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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?

Development and Validation of a Polysorbate 20 Assay in a Therapeutic Antibody Formulation by RP-HPLC and Charged Aerosol Detector (CAD)

WHITE PAPER

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

ABSTRACT

The polysorbate 20 content of a therapeutic antibody formulation was quantified directly by RPHPLC and CAD without prior removal of the high concentration of antibody and excipients containing histidine and sorbitol. Isopropanol was added and thoroughly mixed with the antibody formulation at low temperature prior to analysis. The resulting solution was chromatographed on a C4 RP-HPLC and eluted with multiple gradients of methanol to allow for the complete separation of the antibody from the polysorbate 20. The other formulation excipients were eluted at the void volume of the chromatogram. The antibody was eluted at lower concentration of methanol while polysorbate 20 was highly retained and eluted at higher concentration of methanol. The fully resolved peak of polysorbate 20 in the chromatogram was quantified using a calibration curve of polysorbate 20 spiked in human IgG and detected by CAD.

Method validation showed assay linearity with correlation coefficient (r) > 0.99 from 0.005% to 0.03% of polysorbate 20. Accuracy by recovery of spiked polysorbate 20 ranged from 92% to 101% at three levels bracketing the nominal concentration of 0.01% of polysorbate 20. Precision of the assay was below 5% RSD at the same three levels of spiked polysorbate 20. Specificity was evaluated in spiked placebo and formulation buffer and showed no interference in the determination of polysorbate 20. Quantitation limit of the assay was 0.0012% of polysorbate 20. The current HPLC-CAD method was determined to be suitable for the quantitation of polysorbate 20 in drug formulation containing high concentration of antibody.

BACKGROUND

Polysorbate 20 is typically used in the formulation of antibody therapeutics for its stabilizing effect on the molecule. Most methods used to quantify polysorbate 20 in the presence of high concentration of antibody involved removal of the protein by precipitation or solid phase extraction. In the current method, polysorbate 20 was quantified by direct analysis of the antibody sample without prior removal of the protein. The poster describes the development and validation of a polysorbate assay using RPHPLC and CAD.

MATERIALS AND 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 charged aerosol detector (CAD).

Sample and Standard preparation

For samples and blanks, the antibody or blank was diluted with sample buffer and mixed with pre-chilled isopropanol. All steps were conducted at 2–8 °C. For standard, commercially-available Polysorbate 20, NF grade, was diluted with buffer and spiked with human IgG as surrogate for the antibody. The resulting solution was mixed with pre-chilled isopropanol. All steps were carried out at 2–8 °C.

RESULTS AND DISCUSSION

I. Linearity

Linearity was evaluated with triplicate preparations of five (5) different concentrations within the range equivalent to about 0.005% to 0.030% of spiked polysorbate 20 in matrix.

Figure 1. Linearity Plot

Table 1. Results of the Evaluation of Linearity

II. Accuracy by Recovery

Accuracy by recovery was evaluated by spiking polysorbate 20 equivalent to about 0.005%, 0.010%, and 0.020% in matrix and determining the recovery.

Table 2. Results of the Evaluation of Accuracy by Recovery

III. Precision

Precision of the assay was evaluated in one day. Triplicate preparations of spiked polysorbate 20 equivalent to about 0.005%, 0.010%, and 0.020% in matrix were analyzed, and percentage RSD was evaluated at each level. In addition, one replicate with six injections for unspiked antibody drug product and placebo was evaluated. RSD was evaluated.

Table 3. Results of the Precision Evaluation of Polysorbate 20 in Matrix

Table 4

IV. Specificity

Specificity was evaluated in triplicate in spiked and unspiked placebo and spiked and unspiked formulation buffer. Polysorbate 20 equivalent to 0.010% was added to “spiked” preparations. Average Recovery was evaluated.

The chromatograms for placebo and formulation buffer which both contains polysorbate 20 showed a peak corresponding to Polysorbate 20 as expected. The spiked polysorbate 20 in either placebo or formulation buffer resulted in a corresponding increase in the polysorbate 20 peak (Fig. 2 and 3).

Table 5. Results of the Evaluation of Specificity

V. LOD Determination

The LOD of the assay was evaluated using the following formula:

Where SD is the standard deviation of the Y-intercepts and S is the slope of the multiple calibration curves.

Table 6. Results of the Determination of LOD

VI. LOQ Determination

The LOQ of the assay was evaluated using the following formula:

Where SD is the standard deviation of the Y-intercepts and S is the slope of the multiple calibration curves.

Table 7. Results of the Determination of LOQ

Figure 2. Chromatograms for Specificity: Placebo and Spiked Placebo (Low Resolution, Full Analysis Time)

Figure 3. Chromatograms for Specificity: Formulation Buffer and Spiked Formulation Buffer (Low Resolution, Full Analysis Time)

CONCLUSIONS

The current HPLC-CAD method was validated to determine its suitability for the quantitation of polysorbate 20 in a therapeutic antibody formulation. The results of the evaluation of linearity, accuracy by recovery, precision, specificity, LOD determination, and LOQ determination met the acceptance criteria specified in the validation protocol. The current method is deemed validated and suitable for the specified intended use at EAG Laboratories.