Site-Specific Conjugation

Site-Specific Conjugation

Site-specific conjugation is an important aspect of antibody-drug conjugates (ADCs). Compared with non-specific conjugation, site-specific bioconjugation can produce more uniform, well-defined, and developable antibody conjugates. At BOC Sciences, we offer a range of site-specific conjugation services to meet the diverse needs of our customers in the ADC space. Our experienced team of scientists is well-versed in the latest conjugation technologies and can work with a variety of antibody formats, including monoclonal antibodies, antibody fragments, and engineered antibodies.

Site-specific Conjugation for ADC

Although non-specific chemical couplings such as lysine amine acylation or cysteine thiol alkylation are widely used, they still have a series of shortcomings. For example, a lack of site specificity leads to product heterogeneity and process irreproducibility; potential modifications in complementarity-determining regions may reduce the binding affinity and specificity of the antibody. Site-specific conjugation can produce more uniform antibody conjugates, thereby ensuring product development and clinical application. Recent comparisons between site-specific and random modifications have shown that site-specific modifications can achieve the desired characteristics and functions, such as improving plasma stability, reducing variability in dose-dependent studies (especially at low concentrations), enhancing binding affinity, and increasing tumor uptake and absorption.

Categories of site-specific ADCsFig. 1. Categories of site-specific ADCs (Biomedicines. 2017, 5(4): 64).

In recent years, many research groups have reported site-specific conjugation strategies for the preparation of homogeneous ADC drugs. Some studies use genetic engineering technology to introduce some specific reaction sites at a certain position of the antibody, and achieve site-specific coupling of antibodies and drugs through the selective reaction of drug-loaded groups with specific sites on the antibody. Among them, Thiomab technology may be the earliest strategy to realize site-specific conjugation of antibodies and drugs. It inserts two cysteine residues into the light and heavy chains of the antibody through genetic engineering technology. The sulfhydryl group of cysteine can be coupled with the microtubule-related inhibitor MMAE to obtain a homogeneous ADC drug with a DAR of 2.

Site-specific Conjugation Services

With our state-of-the-art facilities, cutting-edge technology, and stringent quality control measures, BOC Sciences is committed to providing high-quality site-specific coupling services for ADC development. Whether you want to improve the stability, efficacy or safety of your ADC, we have the expertise and capabilities to help you achieve your development goals.

Site-specific Conjugation of Amino Acids

Site-specific conjugation of amino acids often requires the selection of several natural or engineered amino acids, such as cysteine and glutamine, as conjugation sites. Among them, unpaired cysteine-mediated conjugation was the first site-specific ADC to be described. Cysteine residues are engineered into different positions on the antibody heavy chain (HC) or light chain (LC) for conjugation. Because engineered cysteines are always capped by glutathione or other substances during expression, partial reduction of the antibody is required to remove the cap. The uncapped cysteine is then coupled to a cytotoxic-containing thiol-reactive linker using thiol-maleimide chemistry.

Site-specific Conjugation of Glycans

There are also researchers constructing ADC drugs by modifying different types of glycosyl-groups on natural antibodies. These glycosyl-groups on the antibody can be exposed to N-acetylglucosamine under the action of endoglycosidase, and then N-acetylgalactosamine modified by azide is linked to the N-acetylglucose of the antibody under the action of glycosyltransferase. Finally, azide-modified antibodies can achieve site-specific coupling of antibodies and drugs by clicking chemical reactions with alkynyl linker toxins.

Site-specific Conjugation of Unnatural Amino Acids

In addition to modifying natural antibodies, non natural amino acids (such as acetylphenylalanine, azidylmethyl-L-phenylalanine with bioorthogonal functionality, and azidyllysine) can be integrated into antibodies by using enzymes that can specifically recognize non natural amino acids. These unnatural amino acids contain specific functional groups (such as ketone groups, azide groups, etc.) that can react specifically with other functional groups in the drug linker (such as hydroxylamine groups, alkynyl groups, etc.) to obtain ADC drugs with uniform DAR. In addition, enzymes have high specificity and efficiency. Some researchers recognize specific sequences or sites of antibodies through some enzymes (such as transglutaminase, formylglycine synthetase and sortase A, etc.), and then introduce some specific functional groups through the catalysis of enzymes to achieve site specificity.

