Separation of Small Hydrophobic Ligands from Proteins via Tangential Flow Filtration

Novel strategy to separate hydrophobic ligands from proteins through the use of Tangential Flow Filtration in which efficiency, safety, and scalability are improved. Enhanced production of proteins in this native state holds promise both for improved research and biomedical applications.

The Need

Successfully isolating proteins in their non-liganded state (i.e. apoprotein form) allows for an in-depth study of proteins from a research perspective. From an application standpoint apoproteins hold tremendous promise as both potential therapeutics and as drug delivery vehicles given their natural biocompatibility from a toxicity standpoint. The unoccupied ligand binding pockets in an apoprotein protein can be utilized to scavenge for particular biomolecules or transport and deliver desired therapeutic or diagnostic agents. Furthermore, either via natural clearance pathways or via conjugation to receptor specific ligands, for instance, the apoprotein-drug complex can be delivered to specific sites.

Existing strategies for separation of hydrophobic ligands from proteins fail to meet the high standards for proper scale up. Many of these processes use unsafe materials. Additionally, many existing techniques require costly, specialized equipment, and long processing times to yield large quantities of ligand-free protein. These inefficiencies restrict the expansion of apoprotein applications. Thus, there exists a need for an approach applicable to a diverse range of proteins that overcomes these barriers and can produce high-quality apoproteins at large-scales.

The Technology

Researchers at The Ohio State University, led by Dr. Andre Palmer, have created a novel strategy to separate hydrophobic ligands from proteins. Compared to existing methodologies, their approach uses safer reagents, can be carried out more expediently, and uses readily available, industry standard equipment. Improving on the aforementioned barriers to scale-up, this approach offers the opportunity to scale up ligand-free protein production for a wide variety of applications.

The investigators have validated this approach through demonstrated purification of apohemoglobin (apoHb) from hemoglobin (Hb). Current methods of apoHb production are often expensive, time-consuming, and inefficient, but with the new technology, apoHb is produced at large-scales in an efficient all-in-one system.

ApoHb has the potential to transport therapeutics in its’ unoccupied heme-binding pockets, directly enhancing their solubility in vivo. Furthermore, the researchers confirmed the heme-scavenging capabilities of their purified apoHb product in vitro. Building on their proof of concept experiment, this approach holds promise for a variety of other proteins used for drug delivery and therapeutic applications. For example, serum albumin may be made free of metabolites, lipids, and/or conjugated drugs. Overall, this novel method offers an opportunity for research and high-yield, scalable production of ligand-free proteins for biomedical and research applications.

Commercial Applications

  • Protein and peptide-based therapies
  • Biomolecule scavenging
  • Drug delivery
  • Biomedical research
  • Apoprotein purification


  • Increased specificity-targeting for drug delivery
  • Enhanced safety
  • Reduced cost
  • Greater solubility of hydrophobic ligands or drugs

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