Recent application of immunoglobulin-M (IgM) for treatment of life-threatening diseases such as cancer and in stem cell research is based on the recognition between native or genetically engineered antibody and cell-surface receptors. Although naturally produced by the immune system, the need for IgM with unique specificities and designed for single application, has encouraged the search for novel IgM purification strategies. As purity requirements for the final antibody preparation are very stringent, particularly for in vivo applications, there has been the need to search for more efficient purification strategies. Among the available purification techniques, affinity chromatography is a promising technique as it allows for large purity gains in a single-step, reduction in non-specific interactions, increase in operational yields and facilitates the elimination of undesirable contaminants.

Our research aimed at investigating the suitability of peptidic ligands based on Domain 1 of the polymeric immunoglobulin receptor (pIgR-D1) for affinity purification of IgM molecules. pIgR is a glycosylated type I transmembrane protein, consisting of a 620 residue extracellular region, a 23 residue transmembrane region, and a 103 residue cytoplasmic tail. The extracellular region contains five domains (D1–D5) that share sequence similarity with Ig variable regions. Studies suggest that 1) binding between IgM and SC depends preferentially on a strong interaction with domain 1 of SC and 2) domain 1 of SC consists of three CDR (complimentary determining region) like loops and the region containing the CDR2-like loop (SSEGY) is most important in the pentameric IgM-binding process. These findings prompted us to investigate peptidic ligands incorporating the CDR-2 like loop, as affinity ligands for IgM purification.

In this regard, three peptides: pep12 (ITLISSEGYVSS), pep13 (ITLISSEGYVSSK) and pep14 (CITLISSEGYVSSK), incorporating the CDR-2 region of domain 1 were synthesized. Qualitative characterization of the binding of synthesized peptides to IgM, IgG1 and bovine serum albumin (BSA) was performed by Surface Plasmon resonance (SPR) biosensor assays on a CM5 chip. SPR results showed that pep14 bound specifically to IgM with no binding observed for IgG1 and BSA. On the other hand, pep12 and pep13 showed no binding to IgM, IgG1 and BSA. Circular dichroism (CD) studies were performed to ascertain the role of structural conformations for the differential binding behavior exhibited by the three peptides. CD results suggested a random coil structure for pep12 and pep13 while pep14 exhibited a mix of beta sheet and random coil conformation. The above study gives an insight to the crucial role of structural conformation of pep14 for IgM binding and establishes pep14 as an affinity ligand for IgM purification.