Stuart R. Gallant, MD, PhD

In the first part of this two-part post, we looked at the biochemistry of clotting factor Factor VIII and the production of Factor VIII by plasma fractionation.  We have now reached the late 1970’s, and the era of recombinant DNA technology.  Our story continues…

Recombinant Factor VIII

Pharmaceutical companies had been thinking about which proteins made the most sense as potential products of recombinant DNA techniques.  Factor VIII had a number of features to recommend it:  1) as mentioned in the first part of this post, a wide range of Factor VIII blood levels provide therapeutic benefit, so dosing should be relatively easy, 2) a good deal was already known about how to treat the disease based on experience with fractionated blood factors, and 3) it was a sexy problem—it’s a large, labile, glycosylated protein—a company which could bring this product to market could really produce any protein.  On the other hand, as a business decision, it had some risks:  1) fractionated blood factors were relatively cheap—it was unclear how much of the market might shift to a safer but more expensive product and 2) the market was fairly small (with fewer than 20,000 US patients at the time), much smaller than the insulin market (with 10s of millions of Type 1 diabetics worldwide).

Then, one event tipped the balance in favor of the Factor VIII project.  In 1981, the CDC became concerned about the spread of HIV in the blood supply, and the sense of urgency to this project dramatically increased.  Several groups were working in parallel at different institutions and corporations to make recombinant Factor VIII a reality.  Here are some of the key players in this drama:

• Royal Free Hospital:  Led by Dr. Tuddenham, this group included Frances Rotblat and Don O’Brien.  They produced purified Factor VIII to allow Genentech to being the process of cloning the Factor VIII gene into BHK cells.  Because Factor VIII exists in such low levels in blood, they had to create a kind of cottage industry inside their lab.  Dr. Tuddenham recounts, “Altogether Don and Frances made and shipped over to Genentech 20 milligrams of FVIII, derived from 2000 L of plasma or about 10 000 blood donations.”  However, Dr. Tuddenham’s group was not alone in the purification of Factor VIII, and that fact becomes important in how the intellectual property was distributed eventually.
• Genentech:  The Genentech team had made a successful pitch to the Royal Free Hospital team to obtain purified Factor VIII.  With a supply of purified Factor VIII, over an 18-month period leading up to April 1984, they were able to transfect the Factor VIII gene into BHK cells.  The plasmid as depicted in the Genentech publication is shown below [1].  Subsequent characterization showed the cell line to have 150 copies of the Factor VIII gene per cell and to produce the correct 2332 amino acid mature protein with a molecular weight of 330 kDa, including glycosylation.

• Genetics Institute:  Genetics Institute was also in the race.  They had a somewhat different approach to transfection [2].  As seen in the figure below from R. J. Kaufman’s paper, the CHO cells were transfected with the Factor VIII gene initially.  Subsequently, they were transfected with the von Willebrand factor gene.  Expressing vWF stabilized Factor VIII, improving cell culture productivity.

Genentech’s cell line would go on to be the production cell line for Bayer’s Kogenate and Kogenate FS.  Genetics Institute’s cell line would be used to manufacture Baxter’s Recombinate.  These were marriages of convenience between startup pharmaceutical companies (Genentech and GI) hungry for cash and large pharmaceutical companies with decades of experience in plasma fractionation (Bayer and Baxter).

Purification

Production in mammalian cell culture allowed large quantities of Factor VIII to be produced free from the risk of viral contamination; however, purification and stabilization of Factor VIII were also important.  High-concentration, high-purity had only been produced at the Royal Free Hospital laboratory of the Tuddenham group and at a small number of other sites in the world.  As a result, the knowledge base on purification and formulation of FVIII was fragmentary and limited.  Let’s consider how Factor VIII was purified [3]:

On the left is Bayer’s original purification process.  It has three orthogonal purification steps (anion exchange, immunoaffinity, and size exclusion).  But, two of them are repeated (immunoaffinity and anion exchange)—the reason is that as Factor VIII is made more pure, preserving its activity was difficult because of its instability.  In the mid-1980s, one of the few known ways to stabilize Factor VIII was by addition of the protein albumin.  So, the first-generation Bayer process preserves Factor VIII activity by repeatedly adding back the very albumin that its purification process removes.  Nevertheless, the process worked, and Bayer launched Kogenate using the process on the left of the figure.

