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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Validation of a Dual Wavelength Size Exclusion HPLC Method with Improved Sensitivity to Detect Protein Aggregates of a Monoclonal Antibody Biotherapeutic
By J. Tompkins¹, T. Spurgeon1, R. Tobias¹, J. Anders¹, E. Butler-Roberts², and M. Adomaitis²
¹EAG Laboratories and ²Morphotek, Inc.
Purpose: The objective of the current study was to validate a dual wavelength SE HPLC UV214, 280 method to enhance the sensitivity for detection of aggregate forms of a monoclonal IgG drug product.
Methods: Concentrated solutions of IgG antibody were chromatographed onto a TSKgel G3000 SWXL SEC column by isocratic elution at 0.7 ml/minute at ambient temperature with a mobile phase composed of 0.2 M Sodium Phosphate, pH 6.8. A Waters Alliance 2695 HPLC with dual wavelength detector was used at 214 and 280 nm. The response ratio for the IgG monomer at 214:280 nm was determined by injection of 10 g of reference standard. In test samples, low abundant aggregate forms of this IgG were best detected at 214 nm when injected at high amounts (300 μg), but the monomer peak was off scale under those conditions. Therefore, the peak area of the monomer at 214 nm was estimated by multiplication of its 280 nm response by the 214:280 ratio determined previously. The % purity of the IgG sample (300 μg/injection) was then calculated based upon the peak area normalized responses at 214 nm for all forms of the IgG.
Results: Determination of the 214:280 ratio was found to be consistent between 5 to 15 μg/injection with an average ratio response of 14.1 (0.17 % RSD) at 10 μg/injection. The method was found to be precise with intraday and inter day results of 1.5% and 1.9% RSD, respectively. It was also linear and accurate for monomer at nominal levels (300 μg/injection) with a correlation coefficient (r) of 1.00 and an average recovery of 99.1% (0.27% RSD), respectively. In addition, the method was found to be specific with no interferences detected in the formulation buffer that would interfere with the detection of all forms of the IgG. Sensitivity for quantitation (LOQ) and detection (LOD) was estimated to be 0.66 mg/mL and 0.22 mg/mL, respectively. Lastly, the method was found to be robust following purposeful small changes in key method parameters.
Conclusions: The DW SE HPLC method was found to be accurate, precise, linear, specific, sensitive, and robust and, therefore, suitable for its intended use. Sensitivity was increased approximately 6.4-fold by SEHPLC analysis with the use of concentrated IgG samples. This was made possible by normalizing the 214 nm response of the IgG monomer by the 214:280 ratio as determined in each analytical run.
The use of dual wavelength SE HPLC to increase the sensitivity to detect protein aggregates was first proposed by investigators, Bond et al. (2010), at Centocor R&D, Inc. In this presentation, the validation of a DW SE HPLC method for another biotherapeutic IgG monoclonal antibody produced by Morphotek Inc. is presented. In brief, the method utilizes UV detection of the eluents from SE HPLC at two different wavelengths, 214 nm and 280 nm. The method relies upon the difference in absorptivity of the amide bond at 214 nm versus aromatic residues, such as tryptophan and tyrosine, at 280 nm. Since absorptivity of proteins is generally much greater at 214 nm than at 280 nm, one can detect aggregate forms of proteins more readily at 214 nm. However, detection of low abundant aggregates at 214 nm often results in maximizing the capacity of the UV detector for the monomer peak. With the monomer o-scale, accurately determining the relative abundance of aggregate(s) vs. the monomer is impossible. This is overcome by determining the 214:280 ratio for the IgG at a lower concentration, where the monomer peak is within the dynamic range of the detector for both wavelengths. Once the 214:280 ratio is established, the peak area of the monomer at 214 nm in more concentrated samples can be determined by multiplying the 214:280 ratio by the peak area response at 280 nm. Once normalized, the monomer peak in concentrated sample preparations detected at 214 nm can now be used to determine the relative abundance of IgG monomer, dimer, aggregates, fragments, and other product forms where they can be readily detected.
