As a bright pearl in the field of modern biomedicine, the quality analysis of antibody drugs is not only the key to ensure the efficacy, but also the cornerstone to ensure the patient safety. Accurate quality control means, like a sharp scalpel, analyze the microscopic world of drugs and reveal its inner mystery. In 2025, with the continuous progress of science and technology, the quality analysis technology of antibody drugs has been improved. From molecular structure to biological activity, every detail needs to be strictly controlled to ensure its excellent performance in clinical application. This article introduces in detail the quality analysis and quality standards, including physicochemical properties analysis, biological activity assessment, content determination, purity and impurity detection and other key links. Also, the specific requirements and methods of identification test, purity analysis, residual DNA content determination and residual host protein content determination are described. Through these analyses, this paper aims to build a rigorous antibody drug quality assurance system to ensure that each batch of products can meet the expected efficacy and safety standards.
Table 1. Common Analytical Tests Employed for ADCs on Stability
| Attributes | Tests |
| Physical | Appearance pH Particulates Osmolality |
| Purity | Size Variants Charge Variants Drug Related lmpurities(RP-HPLO |
| Potency | Binding Assay(Enzyme-Linked lmmunosorbent Assay,ELSA Cytototoxity Assay (Cell-BasedBioassay) |
| Purity/Potency Tests Uniqueto ADCs |
DAR Unconjugated Antibody Free Drug(RP-HPLC) |
| Microbiologica | Sterility Endotoxin Bioburden |
Key Components of Antibody Drug Quality Analysis and Standards
Antibody drug quality analysis is a multifaceted process that ensures both safety and efficacy. It involves a range of crucial steps, each contributing to the overall quality assurance framework for antibody products. These steps include the analysis of physicochemical properties, biological activity assessment, content determination, and purity and impurity testing. Additional routine testing also covers stability studies, immunogenicity evaluation, safety testing, and production process control. Together, these components form a comprehensive system that ensures each batch of antibody drugs meets the expected therapeutic and safety standards.
Quality Control Standards for Recombinant Protein Products
For recombinant protein products, such as recombinant collagen, specific quality control standards must be established for raw materials, intermediate products, and finished goods. These standards address key parameters including physicochemical properties, biological activity, content, and purity, and are evaluated through various analytical techniques. Methods like ELISA, SDS-PAGE, Western blotting, and scanning electron microscopy (SEM) are commonly employed to ensure product quality and effectiveness.
Testing Items and Methodologies
The quality control process typically includes dozens of tests, such as:
- Identification tests
- Precise content determination
- Accurate molecular weight measurement
- Peptide mapping analysis
- Purity analysis and related substance testing
- Activity evaluation and specific activity calculation
- Residual DNA content measurement
- Residual host protein quantification
- Screening for potential harmful impurities
It is essential to provide detailed testing records and results for multiple batches to ensure consistency and reliability across the production process.
Requirements for Identification Testing in Antibody Drug Quality Control
Identification testing is a critical component in ensuring the quality and authenticity of antibody drugs. The specificity of these tests is paramount, as they must reliably distinguish the drug from other substances and confirm its identity. Depending on the characteristics of the product, a combination of physicochemical, biological, and/or immunological methods may be used. In practice, immunoblotting (Western blot) is commonly employed for identification purposes in most current antibody products.
Standardized Testing Methods
To ensure the reliability of the identification test, standardized protocols must be followed. This includes the use of a molecular weight marker, and it is advisable to incorporate an electrophoresis control before transfer. Such controls help maintain the accuracy and reproducibility of the results.
Advanced Techniques for Protein Characterization
As our understanding of protein characteristics continues to evolve, newer technologies have been integrated into identification testing. These include:
- Capillary zone electrophoresis (CZE)
- Capillary isoelectric focusing (CIEF)
- High-performance liquid chromatography (HPLC)
These techniques enable precise comparison between the product and a reference sample, enhancing the specificity of the identification process.
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Key Considerations in Quality Standards
In establishing quality standards for antibody drugs, it is crucial to specify control parameters for the major peaks or bands observed in the testing process. This includes defining their relative proportions, ensuring consistency and reliability across batches. By adhering to these standards, the identification process can effectively confirm the product's authenticity and ensure its suitability for clinical use.
