Two things are infinite: the universe and human stupidity; and I'm not sure about the universe. (Albert Einstein)

A story of EPO: based on true events. More particularly, the love of money is the root of infinite human stupidity.

Every young scientist should know the story of EPO. The research surrounding EPO is an unprecedented learning opportunity for human biotherapeutic research which for ethical reasons will probably never be repeated exactly. And this research was the primary contributing factor to the development of the idea of biosimilars.

What really happened? As with any historical event, no one really knows. Why did the Roman Empire fall? Did Christianity push it down (a review of Gibbon’s book Decline and Fall of the Roman Empire claimed blaming Christianity was a justifiable summary), or did the internal corruption of moving from a republic to an empire doom Rome to failure? Did Rome fall because of lead pipes? Maybe the emperors who stayed at home were damaged by lead poisoning and went insane, leaving only the warmongering emperors who drank pristine water out in the battle fields sane enough to govern. Or was it a combination of a myriad of factors too complex for the human mind? No one really knows.

Scientific and technical history has an added complexity. The Copenhagen interpretation of quantum theory led to multi-year discussions between Albert Einstein, Werner Heisenberg, Richard Feynman, and other geniuses and near-geniuses. The greatest geniuses demonstrate Richard Feynman’s adage: “If we can’t explain it to the Freshmen, then we don’t really understand it.” I have read books by Einstein, Heisenberg, and Feynman that do a good job of explaining some parts of quantum mechanics to average college students. At St. Johns College of Sante Fe, New Mexico, and Annapolis, Maryland, and for about half of the lifetime of Magdalen College of Bedford and Warner, New Hampshire, students read these books. But even translated by geniuses who really understood the concepts, not every student was able to understood quantum mechanics and why quantum mechanics is a revolution of human thought. The books Einstein and others wrote contain only part of the conversation that led to the acceptance of quantum mechanics. In fact, Richard Feynman wrote a book for a friend (literature professor, not a freshman but a PhD) who was not a physicist to try to explain quantum mechanics to her. The book was entitled QED: The Strange Theory of Light and Matter. In the book, Feynman starts the introduction by saying:

What I am going to tell you about is what we teach our physics students in the third or fourth year of graduate school... It is my task to convince you not to turn away because you don't understand it. You see my physics students don't understand it... That is because I don't understand it. Nobody does.

How could Feynman write a book about something he doesn’t understand? Because he doesn’t understand the whole thing, but he understands some things. To pass on what he does understand Feynman teaches classes to graduate students and he writes books. Understanding something is practically helpful, but more importantly, glimpses of the truth are beautiful and enrich every soul that listens. Speaking of Helen Keller, Edward Everett Hale famously said:

I am one, but only one. I cannot do everything, but I can do something. Because I cannot do everything, I won’t refuse to do what I can.

It is reasonable to think that a biotherapeutic drug, like EPO, would be almost as difficult to understand as quantum mechanics. And as a patented drug for many years, the inventors were not completely forthcoming in providing details about how they invented EPO and even less forthcoming about the issues with EPO that led to severe adverse events for some patients who received the drug. Scientist wrote for years conflicting opinions about what really happened, and I have read some but not all of these scientific papers. I cannot explain everything about EPO, but I can tell you the part of the story that I heard and understood. I am not a genius, but I do have a PhD in biophysics and I am likely able to understand more of the details of this story than the average PhD, college student, or high school student.?

This story I will tell is not about the deep and difficult parts of immunology and biotherapeutic drug research and development, but it is a story about another, perhaps infinite, thing. Albert Einstein said:

Two things are infinite: the universe and human stupidity; and I'm not sure about the universe.

The story I want to tell is about human stupidity. I believe just about anyone can understand this story. And as in any history, my sources are limited, some sources may have passed on exaggerations, and there are conflicting opinions as to causes and as to some details. To make the story readable, I am giving you my opinion about the most probable explanation for a number of the causes involved in the events of the story of EPO. At times, I also relate written or spoken opinions from others that I think are particularly insightful. Scientific authors often avoid opinion altogether and often feel compelled to give both sides of the argument for every point in the story that may be controversial. But this spoils the flow of the story and makes the larger ordering principles more difficult to understand. And like the fall of the Roman Empire, it may be impossible to know for sure the number and the relative impact of every event or motive.

