Detailed characterization of therapeutic biological molecules is required before they can be considered acceptable for its intended use. As suggested in the ICH Q6B guidance, extensive characterization usually should include determination of physicochemical properties, biological activity, immunochemical properties, purity and impurities. The guidance also suggests that as new analytical technology and modifications to existing technology are continually being developed, and they should be utilized when appropriate to allow relevant specifications to be established.
A well characterized product requires characterization of product contaminations as well as establishing specifications for each step of process. Setting specification to in-process stages ensures a consistent drug substance, drug product or materials that can be regarded as safe for its intended use.
The team of scientists at Zelle Biotechnology has experience in Upstream and downstream development, manufacturing as well as assay development and quality control. We have worked with a broad spectrum of molecules, including small molecules, peptides, enzymes and glycoproteins.
A range of analytical services are available in support of:
Amino Acid Analysis is the used to determine protein concentration and also provides amino acid composition and free amino acids. The amino acid composition gives a characteristic profile for proteins which is used to identify a protein.
Sodium dodecyl sulfate (SDS) is an amphipathic detergent which binds non-covalently with the protein and masks intrinsic charge of a protein. Polyacrylamide gel electrophoresis (PAGE) is the most common analytical technique used to separate and characterize proteins. Due to SDS proteins have very similar charge-to-mass ratios, and similar shapes. During PAGE, the rate of migration of SDS-treated proteins is determined by molecular weight.
IEF is used to separate proteins according to their net charge. This method is used to characterize contaminants as well as product variants.
Western Blotting can be used in conjunction with the electrophoretic separations listed above to prove the identity of a protein by detection with a specific antibody. In combination with a polyclonal antibody e.g. raised against a cell lysate, western blotting can be used to assess the specificity of HCP-ELISAs.
The main application of Size-Exclusion chromatography (SEC) is to detect protein aggregation and protein fragmentation.
Reversed-Phase HPLC is often used in combination with UV or mass spectrometry as a proof of identity and purity. As examples, protein fragmentation or oxidation can be monitored.
Peptide mapping creates a fingerprint that is unique and is used to confirm the identity of a protein. It is usually uses RP-HPLC with either UV or MS detection.
N- and O-glycans, neutral monosaccharides and sialic acids are detected by MS or NP-HPLC. The NP-HPLC method requires labelling of the glycans.
Capillary electrophoresis is principally separation of charged analytes in presence electrical field.
The Ion Exchange HPLC (IEX) technique detects variation in the protein due to charge. These charged variants are due to deamidation or C-terminal lysine variants in monoclonal antibodies.
The measurement of protein concentration in an aqueous sample is an important assay in biochemistry research and development labs for applications ranging from enzymatic studies to providing data for biopharmaceutical lot release. Spectrophotometric protein quantitation assays are methods that use UV and visible spectroscopy to rapidly determine the concentration of protein, relative to a standard, or using an assigned extinction coefficient.
Sialic acids are negatively charged, terminal monosaccharides. Variation in the quality and quantity of sialic acid impacts activity and stability of glycoproteins. To produce a consistent product it’s important to be able to monitor the sialic acid in the structure.
The application of mass spectrometry in protein research has revolutionized the analysis of the large and complex molecules. Different ionization techniques and mass detectors were developed that dramatically has increased the sensitivity, accuracy and resolution of the mass spectrometers in the last 25 years. Also, the mass spectrometers were interfaced with liquid chromatography separation, further expanding the range of applications. LC-ESI-MS is a technique that combines the resolving power of HPLC separation with high mass accuracy of a mass spectrometer. As such, it is useful for the determination of intact protein mass or the in-depth analysis of protein or peptide mixtures. Coupling to a UPLC allows quantifying the detected features either by UV or by the MS signal intensity. More