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.
  • 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 AnalyticsQuality AttributePlatform
Intact Mass
(non-reduced, reduced, +/-PNGaseF)
Primary Structure – MWLC – MS /QTOF (Agilent)
PepMapIdentity an Primary Structure PTMsLC – MS /QTOF (Agilent)
PepMap UVIdentity LC – MS /QTOF (Agilent)
N-Glycan profiling by RP-MS or HILIC-MSPrimary Structure – PTMsLC – MS /QTOF (Agilent)
Glycation Primary Structure – PTMsLC – MS /QTOF (Agilent)
Absolute mass for monomer multimers, and aggregatePurity – Size variants SEC – HPLC – MALS (Wyatt)
Tm, Tagg, Size and PDIThermal Stability, Higher Order Structure (HOS)SLS /DLS (Unchained labs)
Sialic Acid ContentPrimary Structure – PTMsUPLC (Agilent)
N -Glycan profilingPrimary Structure – PTMsHILIC -FLD /UPLC
Charge variantsPurity – Charge variantsIEX/HPLC (Agilent)
Size variants (aggregates )Purity – Size variants SEC-HPLC (Agilent)
Hydrophobicity variantsPurity – Protein related substances and impuritiesRP or HIC/UPLC (Agilent)
GlycationPrimary Structure – PTMsRP or HIC/UPLC (Agilent)
Protein concentration by Protein-ARPTiter/QuantitationBAC – HPLC (Agilent)
Free – thiolPrimary Structure – Free thiolFLD – UPLC
Protein concentration by A280QuantityUV – VIS
A280, A320, AppearanceGeneral – Appearance, TrubidityUV – VIS
Free-thiol by Ellman’s assayPrimary Structure – Free thiolUV – VIS
Protein AnalyticsQuality AttributePlatform
N – Glycan profilingPrimary Structure -PTMsCE – SDS (SCIEX)
Charge variantsPurity cIEF(SCIEX)
Charge variantsPurity CZE (SCIEX)
Slab gel – charge variantsPurityIEF, Gel Electrophoresis
Size variantsPurity SDS-PAGE, Gel Electrophoresis
Western blotIdentityGel Electrophoresis
Binding kinetics (kon, koff, Kd)Potency, IdentityBLI (Sartorious /ForteBio) or SPR (Cytiva /Biacore)
FCR and C1Q screeningPotency, Half – life, SafetyBLI (Sartorious /ForteBio) or SPR (Cytiva /Biacore
Protein/antigen titerQuantityBLI (Sartorious /ForteBio)
Epitope binningEpitope characterizationBLI (Sartorious /ForteBio) or SPR (Cytiva /Biacore)
Functional blockingPotency, IdentityBLI (Sartorious /ForteBio) or SPR (Cytiva /Biacore)
Yes /No bindingIdentity BLI (Sartorious /ForteBio) or SPR (Cytiva /Biacore)
BindingPotencySPR (Cytiva /Biacore)
Host Cell Proteins (Cygnus)Safety ELISA /Multimode plate reader (SpectraMax)
Residual Protein A/G/LSafety ELISA /Multimode plate reader (SpectraMax)
Endotoxin TestingSafety LAL /Charles River EndoSafe
Bioburden, Sterility TestSafety Culturing

Comprehensive Biologics Characterization




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

Key Features

  • Binding kinetics (1-3wks) 
  • Fc Receptors 
  • Ligand binding 
  • Competitive inhibition 
  • FcR and C1q screenings (1-4wks) 
  • Functional blocking (1-4wks) 
  • Epitope binning (3-4wks)

Case Study : Octet-based FcR and C1q screen

Agilent 1290 UPLC + 6530B QToF 

Case study : Sequence of target molecule – peptide mapping

Case Study : Determination of the sugar content of bioreactor antibodies – N-glycan analysis


Case study : Assessment and rigorous testing of the quality of purified target molecules


Case study : Determining the sequence of target molecule – peptide mapping

Formulation Development using DLS/Uncle 

Key Features

  • Tm, Tagg, Size, PDI (1-3 weeks) 
  • Monomers 
  • Aggregates 
  • Oligomers 

Measuring various parameters on multiple samples simultaneously using Uncle

  1. Tm
  2. Tagg
  3. Isothermal Stability
  4. Thermal Recovery
  5. Sizing
  6. Polydispersity
  7. Sizing with Thermal Ramp
  8. kD diffusion interaction parameter
  9. B22: 2nd Virial Coefficient
Preliminary formulation Selection
Verify with stability studies at intended and stressed conditions using comprehensive analytics.
Fast Final Formulation Selection.

Developability Process to obtain Insight into Structure/Function relationships and potential Critical Quality Attributes (pCQAs)


Characterization by Multimode Plate Readers

  • ELISAs
  • Enzyme Kinetics