Identity and Purity

Protein identity testing confirms that a biopharmaceutical product is the intended molecule. Purity testing identifies and quantifies any unwanted impurities or product-related variants present in the sample. Both are regulatory requirements under ICH Q6B for proteins, peptides and glycoproteins, and must be demonstrated using appropriate analytical methods before a biopharmaceutical can progress through development and towards regulatory submission.

Identity testing in biopharmaceuticals typically uses a combination of chromatographic and electrophoretic techniques, chosen based on the structural properties of the molecule. Common methods include reversed-phase HPLC, ion-exchange chromatography (IEX), imaging capillary isoelectric focusing (icIEF), size exclusion chromatography (SEC), CE-SDS and SDS-PAGE for purity assessment. Peptide mapping using LC/ES-MS with MSe or MS/MS fragment ion generation is widely used as an identity test, providing a highly specific structural fingerprint of the protein.

In peptide mapping identity testing, the protein is enzymatically digested and the resulting peptides are analysed by online LC/ES-MS with MSe or MS/MS fragment ion generation. Each peptide is identified by its molecular weight and fragment ion data. Once all released peptides have been assigned and a reference trace qualified, the UV chromatographic profile alone can serve as the routine identity test — without the need for mass spectrometric detection on every run. This approach provides a highly specific and reproducible identity fingerprint aligned with ICH Q6B requirements.

Imaging capillary isoelectric focusing (icIEF) is a charge-based electrophoretic technique that separates protein variants according to their isoelectric point (pI). It provides high-resolution charge profiles that can be used to confirm product purity and assess charge heterogeneity, including acidic and basic variants arising from post-translational modifications such as deamidation or sialylation. icIEF is typically developed in parallel with ion-exchange chromatography (IEX) to provide complementary charge-based data. fraction collection for further structural analysis can be performed from both these techniques.

Protein purity in a biopharmaceutical context is determined by characterizing both the main peak and the minor peaks in chromatographic or electrophoretic profiles. The main component is confirmed as the intended product through intact mass analysis, peptide mapping and glycan characterization where applicable. Minor peaks are identified as product or process-related impurities and their relative proportions calculated to give an overall purity assessment. Multiple orthogonal methods are used to ensure a comprehensive and regulatory-compliant picture of purity. Chromatographic separation using different dimensional techniques provides a detailed picture of the impurity profile of the product. Fractions can be collected for specific analysis and identification of the impurities.