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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 or 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?

Determination of the Glycoforms and Deamidation of a Monoclonal Antibody by Ion Exchange Chromatography and Mass Spectrometry

APPLICATION NOTE

By R. Tobias¹, C. Detchou¹, J. Pan1, J. Anders¹, E. Butler-Roberts², and M. Adomaitis²

¹EAG Laboratories, ²Morphotek, Inc.

ABSTRACT

Purpose: The objective of the current study was to evaluate the glycoform composition and the potential deamidation species of an IgG antibody by cation exchange chromatography and mass spectrometry.

Methods: A concentrated solution of an IgG antibody was chromatographed onto a cation exchange column (Biosuite CM) and eluted with NaCl gradient in phosphate buffer on an HPLC to evaluate the glycoform composition of the sample. For determination of deamidated species, the antibody was incubated with basic solutions of either ammonium carbonate or Tris-HCl at different temperatures and time intervals. The resulting forced-deamidated antibody was chromatographed onto the same cation exchange column. Multiple fractions of the eluted antibody peak were concentrated, desalted, and analyzed by LC-TOF mass spectrometer. The protein concentration was determined by UV mass spectrometry at 280 nm.

Results: Three major glycoforms of the test antibody were sequentially eluted on the cation exchanger; the three glycoforms corresponded to the degree of terminal galactosylation as determined by mass spectrometry. The intact mass of the three major N-glycoforms differed by about 162 Da representing each additional terminal galactosyl moiety in the antibody. The high galactose glycoform showed a longer retention timeas compared with the low galactose species on the cation exchanger. With increasing deamidation temperature or time, the main antibody peak shifted to shorter retention times on the cation exchanger, which corresponded to the presence of acidic or deamidated glycoforms of the antibody. By mass spectrometry, the dominant mass of these acidic glycoforms increased by at least 1 Da as compared with the control. These findings are consistent with the presence of multiple deamidated species of the antibody glycoforms in forced-deamidated samples.

Conclusions: A cation exchange method coupled with mass spectrometry was developed to determine the major glycoforms and the corresponding deamidated species of the test antibody. The deamidated species of the antibody were resolved from their corresponding native forms as determined by mass spectrometry.

BACKGROUND

Glycosylation is an important post-translational modification of proteins. Monoclonal antibodies, which are inherently glycosylated, represent a major component of protein therapeutics and biologics in the biopharmaceutical industry. The glycoform profile and deamidation play a major role in the stability, safety, and efficacy of a monoclonal antibody molecule. The sugar chains on glycoproteins can mediate biological activity and can, therefore, be associated with the safety and efficacy attributes of many biopharmaceuticals. Deamidation, or the conversion of asparagine and glutamine to aspartic/isoaspartic acid and glutamic acid, respectively, has been linked to changes in the physicochemical and functional stabilities of the protein molecule. As such, the heterogeneity of the glycosylation patterns and deamidation are typically characterized and monitored at multiple stages of biopharmaceutical drug development.

OBJECTIVE

The objective of the current study is to evaluate the glycoform composition and the potential deamidation species of the test antibody by cation exchange chromatography and mass spectrometry.

METHODS

Materials: The main sample used in this project consisted of a monoclonal IgG antibody, obtained from Morphotek, Inc.

Equipment: The primary equipment used in this project included an HPLC equipped with fraction collector, a Tangential Flow Filtration (TFF) system, and a UPLC- LCT Premier XE TOF MS system.

Preparative HPLC Conditions: The sample was chromatographed onto a BioSuite CM Cation Exchanger 13 μm, 21.5 × 150 mm at ambient temperature. The monoclonal glycoforms were eluted in a sodium chloride gradient with phosphate buffer, pH 6.0. Eluate fractions of the peaks were collected and stored at 2 – 8 °C until further processing.

LC-TOF MS Analysis: The individual eluate fractions were analyzed on ESI-LC-TOF MS for intact mass using a micro desalting column and eluted with an acetonitrile gradient in 0.1% formic acid. The representative parameters for the mass spectrometer were set as follows:

  • Ion Mode: Positive ion detection
  • Scan Range: 500 to 4000 m/z
  • Capillary: 3200 V
  • Sample Cone: 40 V
  • Desolvation Temp: 350 °C
  • Source Temp: 120 °C
  • Cone Gas Flow: 40 L/h
  • Desolvatio Gas Flow: 800 L/h
  • Lockmass: 0.5 ppm leucine enkephalin

Ultrafiltration/Diafiltration (UF/DF): The selected eluate fractions were pooled, concentrated, and buffer-exchanged using a TFF system equipped with a 5 K cut-off filter. The retentate was repeatedly circulated, diafiltered, and concentrated with PBS. The final concentration of the protein sample was determined by extinction coefficient at A280 nm. When applicable, a Vivaspin micro-spin filter with 3K MWCO was used to further concentrate the protein samples.

