Background and overview

Antibody-drug conjugates (ADCs) services include one-stop solutions from monoclonal antibody development, to cytotoxin synthesis, linker design and preparation, ADCs synthesis, purification process development, analytical method research, and quality standard establishment. Under the premise of drug safety, it will maximize the killing ability of drugs. The idea of ​​using antibodies to achieve targeted delivery of cytotoxic drugs can be traced back to the beginning of the last century. With the maturity of genetic engineering technology, antibody preparation technology, and the emergence of new chemical connection technology, this field has achieved milestone development in recent years, and the clinical transformation of drugs has been achieved. As early as 2000, Gemtuzumabozogamicin (trade name Mylotarg) had become the first antibody-conjugated drug approved by the FDA for the treatment of acute myeloid leukemia (AML). However, its phase III clinical trial found that it was hepatotoxic and compared with the control group, the curative effect was not significant, and Pfizer applied for delisting in June 2010.

In August 2011, the ADC drug Brentuximabvedotin (trade name Adcetris) was approved by the FDA. At the same time, it is also the first new FDA-approved drug for the treatment of Hodgkin’s lymphoma in the past 30 years and the first for the treatment of rare disease systemic diseases, for degenerative large cell lymphoma. In February 2013, another antibody-conjugated drug Ado-trastuzumabemtansine (trade name Kadcyla) was approved by the FDA for the treatment of HER2-positive metastatic breast cancer. The launch of these two ADC drugs is just a microcosm of the upsurge in antibody-conjugated drug research. Currently, about 80 ADC drugs are in the R&D stage. As of November 2013, 35 have entered the clinical trial stage. Compared with traditional antibody drugs or cytotoxic drugs, antibody drug conjugate have more complex structures and quality attributes. Antibody drug conjugate is composed of two parts, namely antibody and drug, connected by linker molecules. The development of ADC drugs with high safety and good curative effect depends on the joint development of three aspects: the selection of antibodies and drugs, and the optimization of linker technology. At the same time, its complex quality attributes also put forward higher requirements for the quality control of antibody-drug conjugates.

The preparation methods of ADCs involve random modification of antibody amino acid residues and selective introduction of drug molecules into antibody molecules. The random modification process includes amination of carboxylic acid and acylation of amino group of lysine side chain. Antibody molecules contain many groups that can be used to modify cross-linking, and cross-linking groups include amino and carboxyl groups. Methods for drug molecules to selectively bind antibodies include gene recombination technology to express fusion proteins, reductive amination of oxidized polysaccharides of antibodies, and reductive alkylation of disulfide bonds between antibody chains.

The preparation of antibody drug conjugate is as follows:

1) Antibody reduction

Using the disulfide bond on the antibody for drug coupling to generate the antibody drug conjugate, firstly, the disulfide bond of the antibody needs to be reduced to generate a sulfhydryl group. The commonly used antibody type is IgG, or immunoglobulin. The molecular weight of IgG antibody is about 150KD. It is difficult to penetrate the capillary endothelium and extracellular space such as its huge antibody and its coupling molecules. Therefore, the ratio of the antibody reaching the tumor cell to the actual dose is small. For IgG antibody, it is usually smaller than 0.1% of the injection volume will reach the tumor site. According to the difference of the heavy chain constant region, there are five main types of antibody molecules, denoted as IgG, IgM, Ig, IgE, and IgD. Among them, IgG, IgE and IgD 3 types of antibody molecules all include Ig monomers composed of two heavy chains and two light chains. Through X-ray diffraction analysis, the antibody molecule is mainly composed of three parts, two identical antibody fragments-antigen binding arms and one fragment that is easy to crystallize.

2) Antibody reduction test

A certain amount of sulfhydryl groups can be produced by controlling the amount of reducing agent. If the number of required drug couplings has been given, it is necessary to design experiments to control the reducing agent reaction ratio, reaction temperature, reaction time, and optimize the reduction reaction parameters to achieve the required product. Studies have shown that when four drugs are attached to each antibody molecule, the drug is most effective and has the highest survival rate in vivo when the drugs are coupled to the hinge region. Therefore, it is necessary to control the antibody reduction reaction by chemical methods, so that the disulfide bond in the hinge region of the antibody is reduced to produce four sulfhydryl groups to couple the drug with the linker. The free reducing agent is dispersed in the solution. As long as the amount of reducing agent is sufficient, it can not only reduce the disulfide bonds exposed to the outside of the protein, but also increase the amount of reducing agent to reduce the disulfide bonds wrapped in the protein. The reducing agents for protein disulfide bonds include thiol-based reducing agents and phosphine-based reducing agents, including tri-n-butyl phosphine and tricarboxyethyl phosphine. The immobilized reducing agent, as the name suggests, is to fix the commonly used disulfide bond reducing agent on some medium, usually on agarose. The use of an immobilized reducing agent to reduce the disulfide bond can improve the use efficiency of the reducing agent and has a wider application range. At the same time, since the reducing agent is fixed on the medium, after the reduction reaction is completed, there is no need to remove the reducing agent, which reduces the loss of protein. It has been reported that many methods can be used to detect antibodies and antibody fragments, which can be used as reference methods for the detection of antibody reduction, including sodium dodecyl sulfate polyacrylamide gel electrophoresis, capillary electrophoresis, molecular exclusion chromatography resistance chromatography-mass spectrometry, etc.

3) Antibody drug conjugate

The body is reduced to a suitable degree, and the coupling reaction with the drug can be carried out. There are many ways of coupling, which can be combined through strong covalent bonds, or through scattered ionic bonds and hydrophobic bonds. For ADC drugs, the stability in the aqueous solution must first be ensured for easy use. Secondly, the linker must not only remain stable in the blood circulation to reduce the toxic side effects of the drug, but also quickly release the drug to exert its activity after entering the target cell. Nowadays, the connectors connecting antibodies and toxic drugs include disconnectable connectors and non-disconnectable connectors. According to different target cells, the connection method suitable for each drug is screened through in vivo and in vitro experiments. Generally speaking, the antibody-calisporin conjugate is connected by acid-sensitive hydrazone, the antibody-maytansinol conjugate is connected by disulfide bond or thioether, and the connection of the antibody-auristatin conjugate is soluble in the enzyme cleaved peptide link or unbreakable thioether link in the enzyme body.

4) Purification method

After the antibody-drug conjugate reaction is terminated, excess drug linkers, organic solvents, EDTA and other additives need to be removed. According to the scale of the reaction, there are many methods to choose from. For laboratory scales such as micrograms or less, ion exchange chromatography, affinity chromatography, gel filtration methods, centrifugal ultrafiltration, ion exchange chromatography and affinity chromatography can be used. For scales of several hundred milligrams or more, a combination of ultrafiltration/dialysis can be used. Ion exchange chromatography includes cation exchange chromatography and anion exchange chromatography. Proteins of different molecular weights have different amounts of charge, so their binding ability to the exchanger is strong or weak, and they will be eluted sequentially with the flow of the mobile phase, thereby achieving separation between different proteins. Affinity chromatography usually uses only one step to separate the target protein from a large amount of impurities, can complete the separation that is difficult to complete by ordinary methods, and can achieve a purity of greater than 90%.