Analytical characterization of proteins is required according to the ICH Q6B Guideline: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products, when proteins are developed for human clinical use.

Many questions and quality control issues are important when working with protein expression, purification and functional in-vitro and in-vivo assays. Using our advanced protein analysis methods we can assist you with analytical characterization of your protein product and protein contaminants to help you improve and speed up the process development. The results can be used as part of CMC documentation for regulatory authorities.

Typical tasks that we solve:

• Mass spectrometric peptide mapping to get more than 90% sequence coverage of a specific protein
• Compare protein preparation from reference and batch material
• Confirm correct N-terminal processing from different cell clones
• Molecular weight determination to confirm full length expression and purification
• Identification of contaminating host cell proteins
• Identification of drug substance variants
• Develop absolute quantification assay for a specific protein in a complex protein mixture and matrix
• N- and C-terminal protein sequencing

Contact us to discuss your specific project in more detail.  We will provide you with a full project proposal within 2 days that includes analysis protocols, timelines and costs.

Alphalyse Protein Analytical Methods:

N-terminal Edman Sequencing
Determination of the amino acids sequence from the N-terminus of the protein. Typically 6-15 amino acids for sequence confirmation.

Molecular Weight Determination of Peptides and Proteins by MALDI MS and ESI MS
Analysis of unknown proteins and peptides, or confirmation of expected sequence by Mw.

N- and C-terminal Sequencing by MALDI ISD
Confirmation of full length protein sequence and correct expression. Detection of sequence truncations and terminal modifications.

SDS PAGE (1D and 2D)
Characterisation of protein Mw-isoforms and pI-isoforms. Separation and purification of simple and complex protein mixtures for MS protein identification, N-terminal Edman sequencing and Western blotting.

Isoelectric Focusing (IEF)
Characterisation of protein isoforms with different isoelectric points (pI).

Chromatography, RP-HPLC
Separation and purification of proteins based on hydrophobicity. Quantity and impurity profile.

Peptide Mapping by MALDI MS and LC-ESI MS
Protease cleavage by trypsin, Glu-C, Asp-N, Lys-C, chymotrypsin followed by mass spectrometry. Confirmation of protein sequence. Detection of post-translational modifications.

Mass Spectrometry Protein Identification Service
Mass spectrometric peptide mapping and database searching for identification of proteins in gel bands and chromatographic peaks.

Glycosylation/Phosporylation/Post-Translational Modification Analysis
Using affinity purifications, specific enzymes, chemical reactions combined with mass spectrometric peptide mapping and sequence correlation to detect, identify and compare modifications of different samples. Modifications include: deamidation, pyroglutatamate, met oxidation, methylation, acetylation, free thiol, disulfide bonds.

Amino Acid Composition Analysis (AAA)
Hydrolysis and quantification of individual amino acids to determine amino acid composition of protein/peptide.

Quantification and impurity assessment of proteins with known sequence.
Absolution Protein Quantification Assay by MS
Absolute quantification of specific protein in complex samples using stabile isotope labeled peptides (AQUA peptides) as internal standards for MRM measurements by LC-MS/MS.

Host Cell Protein (HCP) Identification Assay
Separation and identification of Host Cell Protein (HCPs) contaminants in protein drug substance by 1D SDS PAGE and mass spectrometric protein identification.