Author: Dr.Richard Easton Date: 5th August 2026
For any protein-based biopharmaceutical product, it is essential to confirm that the primary amino acid sequence is correct. Errors in the transcription or translation process, or any modifications to the DNA itself following uptake by the cells, may give rise to mutations in the protein sequence. Therefore, sequence verification is a key requirement outlined within ICH Q6B analytical guidelines (1, 2) adopted by the regulatory agencies and referenced within biosimilarity guidance issued by both the EMA (3) and FDA (4). The UK MHRA largely follows the EMA principles for demonstrating biosimilarity, which include detailed comparative structural investigations (5).
As biosimilarity assessments require direct comparison between the biosimilar and the innovator product, primary amino acid sequence assessment should be performed on samples from both biosimilar and innovator (typically one batch of each). This should be performed early in product development to identify any sequence-related discrepancies at an early stage, allowing sufficient time for rectification, since a biosimilar must have the same primary amino acid sequence as the reference innovator product.
Protein molecules vary considerably in size and complexity. While some proteins are relatively small (low kDa in mass), monoclonal antibodies can exceed 150 kDa and contain several hundred amino acids across more than one chain. Each protein has a unique amino acid sequence, although certain regions of similarity may exist between related proteins, such as those observed across different monoclonal antibody classes.
Consequently, protein sequencing strategies are most effectively designed with knowledge of the expected sequence, where available. However, de novo sequencing approaches can also be applied when the protein sequence is unknown. While general sequencing principles can be applied, the specific approach must be tailored to the characteristics of the protein being analysed.
The key question therefore becomes: What is the most efficient and effective strategy to sequence a protein?
The key tool in the protein sequencing workflow is the mass spectrometer. These instruments, particularly Q-TOF platforms, offer high sensitivity and mass accuracy and can fragment enzymatically generated peptides along the protein backbone through well-established mechanisms, producing a series of sequence-specific fragment ions. The masses of these ions depend on the masses of the amino acid side chains, and thus, by knowing the masses of the naturally occurring amino acids, the peptide sequence can be determined.
The precise mechanisms for generating these fragment ions were elucidated by BioPharmaSpec’s Chairman and CSO, Professor Howard R. Morris FRS, in the early 1980s (6). By applying a protein digestion strategy to generate defined peptides, followed by their separation using on-line liquid chromatography and direct introduction into the mass spectrometer, sequence information from a protein can be generated.
To achieve comprehensive sequence coverage, multiple digestion strategies are typically employed. These precise strategies are designed and executed to generate clearly identifiable fragment ions from each digest so that the full linear amino acid sequence can be identified with confidence.
The simplest ways to generate the majority of the sequence information is through in-source fragmentation of all ions in a non-selective manner or through collisional activation of selected masses following a mass selection event within the instrument. These are standard fragmentation methodologies for certain types of mass spectrometers.
The mass spectrometry-based sequencing strategy works because most amino acids have unique masses and hence generate unique mass fragment ions within the peptides. However, one important exception exists.
Leucine and isoleucine are structural isomers and are therefore isobaric, meaning they have exactly the same mass.
Lysine and glutamine also have essentially the same mass, although they are not isomers. These two amino acids can be distinguished by accurate mass values and by their susceptibility to proteolytic digestion; for example, trypsin cleaves at the C-terminal side of lysine but not glutamine. Leucine and Isoleucine differentiation lacks a similarly reliable enzymatic discriminant, making it a persistent analytical challenge.
Discrimination between Leucine and Isoleucine is essential because unless a definitive identification is made, the precise nature of the amino acid at specific position within the protein chain remains ambiguous. Consequently, complete primary sequence identification at the protein level cannot be claimed.
This raises an important question:
How can mass spectrometry-based approaches be used to differentiate between leucine and isoleucine?
Figure: I/L discrimination by ECD w-type ions
Recent advances in mass spectrometry have expanded the ability to differentiate leucine and isoleucine. State-of-the-art platforms such as the Waters Cyclic Ion Mobility Separation Mass Spectrometer and the SCIEX ZenoTOF 7600, both used within BioPharmaSpec laboratories, can use Electron Capture Dissociation (ECD) and Electron Activated Dissociation (EAD) respectively, to induce fragmentation through mechanisms other than the standard energy- and collision-based backbone fragmentation described previously.
Unlike traditional fragmentation methods, these electron-based fragmentation techniques induce side-chain fragmentation of amino acids, producing additional sequence ion information. For leucine and isoleucine, this side-chain fragmentation generates specific and unique fragment ions that allow these two isobaric amino acids to be differentiated. As side-chain fragmentation occurs alongside backbone fragmentation, these data enable both the identification and localisation of leucine and isoleucine residues within the protein sequence.
Importantly, these fragmentation techniques can be applied during on-line liquid chromatography (LC) separation of a proteolytic digest mixture, allowing real-time generation of sequence information.
Mass spectrometry is a highly powerful tool for protein sequencing owing to the range of fragmentation mechanisms that can be induced. Readily produced backbone fragment ions allow peptide sequences to be identified, and any anomalies can be assigned quickly based on mass discrepancies from expected values and on the overall mass of the observed proteolytic digest products. In addition, newer mass spectrometric technologies have introduced alternative fragmentation mechanisms and, as a result, the possibility of side chain fragmentation, allowing the isobaric amino acids leucine and isoleucine to be distinguished and definitively identified within the protein sequence.
These technologies enable rapid data generation and protein sequencing, while reducing the need for other sequencing approaches by extending the effective capabilities of mass spectrometry. Nevertheless, mass spectrometry alone may not always generate clear fragment ions across the entire protein sequence. There may be regions that are not amenable to producing distinct fragment ions due to peptide size or specific vagaries of the sequence, such as high proline content biasing the formation of specific fragment ions.
In these instances, BioPharmaSpec’s approach is to supplement the MS data with Edman based N-terminal gas phase sequencing of isolated peptides to provide the complete amino acid sequence of the molecule, irrespective of its size.
Our ECD/EAD-enabled MS platforms resolve isobaric residues that standard fragmentation can't.