What is EPO?

EPO is the short name for a protein called Erythropoietin (sometimes spelled without the letter “i” (Erythropoetin). In the Uniprot database, the gene for Erythropoietin is labeled “EPO.” A search for “Erythropoetin” at UNIPROT returns 3005 results including short protein names such as EPO_DANRE, EPO_RAT, EPO_HUMAN, EPO_NANGA, EPO_BOVIN, EPO_EPICO, EPO_PIG, EPO_XELNA, EPO_MACMU, EPO_HORSE, EPO_TETNG, and G3W289_SARHA along with around 800 homologous or similar proteins and around 2000 related proteins not coming from the EPO gene. While some suffixes are self explanatory, like RAT and HUMAN, it is helpful to know that DANRE is the Zebrafish, NANGA is the Northern Israeli blind subterranean mole rat, XELNA is the African clawed frog, EPICO is the Orange-spotted grouper, TETNG is the spotted green pufferfish, and SARHA is the Tasmanian Devil. In other words, lots of creatures have the EPO gene and protein, even Tas.

EPO is a small protein (around 34,000 Daltons in molecular weight), a globular single domain in shape, heavily glycosylated, and has about a 5-hour half-life in the blood stream. In contrast, Humira, an antibody protein, is a large protein (around 150,000 Daltons in molecular weight), complex tri-domain in shape, lightly glycosylated, and has a 2-week half-life in the blood stream. Insulin is a very small protein (around 6,000 Daltons in molecular weight), a compact single domain in shape, and has a half-life of 3 to 10 minutes in the blood stream.

Horomones are molecules that send a message. Insulin is labeled a peptide hormone. A peptide often refers to a part or fragment of a protein. Insulin sends the message that excess sugar is available in the blood, and that cells should absorb that sugar immediately. The function of EPO is to deliver the message to red blood cell progenitor cells to turn into red blood cells. EPO is labeled a protein hormone. EPO comes from the kidney or liver and sends the one signal to mature into red blood cells to the progenitor blood cells where they reside in red bone marrow. Antibodies, including Humira, are proteins, and they are never labeled with the name hormone. An antibody such as Humira is an evolutionary advanced protein with multiple functional groups that hangs around and participates in many functional pathways over a long time period.

Anemia is a disease state in humans where not enough oxygen is transported throughout the body. Anemia may be caused by an excess loss of red blood cells or lack of the nutrient iron that is an essential part of hemoglobin: the protein in red blood cells that transports oxygen. The production of red blood cells in a human (or fish or frog or Tasmanian Devil) body depends on EPO signaling. With no active EPO, a human will have “aplastic anemia.” The prefix “aplasic” means “without cells.” If there is no active EPO, there are no red blood cells.?

The pioneering biotech company, Amgen, was the first company to produce EPO as a drug. Patients with chronic kidney disease make less EPO (and therefore fewer red blood cells) because EPO is produced in the kidney. Also, chronic kidney disease patients often lose more red blood cells because they are on dialysis. Scientists at Amgen researched and developed the process to make EPO in a bioreactor factory. The first “indication” for EPO as a drug was for anemia caused by chronic kidney disease. The technical term, “indication,” is an important part of the story. Drug companies conduct clinical trials to prove the efficacy and safety of drugs for a certain indication. When Amgen conducted its trial for EPO, it enrolled patients with chronic kidney disease and anemia and proved that EPO doses improved their health with an acceptable safety risk. No drug is 100% safe. All drugs have side effects that are possibly harmful. When approved, the regulatory agency confirms that clinical trials have shown efficacy worth the risk of harm. Because there were no cancer patients in the original EPO clinical trial, Amgen cannot say that EPO helps cancer patients. But doctors can use drugs “off label” to help certain patients. It made perfect sense that EPO would be as good or better for cancer patients than it was for chronic kidney disease, because cancer patients tolerate more risk to get rid of the cancer. J&J expected to run clinical trials and produce EPO for more indications.

