Antibody characterization full process guide for testing technology services

Antibody characterization full process guide for testing technology servicesDetails

As a key molecule in basic research and biomedicine, the performance of antibodies directly affects the reliability of experimental results and the effectiveness of downstream applications. The characterization of antibodies in the system helps to clarify their structural characteristics, functional activity, and stability, and is a key step in ensuring the consistency and controllability of antibody quality, especially in fields such as drug development, diagnostic reagents, and treatment.

1、 Why is antibody characterization important?

Antibodies are a class of highly functional and specific immune molecules, but in practical applications, their performance often varies significantly due to structural differences or preparation methods. The core task of antibody characterization is:

• Confirm the antibody sequence and molecular structure.

• Clarify its binding ability and specificity.

• Evaluate its stability, purity, and biological activity.

This is not only a fundamental guarantee for the reproducibility and credibility of results in basic scientific research, but also an important quality checkpoint before entering the clinical stage in fields such as antibody drug development and CDx companion diagnosis.

2、 The core process and technical path of antibody characterization

Antibody characterization typically involves several core steps, each with corresponding technical means and analytical indicators.

1. Antibody Sequence Confirmation

(1) Purpose: To confirm the sequence information of the variable region (VH, VL) and constant region (CH) of the antibody, ensuring the consistency and controllability of the antibody.

(2) Common techniques:

• Mass spectrometry (LC-MS/MS) sequencing: Suitable for antibodies that have been produced but lack original sequence information, it can achieve reverse sequencing from the protein level.

• CDNA sequencing: suitable for hybridoma cells or expression vectors, directly obtaining antibody gene sequences at the nucleic acid level.

2. Antibody Purity&Integrity Analysis

(1) Purpose: To ensure that antibodies do not aggregate, degrade, or modify, and to ensure their structural stability is consistent with downstream experiments.

(2) Common techniques:

• SDS-PAGE/Western Blot: Roughly determine the molecular weight and heavy/light chain ratio of the antibody.

• SEC-HPLC (gel filtration): separate monomer and aggregate, and evaluate the aggregation level.

• CE-SDS: High resolution electrophoresis method for automated analysis of heavy and light chain integrity.

• Mass spectrometry analysis: used to detect slight degradation or modification of antibodies, particularly suitable for quality control scenarios.

3. Antibody glycosylation and modification analysis (Glycosylation&PTM Analysis)

(1) Objective: The glycosylation mode of antibodies significantly affects their structural stability and Fc mediated immune function. Other post-translational modifications (such as oxidation and deamidation) also need to be monitored.

(2) Common techniques:

• Glycotype analysis by mass spectrometry: rapid identification of sugar chain types and relative abundance.

• LC-MS protein/peptide mapping analysis: identifying post-translational modification positions and types.

• Capillary isoelectric focusing (cIEF): evaluates changes in isoelectric point and indirectly reflects the modification state.

4. Binding Specification&Affinity Evaluation

(1) Purpose: To verify whether the antibody specifically recognizes the target and further quantify its affinity parameters (such as KD value).

(2) Common techniques:

• ELISA: Quickly determine whether it binds to the target antigen.

• Western Blot/IHC: Verify the specific recognition ability of antibodies in different samples.

• Surface Plasmon Resonance (SPR)/Biological Layer Interference (BLI): High throughput and high-sensitivity determination of antigen antibody interaction kinetic parameters (ka, kd, KD, etc.).

5. Stability&Thermodynamics Analysis of Antibodies

(1) Objective: The physical stability of antibodies during storage, transportation, and application has a significant impact on their performance.

(2) Common techniques:

• DSC (Differential Scanning Calorimetry): measures the thermal denaturation temperature (Tm) of antibodies to reflect their thermal stability.

• DLS (Dynamic Light Scattering): detects the aggregation tendency of antibodies in solution.

• Accelerated stability experiment: Evaluate the stability of antibodies under different pH, temperature, repeated freezing and thawing conditions.

