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Friday, June 28, 2013

Protein Characterization : Methods & Techniques

Protein Characterization: Protein characterization is primarily focused on the analysis of recombinant proteins for identifying and generating peptide maps of proteins which will aid in the purification process. Protein characterization can be done using the following methods:
  • Amino Acid Composition Analysis (AAA)
Determination of amino acid composition of a protein. Used for confirmation of primary structure.
  • N- and C-terminal Sequencing
N and C terminal protein sequencing

Determination of amino acid sequence from the N- and C-terminus of protein using Edman sequencing and/or MALDI-ISD. Used as a confirmation of primary structure or de novo sequencing.
  • 1D/2D SDS-PAGE
Detection of mono- and multimeric forms of protein and impurities.

SDS PAGE analysis

  • (Capillary) Isoelectric Focusing (IEF + cIEF)
Determination of the isoelectric point (pl) of a protein and assessment of isoform distribution and impurity profiling.
  • Western Blot
Identification of proteins by their immunologic reaction with antibodies of known specificity (from 1D and 2D PAGE gels).
Separation of protein based on size. Determination of degree of aggregation.
  • RP and IEX-HPLC
Identity, content and impurity determination. Detection of oxidation and deamidation products.
  • Peptide Mapping (by LC-MS) and MS/MS (de novo) Protein Sequencing
Used to confirm identity/primary structure. Enzymatic digestion of protein followed by mass spectrometry.
  • Monosaccharide Composition
Quantification of neutral amino sugars: glucosamine, galactosamine, mannose, galactose and fucose.

Carbohydrate Linkage Analysis Determination of monosaccharide linkage variants as part of a glycan structure analysis.
  • Sialic Acid Determination
Quantitation of acidic sugars NANA (Neu5Ac) and NGNA (Neu5Gc).
  • Oligosaccharide Profiling
N-linked and O-linked oligosaccharide profiling including analysis of sialylated and desialylated carbohydrate structures by NP-HPLC (HILIC), HPAEC-PAD and mass spectrometry.
Detection and quantitation of residual proteins including BSA, Protein A and host cell proteins. Proprietary HCP assays for CHO and E. coli; also useful in bioassays and protein characterization.

ELISA Protein detection

  • Extinction Coefficient
Determination of extinction coefficient and content of protein by UV and Amino Acid Analysis.
  • Determination of Protein Content
Routine determination by UV or other methods.
  • Glycosylation/Sulfation/Phosphorylation Site Identification
Mass spectrometry analysis to determine the site of post-translational modifications.
  • Disulfide Bridge Analysis
Cystine linkage sites are identified (de novo assessment and/or confirmation).
  • Oxidation and Deamidation Analysis
Mass spectrometry is used for detailed characterization of extent and site of modification.
  • Presentation-Specific Testing
Physicochemical testing as required for the product type (e.g., osmolality, sub-visible particulates, moisture content).
  • Capillary (Zone or SDS) Electrophoresis
Used for the characterization and separation of protein mixtures based on mass or charge.