Soon after EPO was approved for chronic kidney disease, doctors began to notice that EPO could also be used to great positive advantage with cancer patients who had strong chemo therapy, radiation treatments, or bone marrow transplants. The chemo, the radiation, and the bone marrow transplant protocol all greatly reduced the number of progenitor red blood cells. EPO replacement treatment could help the body produce more red blood cells in a short time frame. Supplemental EPO would improve the health of the patient. Faster recovery to sufficient red blood cell count meant the chemo or radiation treatments could continue on a shorter time frame and increase the odds of curing the cancer before ruining the health of the patient.

The unexpected EPO disaster.

At the time, Amgen was a relatively small company with limited resources in Europe and also was too busy for quickly conducting new clinical trials for cancer indications. Amgen kept making EPO for chronic kidney disease in the US and licensed the rights to produce EPO to the larger company Johnson & Johnson. J&J was to make EPO in Europe and also sell EPO in the United States for indications other than chronic kidney disease.

Amgen gave J&J all the information needed to manufacture EPO in a bioreactor factory. J&J could have manufactured EPO exactly the same way that Amgen did. But exact manufacture practices did not seem like the best course of action for two main reasons. First, manufacturing science was in its infancy when Amgen designed EPO, so J&J reasonably expected to be able to improve the manufacturing process and do so at a lower cost by using their own new scientific discoveries. Second, the European Medicines Agency (EMEA) were concerned about the use of human serum albumin in the formulation of EPO, and requested that J&J find a safer alternative formulation. For these and potentially other reasons I am unaware of, J&J manufactured and formulated EPO differently from Amgen. Extensive tests showed that the active ingredient, EPO protein, as manufactured by J&J, was identical with EPO manufactured by Amgen within the power of the tests used: and the tests used were as advanced as available at the time.

Drugs have an active ingredient, and every other ingredient in the pill or in the liquid solution is called the formulation. EPO protein is the active ingredient. EPO’s formulation was a liquid solution. EPO’s formulation was not just water. It included ingredients to make the injection safer and more tolerable to blood chemistry and it also included ingredients to improve the stability and extend the storage life of the active ingredient.

EPO was known to be a very stable small protein. It was shown to be easily manufactured with the measurable properties of the active ingredient being identical to the innovator version. Small molecule drugs like aspirin had a long history of generic manufacture. For small molecules, drugs are shown by physical tests to be identical to innovator manufactured versions and a cheaper generic version of the drug may be interchanged for the original version manufactured by the innovator company. At the time Amgen out-licensed EPO, EPO was still under patent and there was very little experience in generic biotherapeutic drugs. Insulin was considered to be a good comparison for EPO. Insulin was purified and/or manufactured by a number of companies and in a number of different ways and was a very safe drug product regardless of manufacture site. The formulation of insulin did not seem to affect the safety or efficacy of insulin. In fact, extended-release insulin used a different formulation to slow how quickly the body absorbed the insulin and was a great advance in insulin therapy. J&J decided to go ahead and manufacture EPO their own way, in their own factories, with an improved formulation. J&J began selling EPO in Europe around 1998. By 2004, Amgen’s sales of EPO were 2.6 billion dollars per year while J&J’s sales were 3.6 billion dollars per year.

Beside the use in chronic kidney failure and then cancer, athletes also started using EPO. EPO injections could quickly replace months of high-altitude training and push maximum exertion levels 15 – 25% higher than placebo. Too many red blood cells has the possible side effect of hyper viscosity of blood when an EPO user is dehydrated. The hyperviscosity can lead to strokes and heart attacks which may be fatal. Eventually, EPO started to be used in horse racing also to increase maximum effort.

