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December 15, 2010: Blood Products Advisory Committee Meeting Transcript

FOOD AND DRUG ADMINISTRATION
CENTER FOR BIOLOGICS EVALUATION AND RESEARCH

BLOOD PRODUCTS ADVISORY COMMITTEE

December 15, 2010

The Hilton Washington DC North/Gaithersburg
620 Perry Parkway
Gaithersburg, Maryland

This transcript has not been edited or corrected, but appears as received from the commercial transcribing service.  Accordingly, the Food and Drug Administration  makes no representation as to its accuracy.

Proceedings by:
CASET Associates, Ltd.
Fairfax, Virginia  22030
703-266-8402

 

TABLE OF CONTENTS

 Page
Opening Remarks:  Blaine Hollinger                          1
Statement of Conflicts of Interest:  Dr. Emery              1
Committee Updates 
Update from the HHS Advisory Committee on Blood Safety    
and Summary of November 4-5, 2010 Meeting:  CDR Richard Henry
3
Summary of December 9-10, 2010 Workshop "Product Development
Program for Interventions in Patients with Severe Bleeding Due
to Trauma or Other Causes":  Jaro Vostal
10
FDA Guidance for Industry: Use of Serological Tests to   
Reduce the Risk of Transmission of Trypanosoma cruzi
Infection in Whole Blood and Blood Components Intended for
Transfusion:  Robert Duncan
26
Update on the Study to Further Define the Incidence of   
Trypanosoma cruzi Infection in the U.S. Blood Donor
Population
34
Topic III:  Review of the Research Programs in the Laboratories
of Hemostasis and Plasma Derivatives, Division of Hematology, OBRR
 
Overview of CBER Research:  Carolyn Wilson     45
Overview of OBRR Research:  C.D. Atreya  52
Overview of the Division of Hematology Research Program  
Basil Golding
57
Overview of the Laboratory of Hemostasis:  Basil Golding66
Overview of the Laboratory of Plasma Derivatives         
Dorothy Scott
84
Open Public Hearing111


PROCEEDINGS(8:10 a.m.)
Agenda Item:  Opening Remarks
DR. HOLLINGER:  Thank you all for being here to the second day of the Blood Products Advisory Committee meeting. 
(Remarks off the record regarding travel arrangements.)

Table of Contents

Agenda Item:  Statement of Conflicts of Interest
DR. EMERY:  Good morning.  This is the COI statement for December 15, 2010.
This brief announcement is in addition to the conflict of interest statement read at the beginning of the meeting on December 14, 2010 and will be part of the public record for the Blood Products Advisory Committee meeting on December 15, 2010. 
The committee will hear an overview of the research programs in the laboratories of hemostasis and plasma derivatives in the Division of Hematology, Office of Blood Research and Review.  This is a non-particular matter. In addition, the committee will hear updates on several topics.  There is no conflict of interest involved with the research overview and committee updates.
Dr. Kelso Bianco is serving as the industry representative, acting on behalf of all related industry, and is employed by America's Blood Centers in Washington, D.C.  Industry representatives are not special government employees and do not vote. 
This conflict of interest statement will be available for review at the registration table.  We would like to remind members and participants that if the discussions involve any other products or firms not already on the agenda for which an FDA participant has a personal or imputed financial interest, the participants need to exclude themselves from such involvement, and their exclusion will be noted for the record.  FDA encourages all other participants to advise the committee of any financial relationships that you may have with any firms, its products, and if known, its direct competitors.
Thank you.
DR. HOLLINGER:  Thank you, Brian.  We will start out with some committee updates.  The FDA is not seeking our advice on these updates.  We can ask them questions of clarification and so on, but no recommendations are being asked for.
We will ask the speakers again to watch their time, so we try to stay on time.  I guarantee you we will be through before 7:30 tonight.  No, we will be through a lot earlier than that.
Let's start.  The first one is going to be on the update from the HHS Advisory Committee on Blood Safety and Availability, and a summary of the November 4-5, 2010 meeting.  Commander Richard Henry will give this presentation.

