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Pharmaceutical & Biopharmaceutical Development Services

EAG brings unparalleled expertise to the development and commercialization of small molecule drugs, biopharmaceuticals, antibody-drug conjugates (ADCs), drug-device combination…

EAG brings unparalleled expertise to the development and commercialization of small molecule drugs, biopharmaceuticals, antibody-drug conjugates (ADCs), drug-device combination products and other therapies. From designing IND-enabling studies to delivering full CMC analytical and QC support, we join your R&D team as a true partner. EAG scientists take time to understand both your commercial goals and the unique characteristics of your compound. We provide expert guidance to balance regulatory expectations with expediency and cost, and approach technical challenges with flexibility and resolve.

Materials Testing & Analysis

When it comes to understanding the physical structure, chemical properties and composition of materials, no scientific services company offers the breadth of experience, diversity…

When it comes to understanding the physical structure, chemical properties and composition of materials, no scientific services company offers the breadth of experience, diversity of analytical techniques of technical ingenuity of EAG. From polymers to composites, thin films to superalloys—we know how to leverage materials sciences to gain a competitive edge. At EAG, we don’t just perform testing, we drive commercial success—through thoughtfully designed investigations, technically superior analyses and expert interpretation of data.

Environmental Testing & Regulatory Compliance

Having helped develop the test methods that shape current regulatory guidelines, EAG chemists, biologists and toxicologists have evaluated the environmental impact of thousands of…

Having helped develop the test methods that shape current regulatory guidelines, EAG chemists, biologists and toxicologists have evaluated the environmental impact of thousands of active ingredients and formulations—from pesticides and pharmaceuticals to industrial chemicals and consumer products. Whether you are exploring “what if” scenarios, registering a new active ingredient or formulation, responding to a data call-in or seeking to understand the latest guidance, turn to EAG for technical excellence, sound advice, GLP-compliant study execution and expert interpretation.

Microelectronics Test & Engineering

Whether connecting the internet of things, guiding surgical lasers or powering the latest smart phone, integrated circuits and microelectronics touch nearly every aspect of human…

Whether connecting the internet of things, guiding surgical lasers or powering the latest smart phone, integrated circuits and microelectronics touch nearly every aspect of human life. In the world of technology, innovation and continuous improvement are imperatives—and being able to quickly and reliably test, debug, diagnose failures and take corrective action can make the difference between a doomed product launch and building a successful global brand. EAG offers you the world’s largest and most diverse collection of specialized analytical instrumentation, capacity to perform a variety of microelectronic tests in parallel, and the multi-disciplinary expertise required to draw true insight from data.

Custom Synthesis & Radiolabeling

No contract service provider has more experience performing custom synthesis and producing isotopically labeled compounds to support product development in life science, chemical…

No contract service provider has more experience performing custom synthesis and producing isotopically labeled compounds to support product development in life science, chemical and related industries than we do. From 14C and 3H radiolabeled clinical trial materials synthesized under cGMP, to stable-labeled active ingredients for metabolism and environmental fate/effects testing, turn to EAG. We have extensive experience with multi-step and other complex synthesis projects, and our comprehensive, in-house analytical services ensure quick turnaround of purity and structural confirmation.

Crop Biotechnology & Development

EAG combines biotechnology and protein characterization expertise with more than 50 years' experience analyzing chemical compounds in plant and environmental matrices to address…

EAG combines biotechnology and protein characterization expertise with more than 50 years’ experience analyzing chemical compounds in plant and environmental matrices to address the growing needs of the biotechnology crop industry. We offer a wide range of techniques required to fully characterize the event insertion and expressed proteins, as well as the various studies required to confirm the food, feed and environmental safety of products that represent the trait. From early-stage protein confirmation to GLP-compliant EDSP and allergenicity testing, we help you make faster, more informed development decisions and comply with evolving global regulations of genetically engineered crops.

