Stuart R. Gallant, MD, PhD

Today’s post is a big story involving genetic engineering, pharmaceuticals, intellectual property, and economics.  You will recall from the first installment of Pharmaceutical Design Focus that Genentech was founded in 1976 and expressed its first commercial protein (insulin) in E. coli in 1979.  Genentech could have spent a few decades cranking out products from the E. coli system, and that alone would have made them legendary.  But instead, they transitioned from E. coli to one of the most difficult expression systems (mammalian cells), and they used it to express a gigantic, 330 kDa protein (Factor VIII) [1].  However, the story of Factor VIII, starts long before Genentech’s 1984 triumphant Nature publication announcing expression of Factor VIII, and it continues several decades afterward.

It’s such a long and complex tale, that this will be a two-part post.  Here we go…

Biochemistry of Factor VIII and Hemophilia

It’s easy to forget that our understanding of Factor VIII is relatively new science.  For comparison, insulin had been crystalized in the 1920s, so scientists had decades of experience working with insulin before Genentech attempted to express it with E. coli.  Not so with Factor VIII.  In the mid-1970s, scientists were still trying to figure out of what precisely Factor VIII was and where it was produced in the body.  So, the ultimately successful attempts to treat Hemophilia A with a recombinant protein contained a lot more risk than might have appeared on the surface.

Here’s a quick introduction to Factor VIII.  Every day all humans have tiny leaks in our blood circulation—ones that we aren’t aware of—as well as bigger leaks (cuts, bruises, and other injuries).  The proteins of the clotting cascade stop those leaks to allow our bodies to have enough time to heal.  Without the clotting cascade, a bump or a bruise could become a huge hematoma and might even be fatal.  (Interestingly, all vertebrates have a version of the clotting cascade to allow them to survive injuries, and the clotting cascade has become progressively more complex with evolution.  Factor VIII evolved to become part of the vertebrate clotting cascade after lampreys came into existence (because lampreys do not have Factor VIII) but before sharks evolved (because sharks do have Factor VIII in their clotting cascade [2].)

Factor VIII participates in the clotting cascade in the following way (see illustration above for comparison with the text below):

1. Factor VIII, a glycoprotein of 2332 amino acids, starts out in the liver where it is produced.
2. Once it is released into the blood, it is bound by von Willebrand factor (vWF) which protects it against degradation while it circulates.
3. When bleeding occurs, thrombin, a serine protease, activates Factor VIII to Factor VIIIa.
4. Factor VIIIa goes on to support the activity of another blood factor—Factor IX.  And so on, until a fibrin clot is formed.

When a family possesses a Factor VIII gene which does not produce active Factor VIII (due to a deleterious mutation), members of that family can be affected in two ways:  male children may have difficulty producing clots and maintaining hemostasis (the free flow of blood and the resolution of bleeding events), and female children may carry the erroneous gene passing it on to their own children.  Generally, girls and women do not have trouble with clotting because they have two copies of the Factor VIII gene which back each other up, in contrast to boys and men who have only one copy.

Clearly, this is a complex mechanism designed by evolution to ensure hemostasis.  This biochemistry has several effects on the use of Factor VIII as a drug:

• Factor VIII is a large “labile” protein, easily inactivated by many physical conditions (for example, extremes of pH or temperature) and by the absence of formulating agents which stabilize the protein.  As a pharmaceutical product, Factor VIII is formulated as a dried powder with stabilizing agents present—capable of being stored for almost 3 years when refrigerated [3].
• Fortunately, the body tolerates a fairly broad range of levels of Factor VIII.  This is an ideal circumstance for a drug because it isn’t necessary to check blood levels or tightly control the amount of Factor VIII available at any time.

This has led to two types of treatment for deficiency.  Early on, Factor VIII was given when a patient had a crisis—a large bleed that resulted in discomfort to the patient and might threaten the patient’s life.  Over time, it became clear that maintenance dosing was important to treat small and often asymptomatic bleeding—this prevents damage to the patient’s joints, for example.  Patients typically receive an intravenous dose three times per week to ensure hemostasis, as the half-life of Factor VIII is about 12 hours.

Fractionated Factor VIII

Although humans have known that bleeding diseases occur since ancient times, we don’t want to start our story with the Bible and the Ancient Egyptians.  A good place to start is World War II.

World War II caused massive need for all types of medical care, particularly trauma care.  At that time, and even today, blood was provided as a treatment via transfusion for serious injuries, such as war wounds.  But the question occurred, how can the benefit of donated blood be maximized?

