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BOC Sciences has extensive expertise and capabilities in the synthesis and conjugation of non-natural amino acids (nnAAs). We have a team of experienced chemists and scientists specializing in organic synthesis, peptide chemistry and bioconjugation. We employ efficient coupling chemistries, such as click chemistry or bioorthogonal conjugation, to achieve site-specific and selective conjugation. Our services can be applied to various research areas, including antibody-drug conjugation (ADC), drug discovery, protein engineering, bioconjugation, and molecular biology.
Non-natural amino acid (nnAA) conjugation refers to the process of chemically linking or conjugating non-natural amino acids to antibody, protein or peptide sequences. nnAAs are amino acid derivatives that do not naturally occur in biological systems but can be synthesized or modified in the laboratory. Conjugation of nnAAs can introduce new functional groups or properties into biomolecules, thereby expanding their chemical diversity and potential applications. Genetically integrated nnAAs allow a unique orthogonal coupling strategy compared to those used for the twenty naturally occurring amino acids. Thus, nnAA provides a novel paradigm for creating next-generation ADCs. Furthermore, site-specific conjugation through nnAAs can also enpower unique small molecule, bispecific, multispecific and other conjugates that could be important constructs for therapeutics, diagnostics and research reagents.
By precisely introducing nnAAs at specific sites within the antibody, site-specific drug conjugation can be achieved, enabling precise control over the drug-to-antibody ratio (DAR) and avoiding the heterogeneity issues associated with traditional random conjugation methods. This site-specific conjugation not only enhances the homogeneity and stability of ADCs but also improves their efficacy and pharmacokinetic properties. Furthermore, the introduction of nnAAs provides greater flexibility in ADC design, supporting a variety of bioorthogonal chemical reactions and expanding the potential applications of ADCs. As a result, nnAA incorporation technology offers a powerful tool for developing safer and more effective ADCs.
Fig. 1. Non-natural amino acids conjugation strategies (BOC Sciences Authorized).
In addition to modification of known amino acids in antibodies, nnAAs can also be integrated into the protein sequence of antibodies through genetic code expansion (GCE) to specifically modify the binding site of the antibody. Antibody-drug site-specific conjugation can be achieved through nnAAs, that is, non-natural amino acids can be introduced into specific sites of any target protein in the organism. The residues on these non-natural amino acids can be combined with linkers to form site-specific ADCs. Compared with traditional ADC drugs, the introduction of non-natural amino acid antibody and drug linkers can achieve targeted and quantitative coupling. The obtained ADC has high efficacy, good stability, long circulation half-life, high safety, and uniform DAR.
Drug-antibody-ratio (DAR) is a key quality attribute of ADC compounds, which has a profound impact on the efficacy, safety, stability, anti-tumor and pharmacokinetic properties of ADCs. A moderate DAR value (usually between 2 and 4) balances the drug loading and targeting of the ADC, allowing it to deliver an effective concentration of the drug within the target cell for optimal therapeutic effect. However, too low a DAR may result in insufficient drug efficacy, while too high a DAR may increase hydrophobicity, induce non-specific aggregation, accelerated clearance, and non-targeted toxicity, reducing the stability and targeting ability of the drug. In addition, the uniformity of DAR values is also critical, and uneven distribution can increase product heterogeneity and affect drug consistency and repeatability. The introduction of nnAAs at specific sites can further enhance the uniformity and control of DAR values, enabling more precise engineering of ADCs with improved stability and targeting properties. Therefore, in the design and production of ADC drugs, the accurate control of DAR values and their distribution is a key step to ensure drug efficacy, safety, and quality.
Cell-free expression system is a technology for protein synthesis in vitro, utilizing cellular extracts (such as ribosomes, tRNAs, and enzymes) to carry out transcription and translation in a cell-free environment. This system is characterized by its speed, efficiency, high controllability, absence of cellular toxicity limitations, flexibility, and scalability, enabling protein synthesis to be completed within hours and significantly shortening the development cycle. Since no cell culture is required, the system is particularly suitable for expressing proteins that are toxic or unstable in cells, such as proteins containing nnAAs. Additionally, proteins produced by cell-free expression systems are typically non-glycosylated, simplifying the purification and characterization processes. Currently, a number of cell-free extract or lysate-derived systems have been developed from Escherichia coli, yeast, eukaryotic wheat germ, rabbit reticulocytes, Leymania sp., Thermus thermophilus, HeLa, and CHO cells for the use in nnAAs antibody conjugation. This technology has broad application prospects in areas such as ADCs, enzyme engineering, and protein labeling, providing a powerful tool for protein engineering and drug development.
Our In Vitro nnAA Antibody Conjugation Services Include:
BOC Sciences offers comprehensive analytical support to ensure the success of the conjugation process. We employ various techniques, such as HPLC, LC-MS, and NMR, to confirm the successful conjugation and assess the purity and integrity of the final product. In addition, with low-volume and high-volume infrastructure and production capabilities, we can accommodate projects of different sizes, from small research needs to large-scale production needs, ensuring flexibility and scalability.
Case Study 1 - Various naked antibodies and site-specific conjugation
Case Study 2 - Nanobod y conjugate
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