By the late 1990s, Bayer needed a new manufacturing process—one with larger scale to meet increased demand for its virus-safe Factor VIII.  Bayer’s second-generation process (Kogenate FS) has six orthogonal chemistries (anion exchange, immunoaffinity, metal chelate, gelatin affinty, cation exchange, and anion exchange) and none of these chemistries are run twice.  Also, the state of stabilization knowledge is much improved, so there is no need to add albumin as Factor VIII was purified.

Why six orthogonal chemistries?  As produced in cell-culture, Factor VIII is a large, labile, and somewhat heterogeneous protein.  As a result, it produces wide chromatography peaks which can hide impurities (copurification).  Six separate chromatographies were required to reach the desired product purity of Kogenate FS.  Bayer’s technical expertise in Factor VIII production would only reach its full realization with its third generation product (Kovaltry)—readers are referred to Garger and coworkers for more details about Kovaltry [4].

Intellectual Property

Without patents, the massive investments required to bring pharmaceutical medications to patients could not be raised because investors would have little hope of return.  So, patents are the life blood of pharmaceutical development.  Over the more than 3 decades of recombinant Factor VIII production, there have been a number of patent disputes.  Here are a few of the intellectual property highlights:

• Scripps Clinic & Research Foundation:  Tuddenham’s group from Royal Free Hospital provided the purified Factor VIII to Genentech that allowed Genentech’s effort to get started, but they did not hold the patent for high purity Factor VIII.  Theodore Zimmerman purified Factor VIII to a level above 2000 units per mg and patented not only the process, but high purity Factor VIII in general [5].  In order not to infringe, other manufacturers such as Bayer, had to license this patent.
• Licensed Recombinant Products:  Unlike most pharmaceuticals in which a single branded drug exists for the life of the primary patents, from the very beginning, there were multiple recombinant Factor VIII’s on the market.  By the mid-1990s, Bayer had launched Kogenate (with the BHK cell line from Genentech) and Baxter had launched Recombinate (with the CHO cell line from Genetics Institute).  In addition, Bayer and Baxter supplied Kogenate and Recombinate to separate companies to be sold as Helixate and Bioclate, respectively.  These side deals resulted from the messy intellectual property landscape of Factor VIII.
• Entry of Competitors:  Over time competition entered the FVIII market and existing manufacturers have moved to improve their product offerings [4].  One category is B-domain-deleted Factor VIII [6] which removes a section of the native Factor VIII structure not critical for activity.

Some lessons from the patent disputes over Factor VIII are:  1) sometimes it is impossible to prevent entry of rivals—in those cases, making a deal to allow your product to enter the market is a good strategy; Bayer has always been willing to make such deals, but 2) anticipation of potential future competition is also critically important.  One wonders if anticipation of the B-domain deleted products could have allowed a patent strategy to more adequately defend against their entry.  In the pharmaceutical business, it is vital to guard against rivals who tell the patent office that they are making a different molecule but tell potential customers that their product works just the same.

Market

Here is a table which lists the leading Hemophilia A medications and their shares of the $7.2B world-wide Factor VIII market, as well as the modifications made to non-full length Factor VIII products and the product half-lives:

Generation		Modifications	Half Life	Percent
3rd Gen	Advate		8-12 hr	15%
	Adynovate	PEG	14-20 hr	7%
	Afstyla	Single Chain	14 hr	8%
	Eloctate	B-domain Deleted, Fc Fusion	19 hr	9%
	Esperoct	B-domain Truncated, PEG	19 hr	1%
	Hemlibra	Biospecific Antibody	4 wks	45%
	Jivi	B-domain Truncated, PEG	19 hr	*
	Kovaltry		8-12 hr	13%
	NovoEight	B-domain Truncated	12 hr	***
	Nuwiq	B-domain Deleted	17 hr	0%
	Xyntha	B-domain Deleted	12 hr	1%
2nd Gen	Kogenate FS		8-12 hr	*
	ReFacto	B-domain Deleted	11 hr	**
1st Gen	Recombinate		8-12 hr	2%

* Bayer products taken together (Jivi, Kovaltry, and Kogenate FS) appear on Kovaltry line.

