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Biochromatography - Peptide Mapping - Sequence and Structural Analysis

Peptide mapping is an essential technique for studying the primary structure of proteins.

For recombinant protein pharmaceuticals, peptide mapping is used for the initial proof of structure characterisation. It is also employed for lot-to-lot identity testing in support of bioprocess development and clinical trials. Peptide mapping is used as the current method of choice for monitoring the genetic stability of recombinant cell lines.

Peptide mapping generally involves enzymatic digestion (usually using trypsin) of a protein to produce peptide fragments, followed by separation and identification of the fragments in a reproducible manner, allowing the detection and monitoring of single amino acid chains, oxidation, deamidation and other degradation products.

It also enables the direct detection of common monoclonal antibody variants such as N-terminal cyclisation, C-terminal lysine processing and N-glycoslylation, as well as other post-translational modifications.

Peptide mapping is considered a comparative procedure that confirms the primary structure of the protein and detects alterations in structure. Additionally, it demonstrates process consistency and genetic stability.

A peptide map should include positive identification of the protein, maximise coverage of the complete peptide sequence, and provide additional information and sequence identification beyond that obtained at the non-digested protein level.

 

Product Information

Reversed-phase chromatography is the predominant technique used for peptide mapping. The preferred columns for peptide mapping separations are similar to those used for small molecules with C18 being the phase of choice. For more detailed product information click on the links below:

Agilent AdvanceBio Peptide Mapping

Agilent Zorbax 300Å Stablebond columns

ThermoScientific Accucore 150Å columns

Thermo Scientific Acclaim PepMap columns

Thermo Scientific SMART Digest™ Kits

 

 

 

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Application notes

Here at Crawford Scientific we have gathered all of the most relevant and useful documentation, including application notes and some general guides, relating to protein characterisation using reversed-phase chromatography, see below;

Agilent

Video: Peptide Mapping “Cooking Series” Video

Protein and Impurity Profiling Using Wide Pore Reversed Phase HPLC

Fast mAb Peptide Mapping with Porohell Particles

Reversed Phase Characterisation of Biomolecules with Advancebio Columns

Reduced Peptide Mapping Times using AdvanceBio columns

Rapid Peptide Mappping using UHPLC

Using Temperature to Increase Resolution in Ultrafast Protein Separations

Poroshell300 Fast Protein Digest Analysis

Disulfide Linkage Analysis of IgG1 using an Agilent 1260 Infinity Bio-inert LC System with an Agilent ZORBAX RRHD Diphenyl sub-2 μm Column

Reversed-Phase Optimization for Ultra-Fast Profiling of Intact and Reduced Monoclonal Antibodies Using ZORBAX Rapid Resolution High Definition 300SB-C3 Column

N-Terminal Site-Specific PEGylation and Analytical-Scale Purification of PEG Lysozyme

Ultra High Speed and High Resolution Separations of Reduced and Intact Monoclonal Antibodies with Agilent ZORBAX RRHD Sub-2 μm 300 Diphenyl UHPLC Column

Fast Separation of Recombinant Human Erythropoietin Using Reversed Phased Agilent ZORBAX RRHD 300SB-C18, 1.8 μm

Thermo

Acclaim PepMap and Acclaim PepMap RSLC

ProPac HIC-10 Column

PepSwift ProSwift Capillary Monolithic

 
 

 

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