Protein Analytics

Biophysical characterization of biologics is critical in the development of biopharmaceuticals such as innovator biologics and biosimilars. Many aspects of biologic drug function, activity, and stability are affected by the physical and biological behavior of proteins. Biophysical analytical techniques, for example, can aid in monitoring or confirming conformational integrity, the nature of the protein’s folded state, and how peptides interact to form complex physical structures. Consideration of specific interactions between drug substance and excipients that might potentially alter protein structure and product stability, is critical during formulation development, stability studies, and comparability. Protein degradation and aggregation can also be studied using biophysical techniques.

Aragen has established an advanced state-of-the-art, fully equipped analytical laboratory with highly qualified analytical scientists to perform a comprehensive characterization of biologics.

Analytics in Drug Discovery and Development Process using array of Instruments.

Assess and rigorously test the quality of purified target molecules.
- CE-SDS, cIEF, SEC-HPLC/MALS, SDS-PAGE, LC-MS, DLS/SLS, LAL, HIC, HILIC
Establish assays and screens for interactors and/or inhibitors.
- SPR, BLI, Multimode plate readers
Validate hits and characterize various interactions of drugs.
- SPR, BLI, Multimode plate readers
Rigorously characterize final drug products for FDA approval or patent preparation.
- cIEF, CE-SDS, SEC-HPLC/MALS, DLS/SLS, LAL, HILIC, LC-MS, SPR, BLI, Multimode plate readers

Protein Analytics	Quality Attribute	Platform
Intact Mass (non-reduced, reduced, +/-PNGaseF)	Primary Structure – MW	LC – MS /QTOF (Agilent)
PepMap	Identity an Primary Structure PTMs	LC – MS /QTOF (Agilent)
PepMap UV	Identity	LC – MS /QTOF (Agilent)
N-Glycan profiling by RP-MS or HILIC-MS	Primary Structure – PTMs	LC – MS /QTOF (Agilent)
Glycation	Primary Structure – PTMs	LC – MS /QTOF (Agilent)
Absolute mass for monomer multimers, and aggregate	Purity – Size variants	SEC – HPLC – MALS (Wyatt)
T_m, Ta_gg, Size and PDI	Thermal Stability, Higher Order Structure (HOS)	SLS /DLS (Unchained labs)
Sialic Acid Content	Primary Structure – PTMs	UPLC (Agilent)
N -Glycan profiling	Primary Structure – PTMs	HILIC -FLD /UPLC (Agilent)
Charge variants	Purity – Charge variants	IEX/HPLC (Agilent)
Size variants (aggregates )	Purity – Size variants	SEC-HPLC (Agilent)
Hydrophobicity variants	Purity – Protein related substances and impurities	RP or HIC/UPLC (Agilent)
Glycation	Primary Structure – PTMs	RP or HIC/UPLC (Agilent)
Protein concentration by Protein-ARP	Titer/Quantitation	BAC – HPLC (Agilent)
Free – thiol	Primary Structure – Free thiol	FLD – UPLC (Agilent)
Protein concentration by A280	Quantity	UV – VIS
A280, A320, Appearance	General – Appearance, Trubidity	UV – VIS
Free-thiol by Ellman’s assay	Primary Structure – Free thiol	UV – VIS

Protein Analytics	Quality Attribute	Platform
N – Glycan profiling	Primary Structure -PTMs	CE – SDS (SCIEX)
Charge variants	Purity	cIEF(SCIEX)
Charge variants	Purity	CZE (SCIEX)
Slab gel – charge variants	Purity	IEF, Gel Electrophoresis
Size variants	Purity	SDS-PAGE, Gel Electrophoresis
Western blot	Identity	Gel Electrophoresis
Binding kinetics (kon, koff, Kd)	Potency, Identity	BLI (Sartorious /ForteBio) or SPR (Cytiva /Biacore)
FCR and C1Q screening	Potency, Half – life, Safety	BLI (Sartorious /ForteBio) or SPR (Cytiva /Biacore
Protein/antigen titer	Quantity	BLI (Sartorious /ForteBio)
Epitope binning	Epitope characterization	BLI (Sartorious /ForteBio) or SPR (Cytiva /Biacore)
Functional blocking	Potency, Identity	BLI (Sartorious /ForteBio) or SPR (Cytiva /Biacore)
Yes /No binding	Identity	BLI (Sartorious /ForteBio) or SPR (Cytiva /Biacore)
Binding	Potency	SPR (Cytiva /Biacore)
Host Cell Proteins (Cygnus)	Safety	ELISA /Multimode plate reader (SpectraMax)
Residual Protein A/G/L	Safety	ELISA /Multimode plate reader (SpectraMax)
Endotoxin Testing	Safety	LAL /Charles River EndoSafe
Bioburden, Sterility Test	Safety	Culturing

Comprehensive Biologics Characterization

Instrumentation

Octet

Biacore

For characterizing antibody epitopes and multiprotein complexes of biological significance – detect presence of specific proteins with minimal interference from complex matrices.

Key Features