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Agenda Item:  Update from the HHS Advisory Committee on Blood Safety and Summary of November 4-5, 2010 Meeting
CMDR. HENRY:  Good morning.  Richard Henry from the Secretary's Advisory Committee on Blood Safety and Availability.  Thanks for having me.  I will give you a quick update on our Advisory Committee meeting that took place last month on November 4-5.
The ACBSA charter provides that the Advisory Committee gives the Secretary and the Assistant Secretary advice on a range of broad issues.  They include the definition of public health parameters around safety and availability of the blood and blood product supplies, broad public health, ethical and legal issues related to the transfusion and transplantation safety throughout the country, and implications for safety and availability of various economic factors affecting product cost and supply.
Our topic for November 4-5 was to meet and discuss the strategic priority of HHS, including the new 2010 Affordable Care Act.  The purpose of the meeting was to review and comment on the current status of transfusion and transplantation safety, to review and comment on progress made in transfusion and transplantation safety, to review and comment on previous ACBSA recommendations regarding elements of the strategic plan, and to recommend current priorities and how they align with the Secretary's strategic initiatives and key interagency collaborations.
The ACBSA made several recommendations, 14 in all, surrounding blood, organs, tissues and some cross-cutting issues.  For blood, the ACBSA recommended under patient safety to promote and establish high value and effective evidence based patient blood management; under product safety, to fund safety technology development and implementation; to establish evidence based donor history questionnaires for blood, there is some work being done for organs and tissues as well, and to fully reimburse cost of safety measures. 
For biovigilance, to fully establish biovigilance for blood donors and recipients to improve outcomes. 
Under donor wellness, to promote and protect donor health.
For risk management, to improve risk based decision making and communication.
For cells and tissues, the ACBSA recommended under product safety to determine the elements that contribute to product safety, specifically to assess the intrinsic quality of cell and tissue products, to determine the optimal processing and testing of cells and tissues to mitigate disease transmission, and to  establish an evidence based donor history questionnaire.
Under product safety and risk management, the ACBSA recommended to improve risk based decision making and communication for optimal tissue utilization. 
For biovigilance, the committee recommended to establish Biomarkers Consortium for cell and tissue recipients to improve communication, traceability and outcomes.
For organs, the ACBSA recommended under product safety to support the development of technologies for appropriate rapid screening of transmissible markers to quality organ availability, and to establish an evidence based donor history questionnaire.
Under patient safety and risk management, the ACBSA recommended to improve risk based decision making and communication for optimal patient management.  For biovigilance, the committee recommended to establish biovigilance for organ recipients to improve communication, traceability and outcomes.
The committee recommended several cross-cutting issues.  The first, to improve flexible funding mechanisms for emerging public health priorities, and to increase public awareness of issues affecting the safety and availability of blood, cells, tissues and organs, and last, to support international collaborations that additionally leverage international experience.
Any questions, comments, concerns?
DR. HOLLINGER:  Just some questions.  These are all laudable goals in there.  Some questions come up.  You mentioned something about, fully reimburse the cost of safety.  Does that mean to fully reimburse the blood banking community for the cost of a test that they are doing or would ask to be done?  If so, where would this reimbursement come from?
CMDR. HENRY:  Listening to the committee, it did not appear that they intended that those reimbursements would specifically happen from the Department to the community.  So it was more of an overall broad fully reimbursed mechanism for CMS reimbursement.  It would take into account the overall costs and not specific costs.