Analysis Requirements for Purity and Related Substances
Analytical Techniques
At least two distinct analytical techniques based on different principles should be employed for identification. Current pharmacopeia typically includes non-reductive SDS-PAGE and HPLC methods. When performing SDS-PAGE analysis, if silver staining is used, the sample loading amount should be no less than 5 μg. If Coomassie Brilliant Blue staining is employed, the sample loading amount should not be less than 10 μg.
Analysis Results and Purity Standards
The analysis should reveal the expected band pattern. Through scanning analysis, the product purity is generally expected to exceed 95%. If minor bands (representing less than 5% of the total content) are observed, their characteristics should be analyzed, and their presence should be clearly documented in the quality control method description.
HPLC Analysis
For HPLC analysis, it is recommended to use chromatographic media based on various separation principles, such as molecular sieve chromatography, reversed-phase chromatography, or ion-exchange chromatography. The specific model and selection criteria of the chromatographic column should be clearly stated.
Heterogeneity and Impurity Profiling
If the chromatographic separation reveals multiple peaks, this indicates protein heterogeneity. Efforts should be made to analyze and confirm the identity of these peaks, which may correspond to different isoforms. The relationship of these isoforms to product activity, in vivo metabolism, and immunogenicity should be investigated. In cases where necessary, content control should be considered.
Requirements for Measuring Residual DNA Content
Analytical Methods
Current pharmacopeia methods for residual DNA analysis include DNA probe hybridization and fluorescence staining techniques. Research is ongoing to explore quantitative polymerase chain reaction (qPCR) as a potential alternative method. The sensitivity of detection should be determined based on process validation, clinical batch data, and the level of exposure of the product in humans. Typically, the sensitivity should be below 10 ng per dose.
Stricter Standards for High-Risk Products
For products produced using mammalian cell lines with a higher risk of tumorigenicity, as well as for products with higher levels of human exposure, more stringent residual DNA content control standards should be established.
Regulatory Requirements for Residual Host Protein Content
Detection Methods
Enzyme-linked immunosorbent assay (ELISA) is typically employed to detect residual host proteins. Most biopharmaceutical products use commercially available assay kits for this purpose. It is recommended that the suitability of the chosen kit be evaluated, and, when necessary, more specific detection methods should be developed based on the particular needs of the product.
Residual Host Protein Standards
For most biopharmaceutical products that have been approved for market, the residual host protein content is generally limited to no more than 0.1%. However, these products typically involve low single-dose clinical administrations. For new products with higher intended dosages, where multiple administrations may lead to significant human exposure, it is important to adjust this standard according to clinical requirements.
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Conclusion
In summary, the quality control of biopharmaceutical products requires the integration of various analytical techniques to ensure product purity and safety. Residual DNA and host protein content standards should be set based on the product characteristics and clinical needs to safeguard patient safety. Continuous optimization of detection methods, improving sensitivity and specificity, is critical to advancing quality control practices. Future research should focus on the application of new technologies to balance efficacy and safety in biopharmaceuticals. In practice, quality control strategies should be adjusted flexibly, taking into account the unique properties of the product and manufacturing processes to ensure compliance with regulatory requirements at every step.
This article provides a comprehensive framework for the quality analysis of antibody-based drugs. It underscores the critical role of stringent quality control in ensuring both patient safety and the therapeutic efficacy of drugs during the development and manufacturing processes. Additionally, the article highlights the current challenges in quality control and outlines future directions for improvement. This discussion offers valuable insights for researchers and practitioners in the field, contributing to the ongoing advancement of biopharmaceutical quality standards.
Our Expertise in Antibody Drug Quality Characterization
At our company, we specialize in protein and antibody drug characterization services, offering comprehensive support to clients in analyzing the quality of antibody-based therapeutics. Our services cover a broad spectrum of quality control aspects, including stability assessments, purity analysis, content determination, and the evaluation of related substances. Using advanced techniques such as ELISA, SDS-PAGE, HPLC, and differential scanning calorimetry (DSC), we help clients ensure their antibody drugs meet the rigorous standards necessary for clinical efficacy and safety. Through our precise and reliable analyses, we empower our clients to optimize their antibody products, ensuring that each batch is compliant with regulatory requirements and prepared for successful clinical trials and eventual market approval.
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