** Pfizer products taken together (Refacto, Xyntha) appear on Xyntha line.

*** Novo Nordisk products taken together (Esperoct, NovoEight) appear on Esperoct line.

Some insights from the table include:

• Based on the incentives of such a large and growing market, companies have continued to innovate offering better and better products.  We discuss two generations of Bayer Factor VIII treatments above (Kogenate and Kogenate FS).  Bayer has continued to innovate offering a third-generation full length Factor VIII (Kovaltry) which is arguably of higher quality than Kogenate FS [4].  Bayer also offers Jivi which is a truncated Factor VIII with a covalently attached polyethylene glycol molecule to enhance circulation time.
• About 30% of patients affected by severe hemophilia develop antibodies to Factor VIII (inhibitors).  Hemlibra was introduced as a treatment for patients with inhibitors because its participation in the clotting cascade uses a bispecific antibody.  Hemlibra can be given to patients with inhibitory antibodies; however, even patients without inhibitors have been transitioning to Hemlibra because of the extended circulatory time which greatly reduces the frequency of administration.  Hemlibra has a large and growing market share of the Factor VIII and related products market.

In addition, to the products listed in the table, there is a raft of other new products in the development pipeline:

• Novel Molecules:  Sanofi’s BIVV001 fuses Factor VIII with von Willebrand factor and the XTEN polypeptide to created a longer lived form of Factor VIII (38 hr half-life).  Pfizer’s marstacimab treats Hemophilia A and B by blocking TFPI (tissue factor pathway inhibitor).  And, Novo Nordisk’s Concizumab Factor Xa preventing TFPI from inhibiting Xa.
• Fitusiran is an siRNA treatment for Hemophilia A and B from Alnylam to be marketed by Sanofi.  Fitusiran targets antithrombin in the liver, binding messenger RNA of antithrombin and silencing the antithrombin gene.
• Biomarin (Valoctocogene Roxaparvovec), Sangamo Therapeutics (SB-525), and Spark Therapeutics (SPK-8011) are all trialing Factor VIII gene therapy using adeno-associated virus (AAV) vector.

Conclusions

We talked a little bit about lessons in the section on intellectual property.  Some further lessons can be drawn as well from the Factor VIII experience:

• Recall that the situation in the early 1970s was one in which the prevailing treatment (really the only treatment) for Hemophilia A was Factor VIII from fractionated plasma.  It had poor quality (with variable potency, susceptibility to inhibitors, and contaminating viruses).  There was need for new medications, but no incentive because the existing treatment was quite cheap.  New medications require incentives.
• Development of the Factor VIII market has created incentives in two ways:  1) Hemophilia A is much more well understood than it was in the mid-1970s.  As a result, a startup with an idea for a new treatment has medical studies, assays, and other information that reduce risk, making investment in a new drug more attractive.  2) With a well-developed market like Factor VIII, it is much easier to project the possible value of a new treatment.  So, paradoxically, a market which is already served by a number of high-quality medications continues to draw new innovation.
• As an orphan disease, Hemophilia A, can be a model for the development of other orphan drugs.  Of course, there are many case studies now for orphan indications, but Hemophilia A is unique because of the breadth of medications that have been developed and the time period over which Hemophilia A has been treated (4 decades of recombinants).
• Let’s let Dr. Tuddenham have the last word [5].  He has described his motivation for participating in Factor VIII research.  Hearing his words will help us remember how far we have come in the last 4 decades.  “As a senior house officer in Pathology at Royal Liverpool Infirmary in 1970, I had to thaw and draw up into large syringes a viscous yellow liquid called cryo-precipitate and then inject it into the veins of patients with hemophilia to stop them bleeding. A rule of thumb was to use about five bags for a joint bleed and hope for the best. I do not think we checked the dose or the response but I do remember one patient whose bleeding failed to stop. After several doses we checked for an inhibitor and sure enough Alan Smith, then as now the coagulation technician in the hematology laboratory, found one. This, of course, was bad news for the patient as we had no way of treating him after that, but it sparked my interest in the whole area of hemostasis.”

[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]  Kaufman, R.J. “Expression and Structure-Function Properties of Recombinant Factor VIII,” Transfusion Medicine Reviews, VI (1992) 235-246.

[3]  Lusher, J.M. and Scharrer I.  “Evolution of recombinant factor VIII safety: KOGENATE and Kogenate FS/Bayer,” Int J Hematol (2009) 90:446–454

[4]  Garger, S., et al.  “BAY 81-8973, a full-length recombinant factor VIII:  manufacturing processes and product characteristics,” Haemophilia (2017), 23, e67–e78.

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

[6]  Eriksson, The Manufacturing Process for B-Domain Deleted Recombinant Fact& VIII,” Seminars in Hematology, Vol 38, No 2, Suppl 4 (April), 2001: pp 24-31. 

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