In February of 2002, a group of doctors in Paris, led by a specialist in anemia, published an article in the New England Journal of Medicine. The article identified 13 patients who had developed Pure Red-Cell Aplasia (PRCA) most probably due to treatment with recombinant Erythropoietin. During the publication process, 9 more patients with PRCA were identified but there was not sufficient time to include the follow up testing data for them. The title of the article was: Pure Red-Cell Aplasia and Antierythropoietin Antibodies in Patients Treated with Recombinant Erythropoietin.

The initial response by J&J was: EPO is safe; this is impossible! In the first 10 years of clinical use of EPO, 1988 - 1998, 3 cases of PRCA caused by anti-EPO antibodies were reported. After 10 years of experience, how could 22 patients contract PRCA in just 3 years? If a person develops blocking anti-EPO antibodies, for the rest of their short and miserable lives, they will not make any natural red blood cells on their own. They will survive for a while with transfusions, and their lives may be prolonged with strong immune-suppressive drugs: but immune-suppression has risks of developing serious infections and other side effects.

Cases of PRCA kept appearing at a high rate, but only in Europe and only with the EPO manufactured and distributed by J&J. EPO manufactured by Amgen seemed safer and the first reported case of PRCA linked to Amgen EPO was reported after 2004. After 2004, the anemia specialist group reported a case of PRCA in a patient who had never taken EPO of any kind. EPO is not the sole cause of PRCA, but something about the J&J product caused it to have a much higher incidence of anti-drug antibodies causing PRCA.

In the literature and in science talk at conferences, conflicting reports flew around. J&J lawyers tried to blame Amgen for everything. You (Amgen) designed this dangerous drug: we just out licensed it. J&J sold more units of drug than Amgen had: maybe it was a simple statistical aberration that made it look safe in the US. But more and more cases came in so that blaming the active ingredient was untenable. One scientist found that in Europe there was a non-random correlation between cases of PRCA and the average temperature of the country where the unfortunate patients lived. Amgen said nothing in print since their out-licensing agreement forbid them to comment. My friends who had worked at Amgen said nothing. But they could not be sued for harrumphs and growling whenever J&J and EPO were mentioned.

Since the active ingredient, the EPO protein, could not be blamed everyone had to admit that it was the formulation change that was the likeliest cause of excess PRCA cases. J&J published a number of changes to the formulation and the delivery; implying that the major cause was the silicon oil coating in the pre-filled syringe stoppers. An excellent scientist said silicon in the syringes did not fully explain the correlation to country temperature and there must be more reasons. J&J started to use silicon free syringes. They also hired a fleet of refrigerator trucks and drivers. Drivers were trained to accompany shipments of EPO into the hospitals and could not sign that the shipment was delivered until they saw, with their own eyes, the box of EPO in a hospital refrigerator. They increased release testing and made some other ingredient tweaks to the formulation. The cases of PRCA fell drastically. Because more and more people were using EPO and relatively few people used EPO in the early years, statistics are inconclusive as to whether or not the J&J manufactured EPO was ever as safe as the Amgen manufactured EPO. J&J’s success proved that it was formulation differences and not differences in the active ingredient that caused a change in the safety of a biotherapeutic.

Why did Amgen take the expensive and unusual tactic to add human serum albumin to a protein drug formulation? It’s a trade secret. My guess is that Amgen knew the human serum albumin had a protective role in the formulation. Some scientists suggested that it was particle formation that caused excess immunogenicity. And particles are known to form more quickly at higher temperatures. Serum albumin may slow down particle formation.?

We will never know exactly why J&J EPO was not as safe as Amgen EPO. And it would be immoral to test it. You cannot morally reproduce J&J EPO as it was originally produced, inject it into 30,000 people and observe that it caused a few hundred cases of PRCA while the control group of Amgen EPO produced only 5-10 cases of PRCA. Morally, you have to inject the best and safest drug available into large numbers of patients. Clinical trials are moral when you don’t know and you try to find the best and safest variant and dose. Later, when you know something is not safe, you can’t study exactly why it harms.

