Production & Purification.

As a standard, sdAbs are produced as recombinant proteins in E. coli. For this purpose, sdAbs are equipped with a leader sequence that directs them to the bacterial periplasm and is cleaved off in the process. From the periplasm, the sdAbs are then purified via a C-terminal 6xHis-tag using immobilized metal affinity chromatography (IMAC).

For larger batches, or to incorporate an extra cysteine for conjugation, sdAbs are produced in yeast (Saccharomyces cerevisiae). Here, the sdAbs are equipped with a signal peptide for secretion into the culture medium.

For some sdAb-containing fusion constructs, expression in mammalian cells is most efficient. For instance, we produce sdAbs fused to Fc domains of IgG antibodies or multivalent sdAbs in ExpiHEK or CHO-K1 cells. Furthermore, expression in mammalian cells can optimize the folding and post-translational modification of the expressed proteins.

In case of production by yeast or mammalian cells, the sdAbs are purified from the culture medium using affinity chromatography. Approximately half of all sdAbs have intrinsic affinity for protein A . The rest is either purified via a C-terminal EPEA sequence in our proprietary C-Direct tag or can be engineered to incorporate protein A binding affinity. sdAb-Fc fusion molecules are also purified by protein A affinity chromatography.

After purification, all sdAbs are buffer-exchanged to PBS unless requested otherwise and are quality controlled.