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Peptide Analytical
The hard measurement is epimerization.
Analytical development
A dedicated analytical team covers peptide analysis across all development phases from preclinical through commercial manufacturing, for both drug substance and drug product.
The team is co-located with the API process development team.
Enantiomeric purity
Determining the enantiomeric purity of a peptide accurately is difficult. Peptides with different amino acid isomers share the same physicochemical properties, which makes them hard to analyze intact. Free amino acids do not retain well for HPLC-UV analysis, and peptides containing multiple amino acids interfere with each other during analysis.
We quantify D-isomer content by acid hydrolysis and derivatization followed by LC-MS, covering all nineteen isomeric natural amino acids plus ornithine. Asparagine and aspartic acid, and glutamine and glutamic acid, are reported together.
Identity is confirmed by tandem LC-MS/MS and by peptide mapping after enzymatic digestion.
Drug substance analytical services
- Analytical development for drug substance specific methods (purity, sequencing) and feasibility for peptide generic methods
- Forced degradation study with impurity qualification and quantitation
- Release testing and stability study under R&D conditions
- Reference standard and impurity marker qualification and retesting
- Physicochemical property study and structure elucidation
- Microbiological safety: endotoxin limit and quantitation testing, bioburden for microbial limit test, microbe identification and strain typing
Drug product analytical services
- Container-closure integrity testing, including quantitative vacuum decay and high-voltage leak detection and qualitative microbial challenge and tracer liquid methods
- Foreign and particulate matter observed in situ, verified by flow imaging and identified by IR, Raman or EDX, with osmolality
- Purity by LC-MS, including identification and quantitation by Q-TOF HRMS and quadrupole LC-MS, for hard-to-resolve parenteral impurity analysis
Special capabilities
- Process-related impurity identification by 2D UPLC coupled with Q-TOF HRMS
- Identification by tandem LC-MS/MS for sequence confirmation
- Amino acid analysis and enantiomeric purity by acid hydrolysis and derivatization followed by LC-MS, giving the amino acid ratio and the D-isomer impurity amount in the sequence
- Identity by peptide mapping, with enzymatic digestion followed by LC-MS
Quality control
- Method qualification and validation for IND, NDA, and commercial use
- Transfer of GMP validated methods according to USP <1224>
- Full release testing across raw materials, reagents, intermediates, drug substance, and drug product
- Accelerated and long-term stability studies under ICH conditions
Case study: main peak purity by 2D-UPLC-TOF
Multi-heart cutting 2D-UPLC-TOF is used routinely for quantitation and qualification of impurities that regular reverse-phase HPLC struggles to separate, which improves both the efficiency of impurity analysis and the accuracy of the data.
Case study: sequencing a cyclic peptide
A method was developed for sequencing peptides with structures more complex than linear peptides, for identification and release of the peptide drug substance.
Case study: chiral amino acid analysis
The enantiomeric purity of all nineteen isomeric natural amino acids, plus ornithine, is analyzed despite the shared physicochemical properties of the isomers and the interference between multiple amino acids in one sequence.
Instrumentation
- Chromatography: UPLC, HPLC, GC, IC, 2D-UPLC, HPLC-CAD
- Mass spectrometry: Q-TOF, MALDI-TOF, LC-Orbitrap, LC-MS, LC-QQQ, 2D LC-MS, GC-MS
- Physicochemical testing: FT-IR, UV-Vis, XRPD, DVS, DSC, TGA, polarimetry, 400 and 600 MHz NMR, circular dichroism, polydispersity
- Elemental analysis: ICP-OES, ICP-MS, CHN elemental analysis, Kjeldahl analysis
- Other testing: amino acid analysis, enantiomeric purity, polarimetry, refractometry, titration, turbidimetry, osmolality, total organic carbon, total viable aerobic counts, endotoxins by LAL test, heavy metals, sulfated ash, residue on ignition, polydispersity