The Need

Novel protein and apoprotein purification strategies are valuable tools with a wide variety of medical and research applications. While there are existing techniques for protein and apoprotein purification, some of these have flaws such as the use of harsh denaturants, highly flammable solvents, low yields, cumbersome manufacturing processes, and high capital and operating expenses. Furthermore, they are not easily scalable, which inhibits their applications for expanded use. Therefore, effective, scalable, and safe methods for protein and apoprotein purification are needed.

New therapeutic protein complexes and cocktails can provide comprehensive treatment of complex disease states such as hemolysis. For example, although hemolysis is a multifaceted process, current approaches have mainly focused on single protein therapies towards neutralizing each of the toxic species produced during hemolysis. Thus, efficient therapeutic solutions need to be developed to treat all the major toxic byproducts of hemolysis.

The Portfolio

Dr. Andre Palmer and colleagues at The Ohio State University and University of California at San Diego have developed a novel method for apoprotein manufacturing and several methods for general protein purification, along with a novel, deliverable protein complex for use in various medical applications. These methods utilize various techniques to purify individual protein components, protein cocktails (i.e., protein mixtures), and complexes of interest. For example, some of the proteins purified include apohemoglobin (apoHb), haptoglobin (Hp), and the apoHb-Hp complex.

Separation of Small Hydrophobic Ligands from Proteins

With this invention, the Palmer group have developed a method for separating small hydrophobic ligands from proteins, such as heme from hemoglobin (Hb) or metabolites and lipids from human serum albumin (HSA). This method allows for purification of apoproteins, such as apoHb and ligand-free HSA.

This novel technology does not require the use of specialized equipment, is relatively inexpensive, and easily scalable. Furthermore, this technology has the potential to be applied to any ligand-associated protein to produce the resultant apoprotein.

With a wide variety of medical applications, this method represents a breakthrough in the manufacturing of apoproteins. Large-scale manufacturing of apoproteins opens-up a new approach to facilitate small molecule drug transport in vivo. These proteins may be used for targeted drug delivery, bioimaging, and/or scavenging of toxic molecules during various disease states. Furthermore, these apoproteins could be used in various medical treatments, such as drug delivery for certain cancers. There are also other prospective applications for this technology: currently purified serum proteins may be made free of lipids and metabolites, thus improving their therapeutic index.

Isolation of Plasma Proteins and Plasma Protein Cocktails

Haptoglobin (Hp) is a valuable plasma protein with various medical applications. The Palmer group have refined a method to readily obtain isolated polymeric forms of Hp from plasma or plasma fractions with minimal contamination from other serum proteins or components. Hp can bind cell-free Hb, which allows Hp to be used as a treatment for a variety of medical conditions characterized by states of hemolysis. Therefore, since the Hp-Hb complex can target CD163+ macrophages and monocytes, Hp may be used for targeted drug delivery in combination with Hb and/or Hb-like molecules such as apoHb (see Therapeutic Uses of Protein Complexes below).

Furthermore, with the use of their method of separation, Dr. Palmer’s group is capable of isolating protein cocktails (i.e. protein mixtures) for enhanced treatment of states of hemolysis. One of these cocktails is capable of binding and detoxifying cell-free Hb, cell-free heme and free iron. During disease states characterized by hemolysis, the rupture of red blood cells (RBCs) leads to the build-up of these toxic species in the circulation. Previous therapies have mainly focused on scavenging single toxic species (i.e. Hb, heme or iron). Yet, with the use of the protein scavenging cocktail, the treatment would detoxify the entire set of toxic components of hemolysis (i.e. Hb, heme and iron).

The technology developed for isolation of Hp from plasma or plasma fractions can also be applied to purify other plasma proteins, concentrates and cocktails including Factor I, Factor VIII, Factor X, Von Willebrand factor, Fibrinogen, Fibronectin, Protein C, Ceruloplasmin, serum albumin, prothrombin, and immunoglobulins.

This method also provides an advancement in the field of protein purification. The main technique for purifying proteins is column chromatography. However, chromatography is an expensive technique with highly specialized equipment and specific separation conditions for each protein. Furthermore, in some types of chromatography, such as affinity chromatography, the process may require the use of harsh solvents to purify the target protein. With the use of their method, the process has the benefit of being easily scalable to accommodate the large volumes of plasma processed by the plasma protein industry. Therefore, this novel method represents a more effective, less expensive, and more easily scalable protein purification method versus column chromatography.

Selective Protein Purification

With this invention, the Palmer group have developed a method for isolating specific target proteins from complex protein/particulate mixtures. The method uses a target protein binding molecule (TPBM) that allows for the purification of the desired protein. This process yields purified proteins, which can then be used in various medical or research applications.

This method is an improvement on current technology for several reasons. First, the use of specific TPBMs allows for the targeting and isolation of proteins specific to the researcher or manufacturer’s needs. Also, the method is inexpensive and less cumbersome compared to current technologies, promoting ease of use and wider applicability. This technology is versatile and can be used to purify a variety of proteins.

Therapeutic Uses of Protein Complexes

The researchers have created unique protein complexes that have a wide variety of medical applications. One product is synthetically made apoHb-Hp protein complexes. One medical application of this technology is for treatment of various states of hemolysis, where these complexes can be administered to detoxify both cell-free Hb and cell-free heme. Moreover, this complex can be used as a drug or bioimaging agent carrier. For example, apoHb-Hp can target CD163+ macrophages and monocytes, thus potentially delivering chemotherapeutic drugs or other types of drugs more effectively to these cells.