3、 Differences in characterization strategies for specific antibody types

The characterization strategies of antibodies from different sources or uses also vary:

4、 Common challenges and optimization suggestions in antibody characterization

1. Antibody aggregation is difficult to separate

Suggest combining SEC-HPLC and DLS methods to confirm the proportion of individual units in multiple dimensions.

2. Difficult to identify post translation modifications

LC-MS/MS analysis at the d à nb á izh ì omics level can be used to improve the detection rate of modifications.

3. It is difficult to control the quality of commercially available antibodies with unknown sequences

It is recommended to use de novo mass spectrometry sequencing combined with recombinant expression for quality closed-loop validation.

5、 Baitai Paike Biotechnology: One stop Antibody Characterization Service Platform

At every critical stage of antibody characterization, Biotec Biotech can provide you with customized solutions:

• High resolution Orbitrap and Q-TOF mass spectrometry platforms.

• Whole sequence analysis of antibodies and panoramic map of PTM modification.

• Affinity Dynamics SPR/BLI Assessment Service.

• Antibody drug analysis meets cGMP standards.

Whether you are conducting basic scientific research, developing diagnostic products, or promoting the IND application of antibody drugs, we can build a scientific and reliable antibody characterization data system for you.

Antibody characterization should not only be a "validation" step, but also an important prerequisite for promoting innovation and quality assurance. Through a systematic, comprehensive, and data-driven antibody characterization process, researchers and biopharmaceutical companies can more confidently advance downstream research and clinical translation. Baitai Paike Biotechnology is always committed to becoming your trusted partner for antibody analysis, supporting high-quality scientific research and medical development.

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Baitai Paike Biotechnology Characteristic Project

1、 Protein sequencing

Biotec Biotech uses Thermo's newly launched Obitrap Fusion Lumos mass spectrometer and Shimadzu's Edman degradation sequencing system to analyze the d à nb á izh ì sequence, providing mass spectrometry based protein sequencing analysis services, including amino acid composition analysis of d à nb á izh ì, Ndu ā nc è x ù, Cdu ā nc è x ù, and full sequence analysis, as well as d à nb á izh ì N-terminal sequence analysis services based on Edman degradation. For unknown theoretical sequences, we provide de novo sequencing services based on de novo sequencing to analyze protein sequences.

※ Service advantages:

1. Adopting the world's advanced mass spectrometer Obitrap Fusion Lumos;

2. It can achieve 100% coverage of the measured target protein sequence;

3. It can determine up to 70 amino acid sequences at the N-terminus of proteins;

4. Can measure various forms of samples: protein solution, PVDF protein bands;

5. Low sample usage: Protein samples only require 5-10ug to complete detection;

6. Sequencing is not affected by N-terminal modifications such as blocking, PEC, and glycosylation.

2、 D à nb á izh ì omics

Biotec Biotech uses Thermo Fisher's Orbitrap Fusion Lumos mass spectrometry platform combined with Nano LC to provide a variety of d à nb á izh ì omics analysis services, including quantitative d à nb á izh ì omics, targeted d à nb á izh ì omics, proteomics, post-translational modified proteomics, and more. In addition, Baitai Paike Biotechnology has launched a 4DD à nb á izh ì omics service based on timsTOF Pro, which helps with research in micro sample proteomics, large sample population medicine, and high-throughput modification genomics.

※ Service advantages:

1 . High throughput quantitative protein analysis: Large scale experimental analysis with multiple control groups, discovering new biomarkers;

2. Multiple in vitro and in vivo labeling methods are suitable for analyzing various samples such as tissues, cells, and blood;

3. High sensitivity of mass spectrometry analysis and high repeatability of experimental results;

4. Can detect low abundance proteins with a wide linear range;

5. Bioinformatics analysis is more systematic and accurate.

3、 Single cell mass spectrometry flow cytometry analysis

Baitai Paike Biotechnology uses the Fluidigm mass spectrometry flow cytometry system for single-cell mass spectrometry analysis. Metal element markers (usually metal element labeled specific antibodies) are used to label molecules on the surface and inside of cells. Then, individual cells are separated using flow cytometry principles, and the atomic mass spectrum of individual cells is analyzed using inductively coupled plasma mass spectrometry (ICP-MS). The atomic mass spectrum data is then converted into signal molecule expression levels on the cell surface and inside.

