Post on 13-Jun-2020
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).
biologics@ccamp.res.in
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
10
20
30
40
50
60
70
80
90
100
110
120
130
140
150
160
170
180
190
200
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
biologics@ccamp.res.in
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: biologics@ccamp.res.in services@ccamp.res.in
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