Centre for Cellular and Molecular Platforms (C-CAMP), is a Dept. of Biotechnology (Govt. of India) initiative and a not for profit organisation, that also forms an important part of the Bangalore Bio-cluster. We are acting as an enabler of bioscience research and entrepreneurship by providing research, development, training and service in state of the art technology platforms.
C-CAMP is envisioned to be a major platform technology, industry-interaction and incubator unit. Considering the vacuum that is present at the interface of academia and industry, such an organization is much needed in the country to be able to assure the success of scientific talent.
At C-CAMP, we are generating state of the art platform technologies which are essential requirements for success and leadership in the field of life science. C-CAMP will allow investigators to use techniques as tools and not be limited by technological barriers while pursuing challenging scientific questions.
As a mandate from Dept. Of Biotechnology (DBT), Govt. of India, C-CAMP has set up a comprehensive physicochemical characterization facility for biologics and biosimilars using state-of-the-art instrumentation and standard operating protocols (SOPs).Biological entities, unlike small molecule based pharmaceuticals are very complex in nature. Biopharmaceuticals, can exhibit tremendous heterogeneity in terms of posttranslational modifications (PTMs) such as glycosylation, oxidation, deamidation, phosphorylation etc. and structural modifications. In addition to this, changes in upstream and downstream processes during recombinant production of biologics also influence structural heterogeneity. Therefore, comprehensive characterization of biologics is necessary to ensure safety and efficacy.
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.
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.
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.
C-terminal sequencing is necessary for the complete characterization of biopharmaceuticals. Bottom-up peptide mapping strategy is utilized for C-terminal sequence.
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.
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.
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)
Separation of biopharmaceutical products using SDS-PAGE under reducing and non-reducing conditions is important for establishing approximate molecular weight of heavy and light chains, relative concentration and identification of degradation products between biologics and biosimilar.
Estimation of concentration of a biologics/biosimilar is essential for knowing protein component in the solution or formulation. Bradford method will be used to quantify proteins.
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