PROTEIN AGGREGATION

ICH Q6B – Aggregates includes dimers and higher multiples of the desired product. These are generally resolved from the desired product and product related substances, and quantitated by appropriate analytical procedures (e.g., size exclusion chromatography, capillary electrophoresis)

PROTEIN AGGREGATION

CONSEQUENCES OF AGGREGATION

PROTEIN AGGREGATION

Aggregates can pose a risk to patient safety and product efficacy

REGULATORY EXPECTATIONS FOR SOLUBLE AGGREGATES

SIZE EXCLUSION CHROMATOGRAPHY - PRINCIPLE

Separation occurs based on size

SIZE EXCLUSION CHROMATOGRAPHY -INSTRUMENTATION

PROTEIN AGGREGATION

SEC MALS

  • SEC-MALS combines multi-angle light scattering detector with size-exclusion chromatography.
  • SEC-MALS determines molecular weight from 200 g/mol to 1 billion g/mol. 
  • It can also determine molecular size – the rms radius, or radius of gyration Rg – from 10 nm to 500 nm and beyond. 
  • By combining molar mass and size, it can also assess molecular conformation. 

Applications:

  • Absolute determination of molar mass
  • Characterization of conjugated proteins like glycoproteins, pegylated proteins or surfactant-bound membrane proteins
  • Determination of the stoichiometry of protein-protein and protein-nucleic acid complexes as well as native oligomers and aggregates

SEC MALS – Case Studies

SEC MALS – Case Studies

Figure 1:  Chromatograms of a) BSA and b) NIST mAb (Blue color chromatogram indicates UV response and red color chromatogram indicates MALS detector response)

SEC MALS ANALYSIS AT CBA

 

Aggregate/Multimer analysis of proteins

Sample requirement

Amount- 1-2 ml (0.5-1 mg/ml)

Formulation buffer- 5 ml

Deliverables

Size exclusion chromatogram of sample in UV and MALS mode, Percentage of monomer, dimer and other aggregates

 

Information required from client

  • Concentration 
  • UV Extinction coefficient and λmax
  • Buffer composition (in case of formulation)

SERVICES OFFERED BY CBA

  • Protein Intact Mass Analysis using HRMS
  • Peptide Mapping using HRMS
  • Post Translational Modifications Analysis using HRMS
  • Host Cell Protein Impurity Identification using HRMS
  • Host Cell Protein Impurity Profiling using SDS-PAGE
  • Secondary Structure Analysis using far-UV CD Spectroscopy
  • Tertiary Structure Analysis using near-UV CD Spectroscopy
  • Tertiary Structure Analysis using Fluorescence Spectroscopy
  • Higher Order Structure Analysis using Fluorescence Spectroscopy
  • Secondary Structure Analysis using FTIR Spectroscopy
  • Higher Order Structure Analysis using DSC
  • Aggregate/Multimers Analysis using SEC-MALS
  • Protein Protein Interaction Analysis using SPR
  • Biosimilarity assessment Project

SERVICES OFFERED BY CBA

Dr. Smita Kale

PI- Center for BioPharma Analysis

Venture Center

100, NCL Innovation Park, Dr. Homi Bhabha Road, Pune – 08

Phone: 020-2586-5877 | Mobile: +91-9923147592

Email: smita@venturecenter.co.in

Mr. Yashodhan Shende

Business Development Executive

Venture Center

100, NCL Innovation Park, Dr. Homi Bhabha Road, Pune – 08

Phone: 020-2586-5877 | Mobile: +91-7755920096 

Email: yashodhan@venturecenter.co.in

For more information, please contact:

Website: https://bioanalysis.in/