In all variations, an antibody binds to the protein of interest and the presence of the protein is visualized by a colorimetric reaction. Indirect, direct and sandwich ELISA are the most common variations of ELISA. ELISA (Enzyme-Linked Immunosorbent Assay)ĮLISA is used to detect proteins and determine the concentration of the protein of interest. This method can be used to identify the protein product or product related impurities. A conjugated secondary antibody is then added which will bind the primary antibody and allow the protein of interest to be visualized. An antibody specific to the protein of interest is added to the membrane which will bind to the protein. Western blots are conducted by first running an SDS-PAGE gel and then transferring the proteins onto a membrane.

Clipped or degraded proteins can be detected by viewing bands that differ from the band produced by the biological product. Larger proteins have a harder time moving through the gel, therefore proteins of different sizes become separated. SDS coats the proteins in a negative charge and the molecules are pulled through a gel matrix by running a current through the gel. SDS-PAGE separates out proteins in a polyacrylamide gel based on size. SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis) TOF-MS is a powerful platform that can also be used to examine for post–translational modifications of proteins. The TOF-MS is also commonly used for intact mass analysis which is sometimes added to identity and purity tests for biologics. For large proteins, peptide mapping following trypsin digestion is commonly used to verify their identity. PBL has a recently purchased Triple Quad 6500 + and a QTOF-MS along with extensive experience performing lot release tests for biologics. Reference standards of known impurities can be purchased to positively identify the impurity.

Mass spectrometry can be used to identify biological products and alert the manufacturer of any impurities. Alternatively, SEC can be coupled with RI and light scattering detection to measure the molecular mass of larger, intact biological compounds. Protein aggregates and 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). SEC can be used with dextran mass standards and refractive index (RI) detection to estimate the molecular mass. Following validated methods, Pacific BioLabs can perform lot release test for identity and purity in a dependable and efficient manner. Size exclusion, ion exchange and reverse phase chromatography are often used in identity and purity lot release tests of large molecules by HPLC. HPLC (High-Performance Liquid Chromatography) ELISA (Enzyme-Linked Immunosorbent Assay).

SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis).HPLC (High-Performance Liquid Chromatography).Pacific BioLabs provides manufacturing support and lot release testing for biologics. When manufacturing biological products (large molecules), it is imperative that each lot produced conforms to predetermined specifications. Large Molecule – Identity and Purity Testing

Sds page vs western blot vs elisa