The Biological Analysis of Bispecific Atibody

With the development of bispecific antibody technology, bispecific antibody now presents a good development prospect. When major companies are deploying bi-antibody product research and development, they also need to pay attention to the detection and analysis of bi-antibody. Points to note and consider. Dr. Zhang Dengji, Director of Pharmaceutical Analysis Department of Medicilon Biotechnology, brought us a three-in-one bi-antibody biological analysis lecture in the cloud lecture hall.
The Pharmaceutical Analysis Department of Medicilon Biotechnology flexibly uses ELISA, ECL, TRFIA, CLIA, IF, IP, CoIP, qPCR, FACS, ELISpot, enzymology and other methods to support cutting-edge biological drugs such as proteins and antibodies (monoclonal antibodies, dual Or multispecific antibodies, antibody fragments), ADC, peptides, nucleic acids, vaccines and cell gene therapy and other drugs in the early development, preclinical and clinical stages of PK/TK/Immunogenicity (Total ADA& Nab)/Biomarker&Cytokine and other research evaluations. Many ADC drugs involving Her2, Trop2, Muc1 and other targets have been supported, and popular targets such as EGFR, PCSK9, IL-17A, IL-6, IL-23, VEGF, CD47, TNF-α, CD20, TIGIT, etc. And 4-1BB, PD1, PDL-1, CTLA4 and other immune checkpoint monoclonal antibodies or polyclonal drugs, CAR-T targeting CD19, various fusion proteins, nucleic acids, and many related to diabetes, cardiovascular, bone Research work in different stages of peptide drugs for related diseases such as, tumor.

Theory: 1+1>2 bispecific antibody

Monoclonal antibodies play a certain biological role by binding to a single specific epitope, such as blocking protein-related effects, activating or regulating receptor functions. The Fc fragment of the antibody can also induce antibody-dependent cell-mediated cytotoxicity or complement-dependent cytotoxicity. But it also limits its application in some fields, such as the treatment of tumors and autoimmune diseases that need to block multiple signal pathways, and the treatment of infectious diseases that require the combination of multiple antigen sites to prevent the virus from escaping due to the high mutation rate of the virus. Therefore, bispecific antibodies came into being.
Bispecific antibody (BsAb) refers to an antibody molecule that can specifically bind to two antigens or two epitopes at the same time, and can exert the synergistic effect of the combination of two monoclonal antibodies. Bispecific antibodies do not exist in the natural state and need to be artificially prepared through recombinant DNA or cell fusion technology.
The main advantage of bispecific antibodies over monoclonal antibodies is that they can mediate temporal or spatial effects. Specifically, dual target dual antibodies or single target dual epitope dual antibodies may be more effective than two single targets The combined use of monoclonal antibodies; a dual-target fusion of an antibody drug molecule may have less side effects than a single-target antibody drug; a dual-target fusion of an antibody drug molecule may solve the problem of the onset mechanism that cannot be solved by the combination. But at the same time, the technical threshold and R&D cost are high. The construction stage may encounter problems of low expression and poor stability, and the development evaluation stage may encounter problems with model establishment. Therefore, the entire development process needs to consider the target biology, Structural biology, antibody engineering, screening strategies, etc.
The world’s leading dual antibody drug R&D companies mainly include Amgen, Roche, MacroGenics, Xencor, Genmab, AstraZeneca, and domestic companies mainly include Corning Jerry, BeiGene, Anmai Bio, Cinda Bio, etc. The number and development progress are in the first echelon.
The main mechanisms of action of bispecific antibodies are as follows:

• Bridge cell: T cell redirection;

• Inhibition of receptors: targeted inhibition of oncogene receptor tyrosine kinases (RTKs), such as EGFR and HER2, is a successful anti-cancer method;

• Targeted ligand-rich area: for multiple growth or angiogenic factors redundancy or bsAbs of interest;

• Target non-overlapping epitopes of the same antibody;

• Synergy factor or cofactor mimetic.

Policy: Read the policy without getting lost

The FDA has not issued many guidelines for bispecific antibodies, only the Bispecific Antibody Development Programs, draft Guidance issued in April 2019.
Other guidelines for bispecific antibodies that can be referred to include Guidance for Industry – Bioanalytical Method Validation of the U.S. FDA, ICH Guideline – Bioanalytical Method Validation M10 issued by ICH, and Immunogenicity Testing of Therapeutic Protein Products – Developing and issued by the FDA in 2019. Validating Assays for Anti-Drug Antibody Detections and Guideline on Immunogenicity assessment of therapeutic proteins issued by EMA in 2018.
Domestic regulations and guidelines include bispecific antibodies, 9012 biological sample quantitative analysis method validation guidelines, “Therapeutic protein drugs clinical pharmacokinetic research technical guidelines (draft for comments)” and “Pharmaceutical immunogen Technical Guiding Principles for Sex Research (Draft for Comment).

Practice: Factors to be considered for method establishment

Biopharmaceutical analysis methods need to be designed specifically according to different research contents, and bispecific antibodies are no exception. According to the different composition, properties, mechanism of action and the content of monitoring and analysis of molecules, we should also design and select unique and specific methods to obtain reliable and high-quality data in practice. When designing and selecting the analysis method of double antibodies, we need to focus on the following perspectives:
In terms of structure, according to whether the synthesized bispecific antibody contains Fc-segment protein, it can be divided into IgG-like bispecific antibody (containing Fc part) and non-IgG-like bispecific antibody (without Fc part). IgG-like bispecific antibodies have FC-mediated effector functions, with relatively large molecular weights, which are helpful for the purification of the antibody and improve its solubility and stability. The sensitivity of Assay is not high. It can be based on IgG Fc antibody in the early stage. To establish a method; non-IgG-like bispecific antibodies lack the Fc segment and only exert therapeutic effects through antigen binding capacity, and have the characteristics of low immunogenicity, easy production, and small molecular weight. Because of its small relative molecular weight, it has a higher permeability in tumor tissues, so it has a stronger therapeutic effect. When choosing a biological analysis method, the sensitivity of the Assay is high, and the method of detecting antibodies is generally not available.

A bispecific antibody is an antibody protein with multiple domains. It has multiple different binding sites and a more complex mechanism of action. These add more consideration points to the design of the analysis method of bispecific antibody drugs. For more complexity, the molecular forms that need to be measured include total, free, total bound, and partial bound. The analysis of these forms requires the consideration of Case by Case.

This multi-domain feature also makes potential considerations of immunogenicity more complicated. The immune response to one domain can inhibit specific functions while keeping other domains intact; the generation of potential new epitopes may also cause significant Immunogenicity. Therefore, investigating the immune response to bispecific antibodies may require the development of a variety of assays to measure the immune response to different domains of bispecific antibodies, and the consideration of immunogenicity analysis methods needs to be more complex and comprehensive.

Solution: Medicilon Biotechnology Pharmaceutical Analysis Department

Medicilon Biotechnology Drug Analysis Laboratory is committed to tailoring case by case biological analysis methods for customers, and uses advanced instruments and technologies to complete high-quality and efficient test services.

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