Chromatographic assay to probe the binding energy and mechanisms of homologous proteins to surface-bound ligands.

Probing the affinity of a ligand for homologous protein targets currently relies on laborious assays that need special equipment and high amounts of isolated, highly pure proteins. Herein we present the use of pISep, an integrated buffer system and modeling package, as an analytical method to rapidly and accurately probe the binding strength and mechanisms of homologous proteins to surface-bound ligands. To demonstrate our method, we utilized the four subclasses of human immunoglobulin G (IgG) as model homologous protein targets and the IgG-binding peptide HWRGWV as model ligand. Following IgG adsorption on a HWRGWV-Toyopearl adsorbent, the pISep buffer system was used to run uncoupled dual elution gradients of pH (from pH 8.5 to 2.5) and either isocratic or time dependent salt concentration. Both the sequence and partial overlap of elution times (IgG4 > IgG3 ≥ IgG1 > IgG2) was found to match closely the values of binding strength (KD) determined with both in silico docking simulations and isothermal titration calorimetry experiments. pISep gradients performed at different values of ionic strengths provided a means to compare the contribution of hydrophobic vs. electrostatic interactions to the IgG-peptide affinity. The shifts in retention times indicated that, among the various components of the binding energy, the hydrophobic interaction dominates in the binding of IgG2 and IgG4, whereas the binding of IgG1 and IgG3 features a balance of electrostatic and hydrophobic modes. These findings were also confirmed by the in silico analysis of the complexes formed by HWRGWV and the Fc fragment of the IgG subclasses. Collectively, these results indicate that the retention times on pISep elution gradients - in particular peak max, overlap, and shift under different conditions - directly correlate to the strength and nature of protein-ligand interactions. This work demonstrates the effectiveness of the pISep toolbox for probing the differential binding of homologous proteins to a reference ligand and informing the optimization of platform processes for the purification and fractionation of biotherapeutics.

Isolation and characterization of mesenchymal stem cells from human umbilical cord blood: reevaluation of critical factors for successful isolation and high ability to proliferate and differentiate to chondrocytes as compared to mesenchymal stem cells from bone marrow and adipose tissue.

Human umbilical cord blood (CB) is a potential source for mesenchymal stem cells (MSC) capable of forming specific tissues, for example, bone, cartilage, or muscle. However, difficulty isolating MSC from CB (CB-MSC) has impeded their clinical application. Using more than 450 CB units donated to two public CB banks, we found that successful cell recovery fits a hyper-exponential function of time since birth with very high fidelity. Additionally, significant improvement in the isolation of CB-MSC was achieved by selecting cord blood units having a volume ≥90 ml and time ≤2 h after donor's birth. This resulted in 90% success in isolation of CB-MSC by density gradient purification and without a requirement for immunoaffinity methods as previously reported. Using MSC isolated from bone marrow (BM-MSC) and adipose tissue (AT-MSC) as reference controls, we observed that CB-MSC exhibited a higher proliferation rate and expanded to the order of the 1 × 10(9) cells required for cell therapies. CB-MSC showed karyotype stability after prolonged expansion. Functionally, CB-MSC could be more readily induced to differentiate into chondrocytes than could BM-MSC and AT-MSC. CB-MSC showed immunosuppressive activity equal to that of BM-MSC and AT-MSC. Collectively, our data indicate that viable CB-MSC could be obtained consistently and that CB should be reconsidered as a practical source of MSC for cell therapy and regenerative medicine using the well established CB banking system.