DR. EPSTEIN:  The general problem here is that FDA promulgates recommendations and requirements for safety interventions, but they are not automatically funded through CMS to hospitals.  Blood is sold to the hospital, the hospital gets reimbursed by CMS, but there is no blood line item.  So there is a complex set of things that go on to determine how much the hospital would pay for blood, let alone an additive cost for an intervention.
Also there is the issue of how it is accounted, what is the total cost.  Is it just the test?  What about labor, infrastructure, transportation, et cetera?
So we have a disconnect in our system where we are making safety decisions, but not including in that calculation how it will be reimbursed.
DR. HOLLINGER:  I also see that they had under biovigilance, traceability and outcomes.  We discussed this at some length on occasion.  How is that going to be done because we lose a lot of information by not being able to have the regulations to trace outcomes of diseases that may occur in an individual.  So how can we resolve that? 
CMDR. HENRY:  The quickest way is through a public-private partnership, where industry and government come together and form an alliance, a coalition, regarding biovigilance, and agreements to provide data and mine that data for information.  If we took a regulatory approach, it would be protracted, and perhaps there would be some pushback from industry. 
So on one hand you could regulate that type of collaboration, or you can form a public-private partnership.  At the moment we are forging ahead with the public-private partnership.
DR. HOLLINGER:  Anybody else have any questions? 
DR. TRUNKEY:  Yes.  The military over the last few years has been looking at massive transfusion protocols in theater and the appropriate relationship between category therapy and whole blood therapy, and then using other adjunctive measures. 
My perception is that the work that the military has done, published the data on, is having resistance here in the civilian community to use this in the same type of situation, basically multiple trauma patients, massive transfusion protocols.  I think the data is overwhelming from the military that if you use component therapy at one to one to one, is desirable.  Even more efficacious is using whole blood.  That is very difficult.  Although it was used several years ago in the United States in civilian hospitals, it is no longer done because of availability.
So how are we going to translate that work that has been done in Iraq and Afghanistan to the civilian community?  If it worked for our soldiers, why aren't we using it for these terrible injuries we see in the inner cities and also multiple vehicle accidents?
CMDR. HENRY:  As you mentioned, it could be an availability issue, supply chain collection and supplying whole blood.
DR. TRUNKEY:  I don't accept that.  I really don't accept that.  In 1966 when I was a first year surgical resident, San Francisco General Hospital, the blood bank was run by the San Francisco Medical Society.  They saved one third of every day's procurement to use in the trauma center that night.  If we didn't use it, then they made components out of it the next morning. 
At any one time, 800 policemen were on duty.  We had all of their blood types, and we could call them into the emergency room and they would give blood, and we would use it.  So it is possible to do.
DR. DEMETRIADES:  I think now most civilian trauma centers have the so-called massive transfusion protocol.  This means that after the first six units of blood transfusion, the ration goes one unit of red cells, one FFP, one platelets.    It has been shown that it is associated with superior results. 
Having said that, we did a study where we showed that if you applied this protocol too early, the patient does not need massive transfusion, he needs four or five units.  If you give the higher ratio, one red cells, one FFP, there is higher incidence of organ dysfunction.  So if applied properly, the appropriate case, I think it is life saving.
I do agree with Dr. Trunkey that we need to look into that very seriously. 
DR. HOLLINGER:  The second update is a summary of a workshop that was just held this last week, on Thursday and Friday, on product development program for interventions in patients with severe bleeding due to trauma or other causes. Dr. Vostal.

