Peptide Mapping Analysis

Peptide mapping is widely used in the development and manufacture of therapeutic proteins and peptides in the biopharmaceutical industry to assist in the initial structural identification, identity confirmation, monitoring of degradation events such as oxidation or deamidation, and product comparability testing of protein and peptide therapeutics. The ICH Q6B guideline outlines the use of peptide mapping specifications as a key component of a set of acceptance criteria used in the evaluation of biologics. As an expert in therapeutic proteins and peptides, CD Formulation has developed an in-house protocol method based on LC-MS/MS peptide mapping that can quickly and accurately determine amino acid sequences, detect modified groups in proteins and peptides, and evaluate structural changes or isomer formation.

Peptide Mapping & Why is Peptide Mapping Needed?

Peptide mapping usually adopts a top-down mass spectrometry approach, where researchers digest the proteins in the sample, break them into peptides, and use mass spectrometry to identify and measure the sequence and relative abundance of these peptides to generate a set of unique peptide massl data. The identified unique peptide data are matched with the theoretical mass/spectral data of the sequence in the proteomics database to identify the protein to be tested.

As protein therapeutics become the fastest-growing area in the pharmaceutical industry, it is critical to carefully examine the complete attributes of these products (such as purity, biochemical properties, and stability) throughout the development and manufacturing process. Peptide mapping is a powerful analytical tool for identifying or verifying the primary structure of proteins/peptides, i.e., amino acid sequence and chemical modifications, and can generate multiple information about proteins and peptides in a single analysis. This approach is very important for the comprehensive and accurate characterization of therapeutic proteins and peptides because it can generate both qualitative and quantitative information. In addition, minimizing protein degradation, such as amino acid oxidation and deamidation, during sample preparation is also critical to obtaining an accurate description of therapeutic proteins.

Explore Our Peptide Mapping Analysis Services

Careful and thorough characterization of therapeutic proteins and peptides is essential to ensure product safety and efficacy. With advanced mass spectrometry technology platforms, our experienced protein scientists have developed a peptide mapping protocol based on LC-MS/MS to verify the structure and purity of therapeutic proteins and peptides. This protocol can rapidly determine the composition and purity of proteins and peptides by properly digesting and separating the samples and accurately measuring the molecular weight and amino acid sequence of the peptides using mass spectrometry.

In addition to providing a wide range of peptide mapping services during the development phase, we provide such support in process development, quality control, stability studies, and biosimilar development environments when necessary.

• Identity is confirmed when the chromatographic profile of the peptide map is as expected (e.g., peak retention time, peak height, absence of new or missing peaks) compared to the reference map. Similarly, differences in comparing peptide maps can indicate changes or degradation of the drug substance/product for stability assessment.
• When coupled with mass spectrometry, changes in the profile provide data pinpointed to specific attributes, such as increased oxidation of a certain methionine residue, the appearance of new sequence variants, or altered glycan composition, to assess the biosimilarity of the original drug product.
• Peptide mapping is also an important step in manufacturing process monitoring and quality assurance/quality control (QA/QC) to ensure that unexpected changes to the product have not occurred. In a quality control environment, peptide mapping can track any changes or instabilities that may have occurred during the production of therapeutic proteins and peptides and is routinely used for batch release or stability studies.

Our Peptide Mapping Workflow

Our LC-MS/MS peptide mapping supports primary structure analysis at the intact protein or subunit level as an orthogonal tool and provides additional site-specific information. The workflow includes the following four main stages:

Fig. 1 Workflow of peptide mapping. (CD Formulation)

• Sample preparation (reduction and alkylation of proteins): This step aims to reduce disulfide bonds in proteins while preventing the generation of free thiols through alkylation.
• Enzymatic digestion: The target protein is cleaved separately using specific proteases (Trypsin, Chymotrypsin, Asp-N, Glu-C, Lys-C, and Lys-N). The selection of enzymes is based on theoretical protein sequences and knowledge of enzyme digestion.
• Peptide separation and peptide detection (LC-MS/MS): The samples are analyzed using nano LC-MS/MS, which is based on the characteristics of your sample.
• Sequence confirmation: Peptide identification is performed by searching the data or by comparing it with the theoretical sequence you provide to identify the protein to be tested.

Technology Highlights

• High coverage: The combination of specific and non-specific protein enzymes can achieve 100% coverage of the identification of any protein or peptide.
• High accuracy: The confirmation of each sample is based on the quality of the sample and the MS/MS fingerprint of the sample, and the primary mass spectrometry is more accurate.
• Amino acid level: Overlapping peptide sequences confirm the order of amino acids in the sequence.
• The quantitative detection limit of amino acid mutation sites can reach 0.1%.
• 100% sequence verification service for protein samples.

Applications

• Verify the primary structure (amino acid sequence) of therapeutic proteins and peptides.
• Monitor the presence of post-translational modifications (PTMs) on proteins (e.g., glycosylation, asparagine deamidation, methionine oxidation, aspartate isomerization, C-terminal cleavage, etc.).
• Perform stability studies to identify degradation sites when therapeutic proteins and peptides degrade in active form during storage.
• Perform impurity identification to check for contamination and other major forms in protein samples.
• Determine disulfide bond locations, unpaired cysteines, and free thiol groups, and detect the occurrence of disulfide shuffling.
• Identify point mutations and sequence variations between two or more samples.
• Biosimilar similarity studies.

Fig. 2 Applications of peptide mapping. (CD Formulation)

Why Choose Us for Peptide Mapping Analysis?

• We have extensive experience and hundreds of successful projects in peptide mapping analysis.
• We have state-of-the-art equipment and facilities that are specifically designed for peptide mapping analysis, allowing us to provide high-quality results.
• We provide a complete peptide map analysis report including a list of each detected peptide separated by the protease, total protein coverage, and the confidence associated with each peptide.
• Flexible experimental design and testing options can meet any customer's specific needs.

Publication

CD Formulation has experienced protein chemists dedicated to providing customers with unparalleled peptide mapping solutions to help them succeed in their protein and peptide drug development and manufacturing. Please feel free to contact us if you are interested in our services. We will provide you with the most professional advice and support to ensure the smooth launch and implementation of your project.

How It Works

STEP 1

Submit a service request describing your specific research or development need.

STEP 2

We'll email you to provide your quote and confirm order details if applicable.

STEP 3

Execute the project with real-time communication, and deliver the final report promptly.