Edwin Cohn developed a method of splitting the components of blood to treat different illnesses and injuries [4].  As seen in the figure above, during fractionation, the pH is gradually reduced as ethanol is added to the plasma, and “fractions” of the proteins present in the plasma fall out of solution as precipitates.  Cohn fractionation created multiple individual medications which could be used help many patients.  Albumin could be used to restore circulatory volume following traumatic injury and blood loss without requiring blood type matching.  Immunoglobulins could be used to treat viral illnesses, such as hepatitis (“battlefield jaundice”), which were easily passed between soldiers on the battlefield.

Using “Cohn Fractionation,” it was possible to create a Factor VIII rich solution (“Fraction I”) which could be administered during bleeding crises of hemophilia suffers.  Yet, what was called “antihaemophilic factor” (i.e., Factor VIII) was known to be a temperamental, even fragile, entity.  In the face of the important, but limited success represented by Cohn fractionation, work continued to improve the manufacture and stabilization of Factor VIII to ensure a ready, reliable supply [5].

In the 1970s, work on plasma fractionation, and particularly on plasma fractionation related to Factor VIII, advanced along two parallel but distinct paths:

1. Blood product safety:  Donated blood can carry a host of infectious diseases (hepatitis A, B, and C, as well as HIV being some prominent examples).  In the 1970s, blood product suppliers wanted to increase safety, but there was limited funding for this project.  Fractionated products were sold as commodities with very low net margins.  As a result, the investment in improved manufacturing processes did not attract capital—until the arrival of HIV in the early 1980s. (Caveat:  The story of Factor VIII is inextricably tied with the story of HIV.  Within the confines of this post, I can’t do that story justice.  Suffice to say that many patients who received unsafe blood products prior to their replacement with virus-safe recombinant products contracted HIV.  Readers interested in learning more about this topic may want to read Pemberton’s The Bleeding Disease or access the extensive online oral history collection at Harvard [6, 7].)
2. Factor VIII purity:  It wasn’t very well understood what precisely Factor VIII was and where it was produced in the body.  Scientists wanted to isolate Factor VIII and put it under their figurative microscope to see what made it tick.

Our story will focus on the second of these two paths.  Medical researchers had been working diligently and creatively on the problem of Factor VIII isolation and purification which was critically important to the advent of recombinant Factor VIII, as we are about to see.  One of the central figures is Edward G. D. Tuddenham, a hematologist from the Haemophilia Centre at the Royal Free Hospital in London.  I knew some of the story of the development of Factor VIII into a pharmaceutical, but his personal recollection, “In search of the eighth factor: a personal reminiscence,” allowed me to hear the story told with all the human emotion that this story really deserves [8].  The next chapter of this story coincides with the advent of recombinant DNA technology.  We will pick up there in the second part of this post.

[1]  Wood, W.I., et al.  “Expression of active human factor VIII from recombinant DNA clones,” Nature, 1984 Nov 22-28;312(5992):330-7. doi: 10.1038/312330a0.

[2]  Doolittle, R.F.  “Step-by-Step Evolution of Vertebrate Blood Coagulation,” Cold Spring Harb Symp Quant Biol 2009 74: 35-40.

[3]  Bayer.  “Kogenate FS Package Insert,” www.fda.gov/media/70484/download.

[4]  Ness, P. M. and Pennington R. M.  “Plasma Fractionation in the United States,” JAMA 230, 247-250 (1974).

[5] Pool, J.G., et al.  “High-potency Antihaemophilic Factor Concentrates pepared from Cryoglobulin Precipitate,” Nature 203, 312 (1964).

[6]  Pemberton, S.  The Bleeding Disease: Hemophilia and the Unintended Consequences of Medical Progress, Johns Hopkins University Press (2011).

[7]  Boston Hemophilia Center. Oral History Project.  hollisarchives.lib.harvard.edu/repositories/14/resources/4605.

[8]  Tuddenham, E. G. D.  “In search of the eighth factor: a personal reminiscence,” Journal of Thrombosis and Haemostasis, 1: 403–409.

Disclaimer:  PharmaTopo^TM provides commentary on topics related to drugs.  The content on this website does not constitute technical, medical, legal, or financial advice.  Consult an appropriately skilled professional, such as an engineer, doctor, lawyer, or investment counselor, prior to undertaking any action related to the topics discussed on PharmaTopo.com.