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Agenda Item:  Summary of December 9-10, 2010 Workshop “Product Development Program for Interventions in Patients with Severe Bleeding Due to Trauma or Other Causes”
DR. VOSTAL:  Good morning.  Thank you very much for the opportunity to give you an update on the workshop that took place just last week, so this summary is really hot off the press.  The workshop was focused on severe bleeding due to trauma or other causes. 
Our understanding of trauma physiology is still developing.  Severe bleeding in trauma is associated with coagulapathy, tissue hyperperfusion and shock.  Current treatments such as large volume transfusion may contribute to the coagulapathy and further complicate the pathophysiology of trauma.
Evaluation of new treatments has been hampered in part by a lack of consistent definitions of various trauma-associated pathologic states, for example, the definitions of moderate bleeding versus severe bleeding.  What we need is a uniform approach for evaluation of new therapies in this field. 
The current products for severe bleeding are summarized on this slide.  No products have been approved for severe bleeding.  That is true for systemic products reviewed by CBER and CDER, and also for products reviewed by CDRH.  CDRH does have some products approved for mild and moderate local bleeding, but none indicated for severe bleeding.
Thus, in the current medical practice, products such as Factor VIIA are used off label for severe bleeding, even though these products don't have this indication.  Companies developing products for severe bleeding need a well-defined pathway to evaluate these products.
The workshop objectives were to gather information on evaluation of products intended to be used in patients with severe bleeding from trauma or other causes, to obtain practical information for the FDA that could be used by sponsors to support market application, and to develop a uniform approach to facilitate research and development and ultimately lead to FDA approval of products for severe bleeding.
This is the outline of the workshop.  It started off with the introduction of the severe bleeding issue.  There was then an overview of the currently marketed products with bleeding indications.  As I already mentioned, there were none for systemic agents and some for local agents.
We went through the pathophysiology of trauma, the statistics of clinical trials, and then we had a series of panel sessions that included an introductory talk and a panel discussion by a panel of experts.  These panels covered the definitions of bleeding, the animal models of trauma, end points for clinical trials for local agents or for systemic agents, the ethics of conducting clinical trials in trauma patients, and the options for doing other trials besides trauma clinical trials.            Finally, on the second day there was a summary of the panel sessions by the panel moderators.
I am just going to walk you briefly through the different sessions.  The Session 1 was to define the severe bleeding.  After review of the data and the presentations, it was concluded that based on existing data, it is difficult to identify a single criterion to define severe bleeding.  Many of the doctors and surgeons involved said severe bleeding was obvious when you see it, but it is difficult to define prospectively.
There was some thinking that combination of clinical biomarkers and physiological criteria may be useful in defining severe bleeding, and it was possible to modify the existing trauma scales to fill the gaps, and this could be used to quantitate severe bleeding.  There was a lot of discussion on using some instruments such as TEG and other rapid testing biomarkers that could be used or that should be developed for identifying patients who are going through severe bleeding.
Session 2 discussed animal models.  It was concluded that the animal models should demonstrate the clinical hemostatic challenge for which the drug, device or biologic is to be used.  The models should reflect the coagulapathy and the vascular defect should be severe enough to be lethal, similar to what happens in trauma, be reproducible, have the ability to demonstrate the hemostatic efficacy of the product, and have the ability to apply the hemostatic product as would be potentially indicated.  The hemostatic effects should not be considered separately from the general systemic effects of these products, and it was concluded that a poly trauma model in pigs with uncontrolled bleeding may be the best model at this time.
Session 3 started talking about clinical trials.  This was specifically for local agents.  Local agents would be applied directly to a single bleeding site.  
Some of the things discussed were, local or systemic factors should be taken into account to determine the severity of blood loss in a localized anatomic site.  The panel considered anatomic factors such as external bleeding versus internal bleeding, the area where the bleeding is occurring, and whether it is coming from a large or a small vessel.  There was also a discussion of a variety of patient factors that could influence the outcome, and these were coagulapathy, either to cancer versus trauma versus pharmaceuticals, and these could be different and may not be extrapolatable from one group to the other.  Whether the patient had a history of thromboemboli, whether this was an emergency situation versus an elective surgery, since trauma is more heterogeneous in terms of the injury it produces, whether this was in the field or in the hospital, civilian or military, and also what was the rate of the bleeding at the local site?  However, there was no consensus reached on objective measures of the bleeding at the local site.
The second point that was considered was, what are the clinical meaningful end points of the change in parameters from baseline assessments before or after local agent use to achieve hemostasis or complete cession of bleeding.  It was thought that time to 100 percent hemostasis was an appropriate end point, and the time to measure was considered three minutes.  Another end point was incidence of 100 percent hemostasis also evaluated at three minutes.
The next session looked at the clinical end points in trials for systemic agents.  So these would be agents applied systemically to reduce bleeding from multiple sites.  The clinical end points considered was hemodynamic instability of the patients.  The panelists were not in favor of this end point.  It was thought that the criteria could not be applied uniformly, and it could also be easily manipulated by routine clinical care such as by pharmacological agents. 
Transfusion was thought clinically relevant, but there were varied opinions about the magnitude of production required.  Probably it was considered the best surrogate measure for hemostasis and avoidance of massive transfusion was likely an acceptable end point in these clinical trials.  There was general agreement that mortality was an acceptable end point.  Most thought that the end point should be measured at between six to 24 hours.  However, there are some people who think that a long term mortality should be an end point. In general, there was agreement that a five to ten percent in reduction in mortality would be a clinically meaningful end point.