This technique is an improved method to treat hemolysis because it allows for the scavenging of both cell-free Hb and cell-free heme with a single molecule. There is a need for novel treatments to detoxify the byproducts of hemolysis. This technology therefore represents an advancement in the field that could provide more robust treatment for conditions such as hemolysis, and more efficacious drug delivery for conditions such as cancer.

Commercial Applications

• Drug delivery
• Bioimaging
• Detoxification of byproducts of hemolysis
• Protein purification
• Apoprotein purification
• Detoxification of RBC blood products
• Additive to prolong the ex vivo storage lifetime of blood
• Plasma protein concentrates

Benefits/Advantages

• Inexpensive
• High yield
• Scalable
• More efficacious than current methods
• Less cumbersome than available methods

Patents

PCT/US2020/016267 titled “Methods of Protein Purification”: https://patentscope.wipo.int/search/en/detail.jsf?docId=WO202016050

PCT/US2020/033836 titled “Apohemoglobin-Haptoglobin Complexes and Methods of Using Thereof”: https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2020236952

PCT/US2021/048241 titled “MODELING CONDITIONS FOR TANGENTIAL FLOW FILTRATION PROCESSES FOR PROTEIN PURIFICATION”: https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2022047310&_cid=P21-LKJNLI-13906-1

PCT/US2021/023441 titled “Methods for treating plasma protein imbalances or depletion”: https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2021236227&_cid=P21-LKJO9C-24883-1

Peer-reviewed Publications

• Novel manufacturing method for producing apohemoglobin and its biophysical properties
  • Biotechnology and Bioengineering. 2020;117:125–145.
  • https://doi.org/10.1002/bit.27193
  • Describes the process used to manufacture apohemoglobin from hemoglobin using tangential flow filtration. This process is also generalizable to purify any apoprotein.
• Tangential flow filtration of haptoglobin
  • Biotechnol Progress. 2020; e3010.
  • https://doi.org/10.1002/btpr.3010
  • Describes the process used to purify haptoglobin from Cohn Fraction IV using tangential flow filtration. This process is also generalizable to purify any plasma protein fraction.
• Apohemoglobin-haptoglobin complex attenuates the pathobiology of circulating acellular hemoglobin and heme
  • Am J Physiol Heart Circ Physiol 318: H1296–H1307, 2020.
  • https://doi.org/10.1152/ajpheart.00136.2020
  • Demonstrates the ability of the apohemoglobin-haptoglobin complex to scavenge and neutralize cell-free hemoglobin and free heme.
• Poly(ethylene glycol) surface-conjugated apohemoglobin as a synthetic heme scavenger
  • Biomacromolecules Jun 8;21(6):2155-2164., 2020.
  • https://pubs.acs.org/doi/10.1021/acs.biomac.0c00141
  • Demonstrates the ability of PEGylated apohemoglobin to scavenge and neutralize cell-free heme. Thus the molecule functions as a hemopexin mimetic.
• Apohemoglobin-haptoglobin complexes attenuate the hypertensive response to low-molecular-weight polymerized hemoglobin
  • Blood Advances 4 (12), 2739-2750
  • https://doi.org/10.1182/bloodadvances.2020002045
  • Demonstrates the ability of the apohemoglobin-haptoglobin complex to attenuate the hypertensive response and toxicity to transfusion of low molecular weight polymerized hemoglobins as a red blood cell substitute.

• Enhanced Photodynamic Therapy using the Apohemoglobin-Haptoglobin Complex as a Carrier of Aluminum Phthalocyanine
  • ACS Applied Bio Materials
  • https://doi.org/10.1021/acsabm.0c00450
  • Demonstrates the use of the apohemoglobin-haptoglobin complex as a drug carrier for photodynamic therapy. The apohemoglobin-haptoglobin complex can also be used as a drug delivery vehicle to target CD163+ monocytes and macrophages which play an important role in many disease states.
• Selective protein purification via tangential flow filtration – exploiting protein-protein complexes to enable size-based separations
  • Journal of Membrane Science
  • https://doi.org/10.1016/j.memsci.2020.118712
  • Demonstrates the use of a target protein binding molecule (TPBM) to purify a target protein (TP) of interest. The TPBM-TP complex can then be dissociated to yield the TP, and recycle the TPBM. The separation process utilizes tangential flow filtration.
• Purification and analysis of a protein cocktail capable of scavenging cell-free hemoglobin, heme and iron
  • Transfusion
  • https://doi.org/10.1111/trf.16393
  • Hemolysis releases toxic cell-free hemoglobin, heme, and iron, which overwhelm their natural scavenging mechanisms during acute or chronic hemolytic conditions. This study describes a novel strategy to purify a protein cocktail containing a comprehensive set of scavenger proteins for potential treatment of hemolysis byproducts.
• Apohemoglobin-haptoglobin complex alleviates iron toxicity in mice with β-thalassemia via scavenging cell-free hemoglobin and heme
  • Biomedicine & Pharmacotherapy
  • https://doi.org/10.1016/j.biopha.2022.113911
  • Demonstrates that the apohemoglobin-haptoglobin complex: 1) alleviates iron toxicity and regulates RBC turnover in β-thalassemic mice; 2) reduces hepatosplenomegaly in β-thalassemic mice; 3) increases RBC count, total hemoglobin concentration, and hematocrit over 6 weeks in β-thalassemic mice; and 4) decreases the concentration of iron and increases the concentration of available transferrin over 6 weeks in β-thalassemic mice.
• Engineering therapeutics to detoxify hemoglobin, heme and iron
  • Annual Review of Biomedical Engineering
  • https://doi.org/10.1146/annurev-bioeng-081622-031203
  • Comprehensive review of therapeutics were have developed in the lab to neutralize the toxicity of cell-free hemoglobin, heme and iron.