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NeoProteomics, Inc. offers covalent labeling (CL) and mass spectrometry (MS) services to industry to characterize therapeutic proteins. These services precisely defined epitopes for antigen targets and provide structural characterization of therapeutic proteins suitable for biosimilarity comparisons or any relevant structural definition of the product. Specifically CL-MS (often called protein footprinting) provides a high resolution and sensitive readout of the side chain environment (e.g. solvent accessibility) for virtually all side chains of a protein of interest. Changes in solvent accessibility of side chains, due to ligand binding (e.g. epitope mapping), sequence or process variation, or changes in solvent are easily read out by the technology and are immediately structurally interpretable.

Dr. Mark Chance, NeoProteomics Chief Scientific Officer and Founder, pioneered CL-MS as a fast, high resolution approach to protein characterization by mass spectrometry and has partnered with several major Pharmaceutical and Biotechnology companies to characterize epitopes at the side chain level, providing enhanced patentability, and have characterized antibody structure to assess conformational variability relevant to re-engineering or manufacturing process variation.

Competing technologies for protein characterization, like hydrogen-deuterium exchange (HDX) or NMR may be suitable for assessing conformation variability and/or epitopes. NMR, depending on the technique, (1-D, 2-D or 3-D) may lack specificity or sensitivity, or be prohibitive in cost. HDX is a powerful approach to examining backbone stability, but is more challenging for larger proteins, proteins that have multiple disulfide bonds or proteins that are heavily glycosylated.

CL-MS is a proven approach and NeoProteomics can get you answers rapidly. See the attached FAQ for more information or visit

NeoProteomics Suite of SMS Services Include:

Higher Order Structural Analysis

Epitope Mapping

Protein-drug interaction mapping

NeoProteomics’ Value Proposition:

State-of- the art Covalent labeling based-MS

Turn-key Solutions for Outsourcing or Insourcing

A World-Class Team led by Recognized Leading Experts

For more information, please visit the NeoProteomics website or inquire via e-mail to:

FAQ for protein characterization for biosimilarity or epitope mapping

Q: How much antigen do I need for epitope mapping?

A: For one epitope mapping, minimum 10 nmol of protein. If your antigen is 50 kDa, you need 0.5 mg of the protein. For subsequent epitope mapping of another antibody against the same antigen, less protein may be needed.

Q: What should the antigen sample concentration be for epitope mapping?

A: Minimum 10 µM, preferably 20 µM or higher. If your antigen is 50 kDa, the concentration should be at least 0.5 mg/mL.

Q: How much antibody do you need for characterization or epitope mapping?

A: We typically need 1 mg of antibody.

Q: What should the antibody sample concentration be?

A: Minimum 1 mg/mL, preferably 2 mg/mL or higher.

Q: Fab or IgG?

A: Either works fine.

Q: How pure do the proteins have to be?

A: We usually ask the purities of the antigen and antibody to be better than 80% by LCMS. If you do not have LCMS analyses of the protein samples, the analysis can be performed with extra cost. In general, the purer your samples are, the higher your chances of obtaining good results.

Q: How much does the analysis cost?

A: It depends on a few parameters. You can easily receive a quote without disclosing too much information. To receive a quote, you need to provide us, approximate size of your antigen in kDa, the number of glycosylation sites, the number of other posttranslational modifications (PTMs), the number of disulfide bonds, and the number of antibodies you want to map the epitope.

Q: What information do I have to provide to perform the characterization or epitope mapping?

A: The exact sequence of your antigen construct, the locations and types of glycosylations and other PTMs if any, the locations and linkages of disulfide bonds if any. And buffer conditions for the antigen and antibody. We do not need the sequence information of your antibody, unless you are interested in paratope mapping as well. 

Q: What kind of buffer conditions can I use for the protein samples?

A: Most common buffers, salts and additives are OK. Some exceptions are detergents with oligomeric ethylene glycol chains, such as Brij or Triton. The buffer components must be listed, prior to the initiation of the analysis. 

Q: What kind of exchange conditions can I use?

A: The exchange reaction is initiated by adding the exchange buffer to your protein samples. The exchange buffer can be the same, or different than the protein buffer. For the exchange buffer, most common reagents, salts and additives are OK. Some exceptions are detergents with oligomeric ethylene glycol chains, such as Brij or Triton. If you want the exchange reaction conducted in conditions other than our standard (which is PBS pH 7.0), please provide the exchange buffer. We can help you decide on the best exchange conditions.

Q: How shall I ship the sample?

A: The sample can be in solution, frozen solution or lyophilized. If you send a lyophilized sample, please include the reconstitution buffer.

Q: My antigen has disulfide bonds. Is it going to be a problem? 

A: Usually no, but the location of the linkages should be provided. 

Q: My antigen is phosphorylated. Is it OK?

A: It is OK, as long as you know where they are and let us know. If you do not know the sites, we can search for them, at an extra cost.

Q: My antigen has glycosylation sites. Is it OK?

A: It is OK as long as you know each location and add-on weight. If you do not know the add-on mass, we can still do the experiments, but we may not be able to obtain the information around the glycosylation sites.

Q: Are there any good references?

Deperalta G, et al. Structural analysis of a therapeutic monoclonal antibody dimer by hydroxyl radical footprinting. mAbs. 2013; 5:86-101; 

Kaur P, et al., Characterizing monoclonal antibody structure by carboxyl group footprinting.mAbs. 2015;7:540-52; 

Kaur, P., et al. Covalent Labeling Techniques for Characterizing Higher Order Structure of Monoclonal Antibodies. 2015. 

Current, State of the Art, and Emerging Technologies for the Analysis of Monoclonal Antibodies Editors: Darryl Lee Davis, Oleg Borisov, and John