The objective of this work was to validate a more sensitive DW SE HPLC method for detection and relative quantitation of low abundant dimers, aggregates, and fragment forms of this IgG antibody.
Materials: The test articles used in the method validation consisted of a formulated human monoclonal IgG drug product and a corresponding reference standard; both materials used the same formulation buffer and had an approximate concentration of 5 mg/mL. The formulation buffer consisting of 10 mM sodium phosphate, 150 mM sodium chloride, 0.01% polysorbate 80, pH 7.2 was used as a control. A concentrated sample of the IgG development material, at 20.4 mg/mL, was also used in some validation experiments. A Bio-Rad molecular weight standard preparation containing 5 mg of thyroglobulin, globulin, ovalbumin, 2.5 mg of myoglobin and 0.5 mg of vitamin B12, were also used to approximate the apparent MW of the monomer form of IgG antibody.
Equipment: The primary equipment used in this project included a Waters 2695 Alliance HPLC with a 2487 Dual Wavelength detector and an Empower 2 chromatographic data acquisition system. A Tosoh TSK gel G3000 SWxl 7.8 × 300 mm, 5 m column was also used.
Sample preparation: For system suitability testing and determination of the 214:280 ratio, formulated IgG reference standard was prepared at a concentration of 165 g/mL using water and 10 g (60 L/injection) was analyzed simultaneously at both 214 nm and 280 nm. Formulated IgG samples were analyzed at a nominal concentration of 5 mg/mL and 300 g (60 L/injection) amounts were analyzed at 214 nm. The MW standard was resolubilized with 0.5 mL of water and 20 L were injected onto the SEC column with detection at 280 nm. This corresponds to 200 g of thyroglobulin, globulin and ovalbumin, 100 g myoglobin and 20 g of Vitamin B12 injected onto the SE column.
The mobile phase consisted of 0.2 M Sodium Phosphate at pH 6.8. The flow rate was 0.7 mL/min and the injection volumes were 60 L for IgG material and 20 L for the MW standard. A needle wash solution of water was used and column temperature was ambient. Detection was done at 214 and 280 nm with a 16 nm slit width. Run time was 30 minutes per injection.
Determination of 214:280 nm Ratio: The optimal conditions for determining the 214:280 ratio were determined using nine replicate injections of reference standard IgG at 5.0 μg, 7.5 μg, 10.0 μg, 12.5 μg, and 15.0 μg per injection. The 214:280 ratio were assessed for the monomer peak in all injections. The data are shown Table 1. Response was linear throughout the range tested (Figure 1). For determination of the 214:280 ratios in all subsequent analytical runs, a 0.167 mg/mL preparation of the formulated IgG reference standard was used.
Table 1. Evaluation of 214:280 nm ratio at various levels of lgG
Figure 1. Linearity of Dilute Preparations of IgG Monomer for Determination of 214:280 Ratio Procedure
Figure 2(a). 0.167 mg/mL (10 μg/injection) of IgG Reference Standard at 214 nm, On-Scale and Expanded View
Figure 2(b). 0.167 mg/mL (10 μg/injection) of Iggy Reference Standard at 280 nm
The 214:280 ratios were determined in this manner for each analytical run. A representative chromatogram is shown in Figure 2.
Repeatability and Intermediate Precision of Sample: Repeatability was determined by 10 injections of IgG sample at nominal concentration of 5 mg/mL (300 g/injection) with detection at 280 nm. The resultant precision for injection repeatability was0.1 %RSD for the IgG monomer.
Table 2. Intermediate Precision for lgG Monomer
Figure 3. HPLC size exclusion of IgG sample in formulation buffer, 300 μg injected.
Intermediate precision was performed in triplicate preparations at ± 20% nominal (4 mg/mL, 5 mg/mL, and 6 mg/mL) by two different analysts using separate samples preparations. Precision results are shown in Table 2: the %RSD for a single day analysis was 1.5% and the overall precision between the two analysts was 2.2%. Representative chromatograms for IgG sample injections are shown in Figure 3.