  • Deamidation Conditions: The representative deamidation conditions for the antibody were carried out as follows:
  • Buffer: 0.1 M Tris-HCl, pH 9.0 or 0.1 M ammonium carbonate, pH 8.6
  • Incubation Temp: 25 °C, 37 °C, 45 °C
  • Duration: 2 to 6 days

Sample Designation:

  • CON Untreated IgG antibody
  • De1 treated with 0.1 M Tris-HCl, pH 9 at 25 °C for 6 days
  • De2 treated with 0.1 M Tris-HCl, pH 9 at 37 °C for 2 days
  • De2B treated with 0.1 M Tris-HCl, pH 9 at 37 °C for 4 days
  • De3 treated with 0.1 M Ammonium Carbonate, pH 8.6 at 45 oC for 6 days

RESULTS

Figure 1. Representative IEX Chromatogram of Sample (Full View)
Figure 1. Representative IEX Chromatogram of Sample (Full View)

 

Figure 2. Representative IEX Chromatogram of Sample (Expanded View)
Figure 2. Representative IEX Chromatogram of Sample (Expanded View)

 

Figure 3. Representative IEX Chromatogram of Forced- Deamidated Sample (Full View)
Figure 3. Representative IEX Chromatogram of Forced- Deamidated Sample (Full View)

 

Figure 4. Representative Chromatogram of Forced- Deamidated Sample (Expanded View)
Figure 4. Representative Chromatogram of Forced- Deamidated Sample (Expanded View)

Figure 5. Representative Deconvoluted Mass Spectra of the Control Sample
Figure 5. Representative Deconvoluted Mass Spectra of the Control Sample

Figure 6. Representative Deconvoluted Mass Spectra of Deamidated Sample (De1)
Figure 6. Representative Deconvoluted Mass Spectra of Deamidated Sample (De1)

 

Figure 7. Representative Deconvoluted Mass Spectra of Deamidated Sample (De3)
Figure 7. Representative Deconvoluted Mass Spectra of Deamidated Sample (De3)

 

Figure 8. Representative Deconvoluted Mass Spectra of Deamidated Sample (De2)
Figure 8. Representative Deconvoluted Mass Spectra of Deamidated Sample (De2)

 

Figure 9. Representative Deconvoluted Mass Spectra of Deamidated Sample (De2B)
Figure 9. Representative Deconvoluted Mass Spectra of Deamidated Sample (De2B)

 

Figure 10. Representative Deconvoluted Mass Spectra of De3 at pk 20 min
Figure 10. Representative Deconvoluted Mass Spectra of De3 at pk 20 min

 

Figure 11. Representative Deconvoluted Mass Spectra of De3 at ~pk 60 min
Figure 11. Representative Deconvoluted Mass Spectra of De3 at ~pk 60 min

 

Table 1. Peak areas of the IEX fractions of the IgG Antibody
Table 1. Peak areas of the IEX fractions of the IgG Antibody

 

DISCUSSIONS

A typical preparative ion exchange chromatogram of the IgG antibody displayed the main peak at about 92 minutes, and four early-eluting minor peaks (Figures 1 and 2). The total peak area of the minorpeaks constituted less than 0.1% of the sample (Table I). The fractions of the four minor peaks were too dilute and were not sufficient to obtain intact mass by mass spectrometry.

The forced-deamidation profile of the antibody on cation exchange is shown in Figures 3 and 4. In general, the main antibody peak shifted to a shorter retention time on the cation exchanger with increasing deamidation temperature or time. This shift appears to be consistent with the chromatographic behavior of acidic or deamidated glycoform species on a cation exchanger.

In Figure 5, a representative deconvoluted spectra of the control (untreated) exhibited a typical antibody glycoform profile with the main species corresponding presumably to G0F, G1F, and G2F.

The difference in species mass was about 162 Da, which is consistent with terminal galactosylation. With forced-deamidated samples, the intact mass of the glycoforms appeared to increase by at least 1 Da as compared with the control (Figures 6 to 9). This is consistent with the presence of deamidated species. With increasing deamidation time or temperature, the intact mass of the glycoforms increased progressively, suggesting multiple or further deamidation of the sample.

In Figures 10 and 11, the presence of molecular mass corresponding to the light chain (LC) and heavy chain (HC) subunits of the monoclonal antibody molecule were also observed in the forcedeamidated samples. The light chain and heavy chain were eluted before the main peak, at about 20 minutes and 60 minutes, respectively (Figure 4). The light chain occurred as a sharp and distinct peak as compared with the heavy chain subunit, which was observed to have a more complex spectrum.

CONCLUSION

A preparative cation exchange method coupled with mass spectrometry was developed to determine the major glycoforms and the corresponding deamidated species of the antibody. The deamidated species of the antibody were isolated and resolved from their corresponding native forms based on intact mass analysis, as determined by TOF mass spectrometry.