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Antibody-drug conjugates (ADCs) are complexes that conjugate cytotoxic drugs to monoclonal antibodies. ADC drugs can not only specifically recognize tumor surface antigens through monoclonal antibodies, but also use their own highly efficient small molecule drug toxins to kill tumor target cells. They combine the advantages of targeted drugs and chemotherapy drugs. In the past 20 years, the research and development of ADC drugs has become a hot topic of competition among major pharmaceutical companies. More than 10 ADC drugs have been approved by the FDA, and there are more than 100 ADC drugs in clinical trials or pharmaceutical research and development stages.
However, due to the complex structure, the difficulty of developing and producing the technology is greatly increased. Compared with antibody drugs, the downstream purification process of ADC drugs requires more attention to indicators such as drug-to-antibody ratio (DAR), single linker, and the residual amount of aggregates. Developing a robust ADC platform process is crucial for major pharmaceutical companies.
ADC drugs mainly consist of three parts: an antibody responsible for selectively recognizing tumor cell surface antigens, a payload (such as a small molecule cytotoxic drug responsible for killing tumor cells), and a linker connecting the antibody and the payload. ADC drugs rely on the specificity and targeting of antibodies to tumor cell-associated antigens to reach tumor cells and enter the cells through endocytosis. The linker is broken by intracellular effects such as low pH or lysosomal enzymatic reaction, releasing the payload, resulting in tumor cell death. An ideal ADC drug should maintain stability and integrity in the blood circulation, accurately reach the therapeutic target, and ultimately release the payload within the target (tumor cells).
| Target Antigen | In order to improve the accuracy of targeting, when selecting target antigens, antigens that are expressed only in tumor cells or are highly expressed only in tumor cells and are not expressed or rarely expressed in normal tissues are usually selected, or at least limited to in certain organizations. The corresponding antibody part on the ADC drug should be effectively internalized after binding to the target antigen, thereby achieving precise delivery of the payload into the cell. |
| Antibody | Antibodies targeting the antigen are critical for specific binding of the ADC. The ideal antibody should bind to the target antigen with high specificity and affinity and should also promote efficient internalization. It shows low immunogenicity in the body and maintains a certain half-life of the drug. At the same time, it is also necessary to pay attention to whether the antibody itself has certain anti-tumor activity. |
| Linker | As a bridge connecting antibodies and cytotoxic drugs, linkers have a significant impact on the stability and payload release curve of ADC, thereby affecting drug efficacy and safety. At present, the types of linkers can usually be divided into cleavable linkers and non-cleavable linkers. |
| Payload | The payload is a small molecule drug that works after the ADC is internalized into tumor cells. Currently, the most commonly used are small molecule cytotoxic drugs. The payload is a key factor in determining the lethality of ADC drugs. It is usually advisable to choose hydrophobic small molecule drugs with high toxic potency, stability under physiological conditions, small potential drug interaction (DDI), and the ability to exert a bystander effect. |
| Coupling Method | The coupling mode of payload and antibody is also an important factor in the construction of ADC. The coupling mode will affect the DAR and the distribution characteristics of DAR. When selecting the coupling method, it is usually necessary to take into account the stability of the coupling method and the uniformity of the DAR value. At the same time, it is also necessary to pay attention to whether the coupling site will affect the binding of ADC antibody to the target antigen, the PK and other characteristics of ADC. |
ADC drug purification processes can be divided into two categories: tangential filtration (TFF) (ultrafiltration diafiltration (UFDF)) and chromatography. Chromatography also includes size exclusion chromatography (SEC), hydrophobic chromatography (HIC), hydroxyapatite chromatography (HA) and membrane chromatography (MC).
The ultrafiltration diafiltration (UF/DF) process in tangential flow filtration has been widely used in the purification process of ADC drugs. Because it is used to remove organic impurities, conjugation-related impurities, and for buffer replacement (usually it is necessary to replace monoclonal antibodies into a suitable buffer for conjugated drugs). In addition, the yield of UF/DF as a process in the ADC purification process can also be maintained above 90%. Relevant studies have shown that the molecular weight of small molecular impurities (organic impurities, coupling-related impurities) is lower than that of membrane pore size. Therefore, pH, membrane load, transmembrane pressure and flow rate have little effect on the removal of small molecular impurities during the filtration process, and the process has strong robustness.
It is worth noting that in some special cases, due to the high hydrophobicity of the linker complex, self-association may occur in the buffer solution, resulting in insoluble micelles. These micelles are difficult to remove through the UF/DF step, and usually need to be removed in conjunction with subsequent related chromatography processes. In general, the UF/DF process is a robust and versatile method for the purification of ADC drugs.
Size exclusion chromatography is a chromatography method for separation and purification based on molecular weight differences, which plays a vital role in the purification of ADC drugs. SEC can play the role of buffer replacement (desalting), removal of non-protein impurities (small molecule impurities, such as small molecule cytotoxic drugs), and removal of aggregates during the ADC purification process. In addition, SEC is usually performed under mild conditions (neutral pH buffer and room temperature conditions), which is beneficial to the stability of ADC drugs.
