Developing and Characterization of Bioadsorbents for Extracorporeal Blood Purification

Cynthia M Daniels

doi:https://doi.org/10.21985/N2XQ51

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Developing and Characterization of Bioadsorbents for Extracorporeal Blood Purification

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Chronic kidney disease (CKD) is the ninth leading cause of death in the United States. According to the National Kidney Foundation, more than 20 million people already have CKD, and another 20 million are at risk for developing CKD. The primary causes of kidney disease are diabetes followed closely by hypertension. An unavoidable complication of long-term kidney failure is a debilitating disease called dialysis related amyloidosis (DRA). A key feature of DRA is the formation of amyloid fibrils consisting primarily of b2-microglobulin (b2m) and to a lesser degree glycated b2m or advanced glycation end products (AGE-b2m). Except for kidney transplantation, conventional kidney replacement therapies do not adequately address the removal of elevated concentrations of b2-microglobulin or AGE removal from blood. An anti-human b2m single-chain variable region antibody fragment (scFv) was developed to confer specificity for b2m removal during hemodialysis. The scFv was immobilized onto agarose and characterized for b2m binding capacity, thermal stability, regeneration capacity, storage conditions, and sterility. The immobilized scFv (immunoadsorbent) is thermally stable, can be regenerated, stored short-term in 20% ethanol, lyophilized for long-term storage, and can withstand processing conditions that are similar to that of patients on hemodialysis. Others have shown that the non-enzymatic modification of b2m to form AGEs are not only associated with DRA, but also are implicated in the pathogenesis of cardiovascular disease, diabetic complications, and neurodegenerative diseases. Elevated concentrations of AGEs have an effect on the activation of macrophages and monocytes, cells that are important in acute and chronic inflammation. The effects of AGEs are mediated by the receptor for AGE (RAGE). RAGE, a multi-ligand receptor was expressed in E coli, purified, and immobilized onto agarose via cyanogen bromide activation. The immobilized RAGE (bioadsorbent) was assessed for specificity, thermal stability, regeneration capacity, storage conditions, and its ability to reduce monocyte cytokine and chemokine production, and to inhibit osteoclast formation. The bioadsorbent can be regenerated, is stable under clinically relevant conditions, and can normalize the inflammatory potential of THP-1 cells, a human monocyte cell line. The bioadsorbent can also inhibit osteoclast formation. The bioadsorbent is specific for RAGE ligands that are involved in a persistent and chronic inflammatory cascade, specifically, four moieties of AGE-modified proteins (glycoaldehyde derived AGE-BSA, glucose derived AGE-BSA and b2m, and CML-BSA), S100B, S100A12, amphoterin, and the b-amyloid peptide. The bioadsorbent quantitatively lowered the concentrations of AGEs, S100B, S100A12, and b-amyloid that were present in plasma samples of diabetic patients with end stage kidney disease (ESKD). We envision that the bioadsorbent filters can be used in tandem with a hemodialysis extracorporeal circuit during a patients' weekly hemodialysis session to normalize the plasma concentrations of b2m, AGEs, and other RAGE ligands. We report the first step in the development and characterization of two novel and high affinity bioadsorbents for the extracorporeal removal of undesirable ligands associated or implicated with various diseases and their side effects.

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