Years later, various companies in China, India, and elsewhere also started making EPO. Amgen could not by contract test J&J EPO and publish their criticisms. That is how licensing agreements work. But these new companies had no such legal protection. Amgen tested various sources of these products, did some immunogenicity testing in mice, and reported wide variations in formulation and particle content. A group not at Amgen did extensive immunogenicity testing of blood samples of Amgen, J&J, and “knock-off manufactured” EPO patients. They made the unusual claim that all the blood samples had anti-EPO antibodies to a high extent, but that samples with formulations that used human serum albumin had non-blocking antibodies only while other products had blocking antibodies with a prevalence correlated to the average particle content. I don’t know if I believe this report since it was from a group in China that may have had connections to one of the unauthorized EPO manufacturers.

With billions of dollars in sales involved and hundreds of victimized patients suing various companies, and rich and competitive athletes (and horses) using EPO and worried that they might be at risk for PRCA, the whole mess had a huge impact on regulatory agencies.

I attended a “Biogenerics” conference held by Northeastern University in Boston. I heard with my own ears the number 2 manager at the EMEA say something like this (I can’t find the transcript published, so I have to go by memory): “You (Northeastern University) have misnamed this conference. As long as I work at the EMEA, there will never be a biogeneric drug. Never. Ever. There will only be biosimilars. EPO shows us that no amount of testing can show that one biotherapeutic is exactly as safe as another. EPO showed us that the drug product is not just the active ingredient: it is everything in the vial: formulation and container elements.” (Silicon oil is not an “ingredient” on the label, it is a lubricant on the delivery container.) It made me wonder if this fellow might have been involved in the decision to recommend to J&J that they replace human serum albumin with polysorbate in the EPO formulation for “safety” reasons.

Biosimilars go through abbreviated phase 1 clinical trials, abbreviated phase 2 clinical trials, and nearly full phase 3 clinical trials. Factors like immunogenicity in monkeys, level of particles, and other critical quality attributes are carefully checked. But safety and efficacy has to be proved in the clinic. No one can say, “Humira is safe, so I can just make Humira when the patent expires and start to sell it without a clinical trial.” Humira biosimilars are now on the market and their trials all proved that for Humira, formulation changes cause no measurable difference in safety and immunogenicity. This is actually unfortunate because Humira is around 45% immunogenic in patients and it would have been good for patients if a safer formulation was possible.

The experience with EPO also led to the growth of two or three new industrial endeavors. The company that made syringes worked hard on making silicon free syringes. Checking for aggregated proteins became a well-supported field of study. The detection of particles was identified as an analytical gap in the biotherapeutic industry and new instruments were invented, developed, and marketed to fill in this gap.

The rest of the story.

This essay repeats the claim by Einstein that human stupidity is infinite. The rest of the story of EPO supports Einstein’s conjecture.

The stupidity of changing the formulation of a relatively safe drug can perhaps be forgiven the first time it happens. J&J thought they were smarter than Amgen and their scientists thought they could manufacture a more pure and higher quality protein. The EMEA was worried about using a natural human product in a formulation and had safety concerns based on theory supported by experience. Proteins purified from human blood for hemophiliacs did have a small percent of impurities that caused some hemophiliacs to contract AIDS and die. There were now tests for AIDS, but what about the next yet-to-be-discovered blood disease? Proteins purified from human blood probably will contain contamination before the blood test is developed.

But many years later, a company called Takeda was developing an EPO replacement drug called Peginesatide. Peginesatide was a synthetic peptide. It was smaller than EPO, a bit smaller than insulin in fact, but Peginesatide also stimulated red blood cell production similarly to EPO. A summary journal article tells most of the story about Peginesatide. (Hermanson T, Bennett CL, Macdougall IC. Peginesatide for the treatment of anemia due to chronic kidney disease - an unfulfilled promise. Expert Opin Drug Saf. 2016 Oct;15(10):1421-6.) The abstract of this article relates the basic outline of facts:

The introduction of recombinant human erythropoietin revolutionized the management of anemia in patients with chronic kidney disease (CKD). In order to circumvent costly recombinant DNA technology, synthetic chemistry techniques were used to manufacture peginesatide, a synthetic peptide that bore no resemblance to previous erythropoiesis-stimulating agents (ESAs), and yet was capable of stimulating erythropoiesis. Compared with other ESAs, peginesatide was deemed to have advantages related to immunogenicity, administration schedule, and cost. Marketing approval was restricted to CKD patients on dialysis because cardiovascular events were more common with peginesatide than with darbepoetin in non-dialysis CKD patients. Unfortunately, unexplained serious adverse drug reactions (sADR) led to quick withdrawal of peginesatide from the market.

