Structural and physicochemical characterization are a crucial part of biopharmaceutical product development. The FDA and EMA expectations for the protein characterization methods to be performed are outlined in ICH Topic Q6B.
ICH Topic Q6B provides examples of technical approaches to be considered for structural characterization and confirmation, and evaluation of physicochemical properties of the desired product, drug substance and/or drug product. The specific characterization assays employed will vary from product to product and alternative approaches may be appropriate in many cases. The guideline recognizes that new analytical technology and modifications to existing technology are continuously being developed and should be utilized when appropriate.
ICH Q6B requires data covering the following structural and physicochemical elements for the Active Biopharmaceutical Ingredient:
Data covering the following frequently encountered product-related impurities and any product specific impurities should also be assessed as appropriate:
The techniques used to provide the necessary data to support the regulatory process are outlined in Table 1 below:
Table 1
Analytical requirement | Methodology used for data provision | Instrumentation requirements |
Amino acid sequence | N-terminal sequencing (Edman chemistry) Mass-spectrometric sequencing | N-terminal sequencer On line LC-MS (with MSe and/or MS/MS) MALDI-MS and MS/MS |
Amino acid composition | Amino acid analysis | Amino acid Analyzer (e.g. RP-HPLC with fluorescence detector) |
Terminal amino acid sequence | N-terminal sequencing (Edman or mass spectrometry) Mass spectrometry based sequencing for the C-terminus | N-terminal sequencer Mass spectrometer capable of providing N- and C-terminal sequence information |
Peptide map | Peptide mapping | On line LC-MS (with MSe and/or MS/MS) |
Sulphydryl group(s) and disulphide bridges | Non-reduced peptide mapping | On line LC-MS (with MSe and/or MS/MS) |
Carbohydrate structure | Monosaccharide composition analysis Oligosaccharide population analysis Glycosylation site analysis | GC-MS LC and on line LC-MS (with MSe and/or MS/MS) MALDI-MS and MS/MS |
Molecular weight or size | Intact molecular weight analysis | On line LC-MS MALDI-MS |
Isoform pattern | IsoElectric Focusing (IEF) | Imaged Capillary IEF (icIEF) |
Extinction coefficient | Optical density measurement and protein concentration determination | UV spectrophotometer Amino acid Analyzer (e.g. RP-HPLC with fluorescence detector) |
Electrophoretic analysis | Charge and size based electrophoretic analysis | icIEF Capillary Gel Electrophoresis (CE-SDS) |
Liquid Chromatography | Charge, size and hydrophilicity/hydrophobicity based chromatographic analyses | Ion-exchange chromatography (IEX) Size Exclusion Chromatography (SEC) Reversed Phase Chromatography (RP-HPLC) |
Spectroscopic profiles | Secondary and tertiary structure analysis | NMR (1D and 2D)1 Circular Dichroism (CD) Fourier Transform-Infra Red (FT-IR) Fluorescence (Intrinsic and Extrinsic) |
Analysis of truncated forms | Intact molecular weight analysis Peptide mapping Size based chromatographic and electrophoretic analysis | On line LC-MS (with MSe and/or MS/MS) MALDI-MS SEC CE-SDS |
Analysis of other modified forms | Intact molecular weight Peptide mapping Oligosaccharide profiling Isoform profiling | On line LC-MS (with MSe and/or MS/MS) MALDI-MS icIEF |
Aggregation | Size based chromatography, Analytical UltraCentrifugation (AUC) and Field Flow Fractionation (FFF) | Size Exclusion Chromatography with Multi Angle Laserlight Scattering (SEC-MALS) Sedimentation Velocity-Analytical UltraCentrifugation (SV-AUC) Field Flow Fractionation (FFF; AF4) |
1 HDX-MS data can also be used if NMR data is not available