※ Service advantages:

1. Advanced technology, filling technological gaps

The use of metal labeled antibody technology avoids the problem of few and easily interacting channels in traditional flow cytometry fluorescence. At the single-cell level, multiple indicators can be characterized simultaneously, and Baitai Paike Biotechnology can detect 51 target proteins simultaneously.

2. Large analysis quantity and low cost

Single cell RNAseq is limited by factors such as cost, and the total number of analyzed cells for all samples is generally around 2x10 ^ 4. However, flow cytometry technology can analyze at least 10 ^ 5 cells in one sample, achieving an order of magnitude increase, and the cost is not higher than that of single-cell RNAseq.

3. Great application prospects

① The results of flow cytometry can provide changes in cell subpopulations, which have great research prospects in clinical diagnosis, disease mechanism research, and other fields;

② Combining metal tag technology with other technologies will lead to new application directions. In addition to conventional proteins, mass spectrometry flow cytometry technology can also be used for post-translational modification of proteins;

③ Can detect cell survival rate, cell size, mRNA transcript expression level, DNA synthesis rate, and protease activity.

4、 Immunopeptide omics analysis and novel antigen discovery based on high-precision mass spectrometry

The one-stop solution for high-precision mass spectrometry based immunopeptidomic analysis and new antigen discovery from Biotec Biotech includes our proprietary, highly sensitive immunopeptide enrichment and identification scheme. We can assist you in identifying and recognizing over 10000 type I peptides and over 10000 type II peptides. Through our optimized high-throughput immunopeptidomic analysis platform, immunopeptidomic analysis can be performed with reproducible identification and quantification from small sample materials. This service can be applied to large-scale research, aiming to assist researchers in finding solutions for cancer, immune diseases, and infectious diseases, and to explore unknown targets in depth.

5、 Characterization of Biopharmaceuticals

Baitai Parker is based on high-resolution mass spectrometry technology, MALDI TOF， High performance chromatography separation technology provides a range of biological drug analysis solutions, from amino acid composition and primary structure analysis of biological products such as d à nb á izh ì, peptides, antibodies, vaccines, to product variability and purity analysis. Intended to provide high-quality biopharmaceutical analysis services and assist biopharmaceutical manufacturers in improving the quality of biopharmaceuticals.

Baitai Paike Biotechnology's Seven Major Testing Platforms

Baitai Paike Biotechnology - High quality service provider for characterization of biological products and multi omics of biological mass spectrometry

Beijing Baitai Paike Biotechnology Co., Ltd. is committed to providing quality control testing and project validation services for the biological/pharmaceutical and medical device industries. The company's laboratory follows regulations and guidelines such as NMPA, ICH, FDA, and EMA, and has obtained CNAS/ISO9001 dual quality system certification. It has established a complete quality system, including data cold and hot/remote backup, regular equipment measurement/periodic verification, software audit tracking, and provides customers with integrated solutions and technical services to support new drug research and development, drug application registration, and production release.

1. The company adopts the ISO9001 quality control system and provides CRO detection and analysis services based on mass spectrometry;

2. Recognized by the National CNAS Laboratory, we provide customers with drug quality research services that comply with global pharmaceutical regulations;

3. The business scope covers omics, proteomics, metabolomics, biopharmaceutical characterization, single-cell analysis, single-cell mass spectrometry flow cytometry, bioinformatics cloud analysis, and multi omics integrated analysis of biological mass spectrometry;

4. Seven major quality control testing platforms to meet your one-stop service needs;

5. Providing services to over 3000 enterprises and 10000+customers;

6. Committed to providing you with high-quality biological mass spectrometry analysis services!

Antibody characterization full process guide for testing technology servicesDetails