Litigation Support & Expert Testimony

When you need solid science and investigative engineering to address product failures, inform legal strategy, protect intellectual property or address product liability disputes,…

When you need solid science and investigative engineering to address product failures, inform legal strategy, protect intellectual property or address product liability disputes, turn to EAG. We’ve provided technical consulting, analysis and expert testimony for hundreds of cases involving the aerospace, transportation, medical device, electronics, industrial and consumer product industries. Our team of experts understands the legal process and your need for responsiveness, effective communication, scientifically defensible opinion and confidentiality. From professional consulting to data review to trial preparation and expert witness testimony, ask EAG.

Techniques

Chromatography

Using an array of advanced separation techniques and innovative technology, we conduct highly precise analytical chromatography for various industries. Whether you want a closer…

Using an array of advanced separation techniques and innovative technology, we conduct highly precise analytical chromatography for various industries. Whether you want a closer look at the purity of your pharmaceutical or need to better understand an agrochemical’s components, EAG has the expertise to separate and evaluate any compound.

Mass Spectrometry

Need to evaluate the molecular structure of a compound or identify its origins? EAG knows how. With state-of-the-art tools, we can separate, vaporize and ionize the atoms and…

Need to evaluate the molecular structure of a compound or identify its origins? EAG knows how. With state-of-the-art tools, we can separate, vaporize and ionize the atoms and molecules in almost any pure or complex material to detect and obtain mass spectra of the components. We rely on decades of experience in mass spectrometry to provide our clients with precise analyses and the best detection limits.

Imaging

EAG is a world leader in high-resolution imaging down to the atomic level. We offer unmatched analytical know-how, generating extremely detailed surface and near surface images…

EAG is a world leader in high-resolution imaging down to the atomic level. We offer unmatched analytical know-how, generating extremely detailed surface and near surface images for various industries, from consumer electronics to nanotechnology. Using state-of-the-art equipment and innovative techniques, we conduct expert imaging to aid in failure analysis, dimensional analysis, process characterization, particle identification and more. If you want to investigate a material with angstrom scale resolution, you can count on EAG to get the job done quickly and precisely.

Spectroscopy

EAG offers a vast array of spectroscopic techniques to clients in various industries, from defense contractors to technology pioneers. We combine unparalleled expertise and…

EAG offers a vast array of spectroscopic techniques to clients in various industries, from defense contractors to technology pioneers. We combine unparalleled expertise and methodology with cutting-edge technology to analyze your organic, inorganic, metallic and composite materials for identification, compositional, structural and contaminant information. Whether you need expert spectroscopic analysis to improve your production process or to surmount a technical challenge, EAG is up to the task.

Physical/Chemical Characterization

Need to identify your unique material? Want to analyze the thermal properties of a sample, or measure the success of a process step? If it has to be done quickly and it has to be…

Need to identify your unique material? Want to analyze the thermal properties of a sample, or measure the success of a process step? If it has to be done quickly and it has to be done right, you can count on EAG. We offer a range of adaptable techniques and innovative methods to evaluate the physical and chemical characteristics of any compound. Our highly precise testing and analytical services will improve your production process, expedite R&D and help you conquer any technical challenge.

About

A Global Scientific Services Company

One of the most respected names in contract research and testing, EAG Laboratories is a global scientific services company operating at the intersection of science, technology and…

One of the most respected names in contract research and testing, EAG Laboratories is a global scientific services company operating at the intersection of science, technology and business. The scientists and engineers of EAG apply multi-disciplinary expertise, advanced analytical techniques and “we know how” resolve to answer complex questions that drive commerce around the world.

Our Customers

Science and technology transcend industry boundaries, and so does demand for EAG’s expertise. We partner with companies across a broad spectrum of high-tech, high-impact and…

Science and technology transcend industry boundaries, and so does demand for EAG’s expertise. We partner with companies across a broad spectrum of high-tech, high-impact and highly regulated industries. We help our customers innovate new and improved products, investigate manufacturing problems, perform advanced analyses to determine safety, efficacy and regulatory compliance, and protect their brands.