Site-specific Conjugation of Short Peptide Tags

Several site-specific conjugation methods are being developed by coupling cytotoxins to specific short peptide tags containing four to six amino acid residues. Such methods rely on the introduction of unique short peptide tags into antibodies for enzymatic modification in vivo or in vitro. They allow specific amino acids in the peptide tag to be functionalized and conjugated to drug linkers.

Advantages of Site-specific Conjugation

  • Improving homogeneity and consistency of the final product. By targeting specific sites on the antibody for conjugation, it is possible to produce ADCs with a defined drug-to-antibody ratio (DAR), ensuring a consistent number of drug molecules are attached to each ADC molecule. This results in a more predictable and repeatable product.
  • Improving stability and reducing off-target toxicity. Site-specific conjugation allows precise control of the position and orientation of drug molecules on the antibody, thereby reducing the potential for unwanted interactions with non-target tissues or proteins.
  • Improving pharmacokinetics and pharmacodynamics of ADCs. By controlling the binding site, researchers can optimize drug-antibody interactions to enhance the therapeutic efficacy of ADCs. This could improve tumor targeting, increase drug delivery to cancer cells, and enhance anti-tumor activity.

Why Choose BOC Sciences?

  • Professional knowledge and experience: BOC Sciences has a highly qualified team of scientists and engineers with extensive experience in the development and manufacturing of ADCs. We have a deep understanding of the complex chemistry involved in ADC production and can provide valuable insight and guidance throughout the process.
  • State-of-the-art facility: BOC Sciences has a state-of-the-art facility equipped with the latest technology and equipment for ADC manufacturing. This ensures customers receive high-quality products that meet the highest standards of safety and efficacy.
  • cGMP compliance: BOC Sciences' ADC manufacturing services are fully compliant with current Good Manufacturing Practices (cGMP), ensuring products are produced in a controlled and consistent manner to meet regulatory requirements.
  • Customization services: BOC Sciences provides customized ADC development services to meet the specific needs and requirements of each customer. We work closely with customers to develop custom manufacturing processes that meet their unique specifications and goals.
  • Quality control: BOC Sciences has strict quality control processes to ensure the purity, potency and stability of its ADC products. We perform thorough testing and analysis at every stage of the manufacturing process to guarantee the quality of the final product.

Case Study

A pharmaceutical company is struggling to develop effective ADCs due to a lack of site-specific conjugation expertise. They turned to BOC Sciences for help, and our combination of site-specific services proved to be key to their success.

Case study of site-specific conjugation

Utilizing our cutting-edge technology and state-of-the-art facilities, we are able to engineer cysteine residues into specific locations on antibodies for conjugation. Through precise manipulation and partial reduction, we successfully opened engineered cysteines and coupled them to cytotoxic-containing thiol-reactive linkers using thiol-maleimide chemistry. This has led to the creation of highly stable, effective and DAR consistent ADCs. Thanks to BOC Sciences' expertise and capabilities, the pharmaceutical company was able to achieve its development goals and successfully advance its ADC into clinical studies.


1. What is site-specific ADC conjugation?

Site-specific ADC conjugation is a method used to attach cytotoxic drugs to specific sites on antibody molecules in antibody-drug conjugates (ADCs). This allows for more precise and controlled drug conjugation compared to non-specific conjugation methods, potentially improving efficacy and reducing side effects. Site-specific conjugation can be achieved by various techniques, such as using engineered antibodies with specific conjugation sites or utilizing chemical modifications to target specific amino acid residues on the antibody.

2. What is cysteine conjugation ADC?

Cysteine-conjugated ADCs are a type of antibody-drug conjugate (ADC) in which the drug payload is linked to the antibody through a cysteine residue. Cysteine conjugation involves the use of linker molecules containing reactive groups that can specifically bind to cysteine residues on antibodies, allowing site-specific attachment of drug payloads. This approach could improve ADC stability and efficacy by reducing off-target binding and increasing the specificity of drug delivery to tumor cells.


  1. Zhou, Q. Site-Specific Antibody Conjugation for ADC and Beyond. Biomedicines. 2017, 5(4): 64.
* Only for research. Not suitable for any diagnostic or therapeutic use.
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