IN-HOUSE Laboratory Service
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.
- Drug substance and drug product analysis
- Impurity analysis
- Stability testing
- Biosimilar testing
Intact Mass Analysis
Monoclonal antibodies are one of the most common types of biopharmaceuticals. Knowledge of an antibody’s molecular weight is essential to confirm that the antibody has been expressed correctly; if the molecular weight differs from predicted weight then this indicates that there is a change to the primary structure. One way of screening for these changes is through intact mass analysis by liquid chromatography-mass spectrometry (LC-MS). Analysis of the Intact Mass of a deglycosylated Monoclonal antibody can be done to determine the primary structure molecular weight. N-Deglycosylation of monoclonal antibody is performed using PNGase F.
?Monoclonal antibody (Glycosylated) with no prior treatment (only buffer exchange if required) is resolved from formulation components and detected, using reverse phase chromatography and UV detection on UPLC H-class Bio followed by mass analysis in positive sensitivity-MS only mode using Waters Xevo G2-XS QTOF with UNIFI.
Subunit Mass Analysis
Reduced Peptide Mapping Analysis
Methionine and tryptophan oxidation is investigated in the reduced peptide mapping analysis of monoclonal antibody. Unmodified and oxidized peptides are compared for relative quantification of modified peptides.
Asparagine and glutamine deamidation is investigated in the reduced peptide mapping analysis of monoclonal antibody. Unmodified and deamidated peptides is then compared for relative quantification of modified peptides.
Non Reduced Peptide Mapping Analysis / Disulfide Linkage Analysis
Differential Scanning Calorimetry (DSC)
DSC measures the heat capacity, Cp, of a molecule as a function of temperature. It measures the difference in heat flow between the sample and reference chamber, as both chambers are slowly heated. For some mAbs certain formulation conditions show changes in the thermograms. DSC analysis is done using a nano DSC instrument.
Circular Dichroism (CD)
Circular Dichroism is performed to study far & near UV spectra, protein folding & thermal denaturation of the protein. The secondary and tertiary structure of the monoclonal antibody is characterized using a J-1500 automated CD spectrometer (Jasco). Samples will be dialyzed into buffer and diluted to obtain suitable concentrations for far- and near-UV measurements. The spectra will be obtained for multiple independent replicates, corrected for the dialysate baseline and normalized by absorbance at 192 nm in the far-UV and 280 nm in the near-UV.