Our Company Culture

EAG’s corporate culture is firmly rooted in four guiding principles: “foster a growth mindset,” “find a better way,” “earn more loyal customers,” and “win…

EAG’s corporate culture is firmly rooted in four guiding principles: “foster a growth mindset,” “find a better way,” “earn more loyal customers,” and “win together.” Across all of our 20+ locations, you will find a true passion for science and the power of science to improve the world we live in. Hear what some of our ~1200 scientists, engineers and support personnel say about what it means to be part of EAG Laboratories.

Careers

EAG is growing, and we are always looking for talented, problem-solving oriented individuals to join our company. If you have a “we know how” spirit, we want to hear from you.…

EAG is growing, and we are always looking for talented, problem-solving oriented individuals to join our company. If you have a “we know how” spirit, we want to hear from you. Browse current openings now, and re-visit our careers page often.

How do you make better development decisions faster?

A Cell-Based Assay to Assess the Binding Activity of the Monoclonal Antibody Component of an Antibody Drug Conjugate

APPLICATION NOTE

By Angela Kirik1, Charlie Britten2, Karen Havenith2, Mike Whalon1, Glenn Petrie1, Esohe Edusogie2, Mike Mulkerrin2 and Loretta Sukhu1

1EAG Laboratories, Columbia, Missouri, USA and 2ADC Therapeutics, Murray Hill, New Jersey, USA.

INTRODUCTION

Antibody drug conjugates (ADCs) are cancer therapeutic agents designed to direct a cytotoxic drug to cells expressing a cell-surface antigen recognized by an antibody. The antibody and drug are linked through chemistries that enable release of the cytotoxic drug upon internalization and digestion of the ADC by the cell. The efficiency of any ADC could be evaluated by cell-based Potency and Cytotoxicity Assays.

A competitive cell-based Potency assay was developed for determination of relative binding potency of newly-constructed ADC, containing monoclonal antibody against CD19 antigen, overexpressing on the surface of cancer-modified B lymphocytes, and a Pyrrolobenzodiazepine (PBD) as a cytotoxic DNA damage agent.

The assay utilized CD19 expressing Ramos cells (RA-1, ATCC® CRL1596™); a SULFO-TAG® anti-CD19 antibody was used as a competitor to unlabeled ADC. Conjugated and unconjugated anti-CD19 antibodies were recognized to a similar extent by the antigen. Luminescence was measured by a Mesoscale Discovery (MSD) Sector Imager plate reader and was proportional to the competition by ADC of the binding of SULFO-TAG® antibody to the CD19 antigen.

OBJECTIVES

  1. Establish and qualify a method for the determination of Binding Potency of the monoclonal Antibody component of an ADC.
  2. Demonstration of Assay Stability for determining the Potency of ADC binding activity.

 METHOD

A competitive cell-based binding immunoassay with Electrochemiluminescent (ECL) detection was developed to determine relative binding potency of ADC antibody drug conjugate and antibody intermediate relative to their respective fully-characterized reference standards. The assay utilizes CD19 expressing Ramos cells (RA-1). MSD plates are coated for at least one hour with Concanavalin A. Reference standard, QC and test samples are prepared in dilution buffer containing SULFO-TAG labeled ADCs (anti-CD19), which is used as a competitor for unlabeled ADC, RA-1 cells are pre-incubated with diluted reference standard, QC and test samples at room temperature (RT) for at least 1 hour. Plates then washed 3 times, and content transferred to the Concanavalin A coated plates where incubated for at least two hours at RT. The plates are washed and read buffer is added to the wells. Signals are detected following the application of a voltage to the plate electrodes within the MSD Sector Imager 6000 (Meso Scale Discovery), causing the bound SULFO-TAG to emit light of which the intensity is measured by the Sector Imager.

METHOD QUALIFICATION

During method qualification System Suitability, Accuracy, Precision, Specificity, Linearity and Range were examined.

System Suitability: System Suitability pass in all qualification assays: %CV ≤25%, R2≥0.99, A, B, D values 75%-125% of Reference Standard.

Table 1. Standard solutions prepared at five levels across a range corresponding to the theoretical working concentration.

