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Nanobody recombinant expression

Tek Biotech focuses on providing customers with high-quality and cost-effective antibody drug early discovery technology services. We have 10 years of project development experience and insights in drug antibody discovery, and have accumulated sufficient experience in antibody customization and recombinant antibody production. Based on a complete recombinant antibody expression platform, Tek Biotech can provide customers with recombinant expression preparation services for monoclonal antibodies (including but not limited to VHH antibodies, scFv antibodies, Fab antibodies, chimeric antibodies, humanized antibodies and various types of Fc fusion recombinant antibodies) from different species including but not limited to camel, sheep, and mouse, as well as its supporting complete traceability document system. Tek Biotech's CHO-K1, Expi 293F and other mammalian cell host systems, combined with self-designed secretory high expression vectors and various specifications of amplification expression systems, can provide customers with high-quality recombinant antibody expression services to meet the scientific research and production needs of various customers.

Monoclonal antibodies are complex multimeric proteins. To have biological functions, they require complex folding mechanisms and appropriate post-translational modifications. Therefore, they are more suitable for expression in eukaryotic systems, but large-scale production operations are complex and expensive. Nanobodies (Nbs) have simple structures and are still active in the absence of modification and the presence of Fragment crystallizable (Fc), making them more suitable for production in a variety of microbial expression systems. Nbs have a small relative molecular mass of about 12-15 kDa and a size of only 4 nm×2.5 nm×3 nm. They have a simple structure and consist of 4 framework regions (FR) and 3 complementarity determining regions (CDR).

Recombinant expression of nanobodies - Tekbiotech.png

Figure 1 Structure of traditional antibodies and camel heavy chain antibodies

1. Biological Characteristics of Nanobodies

Nanobodies are smaller in size than monoclonal antibodies and can target intracellular antigens in tumor tissues that are difficult for monoclonal antibodies to reach. Nanobodies also have better extravasation and tissue permeability for the blood-brain barrier that is difficult for large molecules and water-soluble drugs to pass through. But at the same time, nanobodies have a short half-life in serum and are easily cleared quickly, reducing the efficacy of the drug.

The Nbs sequence forms a disulfide bond between the cysteine in the CDR1 region and the cysteine in the CDR3 region. The intrachain disulfide bond makes it more stable and still has high tolerance under extreme pH and high temperature conditions. Some Nbs can even withstand temperatures above 90°C. Four aliphatic amino acid residues in the FR2 region of Nbs are replaced by hydrophilic amino acids, and the hydrophilicity is better than that of traditional antibodies.

2. Commonly used Production and Expression Systems for Nanobodies

Small molecule Nbs do not require post-translational modification (PTM), so they can be expressed and produced in prokaryotic expression systems that lack PTM. Because of their small size, stable single-domain structure and strong water solubility, they can also be produced in mammals, insect cells and fungi. Commonly used production and expression systems are shown in the following table:

Commonly Used Expression Systems

Mammalian Expression System

Escherichia Coli Expression System

Advantages

1. CHO cells are easy to culture and operate, can grow at high density in suspension culture medium, and maintain high vitality in large bioreactors;

2. They can highly express proteins in serum-free culture medium and produce post-translationally modified proteins similar to those expressed by humans, with minimal immunogenicity.

1. This system is suitable for the production of non-glycosylated biotherapeutic proteins, is easy to culture, has fast cell growth rate and high yield;

2. The periplasm of E. coli is maintained in an oxidized state, and less cytoplasm is maintained in a reduced state, which is conducive to the formation of disulfide bonds and can promote the correct folding of exogenous proteins, thereby maintaining structural stability;

3. The periplasmic expression of Nbs in E. coli has more obvious advantages, which improves the solubility of the product and enhances the biological activity of the expressed protein. The periplasmic expressed Nbs can be extracted using a simple method of osmotic shock and is not contaminated by cytoplasmic proteins.

Disadvantages

1. The selection and development process of cell lines is more complicated and time-consuming, and cell growth patterns and stability need to be considered;

2. The cost is high, the difficulty is high, and there may be latent viruses in animal-derived culture medium serum.

1. The space for protein expression in the periplasm is limited, resulting in limited expression;

2. Lack of post-translational modification, low recovery rate after multiple purification processes, and limitations in glycoprotein production;

3. The presence of endotoxins, the prepared recombinant protein shows immune response after treating patients, which has potential safety issues.

Tek Biotech has been committed to the production of alpaca VHH antibodies for more than 9 years, and has an experienced technical team with rich biological technology background, which can provide one-to-one special services. Tek Biotech has a stable mammalian CHO cell and prokaryotic cell nanobody expression platform, which can provide customers with one-stop services from antibody sequence analysis, linker design, label selection design, vector selection design to exploration and optimization of recombinant nanobody expression conditions, recombinant antibody purification, etc. In addition, Tek Biotech also has a complete quality system and has established a variety of purity analysis methods such as SDS-PAGE and HPLC. It has rich experience in nanoantibody quality evaluation systems. At the same time, it also has a variety of nanoantibody purification methods such as affinity chromatography, hydrophobic chromatography, and ion chromatography, which can be flexibly combined and can also provide large-scale production services.

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