Building Orthogonality into Your Analytical and Characterization Plans – Part 1

Dr. Richard L. Easton, Technical Director, BioPharmaSpec

The FDA [1] and EMA [2] guidelines are both comprehensive articles giving clear guidance on what the regulatory agencies expect from structural characterization studies. The use of state-of the-art instrumentation and techniques is expected but what is also clearly required is the use of orthogonal techniques in the structural comparability assessments.

This requirement comes from the need to give a firm structural foundation to any claims, such as the ability to produce consistent batches or biosimilarity between your biosimilar and the reference drug. Thus, the use of characterization techniques that can verify and support conclusions drawn from other techniques will result in a robust package of structural data in which both the manufacturer and the regulatory authorities can have confidence.

In Part 1 of this blog, we describe the first of three examples of orthogonality within required analyses, each examining different aspects of protein structure.

Orthogonality in Glycan analysis

Data on glycan structure can be generated in different ways but it is important to realise the limitations of any techniques that are utilized. For example, as part of an investigation into the structure of a glycoprotein it is necessary to perform a peptide mapping exercise. Peptide mapping is the identification of peptides (through the use of liquid chromatography-mass spectrometry) from a series of specific proteolytic digestions of the protein. The masses of the detected peptides should match the theoretically predicted masses, giving confidence in several aspects of the structure such as sequence, intactness and the presence or absence of post translational modifications. It should be noted that peptide mapping, in its basic form, is not sufficient for confirmation of the primary amino acid sequence unless specific evidence for each amino acid is determined and other techniques are used to eliminate any ambiguities.

Glycopeptides can be readily detected in a peptide mapping study. However, the energy applied during the ionization process (within the mass spectrometer) can result in some breakdown of the glycan structures, producing a profile that contains artificially generated truncated species as well as those naturally present on the glycopeptide (Figure 1A). Furthermore, due to the lower ionization efficiency of glycopeptides over peptides, it is unlikely that all glycopeptides will be detected (with minor species not being observed above the background).

Figure 1: N-glycan analysis using peptide mapping and fluorescent tagging. (A) Peptide mapping signals observed for an IgG glycopeptide, the region containing doubly charged signals is shown (only the glycans are shown for simplicity). A relatively high abundance of truncated glycan species can be seen (marked (*)) in the figure. (B) Chromatographic profile of fluorescently tagged released N-glycans. The truncated species seen in Figure 1A are either absent or significantly reduced, indicating they are a result of in-source fragmentation processes in the peptide mapping experiment.

There is no doubt peptide mapping is very important for assessing glycopeptides and provides important structural evidence for their presence and nature (and also the level of glycosylation site occupancy) but a full assessment of the glycans requires release and analysis in specifically designed experiments (such as on-line liquid chromatography mass spectrometric analysis of fluorescently labeled released glycans). This technique does not suffer from the same issues as the peptide mapping since glycan species are separated and the retention time and peak area of the fluorescently labeled species are used to identify and provide relative quantitation (Figure 1B). Mass spectrometric assessment of the eluate then provides intact mass information and structural information for each released oligosaccharide via fragmentation, the combination allowing identification of each component. The fluorescently labeled data will serve to verify and extend the conclusions drawn from peptide mapping data. This is particularly useful since glycopeptides tend to produce minimal peptide fragmentation in a basic mapping study. Thus, the components observed in the fluorescent data can be used to support the glycopeptide mass assignments made from the mapping data.

Check back with us next month for Part 2 of this blog, where we will consider two further examples of protein structure and how to implement orthogonality within the required characterization methods.


  1. Development of therapeutic protein biosimilars: Comparative analytical assessment and other quality-related considerations. Guidance for Industry. FDA May 2019.
  2. Guideline on similar biological medicinal products containing biotechnology-derived proteins as active substance: quality issues (revision 1). EMA/CHMP/BWP/247713/2012
  3. Beyer, B at el., (2018), Microheterogeneity of recombinant antibodies: Analytics and functional impact. Biotechnol. J., 13, doi. 10.1002/biot.201700476