March 20, 2023
This article originally appeared in AABB News, a benefit of AABB membership. Join AABB today to read the rest of this month’s issue.
The history of transfusion medicine has been a steady march toward getting the safest blood possible to everyone who needs it. From the first human blood transfusion in 1795, through the discovery of ABO blood groups and beyond, to the storage of whole blood and its components, to screening for bloodborne pathogens and testing for bacterial contamination, pathogen reduction, to donor individual risk assessment, blood donation and blood transfusions are safer than a host of other medical procedures.
Yet, getting adequate supplies of whole blood and components to battlefields, massive-bleeding patients, routine surgical procedures and those with transfusion-dependent health conditions remains a limiting factor in providing the right blood to the right patient at the right time. Transfusion medicine is dependent upon altruistic blood donors, the time of year, geography, screening and testing time, storage limitations and the logistics of getting blood where it is needed.
The next advance — the development of a biosynthetic whole-blood surrogate product with a long shelf life that can be freeze-dried for easy portability, storage and reconstituted on site when needed — is closer than ever before.
DARPA Leads Development
In late January 2023, the Defense Advanced Research Projects Agency (DARPA) awarded $46 million to a consortium of more than a dozen universities and biotech companies to fund the development of a shelf-stable whole blood equivalent that can be used to resuscitate trauma patients when donated blood products are not available at the trauma site, during transport or during emergency care. The effort is part of DARPA’s Fieldable Solutions for Hemorrhage with bio-Artificial Resuscitation Products (FSHARP) program.1
The plan essentially adds to the use of freeze-dried plasma on battlefields by the United States military in recent years. In 2018, the U.S. Department of Defense was granted an emergency use authorization (EUA) by the U.S. Food and Drug Administration (FDA) allowing the use of freeze-dried plasma manufactured by the Centre de Transfusion Sanguine des Armées.2
The newly funded initiative is intended to support the development of bio-synthetic red blood cells (RBCs) and synthetic platelets that can be packaged with freeze-dried plasma and reconstituted in the field in the case of the military or in a number of heavy-bleeding scenarios among civilians. The combined product is intended to carry out the key therapeutic functions of whole blood in resuscitation — oxygen delivery, stopping bleeding and replacing volume. The components will be evaluated for efficacy and safety in trauma victims who have complex multiple injuries, including shock and traumatic brain injury, possibly reaching human studies in six to seven years. The project will also need to develop and test the scalability of producing blood surrogates.
The effort is headed by principal investigator Allan Doctor, MD, who is a professor of pediatrics and the director of the Center for Blood Oxygen Transport and Hemostasis (CBOTH) at the University of Maryland School of Medicine, which will manage the $46.4 million four-year research project.3
The bio-synthetic dried whole-blood surrogate product “will be designed for easy use in the field by medics at the point of injury, and will perform like a traditional blood transfusion to, for example, stabilize a patient’s blood pressure or facilitate blood clotting,” Doctor said in a press release.
The four-year project is intended to result in submissions to FDA for investigational new drug applications to study the dried whole blood surrogate. There is the potential for an additional year of funding for any additional studies needed for the investigational new drug (IND) application.
The Need
Beyond the obvious needs of the battlefield, there are an estimated 30,000 deaths per year from traumatic bleeding alone, all of which are preventable. Non-traumatic deaths due to life-threatening bleeding include postpartum hemorrhage and gastrointestinal bleeding.
“Of those 30,000 preventable deaths, 25,000 occur in the prehospital phase — immediately after injury and during transport,” said co-investigator Philip C. Spinella, MD, who is a professor in the departments of surgery and critical care medicine, co-director of the Trauma and Transfusion Medicine Research Center and associate medical director of the Center for Military Medicine Research at the University of Pittsburgh. “So, if you want to improve survival, you have to focus on where the problem is, and that's preventable bleeding in the prehospital period.”
The logistics of providing red blood cells (RBCs), plasma and platelets on helicopters or ambulances make it impossible to get these life-saving components to patients with life-threatening hemorrhage. This is why many EMS systems are implementing low titer group O whole blood (LTOWB). Whole blood, though, may not always be available for multiple reasons. Therefore, the availability of a whole blood surrogate comprised of a dried artificial red cell, dried plasma and dried platelet like products has the potential to save many lives where whole blood or individual components are not available, said Spinella.
