Protein-based therapeutics are produced in living cells, resulting in an inherent degree of structural heterogeneity. Characterizing the inherent structural heterogeneity is an essential requirement for the development of a therapeutic monoclonal antibody. Post-translational modifications can affect in vivo stability, loss of biological activity and cause unwanted immune effects.
Sartorius provides the support and advice required to mitigate these risks by ensuring protein structure and physicochemical analyses are performed at appropriate stages of therapeutic protein development. We offer a comprehensive range of methods to characterize and confirm protein structure, carbohydrate profile and post-translational modifications using state-of-the-art techniques to ensure the ICH Q6B scientific guidelines are met, resulting in an efficient, streamlined development process.
We offer three methods for assessing glycoforms on intact proteins at any stage of your program
We offer structural and physicochemical characterization, combined with complex biological analysis, to fully characterize your protein
For standard molecules such as IgGs, we have pre-qualified our assays for rapid deployment
We can partner with you from early-phase clone characterization to full cGMP-compliant lot release, with tailored solutions at every stage
ICH Q6B Requirements
Amino acid sequence
|Amino acid composition|
Amino acid analysis
Terminal amino acid sequence
Peptide map and post-translational
Sulfhydryl group(s) and disulfide bridges
Non-reduced peptide mapping
The presence and abundance of glycan structures post translationally conjugated to the monoclonal antibody influence a number of characteristics, such as molecule structure, effector function and stability, creates a serious challenge in developing antibody therapeutics.
Released N-Glycan Analysis by LC/MS
Sartorius offers a released N-glycan assay for the in-depth profiling of IgG glycosylation. Glycans are enzymatically removed from the antibody and then labeled before a high-resolution liquid chromatography (LC) separation is performed (see chromatogram graphic).
Fluorescence detection coupled with online ESI mass spectrometry (MS) enables confident assignment and accurate quantification of the different glycan structures. These can then be grouped according to their structural classification (e.g., complex, oligomannose) and monosaccharide composition, such as fucose, galactose and sialic acid profiles.
The chromatogram shows the main glycoforms identified, relative to the most abundant GOF peak set to 100%.
Peptide mapping is the principal technique for confirming a protein’s primary structure (amino acid sequence). The protein is digested using a proteolytic enzyme, and the resultant peptides are analyzed by liquid chromatography–mass spectrometry (LC-MS). Our peptide mapping method combines an efficient digestion strategy, ultra-high performance LC separation and high-resolution MS for in-depth protein characterization.
Commonly, trypsin is used as a protease for peptide mapping because of its high specificity for lysine and arginine residues. However, there may be a requirement to use other enzymes in cases where trypsin alone does not provide 100% sequence coverage, or the protein of interest is resistant to trypsin activity.
Peptide mapping can be performed using a variety of proteolytic enzymes, with orthogonal specificity to trypsin, to obtain different peptide fragmentation pathways and achieve additional confidence for sequence verification through the use of overlapping peptides.
When confirming the amino acid sequence that makes up the primary structure of a protein, Peptide mapping is the method of choice. After using a proteolytic enzyme to digest the protein, the remaining peptides are ready for analysis by liquid chromatography–mass spectrometry (LC-MS). Our peptide mapping method combines an efficient digestion strategy, ultra-high performance LC separation and high-resolution MS for in-depth protein characterization.
Peptide mapping forms an important part of protein structure analysis as it also allows for the analytical characterization of a variety of post-translational modifications (PTMs), including: deamidation, oxidation, N-terminal pyroglutamic acid, C-terminal lysine clipping and glycosylation.