Linearity of Sample: Preparations of IgG sample ranging from 2.5 mg/mL to 7.5 mg/mL (150 g/injection to 450 g/injection) were analyzed at both 214 and 280 nm in triplicate (example shown in Figure 3). All relevant IgG related peaks were evaluated to include the monomer, dimer, aggregate, and fragment peaks observed in these chromatograms. The resultant linear regression analyses for all IgG components are shown in Figure 4. All IgG components were found to behave in a linear fashion and intercept near the origin (Figure 4, panels A, B, D, E and F) except the monomer when measured at 214 nm (Panel C). For monomer, the 214 nm monomer peak response displayed considerable bias and did not pass near the origin.
Figure 4. Linearity for Peak Area Responses of the IgG monomer (at both 280 nm and 214 nm) as well as Dimer, Aggregate, and Fragments at 214 nm Observed in IgG Sample Preparations
This bias is due to maximizing of the response of the monomer, the most abundant peak in the IgG, as expected.
Accuracy: Accuracy was assessed at multiple concentrations of IgG in triplicate ranging from 2.5 mg/mL to 7.5 mg/mL of product. At each level tested, the amount of IgG monomer detected at 280 nm was compared to the theoretical dilution using linear regression as shown in Table 3.
Figure 4 (Continued). Linearity for Peak Area Responses of the IgG monomer (at both 280 nm and 214 nm) as well as Dimer, Aggregate, and Fragments at 214 nm Observed in IgG Sample Preparations
Precision for the monomer peak did not exceed 1.7% RSD at all concentrations. Additionally, determination of the apparent molecular weight of the IgG monomer by comparison to elution of known MW standards is shown in Figure 5.
Specificity, Robustness and Reagent Stability: Specificity was evaluated by analysis of duplicate preparations of formulation buffer. No peaks were found to be present in the placebo formulation buffer that could interfere with the IgG monomer or other primary forms such as dimer, aggregates, and fragments (data not shown).
Table 3. Accuracy of Monomer at 280 nm
Figure 5. Verification of lgG Monomer MW by Comparison to Known MW Standards
Robustness was evaluated by purposeful changes (} 5%) to critical steps in the SE HPLC method. These included adjustments of } 5% in concentration and pH of the SE HPLC mobile phase nominally at 0.2 M Sodium Phosphate, pH 6.8. Additionally, robustness was challenged using }5% change in injection volume and testing an additional lot of column. All such purposeful changes did not affect the performance of the method to any significant degree. Triplicate preparations of the formulated reference standard were also evaluated for stability at 2-8 °C for up to 48 hours and were found to be stable under those conditions.
Sensitivity: Sensitivity of the method was determined by evaluation of IgG samples ranging from 2.5 mg/mL to 7.5 mg/mL in triplicate. The Limit of Quantitation (LOQ) and Limit of Detection (LOD) were determined using the following equation:
LOD = 3.3 x Standard Deviation of Intercepts/Average of Slopes
LOQ = 10 x Standard Deviation of Intercepts/Average of Slopes
Table 4. Results for Evaluation of Sensitivity for IgG by detection of the Monomer Peak at 280 nm versus the same peak when normalization was not performed.
Since purified preparations of the various IgG forms, dimers, aggregates, and fragments were not available, the IgG monomer was used as a surrogate for evaluation of sensitivity of all the known IgG forms.
The IgG monomer response for concentrated sample preparations at 280 nm was normalized using the 214:280 ratio as per this method. For comparison, the IgG response at 214 nm without normalization was also evaluated. This was done to determine differences in sensitivity using both analytical approaches. It was determined that use of the 214:280 ratio to normalize IgG responses in concentrated samples provided an approximate 6-fold increase in sensitivity as shown in Table 4.
Bond M.D., et al., “Evaluation of a Dual-Wavelength Size Exclusion HPLC Method With Improved Sensitivity to Detect Protein Aggregates and Its Use to Better Characterize Degradation Pathways of an IgG1 Monoclonal Antibody”, Journal of Pharmaceutical Sciences, vol. 99, No. 6, June 2010.