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. 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. 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.
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 two triplequads (LC-MS/MS units) and a TOF-MS along with extensive experience performing lot release tests for biologics. In additional to identity and purity tests, the TOF-MS is commonly used for intact mass analysis.
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.
Read More About Large Molecule Identity and Purity Tests
- ICH Q6B Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products