As a global leader of sdAb discovery and manufacturing, Creative Biolabs will briefly introduce a series of sdAb development strategies for our customers all over the world.

pI Influence of sdAb Permeability

The theoretical isoelectric points of sdAb cover a wide range (pI value between 5 and 10), which portends different degrees of physical and/or biological stability between the sdAbs. In general, an extremely high or low isoelectric point will render some of the sdAbs unsuitable for in vivo use. However, the reduced isoelectric point of sdAb could not only enhance the solubility but also result in improved levels of production. Furthermore, the basic pI also has appeared to be an essential parameter to influence the permeability of sdAb. The sdAb with a highly positively charged pI (e.g., pI = 9.4) enhances permeability to cross the blood-brain barrier (BBB) spontaneously. Such sdAb not only gains access to the brain but is even found to penetrate cells and bind intracellular proteins. Shifting the pI of therapeutic sdAbs to a basic level facilitates these sdAbs to cross the BBB.

Disulfide Bond Influence of sdAb Stability

Compared to the conventional antibody, sdAb often has noncanonical Cys pairs that conform additional disulfide bonds to make sdAb with optimal biochemical and biophysical properties, including thermal tolerance, proteolytic resistance, and extraordinarily resistance to various stress conditions. An elegant solution to increase the sdAb stability, including the thermodynamic stability and protease resistance, is introducing the non-canonical disulfide bonds. sdAb with increased stability will provide superior reagents for a myriad of biotechnology, detection, and diagnostic applications, as well as promising for the novel administration (e.g., oral administration). However, for some pharmaceutical purposes, sdAb clones without the additional disulfide bond might be more preferable, which can avoid the potential risk of disulfide bond mismatch during the downstream manufacture.

Site-Specific Labeling of sdAb

In recent years, several versatile and standardized strategies for the site-specific functionalization of proteins have been applied to modify sdAb homogeneously. Reliable site-specific attachment of functional moieties with improved biophysical properties can overcome the limitations of random conjugation. The introduction of a tag for site-specific labeling at the C-terminal end of the sdAb is a valid alternative to obtain a better, more controlled probe since the C-terminal end and the CDR loops are located on opposite sides of the domain, thereby avoiding antigen-binding interference. Using site-specific attachment of tracer to sdAb, a homogeneous tracer population with a well-defined stoichiometry and label position can be obtained. The versatile techniques for site-specific labeling manage to couple various molecular reagents, such as biotin, fluorescent dye, bifunctional chelators, etc., to the sdAbs.

Reference

1. Rissiek, Björn, Friedrich Koch-Nolte, and Tim Magnus. "Nanobodies as modulators of inflammation: potential applications for acute brain injury." Frontiers in cellular neuroscience 8 (2014): 344. Distributed under Open Access license CC BY 4.0, capture the part A of original Figure 1.

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