an expectation that the analytical method will be further scrutinised by LC-MS to confirm that the main peak is pure , and that no co-eluting impurities are present . This typically involves an algorithmic assessment of the mass spectra of the main peak , coupled with further scrutiny of the data by an experienced mass spectrometrist to check for artefacts , such as multiply charged species . Where required , an orthogonal method is used to test for a specific impurity . Analytical development continues to evolve and 2D LC is now becoming more common as a further means of improving the analysis of peptides , making the control
of individual impurities in peptide manufacture increasingly important . The US FDA issued and later withdrew guidance on synthetic peptides , before guidance on ANDAs for peptides produced synthetically was introduced . 1 In Europe , the expectation for control of individual impurities in peptides is based on the information in the EP , that is : a reporting threshold of > 0.1 %; an identification threshold of > 0.5 %; and , a qualification threshold of > 1.0 %. 2 In general terms , control of impurities should increase through clinical development . Specifically , individual impurities should not exceed their values as qualified in the toxicology batch or be greater than 1.0 %, whichever is higher .
Impact of analytical method
Naturally , the impurity profile and hence the specification of a peptide are related to the analytical method used at that time . As additional development is performed , an analytical method may change . This can have a significant impact on the impurity profile and hence the specification limits of impurities that a process has to deliver against . In the example shown in Figure 1a , two impurities were present at a level of > 1.0 % area when this peptide underwent pivotal toxicology studies . Hence , the limit for impurities according to the EP guideline was set at a level equal to or lower than the values shown in any future batches intended for clinical studies . However , this same batch of peptide had a different impurity profile when a newly developed analytical method was used ( Figure 1b ). Now it was noted that more impurities were resolved and the reported levels of impurities was lower . This meant that the process had to be developed such that their levels were kept below 1.0 %. LC-MS is the common technique used to identify impurities . In this case , LC-MS analysis determined that the impurities originally reported as > 1.0 % were mixtures of isomeric impurities . These impurities can be harder to characterise . Chiral amino acid analysis is typically used to aid characterisation after enrichment of the impurity via preparative chromatography , followed by synthesis of the specific isomeric impurity if more than one of the amino acids is present in the sequence .
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Figure 2 – Typical impurities that form & potential controls in solidphase synthesis ( a ), cleavage / deprotection ( b ) & purification / ionexchange ( c )
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