Keeping blood cold is only one of the logistic burdens involved in getting it to trauma victims in the prehospital setting. This complicates implementing prehospital blood product availability. In addition, the supply of whole blood is entirely reliant on donors and ABO compatibility. If the number of donors drops — as it did during the COVID-19 pandemic, for example — so does the supply.
A whole blood equivalent with a shelf life of several months to years could be ready for immediate reconstitution in ambulances and helicopters, on the ground in mass shootings, and of course, on the battlefield. Such a product would provide oxygen delivery, clotting ability and volume replacement, serving as a bridge until patients could be treated in emergency facilities. It also would not require ABO compatibility which would improve both safety and availability.
Whole Blood
“In a way, we've deconstructed whole blood back into three main products — red cells, platelets and plasma,” Spinella said. “It’s simple and logistically much easier to give.” Essentially the components will be co-administered or given serially and will “mix in the patient.” The ultimate goal is to co-formulate all products in one bag with one reconstitution fluid.
“One of the main goals of this project is to determine whether there are any incompatibility issues between the individual products and how to address them if they occur,” he said.
No one is quite sure what to call the finished product. The terms artificial blood product, biosynthetic whole-blood product and whole blood equivalent have all been used.
Freeze-Dried Plasma
The history of freeze-dried plasma goes back to the 1930s, when lyophilization (freeze-drying) and successful infusion were reported.4
Dried plasma was used on a large-scale during WWII, where it was largely successful in stopping hemorrhage. It was used as bridge therapy until whole blood could be transfused. However, by war’s end it was determined that dried plasma could potentially transmit hepatitis.
By 1968, the U.S. military had abandoned the use of lyophilized plasma, though the French Military Blood Institute continued to produce dried plasma until 1984, when it was temporarily discontinued because of concerns about the potential risk of HIV transmission.
The advent of better screening methods for transfusion transmitted diseases and pathogen reduction have dramatically reduced the risk of transmitting infectious diseases with dried plasma. Today there are three commercially available dried plasmas, including the freeze-dried plasma granted an EUA from FDA for the U.S. military. None are available for civilian use in the U.S.
Red Blood Cells
The logistics and limitations of donor-derived RBCs have long highlighted the need for artificial red blood cells, particularly in the trauma setting.
“There have been a number of previous efforts to make artificial red cells, but they all failed. We’ve tried to address all those concerns and limitations — to address all of those design flaws with a system that is nanoparticle-based that can deliver oxygen and release oxygen in a trauma situation,” said Dipanjan Pan, PhD, who is a Dorothy Foehr Huck & J. Lloyd Huck Chair Professor in Nanomedicine and professor in the department of nuclear engineering and materials science and engineering at The Pennsylvania State University. He is also the director of Laboratory for Materials in Medicine (MatMed) at The Pennsylvania State University.
Pan is chief technical officer and co-founder of the Baltimore-based Kalocyte, Inc., which developed the ErythroMer technology that is the red blood cell component of the DARPA project. Spinella is the company’s chief medical officer and a co-founder; Doctor is the chief scientific officer and a co-founder.
ErythroMer is a synthetic artificial cell that uses nanoencapusulated human hemoglobin. It is bioengineered to mimic RBCs and is highly effective at O2 delivery.
“It’s really the designer lipids and the nanoparticle design that make it novel. Encapsulation of hemoglobin is a one part, but it's the release of oxygen where it’s needed most that is the key aspect,” Pan said. The particles are context responsive, meaning they release O2 in the body where the requirement is greatest and start releasing oxygen. In trauma, this is the site of bleeding.
“It’s not really a blood substitute in the true sense. It’s a bridge from resuscitation when time is of the essence. That's when this product is going to be used,” Pan said.
So far, the product appears to be shelf stable for several months. ErythroMer is not only shelf stable, but as a biosynthetic avoids the need for blood typing and cross-matching, which is time consuming in emergency situations where every second counts — without carrying the risk of transfusion reactions.
Research is ongoing about how quickly this technology could be scaled up for large production.
Platelets
Platelets are key to reduce and stop bleeding and they do so via several mechanisms. First, platelets “sense” certain proteins exposed at the site of the injury and attach to them (known as adhesion). Next, they turn on certain receptors and secrete chemical messengers (known as activation). Some of these receptors help platelets clump together, forming a platelet plug (known as aggregation). Platelets also amplify the coagulation output of thrombin generation, which results in crosslinked fibrin formation.
