BioPharmaSpec provides a protein sequencing service that allows you to unambiguously confirm the amino acid sequence of a protein. Primary amino acid sequencing is the determination of the correct positions of each of the amino acids in your biopharmaceutical. This is different to peptide mapping, where the sequence is confirmed based only on the masses of peptides created through product digestion.
Peptide mapping or Protein Sequencing?
During peptide mapping, the protein is digested into peptides and these peptides are analyzed by mass spectrometry. Using an appropriate digestion strategy, all peptides can be assigned using mass spectrometry and overlapping these peptides can confirm the sequence against a theoretical amino acid sequence. The exact positions of each and every amino acid is therefore not confirmed during peptide mapping.
In cases where there is no theoretical sequence or it is a regulatory requirement to confirm the sequence unambiguously (e.g. during biosimilar development), protein sequencing or de novo primary amino acid sequencing should be performed.
Applications of Protein Sequencing
One of the main applications of protein sequencing (or primary, de novo, amino acid sequencing) is to fulfil an important biosimilar regulatory requirement. Primary amino acid sequencing is an essential analysis during the development of a biosimilar, because you must ensure that the primary protein sequence of the biosimilar is identical to that of the innovator product. This is the only way to prove that the biosimilar is the same molecule as the target (or innovator/ reference) molecule. It is not sufficient to draw conclusions about the amino acid sequence based on peptide mapping or the DNA sequence (due to transcription or translation errors).
Once the protein sequences of the biosimilar and innovator are proven to be identical, other comparability studies should be performed to show that the molecules are similar in their functionality and their secondary, tertiary and higher order structure.
Protein Sequencing Techniques
The below flow diagram illustrates the steps taken during a protein sequencing study at BioPharmaSpec:
Looking at this flow diagram, we can see that primary amino acid sequencing relies on two main analytical techniques:
- Mass spectrometry (On line LC-MS plus MSe and MS/MS) and,
- gas phase sequencing (also known as Edman chemistry or N-terminal sequencing).
Let’s now consider how each of these techniques is applied during protein sequencing.
Determine the primary amino acid sequence of your product
You can talk to our scientists today about the best analytical strategies to obtain the primary amino acid sequence of your product, whether you have a theoretical sequence or not.