signal (counts) CHO HCP (broad reactivity) AlphaLISA: CHO HCP (broad reactivity) ELISA: CHO Host Cell Proteins Dynamic range: ~ ng/ml ng/ml Sensitivity (LDL): ~ 0.1 ng/ml ~ 0.3 ng/ml Antibodies detect more CHO proteins than CHO-HCP regular kit Commercially available assay 1,000, ,000 10,000 1,000 CHO HCP Detection Assay (Log-Log Graph) LDL (pg/ml): 139 pg/ml 1,000, ,000 10,000 1, ,000, ,000 10, ,000 Log [Analyte] (g/ml) HiBlock Buffer RPMI + 10% FBS Hybridoma-SFM CD Hybridoma Medium HiBlock Buffer RPMI + 10% FB Hybridoma-SFM CD Hybridoma M Log [CHO HCP] (g/ml) Log [Analyte] (g/ml) 19
20 Pharmacodynamics (PD) Cancer Cardiovascular Multitude of biological readouts: Cytokine response (AlphaLISA kits, LACE) Proliferation (ATP Lite) Functional response (Alphascreen SureFire) AFP β-gf CA125 Caspase-3, active CXCL11/I-TAC EPO ERBB2/HER2 HGFR/c-MET IF-β Autophagy p62 LC3B MMP1 MMP2 MMP3 MMP9 MMP9 (mouse) PSA TFF3 TIMP1 TFα Alpha-2 Macroglobulin ctni D-dimer EPO ICAM-1 Myeloperoxidase Myoglobin PAI-1 PCSK9 Plaminogen Renin/Prorenin tpa eurodegeneration Aß 1-40 Aß 1-40 (mouse/rat) Aß 1-42 Aß 1-42 (mouse/rat) Aß 1-x Ab Oligomers Aß 1-15/16 Aß 1-40 (high spec.) Aß 1-42 (high spec.) sappa (C-term spec.) sappb (high sensitivity) Tau 20 Metabolic Adiponectin Adiponectin (mouse) Albumin (mouse) C-peptide C-peptide (mouse/rat) FGF21 GH GLP-1 IGF1 IGF2 Insulin Leptin Leptin (mouse) Prolactin Angiogenesis EPO TF-α TF-α (mouse) VEGF VEGFB VEGFC VEGFD VEGFA (mouse) Virology P24 P24 High sensitivity Biologics & Bioprocess CHO-P CHO HCP (broad) E.Coli IgA IgE IgG IgG1 (PK) IgG4 (PK) IgM SO-P PERC6 HCP Residual Protein A Serum albumin CCL2 / MCP1 CCL3/MIP-1a CCL4/MIP-1b CCL5/RATES CRP CXCL10/IP-10 G-CSF GM-CSF GM-CSF (mouse) IF-a IF-g IF-g (mouse) IL1α IL1β Inflammation IL1β (mouse) IL1β (rat) IL2 IL2 (mouse) IL3 IL4 IL6 IL6 (mouse) IL7 IL7 (mouse) IL8 IL10 IL10 (mouse) IL11 IL12 (p70) IL13 IL15 IL15 (mouse) IL17 IL17A (mouse/rat) IL18 CCL2/MCP-1 (mouse/rat) CCL5 / RATES (mouse) CXCL1/GRO-a CXCL9/MIG EW
21 Thank You Excitation 680 nm AlphaScreen Emission nm AlphaLISA Emission 615 nm 21 Alpha Donor Bead Alpha Acceptor Bead
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