C-CAMP Brochure...Member of the Bangalore BioCluster C-CAMP • NCBS • inStem C-CAMP Centre for Cellular and Molecular Platforms A Dept. of Biotechnology (DBT, Govt. of India) Initiative

Member of the Bangalore BioCluster C-CAMP • NCBS • inStem

C-CAMP Centre for Cellular and Molecular Platforms

A Dept. of Biotechnology (DBT, Govt. of India) Initiative

Biologics Characterization Facility

Biologics/Biosimilar Characterization

Biologics/Biosimilars can exhibit tremendous heterogeneity in terms of structural and posttranslational modifications (PTMs) such as glycosylation, oxidation, deamidation, phosphorylation. In addition to this, changes in upstream and downstream processes during recombinant production of biologics/biosimilars also influence structural heterogeneity. Therefore, comprehensive characterization of biologics/biosimilars is necessary to ensure comparability, safety and efficacy. The Indian biologics/biosimilars market is growing rapidly and currently produces 20% of the world’s generics. Due to this, there is need for a Biologics/Biosimilar testing laboratory, for complete physicochemical characterization of biologics and biosimilars. As a mandate from Dept. Of Biotechnology (DBT), Govt. of India, C-CAMP has set up a facility for comprehensive physicochemical and structural characterization of biologics and biosimilars using state-of-the-art instrumentation and standard operating protocols (SOPs).

[email protected]  

Services Offered

We have established a Biologics Characterization Facility with the aim of providing guidance and expertise for comprehensive characterization of biologics/biosimilars using state-of-the-art facilities and SOPs.

•  Molecular Weight Analysis Intact mass measurement is performed under reducing and non-reducing conditions using a LC-MS system to determine molecular mass and heterogeneity of proteins e.g. monoclonal antibodies, fusion proteins etc.

•  Peptide Mapping

MS and MS/MS data of protease digested peptides are used to identify proteins. This study gives information about sequence coverage, oxidation, deamidation, N- terminal cyclization, C-terminal lysine processing and N-glycosylation.

•  N-terminal & C-terminal Sequencing

N-terminal and C-terminal sequencing is done by peptide mapping based bottom-up method to establish comparability and characterize biological products.

•  Glycosylation Studies Glycan site analysis will be used to characterize the diversity of glycan structures present on a particular amino acid in a given biopharmaceutical. HPLC based glycan profiling of PNGase F released and fluorophore labeled glycans and LC-MS based analysis

•  Post-translational Modifications Analysis of post-translational modifications like oxidation, amidation, acetylation, methylation, sulfation etc. is important to determine structural heterogeneity.

+TOF MS: 9.0968 to 11.0670 min from Sample 1 (Restova_filtered_0.1ug) of Restova_filtered_0.1ug_01.wiffa=7.01593984286807150e-004, t0=-5.66145823085586030e-001 (DuoSpray ())

Max. 202.8 cps.

2350 2400 2450 2500 2550 2600 2650 2700 2750 2800 2850 2900 2950 3000 3050 3100 3150 3200 3250 3300 3350 3400 3450m/z, Da

0

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80

90

100

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Inte

nsi

ty,

cps

2779.12002727.6635

2730.62472832.5394

2678.11382888.0594

2630.2574 2891.19322949.0614

2633.10512945.78222724.4893

2775.9442

2675.0253 3005.92662584.12972884.78542829.3520

2733.42412587.0130 3068.55212894.17482683.8474 2942.51702838.5429

2952.17362542.42883002.7055

2720.8945 3137.12123012.07462841.38062671.32632787.7971 3074.99462496.5597 2623.8125 3201.88982939.15702881.07802768.7221

3140.14513015.40462667.9360 3130.36152900.71492739.40802638.76472578.08872457.7218 3276.56873208.94992958.44502620.5926 2961.39952714.26712414.74272547.6502

M.Wt. Analysis

Peptide Mapping

N- & C- Terminal Sequencing

Glycosylation Studies

•  Sequence Variants Analysis Sensitive LC-MS based methods will be used for quantification of amino acid sequence variants in biological products, as these variants can impact clinical safety and efficacy.

•  Disulphide Bond Analysis Determination of disulfide bridges is a critical atribute to establish proper folding of the recombinantly expressed proteins including biologics/ biosimilars. We use Pepsin digestion followed by LC-MS/MS methodology for identification and relative quantification of normal (expected) and scrambled disulfide bonds. Coming Soon •  Host Cell Contamination involves characterization of impurities from the host cell, at

all stages of the manufacturing process.

•  Protein Aggregation using analytical ultracentrifugation (AUC) and size exclusion chromatography with multiple angle light scattering (SEC-MALS)

•  Biophysical Characterization is necessary to establish and finger-print secondary and tertiary structure of biopharmaceuticals. 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.

Sequence Variants

Host Cell Contamination

Size Exclusion Chromatography

Glycan Structures

Services Offered

[email protected]  

UPLC  with  Diode  Array  and                                              Fluorescence  Detectors  (Shimadzu)  

GC-­‐MS-­‐FID  with  Head  Space  Trap  (Perkin  Elmer)  

Nano  LC  with  Triple  TOF  5600+  (AB  Sciex)      

5800  MALDI-­‐TOF/TOF    (AB  Sciex  )  

Instrumentation

Contact  

Centre for Cellular and Molecular Platforms, NCBS-TIFR, GKVK Post, Bellary Road, Bangalore 560 065, India  

Dr. Taslimarif Saiyed, Director & COO,

C-CAMP

Website : www.ccamp.res.in/biologics Phone: +91 80-67185055/5052 E-mail: [email protected] [email protected]

Dr. P. Babu Associate Director,

Biologics Characterization

Protein Technology Core

Flow Cytometry Facility

High Throughput Screening Facility

Confocal Imaging Facility

Next Generation Genomics Facility

Fly Facility

Mass Spectrometry Facility

Our Technology Platforms

Core Facility Users

Dr. Ramaswamy S. CEO, C-CAMP