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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?

Analysis of the N-Glycan Profile of a Therapeutic Monoclonal Antibody

WHITE PAPER

By Rowel Tobias, William Hanshaw, Alan Leslie, Paul Lightner, Glenn Petrie, and Mike Whalon

Abstract

The objective of this study was to determine the N-glycosylation profile in an antibody reference standard and product formulations.

The N-glycan profile of the antibody was analyzed by releasing the N-glycans from the molecule using PNGase F digestion. The released N-glycans were extracted and enriched by solid phase extraction, followed by 2-AB labeling. The labeled N-glycans were then resolved by HILIC chromatography and detected by fluorescence spectroscopy. N-glycan standards were used to identify the N-linked oligosaccharides present in the antibody formulations.

The fucosylated N-glycan with one terminal galactose was the major N-glycan species in both the reference standard and the product formulations at about 50 to 55% of the total N-glycans observed. The fucosylated N-glycan with two terminal galactose units was found at about 14 to 18% and an N-glycan without galactosylation was also observed at about 27 to 31% in both the reference standard and product formulations. Low levels of sialylated glycans were detected but no significant amounts of Man5 and N-linked oligosaccharides without a fucose moiety were observed in the test materials.

The N-glycan profile of the antibody product formulation was comparable with the reference standard. All the N-glycans observed in both antibody formulations were fucosylated N-linked oligosaccharides, with one terminal galactose as the major component. The N-glycan with two terminal galactose units and an N-glycan without galactosylation were also observed in these antibody formulations.

Background/Objective

Background

The current study describes the procedure for determining the glycan profile of a therapeutic monoclonal antibody and comparing a reference standard lot from a new drug formulation. The method involves labeling with 2-Aminobenzamide (2-AB) and analyzed on UPLC-HILIC with fluorescence detection. The intended use of the method is to provide analytical support for clinical drug development.

Objective

The objective of this study was to determine the N-glycosylation profile in an antibody reference standard and product formulations.

Methods

Materials
The main materials used in this project were a genetically-expressed recombinant monoclonal antibody therapeutic. The antibody is in highly purified form in a drug substance and product formulations.

Equipment
The primary equipment used in this project was a Waters Acquity Binary UPLC equipped with a Fluorescence detector.

Sample preparation
N-glycan release from the antibody was carried out by denaturing the molecule at 65°C for 10 minutes followed by incubation with PNGase F in 50 mM Tris-HCl, pH 8.0 for at least 16 h at 37°C.

Extraction and enrichment of the released N-glycan
The N-glycan released sample was loaded onto a pre-conditioned SPE cartridge and washed repeatedly with acetonitrile using a vacuum manifold. The bound N-glycan was eluted off the cartridge with aqueous solvent and dried on a vacuum evaporator (Speed VAC concentrator).

Derivatization with 2-AB
The 2-AB labeling solution was added to the sample, mixed, and incubated for 3 h at 65°C. Ammonium acetate and acetonitrile solutions were added to the resulting sample and centrifuged. The supernatant was transferred to a HPLC vial for analysis.

Preparation of standards
To the vial of the Performance Standard was added 100 µL of 100 mM Ammonium Formate, pH 4.5 and 100 µL Acetonitrile. The standard is aliquoted in small volumes and frozen at – 20 C until analysis.

UPLC conditions:
Instrument Parameters:
Acquity Instrument Method

Waters Acquity UPLC system


Mobile Phase A (100 mM Ammonium Formate, pH 4.5)
Mobile Phase B (100% Acetonitrile)

Calculations

1.1. Interference of Control Sample

Note: Calculate the %Peak Control for G0F, G1Fa, GlFb, G2F and GlF + SA, and G2F + SA, respectively.

1.2 Ratio of G0F/G1Fa

1.3. %RD of two (2) replicates of Sample injections for ratio of G0F/G1Fa

1.4. Glycan profiling calculation (Round to two (2) decimal points)

Results

Analysis of the N-glycan profile of a therapeutic monoclonal antibody formulations was obtained from N-glycans cleaved from the antibody following N-glycanase digestion. The digest fraction was loaded onto a solid phase extraction cartridge to bind the hydrophilic oligosaccharides and washed off the hydrophobic proteinaceous fraction with high organic solvent. The bound glucosamine sugars were eluted off the cartridge with aqueous solvent and subsequently labeled with fluorescent 2-AB.

The various forms of N-glycans typically found in immunoglobulins were resolved by HILIC chromatography and detected by Fluorescence spectroscopy (Fig. 1). Immunoglobulin N-glycan standards were concurrently labeled and analyzed with the samples to identify the N-linked oligosaccharides present in the antibody formulations. The HILIC-fluorescence method showed good linearity and precision response for N-glycan determination in monoclonal antibodies (Fig. 2). Likewise, injection repeatability was also demonstrated (Fig. 3) in this method. Similar chromatographic profiles for N-glycans were observed between the reference standard lot and the new drug formulation (Fig. 4).

As shown in Table I, similar N-glycan compositions were obtained with the current HILIC method as compared with the previous method employing ion-exchange chromatography from another testing laboratory. In Table II, the N-glycan with one terminal galactosylation and fucosylation (G1Fa, b) was the major N-glycan species in both the reference standard and the product formulations at about 50 to 55% of the total N-glycans observed. The fucosylated N-glycan with both terminal galactosylation (G2F) was observed at about 14 to 18% and an N-glycan without galactosylation (G0F) was present at about 27 to 31% in both the antibody reference standard and product formulations. Other forms of N-glycans such as monosialylated glucosamines were detected at low levels at less than 2%. Man5 and unfucosylated N-linked oligosaccharides were not detected in the test materials.

Figure 1. Representative chromatogram of the expected (A) and observed (B) N-glycan standard profile by HILIC-fluorescence detection.


Figure 2. Linearity and precision assessment of the N-glycan profiling by HILIC-fluorescence spectroscopy.

Figure 3. Injection repeatability of the N-glycan profile of a therapeutic monoclonal antibody.

Figure 4. HILIC chromatograms of N-glycoforms in the reference standard and antibody drug formulation.

Table I. Comparison of Previous and Current Method of N-glycan profiling

Table II. Comparison of Reference Standard and Drug Formulation

Conclusions

Antibody N-glycan profiling was determined by PNGase F digestion. 2-AB labeling, HILIC chromatography and Fluorescence detection. Overall, the N-glycan profile of the antibody product formulation was comparable with the reference standard. All the N-glycans observed in both antibody formulations were fucosylated N-linked oligosaccharides with single galactosylation as the major glycoform species. The N-glycan with both terminal galactosylation and an N-glycan without galactosylation were also observed in these therapeutic antibody formulations.