You are here


Molecular Weight analysis (Intact Mass measurement)

Molecular weight determination of biopharmaceutical product provides information of the molecular mass  and heterogeneity under non-reducing conditions. When performed under reducing conditions, molecular weight of light and heavy chains can be obtained with high accuracy in addition to heavy chain glycosylation heterogeneity.

Intact mass measurement of the biopharmaceuticals will be performed using a LC-MS system consisting of C4 column (0.5 mm x 50 mm)

Peptide Mapping

Peptide mapping is a powerful tool for separation and identification of proteins The sample is digested with trypsin or proteases and resulting peptides are separated and identified by MS/MS data. Peptide mapping provides information about sequence coverage, amino acid changes, oxidation, deamidation, N- terminal cyclization, C-terminal lysine processing and N-glycosylation.

N-terminal sequencing

Ascertaining N-terminal sequence of a biopharmaceutical product is one of the criteria in establishing comparability of similar biologics. In recent years, bottom-up, LC-MS/MS based strategy has been used for N-terminal sequencing, especially when the protein is modified at the N-terminal. Peptide mapping based bottom-up method will be used for establishing N-terminal sequence.

C-terminal sequencing

C-terminal sequencing is necessary for the complete characterization of biopharmaceuticals. Bottom-up peptide mapping strategy is utilized for C-terminal sequence.

Post-translational modifications

Structural heterogeneity in proteins may occur due to various reasons. Post translational analysis is useful for determining these changes such as oxidation, amidation, acetylation, methylation, sulfation etc.

Glycosylation studies

Glycans, repertoire of complex carbohydrates attached to proteins and lipids, mediate a number of functions in immune defence, cell-cell communication, development and disease. Most of the protein drugs are glycoproteins including monoclonal antibodies. Diversity of glycans structures present on biopharmaceuticals impact folding, stability, half-life, immunogenicity, pharmacokinetics and therapeutic efficacy. Moreover, glycosylation is influenced by expression system and culture conditions of antibody production, purification and formulation. Hence, through characterization of glycans and their attachment site and sialic acid content is necessary to ensure immunogenicity and stability of biologics.

Proteins can be modified based on their attachment to carbohydrate groups. This can occur by N-linkage or O-linkage of glycosidic bonds. Therefore determining the site of N-linkage and O-linkage by site analysis and doing a population profile can be useful in biologics characterization.  Linkage analysis can be useful in determining how monosaccharides can be linked to one another. Another important aspect is sialic acid analysis as sialic acid can affect serum half-life. Therefore, this analysis will help determine the sialic acid concentration present in the given sample.

 HPLC based glycan profiling and LC-MS based glycan site analysis will be used for characterization.

Sequence variants analysis

Amino acid sequence variants of biopharmaceuticals may arise due to DNA mutations and mistranslations during recombinant production. These variants can impact clinical safety and efficacy. Hence, sensitive characterization of sequence variants is important for bioprocess development.  sensitive LC-MS based methods will be used for quantification of sequence variants in biologics.

Host cell contamination

Biologics can often be contaminated by proteins from the host cell, which can affect the safety, efficacy and activity of the final product. Characterization of impurities, at all stages of the manufacturing process, is thus very important in defining well characterized biologics.

Protein aggregation

Protein immunogenicity remains an important concern in therapeutic biotech products. Protein aggregates can be formed at various stages of biologics production. Several techniques are available for analysis of protein aggregates such as analytical ultracentrifugation (AUC) and size exclusion chromatography with multiple angle light scattering (SEC-MALS)

Biophysical characterization

Various biophysical tools are used to establish and finger-print secondary and tertiary structure of biopharmaceuticals and biologics conformance compatibility. This can be done using various bioanalytical techniques such as circular dichroism, size exclusion chromatography and fluorescence spectroscopy. Stability of the proteins will be tested using DSC.

© 2018 C-CAMP All Rights Reserved