Peptide profiling (an important tool in biopharmaceuticals) is a very powerful method widely used for protein identification and testing, especially for recombinant proteins. Under analysis

It is often necessary to generate peptide fragments through enzymatic hydrolysis (often using trypsin) of proteins, followed by reproducible fragment separation and identification to detect and monitor changes in single amino acids, oxidation, deamination, and other degradation products. Peptide profiling can also directly detect common monoclonal antibody variants, such as N-terminal cyclization, C-terminal lysine treatment, N-terminal glycosylation, and other post-translational modifications.

Peptide profiling is a fingerprint of a protein and its final product after multiple processing, providing a comprehensive and in-depth understanding of the analyzed protein. It includes four main steps:

1. Protein isolation and purification.

2. Selective cleavage of peptide bonds.

3. Chromatographic separation of peptide segments

4. Validation analysis of peptide segments.

Peptide profiling is considered a comparison process that can determine the primary structure of a protein and detect structural changes. In addition, it can be used to verify processing consistency and genetic stability. The peptide spectrum should include positive identification of proteins, maximum coverage of complete peptide segments, and provide other information and sequence identification in addition to the information obtained at the enzymatic protein level.

When selecting suitable chromatographic techniques for separating peptide segments and generating peptide profiles, it is necessary to consider the protein itself, the experimental purpose, and the expected results. However, the excellent separation ability of reverse phase chromatography has made it the most important HPLC technology in peptide spectrum separation. Because this technology can use volatile mobile phase eluates, it is also an ideal choice for analytical and preparative separation.

It is important to note that the preferred chromatographic column for peptide spectrum separation is similar to the chromatographic column used for small molecule separation, but because most peptide spectrum separation is performed under low pH and heating conditions, chromatographic columns with excellent pH stability and minimal influence from silanols are often used.

To successfully generate a peptide profile, it is necessary to carefully examine the complete characterization strategy. A spectrogram may contain more than 100 chromatographic peaks representing different peptide segments and their derivatives, so it requires analysts to have knowledge of sample pretreatment methods, powerful separation techniques, and validation protocols. Possessing the skills and information to successfully develop peptide profiles will help you obtain the optimal separation of proteolytic digesters and successfully characterize peptide segments reliably.

The purpose of this peptide profiling guide is to emphasize some important issues when using reverse phase chromatography for peptide profiling; Share some basic techniques used in peptide profiling processes; And emphasize the precautions when optimizing peptide spectrum separation to obtain the best results possible.

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From Omizzur Biotech Ltd