Transfusion of donor-derived platelets have a number of limitations, making it especially difficult to use them at the scene of trauma or during patient transport. The supply is limited by the number of donors, and storage poses several challenges. Platelets stored at room temperature lose some of their functions and hemostatic efficacy. Platelet storage at room temperature also carries the risk of bacterial contamination, limiting the shelf-life to at most seven days in the U.S.
The synthetic platelet surrogates under investigation as part of the DARPA project were invented by Anirban Sen Gupta, PhD, who is a professor in the department of biomedical engineering at Case Western Reserve University in Cleveland, Ohio. Currently, the SynthoPlate technology is being developed by Haima Therapeutics, co-founded by Sen Gupta. He serves as Haima’s chief technological officer and chair of their Scientific Advisory Board.
SynthoPlate is a synthetic nanoparticle system that mimics and amplifies platelet adhesion, activation, aggregation and prothrombotic functions.
“What we have developed is a modular platform technology inspired by platelets. Our research focus has been to form the clot necessary to stop bleeding. If we think of platelet-mediated mechanisms that are involved in hemostasis, they are like the gears of a machine,” Sen Gupta said. While all gears are important, certain gears must kick in first.
“The two very first gears are that platelets need to sense and attach to a bleeding site and then cluster with each other at that site to form a plug. Traditionally we called this primary hemostasis. The platelets must know where to act, and once they start sticking there, a certain spectrum of signaling events happens very quickly to allow incoming platelets to pile up,” Sen Gupta said.
The SynthoPlate design combined the first two gears — the sticking and the clustering functions — together on a single nanoparticle platform. This was achieved by “decorating” the surface of a liposomalparticle with a combination of peptides (called ligand motifs) allowing the particle to attach to collagen and von Willebrand factor (VWF) at the injury site.
“Once platelets start sticking together, they form a clot signal to start piling up; this piling property is driven by the binding of the blood protein fibrinogen that has binding motifs on both ends. By virtue of that, it actually creates this bridging between two platelets by binding to the platelet surface glycoprotein GP IIb/ IIIA,” said Sen Gupta.
To mimic this, a fibrinogen-derived peptide is added to the surface of the central SynthoPlate particle. This combination peptide decorated design is the lead hemostatic component in the DARPA endeavor to make the synthetic whole blood surrogate.
The procoagulant property of amplifying thrombin generation can be thought of as another gear of the platelet machinery. To achieve this, the surface of the core SynthoPlate particle can also be further modified with specific anionic lipids that mimic this procoagulant mechanism of platelets.
“The fourth thing that that biologic platelets do is their secretary function,” said Sen Gupta. “They carry a lot of different biomolecules within granules and cytoplasm, and when platelets get activated to form clots, these are secreted to accelerate or augment the clotting process — either modulating the clotting speed or modulating the final clot outcome [the amount of fibrin and fibrin stability].”
The core SynthoPlate particle is a liposomal system with an aqueous core, like a water balloon, that can be used to “carry cargo.” It’s a modular way of using the particle as a platform to carry other compounds that can provide additional benefits in forming hemostatic clots. For example, the researchers have been able to deliver the antifibrinolytic drug tranexamic acid, which is already used in staunching traumatic bleeding.
“That's why I say it's modular because you may or may not need all of the gears depending upon what outcomes you want,” Sen Gupta said.
The scalability of producing SynthoPlate is also promising. The production steps of liposomes are well-established in the pharmaceutical industry and can be easily adapted to advance from research laboratory to large-scale production. The SynthoPlate nanoparticles are also freeze-dryable and currently the freeze-dried product is stable for at least a year across various temperature conditions, without losing its bioactive functions. This enables significant logistical benefits compared to platelets.
The Future
So far, both Kalocyte and Haima Therapeutics have begun pre-INDA talks with FDA, but it is likely that human trials are at least six years away. The approval process is likely to be unique, given that artificial whole blood substitutes combine a number of technologies, making it unclear whether they are drugs, biologics, devices or some combination.
Down the road, there are a number of non-trauma conditions in which synthetic blood components could play a role. An RBC alternative could be immensely useful for patients with sickle cell disease (SCD), for example. Because these patients are chronically transfused, they can develop antibodies to more and more blood groups, making it increasingly difficult to find matching units. However, an RBC substitute like ErythroMer has no blood groups. It could become the go-to treatment for sickle cell crises presenting at the emergency department.
Synthetic platelet substitutes could be used in patients with platelet dysfunction or reduction, such as those with thrombocytopenia following chemotherapy.
REFERENCES
4550 Montgomery Avenue
Suite 700, North Tower
Bethesda, MD 20814
301.907.6977
