Large Molecule – Identity and Purity Testing
When manufacturing biological products (large molecules) it is imperative that each lot produced conforms to predetermined specifications. Pacific BioLabs provides manufacturing support and lot release testing for biologics and identity and purity/impurity tests are important aspects of all large molecule lot release testing programs.
Testing Services for Identity and Purity
- HPLC (High-Performance Liquid Chromatography)
- Mass Spectrometry
- SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis)
- Western Blot
- ELISA (Enzyme-Linked Immunosorbent Assay)
Size exclusion, ion exchange and reverse phase chromatography are often used in identity and purity/impurity lot release tests of large molecules by HPLC. If the chromatogram of the sample differs from the reference chromatogram impurities may be present. Following validated methods, Pacific BioLabs can perform your HPLC lot release test for identity and purity in a dependable and efficient manner.
Protein aggregates and other impurities such as protein fragments or unreacted components remaining from the manufacture of conjugated or fusion proteins can be analyzed by HPLC with size exclusion chromatography (SEC). For conjugated proteins or larger polymeric compounds where mass spectrometry cannot be used to determine molecular mass, HPLC with SEC can be used with dextran mass standards and refractive index (RI) detection to estimate the molecular mass. Alternatively, HPLC with SEC can be coupled with RI and light scattering detection to measure the molecular mass of larger, intact biological compounds.
Mass spectrometry can be used to identify biological products and alert the manufacturer of any impurities. Reference standards of known impurities can be purchased to positively identify the impurity. PBL has a recently purchased QTOF-MS along with extensive experience performing lot release tests for biologics. For large proteins, peptide mapping following trypsin digestion is commonly used to verify their identity. The TOF-MS is also commonly used for intact mass analysis which is sometimes added to identity and purity tests for biologics. TOF-MS is a powerful platform that can also be used to examine for post–translational modifications of proteins.
SDS-PAGE separates out proteins in a polyacrylamide gel based on size. SDS coats the proteins in a negative charge and the molecules are pulled through a gel matrix by running a current through the gel. Larger proteins have a harder time moving through the gel, therefore proteins of different sizes become separated. Clipped or degraded proteins can be detected by viewing bands that differ from the band produced by the biological product.
Western blots are conducted by first running an SDS-PAGE gel and then transferring the proteins onto a membrane. An antibody specific to the protein of interest is added to the membrane which will bind to the protein. A conjugated secondary antibody is then added which will bind the primary antibody and allow the protein of interest to be visualized. This method can be used to identify the protein product or product related impurities.
ELISA is used to detect proteins and determine the concentration of the protein of interest. Indirect, direct and sandwich ELISA are the most common variations of ELISA. In all variations, an antibody binds to the protein of interest and the presence of the protein is visualized by a colorimetric reaction. The absorbance readings from each well are compared with the standard curve to determine the concentration of the protein of interest from each sample.
Case Study – Drug Mixtures
PBL’s analytical team has characterized complex antibody mixtures using SDS-PAGE and ELISA for identity and purity. The team has developed and validated methods and conducted routine analysis for such products. In recent years, antibody mixtures have been considered more effective than a single antibody in establishing an increased effector response and minimizing the risk of variants that can propagate the disease.
Read More About Large Molecule Identity and Purity Tests
- PBL’s Learning Center – CMC Activities for Biologics and Monoclonal Antibodies
- ICH Q6B Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products