A customizable ADCC assay service for antibodies & fusion proteins.

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1 Antibody Dependent Cell- Mediated Cytotoxicity (ADCC) Assay A customizable ADCC assay service for antibodies & fusion proteins. Our ADCC assay service accurately detects cell lysis based on LDH-release. We use freshly-isolated effector cells (PBMCs), and offer flexible assay specifications to build an assay that fit your needs. Choose from a variety of common target cell lines we have available in-house, or just send us your own. Assay Specifications Assays are performed using a 96-well format. Each data point is tested in triplicate, and appropriate assay controls are included. We can also test your antibody s activity using custom effector or target cell populations (e.g. B cells, NK, neutrophils). Please call us for scheduling and availability. Optional Add-ons Repeats using multiple PBMC donors Donor PBMC genotype analysis of FcγRIIIa 158V/F polymorphism FACS binding analysis of antibody to target cells Step 1: Choose your antibody concentrations (e.g. 5X dilution from 1μg/ml.64ng/ml). Step 2: Select up to 3 Effector/ Target ratios for testing (some common ratios are 25:1, 1:1 and 5:1). Step 3: We ll send you a plate layout for your approval. Step 4: Submit your samples & target cell line, along with a completed Sample Submission Form. Specific lysis (%) Specific lysis of SKBR 3 cells induced by antibodies at E/T ratio 25:1 Anti Her2 (mab1) Anti Her2 (mab2) Herceptin Antibody concentration (μg/ml) Step 5: We ll culture your target cell line, test your samples for ADCC activity, and send you a comprehensive report with your assay results. 2 3 weeks 1st 96-well plate: $4,1. Each additional plate: $8. * Timeline & pricing may vary depending on your custom assay specifications. valid through December 31, 211.

2 Complement Dependent Cytotoxicity (CDC) Assay For testing the efficacy of antibodies & fusion proteins in mediating complementdependent cytotoxicity in target cells. Our customizable CDC assays accurately detect cell lysis based on LDH-release, using human serum complement as a probe. Send us your target cell line, or use one of ours, and we ll take care of the rest. Assay Specifications Assays are performed using a 96-well format. Each data point is tested in triplicate, and appropriate assay controls are included. Optional Add-ons Complement binding assays (C1q, C3) FACS binding analysis of antibody to target cells Step 1: Choose your antibody concentrations (e.g. 5X dilution from 1μg/ml.64ng/ml). Step 2: Select up to 3 different complement percentages for testing (some common ones are 2%, 5% and 1%). Step 3: We ll send you a plate layout for your approval. Step 4: Submit your samples & target cell line, along with a completed Sample Submission Form. Step 5: We ll culture your target cell line, test your samples for CDC activity, and send you a comprehensive report with your assay results. Specific lysis (%) Complement Dependent Specific Lysis of Raji Cells Induced by Rituxan Rituxan (1ug/ml) ET91 (1ug/ml) 2% 5% 1% Serum concentration (%) ET91 is a Fully-human IgG1 Isotype Control Antibody (Eureka Therapeutics, Product# ET91) 1st 96-well plate: $3,4. Each additional plate: $ weeks * Timeline & pricing may vary depending on your custom assay specifications. valid through December 31, 211.

3 Glycosylation Composition Analysis (Monosaccharides & Oligosaccharides) Accurately identify and quantify the mono and oligosaccharides present in an antibody or protein sample. Eureka performs glycosylation analysis using HPAEC-PAD (High Performance Anion Exchange Chromatography with Pulsed Amperometric Detection). Comprehensive glycosylation characterization is critical for determining batch-to-batch variability, assessing effects of process changes, and for comparing originator vs. follow-on biologics. We take care of sample preparation simply send us your sample, and we ll deliver results. Monosaccharide Composition Analysis Monosaccharides are quantified by comparison to commercially available standards. includes sample preparation (i.e. hydrolysis and dehydration). Oligosaccharide Population Analysis Eureka s N-linked oligosaccharide profiling identifies the oligosaccharide populations in your glycoprotein sample. includes sample preparation (i.e. N-glycan release, purification, and desalting) 75. nc 5. nc : L-Fucose 2: D-Galactosamine 3: D-Glucosamine 4: D-Galactose 5: D-Glucose 6: D-Mannose min min Figure: Monosaccharide Analysis performed on antibody produced in CHO Figure: Oligosaccharide Analysis performed on antibody produced in CHO 1st sample: $75. Each additional sample: $45. 1st sample: $1,5. Each additional sample: $9. 2 weeks 2 weeks * valid through December 31, 211.

