Monoclonal Antibody Drugs Quality Analysis

Since the invention of monoclonal antibody preparation technology in the 1970s, it has been rapidly applied to the field of clinical diagnosis and treatment, greatly promoting the development of medicine. The emergence and development of monoclonal antibody have brought new challenges to drug analysis. The drug standards are directly related to the quality of the drug. It is a measure to control drug safety, effectiveness and quality reliability from the source. With the development of biotechnology drugs, the safety of biological products has also attracted more attention. Since monoclonal antibodies may contain harmful substances that are not present in traditional production methods, the quality control of monoclonal antibody is fundamentally different from those produced by traditional methods, and the quality control is more complicated and diverse. Beside identifying the final products, it is also necessary to strictly control each production step, such as cultivation and purification to ensure the effectiveness, safety and consistency of the final product.

Recombinant monoclonal antibodies are one of the great achievements of modern biotechnology. There is no doubt that the uses of them are successful in treating various life-threatening and chronic diseases including cancer, autoimmunity, transplantation and infectious ones. Its share in the global bio-pharmaceutical market has also increased rapidly in recent years. Meanwhile, as one of the most successful bio-therapeutics, how to ensure the safety and efficacy of recombinant antibody products through well-established quality control system is always the focus of attention from both the views of regulatory authorities and industries. This article briefly introduce the research contents and techniques for the control of recombinant antibodies.

Physical and chemical analysis

Structure confirmation

Structure confirmation mainly include N-terminal amino acid sequence, peptide map, glycosylation analysis, etc. Terminal amino acid sequencing is one of the ways to identify the primary structure of antibody. N- terminal amino acid determination is most commonly used by Edman degradation.To further identify the primary structure of the recombinant antibody, liquid peptide map or peptide mass fingerprint analysis can be performed. Glycosylation and protein modifications are important not only for drug stability and efficacy, but also they may have consequences for the patient. For example, incorrect modifications, including aggregation may lead to an immune response to the therapeutic monoclonal antibody. At present, the sugar chain structure can be predicted by measuring the relative molecular mass of the sugar chain in combination with the existing literature, it can also be determined by tandem mass spectrometry.

Purity analysis

The purity of the antibody drug directly reflects the level of the purification process and the quality of the product. In order to avoid the deviation of a detection method in protein purity detection, two different principles are generally used at least for detection to separate impurities as much as possible and obtaining relatively accurate results. Common methods include SDS-PAGE, capillary electrophoresis, and high performance liquid chromatography such as reverse phase, molecular exclusion and ion exchange.

Protein content determination

The commonly used methods for protein content determination include spectrophotometry, Lowry method, Bradford method, BCA method, Kjeldahl method. At present, most of the recombinant antibodies are measured by spectrophotometry.

Other physical and chemical characteristics analysis

It mainly includes relative molecular mass and isoelectric point. The measurement is carried out by SDS-PAGE and isoelectric focusing electrophoresis respectively.

Biological activity

Determination of biological activity is an important quality control index to ensure the effectiveness of recombinant antibodies. The Bioactivity Assay mainly establishes the cell evaluation model in vitro; simulates its action mechanism to produce the objective whole-course response, and evaluates its biological activity by comparing with the bioactivity standard substance. According to the mechanism of action, it is mainly divided into complement dependent cytotoxicity(CDC) method, signal pathway blocking and reporter gene methods. If a specific reaction model cannot be established, the binding ability of the recombinant antibody with its target can also be determined as an indicator of its functionality. For example: ELISA,Flow cytometry, Surface plasmon resonnance(SRP), Biomolecular interaction analysis(BIA).

Residual impurity

Recombinant antibodies are generally produced by mammalian cell expression and therefore require limited control of exogenous components that may be present in the expression system and during purification. According to its production process, the limited control of related impurities mainly includes residual DNA, residual host-cell protein, residual protein A and the like. The residue limit detection for the host-cell protein, protein A are mainly carried out by a sandwich ELISA method.

In theory, the DNA of the cell lineage carries the potential risk of being tumorigenic, and the FDA limits the allowable residual DNA limit for biological products to no more than 100 pg per dose. At present, the detection methods of residual DNA include hybridization, fluorescence staining, quantitative PCR and so on. Quantitative PCR has unique advantages in specificity, sensitivity and accuracy. It can quantify residual DNA objectively and reflect the relative molecular weight distribution of fragments. However, the method still needs further optimization and standardization of primer design and standard products.

References

• Jenkins, N. (2007) ‘Modifications of therapeutic proteins: Challenges and prospects.’ Cytotechnology,2007, 53: 121-125.

• QIAN J,LlU T, YANG l , et al. (2007) ‘Struetural characterization of N-linked oligosaccharides on monoclonal antibody cetuximab by the combination of orthogonal matrix-assisted laser desorption /ionization hybrid quadrupole-quadrupole time -of-flight tandem mass spectrometry and sequential enzymatic digestion.’ Jal Biochem , 364(l):8-18.

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