From the start, there was a heavy emphasis on cost of goods sold in the Peginesatide story. Because it was so small, Peginesatide was made in a machine, not grown in a living cell. Machine production was less expensive. Also, the manufacturers refused to purchase the new and improved particle detection instruments but used an old method of detecting particles that had been proven to be deficient in detecting protein particles.

But the infinity of stupidity is proven by this last part of the story. Peginesatide went through all three clinical trial phases planned to be packaged in a single use sterile vial. After all the trials, the company decided to put Peginesatide into a multi-use vial. Because the first use of the multi-use vial breaks sterility, preservatives were added to the formulation buffer for “safety” reasons. All these changes were made with very little testing to see if the new formulation with preservatives worked as well as the original formulation.

Excuse the ranting and mild cursing, but I have to exclaim: “What the Hell?” If Peginesatide binds the EPO receptor protein, part of it has to be the same shape as EPO: that is how binding works! If Peginesatide causes an immune response, anti-Peginesatide antibodies should also bind to the active binding site in EPO. If you block EPO, your patient gets PCRA and for the rest of their short and miserable lives they will not make a natural red blood cell on their own. What the Hell are you doing, changing the formulation after all the trials? This is an EPO mimetic. How could you not be aware of how bad PCRA is? Did you forget that when J&J changed the formulation of EPO, hundreds of patients needlessly died? At MedImmune we froze the formulation before phase I and never changed it without a full clinical retest. And MedImmune was making antibodies where immunogenicity effects are never as severe as PCRA. And why, why, why did you change the formulation? A single use vial might cost say $10, and multiuse vial a little more, say $20. How much money per dose are you saving with the multi-use vial? Even if the multi-use vial has 10 doses, you save 8 dollars per dose? And that doesn’t count the expense of the extra formulation ingredients, the extra testing for those preservatives, and the possibility that the multi-use vial will never be fully used: no one takes the last dose out of the vial and the vial may expire before all 10 doses are needed. You risk death for your patients to save 8 dollars a dose! And the preservatives themselves are an extra risk to the patients even if they had not affected the immunogenicity. For safety reasons, the FDA should ban multi-use containers for any vaccine or drug. The drug was withdrawn because, surprise, patients started to get immune responses not seen in the clinic. So even from a greedy company perspective, even not caring about the patients, all the money for the research and development and initial production of Peginesatide was lost. Hundreds of thousands of dollars lost for a very minor savings that really may not have saved any money even if it had no “unexpected” adverse repercussions.

After the withdrawal, samples of Peginesatide were tested with better particle measuring instruments, and samples with the new formulation containing preservatives were very high in particles compared to the samples in the original formulation.

As a side note, gene therapy companies are complaining about the high cost of analytical ultracentrifuge (AUC) instruments. But AUC instruments are critical in detecting gene therapy vector aggregates and particles. Nothing else works quite as well. An AUC costs $500,000. But a single course of treatment of one of the new gene therapies cost $3,000,000 for one patient. 3 million for one patient. But the poor company can’t afford half a million to buy an instrument that might save a patient’s life by detecting immunogenic particles?

Individually, the scientists at Takeda, even the ones who worked on Peginesatide, are probably smart people: some with PhD’s. It is the company culture that causes them to do really stupid things. Money first, not science. Managers who are clueless about science. Workers who know better but fear for their jobs if they ever criticize the company. And the moral corruption caused by the company constantly pressuring middle managers to lie, to meet deadlines by cutting corners, to never even hint in speech, email, or writing that the current drug product might not be safe.

“For the love of money is the root of all evil.” (I Timothy 6:10)

More particularly, the love of money is the root of infinite human stupidity.