4 Single-Cell Subcloning (High-Titer CHO Cell Line Development) For the generation of stable, clonal cell lines from your early passage stable pool (for high-titer protein expression), under serum-free conditions. Eureka utilizes Genetix s ClonePix TM FL technology for the selection of clonal, CHO cell lines with optimal protein expression levels. Our high-throughput cell line development platform facilitates the efficient screening of up to 5,+ clones per pool eliminating the need for limiting dilution and ensuring the clonality of your final cell line. We can help you save time, reduce uncertainty, and drastically improve the titer of your stable pool or cell line. Please contact us for pricing and timeline information. Case Study 1 Single-cell subcloning from DG44 stable pool Construct: Cyno Fc-fusion protein Original Titer: 2 mg/l (shake flask) Colonies Screened: 5, Clones Selected: 48 : 4 weeks Titer of Top 5 Clones: Sample Name Day 1 Titer (µg/ml) Viability (%) c69 Clone # c69 Clone # c69 Clone # c69 Clone # c69 Clone # Conclusion: 1X increase in titer from stable pool after 1-round of subcloning; able to screen 5X more clones in half the time compared to limiting dilution. Case Study 2 Single-cell subcloning from unstable CHO clone Construct: Human protein (serine protease) Original Titer: 15 mg/l (shake flask) Background: Cell line (isolated by limiting dilution) has unstable protein expression between batches and when scaling-up for production; titer significantly decreases upon expansion to bioreactors. Colonies Screened: 5, Clones Selected: 24 : 4 weeks Titer of Top Clone: 43 mg/l Conclusion: Parental cell line was not clonal; achieved 3-fold improvement in titer after subcloning using ClonePix TM FL.

5 Antibody Fc Receptor Binding Analysis (By FACS) Assess the binding affinity of your antibody to human Fcγ and FcRn receptors. Determining an antibody s binding affinity to different Fc receptor types is important for understanding mechanism of action and predicting therapeutic potential. Fc gamma receptor binding helps evaluate an antibody s ability to recruit specific immune cells, such as NK, neutrophils and macrophages. Analyzing FcRn (neonatal Fc receptor) binding affinity is useful for predicting an antibody s half-life in vivo. Eureka has developed a comprehensive collection of Fc receptor expression cell lines (in CHO) for characterizing antibody/ Fc-fusion protein binding by FACS analysis. Eureka s Fc Receptor Cell Lines FcrRI FcrRIIa The following receptor types are available for antibody binding analyses: FcγRI FcγRIIa CHO-ET11 CHO-1E5-ET CHO-ET11 CHO-1E5-ET FcγRIIb FcγRIIIa (V/V genotype) FcrRIIb FcrRIIIa 158 V/V FcγRIIIa (F/F genotype) FcRn CHO-ET11 CHO-1E5-ET CHO-ET11 CHO-1E5-ET11 Specifications Antibodies are tested at 5 concentrations of your choice. A positive control will be tested concurrently at the highest sample concentration. (per Fc Receptor analyzed) Set-Up Fee: $9. Per Sample Fee: $1, FcrRIIIa 158 F/F CHO-ET11 CHO-1E5-ET FcRn CHO-ET11 CHO-1E5-ET Figure: FcR binding comparison between wild-type antibody (produced in CHO) and Fc-engineered antibody 2.5 weeks ** valid through December 31, 211.

6 Antibody-Antigen Binding Affinity Analysis, Kd (By Bio-Layer Interferometry) Evaluate the binding affinity of your antibody to its target antigen. Kinetic analysis of antibodies is a critical component of lead characterization. Early identification of antibodies with optimal binding profiles saves valuable time, and enables researchers to allocate resources to the most promising candidates. Eureka utilizes Bio-Layer Interferometry (ForteBio s Octet QK System) for accurate analysis of proteinprotein binding interactions (Kd, ka, kd) based on kinetic measurements. What is Bio-Layer Interferometry (BLI)? BLI measures biomolecular interactions by analyzing the interference pattern of white light on a fiber-optic probe in real-time. Molecules binding to or disassociating from the probe shifts the interference pattern, generating a binding affinity profile. nm Separate Fit Model 1:1 A12 (21511 SA_61 Positive) B12 (21511 SA_61 Positive) The Assay.5 time (sec). Step 1: Your antigen (or antibody) is biotinylated and coated onto a streptavidin biosensor Step 2: The coated biosensor is immersed into the antibody (or antigen) solution time (sec) Step 3: The solution is shaken during reading to create an orbital flow Antibody #1 Step 4: As the antibody binds to the antigen, the pattern of white light detected by BLI changes proportionally to the degree of binding Antibody #2 Set-Up Fee: $95. Per Sample Fee: $75. 2 weeks * valid through December 31, 211.

7 Immunoglobulin Gene Cloning (From Hybridoma) Identify the IgG gene DNA sequence from your hybridoma, for recombinant antibody production. The first step to antibody humanization is isolating the IgG-coding gene DNA from your hybridoma. Eureka s IgG gene cloning service accurately determines the variable region DNA sequence of your hybridoma using RACE-PCR. Details mrna preparation from mouse or rat hybridoma cell pellet Antibody leader sequence and variable region amplification by PCR Isolation and sequencing of at least 1 independent gene clones for identification of cdna genes coding for antibody variable region DNA sequence verification of clones Subcloning of antibody variable region DNA into customer-specified vector 3 4 weeks Optional Add-ons Production and purification of 5 mg antibody in CHO cells * Timeline & pricing may vary depending on your custom specifications. valid through December 31, 211. Starting Materials Frozen hybridoma cell pellet (1million cells) Deliverables Plasmids containing IgG DNA (VH and VL) Immunoglobulin DNA sequence Chromatograms with sequencing data from analyzed hybridoma gene clones Cloning vector map $7,5 per hybridoma cell line

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