Therefore, SEC is a golden method for ADC purification. Usually when we choose SEC for purification, we usually assume that there are no secondary interactions, that is, there are no other interactions between the ADC molecules and the SEC packing, such as hydrophobic forces and ionic forces. However, there are many studies reporting the interaction between ADC molecules and SEC fillers. Therefore, during the process development and filler screening stages, we need to select fillers with as small interactions as possible for process development.
In addition to focusing on small molecule impurities and aggregates, the purification of ADC drugs should also focus on the removal of DAR impurities. Since ADC drugs are covalently coupled to hydrophobic drug linkers, ADC drugs have hydrophobic differences with monoclonal antibodies and impurities with different DAR values, so they can be removed by hydrophobic chromatography. For example, cysteine-conjugated ADCs with DAR<8 usually produce five different products (i.e., DAR=0, 2, 4, 6, 8). This situation can be separated by hydrophobic interaction chromatography combined with a linear gradient.
Ion exchange chromatography and hydroxyapatite chromatography are often used in the purification process of monoclonal antibodies, but there are limited examples of applying these two processes to ADC drugs. Among them, ion exchange chromatography separates and purifies through the charge difference between the target product and impurities. It can be used to remove impurities such as small molecule impurities, host DNA, endotoxins, and charge variants. Therefore, ion exchange is a potential candidate process for the purification stage of ADC drugs.
Hydroxyapatite chromatography performs relatively well in removing aggregates from ADC drugs. Studies have shown that using hydroxyapatite chromatography can reduce the aggregate content in antibodies from 60% before purification to 0.1%. In summary, ion exchange chromatography and hydroxyapatite chromatography show certain potential for ADC drug purification.
Compared with the many shortcomings of traditional column chromatography, membrane chromatography has higher yields and usually higher flow rates, which can shorten process time and save costs. Studies have shown that when using Sartobind membrane to purify ADC drugs, aggregates and drug linkers can be effectively removed. Further studies have shown that chromatography modules containing different modes of action can promote each other when connected in series to enhance purification efficiency. For example, when SEC and HIC membranes are connected in series, the desalination effect of the SEC membrane can promote the separation of DAR impurities by the HIC membrane. Similarly, there are studies that connect Sartobind S and Sartobind Phen membranes in series to remove aggregates under high flow rate and low pressure conditions.
| Catalog | Product Name | CAS Number | Category |
| BADC-01165 | BS3 Crosslinker | 82436-77-9 | ADCs Linker |
| BADC-01121 | Amino-PEG6-alcohol | 39160-70-8 | ADCs Linker |
| BADC-00330 | Irofulven | 158440-71-2 | ADCs Cytotoxin |
| BADC-00340 | PBD dimer | 1222490-34-7 | ADCs Cytotoxin |
| BADC-00606 | Deruxtecan | 1599440-13-7 | ADCs Cytotoxin |
| BADC-00780 | Spliceostatin A | 391611-36-2 | ADCs Cytotoxin |
| BADC-01602 | Trastuzumab deruxtecan | 1826843-81-5 | Antibody-Drug Conjugates (ADCs) |
| BADC-01603 | Sacituzumab govitecan | 1491917-83-9 | Antibody-Drug Conjugates (ADCs) |
Based on the mechanism of action, the population selection strategy of ADC drugs mainly focuses on the expression of target antigens on tumor cells. However, different ADC drugs for the same target antigen have different efficacy in different tumor types:
Because ADC's antibodies mainly play a targeting role, they can accurately deliver the payload into the target cells to kill tumor cells. In this process, some drugs can also produce a bystander effect. Therefore, ADC may not only be effective against tumors with high target antigen expression, but also whether it has anti-tumor activity against tumor cells with medium or low target antigen expression is worthy of further exploration.
Rational dosing strategy is a key factor affecting the benefit-risk profile of ADC drugs. Based on the structural characteristics and action characteristics of ADC drugs, payload toxicity is the main dose-limiting factor, and a relatively small increase in payload system exposure may lead to significant adverse reactions. The selection of the optimal dosing strategy needs to comprehensively consider the PK and PD differences between the antibody and the payload. Therefore, it is important to fully understand the PK and PD of ADC drugs and their components in the early stages of research, and to explore their relationship with safety and efficacy outcomes as much as possible, so that dosing regimens can be adjusted appropriately in a timely manner. For example, for ADCs that cause major toxicity due to excessive peak serum concentrations, an effective low-dose fractionated dosing strategy can be used to reduce the peak serum concentration, thereby improving safety and tolerability.
In the clinical development of ADC drugs, its safety management is crucial. For ADC drugs, factors such as the specificity of the antibody, the stability of the linker, and the nature of the payload may affect the safety of the drug, giving it different toxicity characteristics based on the simple payload. Compared with target-related safety risks, off-target related adverse reactions may be the main factor in ADC adverse reactions in most cases. Reasons for off-target toxicity may include: a) Release of payload into the blood circulation; b) Non-specific endocytosis mechanism and receptor-dependent endocytosis mechanism, which can internalize the entire ADC or free payload; c) Bystander effect, etc.
At the same time, off-target toxicity is not necessarily specifically related to the type or mechanism of action of the payload. For example, methylaurestatin F (MMAF) and methylaurestatin E (MMAE) are both tubulin inhibitors, but MMAF ocular toxicity is common, while the incidence of MMAE ocular toxicity is not high. For the above reasons, different ADC drugs may exhibit different safety characteristics when they have the same target antigen or payload.