Accuracy: Accuracy was tested by preparation of the ADC at five levels across a range corresponding to 200%, 150%, 100%, 75% and 50% (each percent denoted as Test Material and 100% as Assay Control) of Theoretical working concentration. Accuracy is assessed as percent recovery, or measured Relative Potency divided by the Theoretical Potency, multiplied by 100. The ADC test solutions were diluted to the five target concentration levels and compared with the ADC or Antibody Intermediate prepared at the 100%. Accuracy was calculated assuming all preparations were at the Theoretical concentrations. Each of five concentration levels was tested three times. Recoveries for individual runs were between 94% and 114% of Theoretical concentrations, and the coefficient of variation between the Recovery data of each tested level was between 1 and 16 percent.

Table 2. Intra Assay Precision for the Antigen Binding Assay was analyzed by determining the percent CV of the Relative Potency of the Assay Control from three individual plates run on the same day by a single analyst.

INTRA ASSAY PRECISION (REPEATABILITY)

The Precision of an analytical procedure is usually expressed as the variance, standard deviation or coefficient of variation of a series of measurements and includes repeatability (intra assay variability) and intermediate precision (inter assay variability).

Intra Assay Precision was assessed using Reference Standard (RS) and Assay Control (AC) ADC and Ab. Intermediate solutions each at 100%, tested by the same Analyst on the same day (repeatability) on three independently prepared plates for each. Intra Assay Precision for the Antigen Binding Assay was analyzed by determining the percent Cv of the Relative Potency of the Assay Control from three individual plates run on the same day by a single analyst.

INTERMEDIATE ASSAY PRECISION

The Intermediate Assay Precision was determined for the Antigen Binding assay for five levels of potency tested experimentally for ADC as well as for Ab. Intermediate. This was done by calculating the percent Cv of Relative Potency values from three individual experiments performed at each level and each tested article (ADC and Ab. Intermediate) to assess 200%, 150%, 75%, and 50% potency levels by two analysts on different days (2 sets of assays were done by Analyst 1 and one set by Analyst 2 for each tested article). The Relative Potency for the 100% Assay Control was used to assess the percent Cv for 100% potency level; six individual runs were used in this determination.

Table 3. The Relative Potency for the 100% Assay Control was used to assess the percent CV for 100% potency level; six individual runs were used in this determination.

SPECIFICITY

Specificity of the method was assayed by use of non-specific anti-CD25 antibody drug conjugate. As shown on the graph, non-specific antibody showed any specific binding and Potency was outside ±50% relative to the Reference Standard.

Figure 1. Specificity of the method assayed by use of a non- specific anti-CD25 antibody drug conjugate.

LINEARITY AND RANGE

Figure 2. The experimentally-acquired mean relative potency of each level charted relative to their theoretical concentrations.

Linearity was evaluated to test the ability of the method to accurately distinguish and quantitate the Relative Potency between and throughout a range of potencies. Five levels of potency for the ADC and Ab. Intermediate; namely, 200%, 150%, 100%, 75%, and 50% were determined and linearity in the range of 50%-200% was seen. The experimentally acquired mean relative potency of each level relative to their theoretical concentrations was charted using linear regression statistics. The results are illustrated in the graph. Linearity was achieved as shown by the coefficient of determination, R2 = 0.98 for ADC and 1.00 for Ab. Intermediate.

EVALUATION STABILITY OF ADC AND ANTIBODY INTER-MEDIATE USING ANTIGEN BINDING ASSAY

The Antigen Binding assay was used for the ADC and Ab. Intermediate stability study. Three assays were run for each time point for ADC as well as Ab. Intermediate. The table above shows collected results of two years stability evaluation.

Table 4. Collected results of two years’ worth of stability evaluations of ADC and Antibody Intermediate using Antigen Binding Assay.

CONCLUSIONS

  • A number of critical assay parameters were evaluated during qualification exercises; including Accuracy, Precision, Specificity, Linearity and Range.
  • The method has been qualified for assessment of the relative binding potencies of an ADC or its corresponding Antibody Intermediate under CGMP guidelines and regulations.
  • Data obtained during the course of method qualification and analysis of system suitability criteria tracked through the course of a two years stability study demonstrated the assay’s suitability and robustness for determining the potency of binding activity.