Morbidity was thought to be not a good single sole end point, but perhaps it was acceptable as a secondary end point.  There was a discussion of composite end points.  It was concluded that these were not preferred for primary end points, but could be acceptable as secondary end points. 
Then we moved on to a session where we discussed whether clinical trials would be required to approve products for trauma indications.  Clinical trials are difficult to conduct and are very costly.  I mean, trauma clinical trials are difficult to conduct and costly.  Some of the reasons for this are that the patients are in the field.  There is variable time before access to medical care.  There is an extremely short period of time to decide if patients meet the study criteria.  There is a wide variety of injuries, comorbidities and patient conditions, and trauma trails contain some unique ethical issues around obtaining consent from the patients involved.
An alternative to doing trauma trials could be doing elective surgery trials, which is a more controlled environment, and clinical trials could be easier to conduct.  There was a lot of discussion on both of these options.  Some of the concerns that came out was that the physiological response in trauma would be different than in elective surgery.  For example, patients in trauma would go through shock or are likely to go through shock and coagulapathy.  The response in surgical patients would be altered because they would be under anesthesia.  So there was some concern whether data obtained in surgical clinical trials could be extrapolated to trauma situations. 
In the end, it was concluded that surgical trials could generate valuable information, but would need further trials to verify results in a target population such as a specific trauma.
The next session considered the ethics of conducting clinical trials in trauma patients.  Now, in trauma patients are frequently unable to consent to participate in the trial due to loss of consciousness or otherwise altered mental state.  Trials can still be conducted in the trauma situations under the 21 CFR 50.24, which allows the whole community to consent to participate in the trial.  If this is the case, the public has to be notified that such a trial is going on, and the public has to have the option not to participate.  Patients who are being enrolled should have the prospect of a benefit from the treatment based on previous studies, and this should be demonstrated by either animal studies or other clinical studies.  After enrollment in this trial, the patient or their relatives should be informed and given the opportunity to have the treatment stopped.
This brings us to the workshop outcomes.  This is the summaries in a nutshell.  For animal studies, it was thought that the polytrauma pig model of uncontrollable bleeding was the appropriate model to evaluate products.  Eligibility of patients for severe bleeding trials, it was not decided what would be the appropriate eligibility criteria.  Trauma scores were considered, biomarkers were considered, but we did not get a uniform consensus on which would be the most appropriate ones. 
Clinical end points for studies in trauma.  The appropriate end points were early mortality and hemostasis.  In terms of conducting a trauma trial versus a non-trauma trial such as a surgical trial, it was concluded that the actual pivotal study of a product that is going to be intended for use in trauma should be done in trauma patients. 
Finally, discussing the ethics, it is possible but difficult to conduct trauma studies in a civilian population and probably is not accepted to conduct such studies in the military setting because of the issue of consent.
Finally, the FDA Office of Blood gratefully acknowledges the efforts of the steering committee that worked long and hard to organize and plan the workshop, the speakers, the panel members and the panel chairs, and also the logistics staff that organized the workshop.  We also gratefully acknowledge the funding for the workshop, which was donated by the DoD and NHLBI, and the HHS Office of the Assistant Secretary of Health.
Thank you very much.
DR. HOLLINGER:  Thank you, Dr. Vostal.  Any questions for clarification? 
DR. DEMETRIADES:  Dr. Vostal, thank you very much for this update.  I want to come back to the blood transfusion issue.  There is a lot of evidence which is growing day by day that in trauma patients, if you transfuse old blood, older than two weeks, there is increased mortality and significantly increased complications, organ dysfunction, sepsis, et cetera.
For some reason, this has been ignored.  How is it this issue wasn't discussed, wasn't brought up?
DR. VOSTAL:  That issue actually did not come up because we were focused on looking at how to design clinical studies to evaluate products to be used in severe bleeding. 
There is a separate issue about the quality of blood.  As far as I know, the studies that suggested there may be a problem with stored blood are all retrospective at this time.  There are ongoing prospective trials to evaluate the quality of fresh and stored blood.
DR. TRUNKEY:  But the fact remains that in some blood banks, as the patient needs more and more blood, then the blood bank tends to send the 41-day old blood.  There is no question, I agree completely with Dr. Demetriades, it does cause complications.  We see the immediate hemolysis of those old red cells contributes to the multiple organ failure. 
Another issue that vexes all of us is the tendency for some early studies done in developing countries, specifically Sub Saharan Africa.  That is what Factor 8 was tested initially and also the bovine hemoglobin.  There were higher death rates.  There was unacceptable mortality in some of those studies.
I think this is a real ethical issue.  We should not be doing these studies in developing countries.  I don't know if that was specifically discussed.
We have a problem in our military right now, that it takes approximately nine to 11 days to get a unit of blood into theater.  The military has recognized, as we have in the civilian community, that old blood is bad.  So they discard blood more than 21 days of age.  I support that completely.
We have a huge problem here in blood transfusion in trauma.  I don't think it is being addressed in a timely way.
DR. HOLLINGER:  Was this discussed at the meeting?  Were these issues discussed at the meeting?
DR. VOSTAL:  One of the issues that came up is whether we would accept studies done in other countries.  That was discussed.  It really depends on whether the quality of the medical care is comparable to medical care in the U.S.
DR. TRUNKEY:  Another example is that Bastion, which is a level three hospital in Southeastern Afghanistan, is primarily manned by British doctors.  They are using a plasminogen inhibitor.  The Marines go to that hospital.  Is this what we really want to do?
DR. VOSTAL:  I think any use of products in the situation should be done after it has been tested for that particular indication. 
DR. TRUNKEY:  I don't believe it has ever been tested in this country. 
DR. HOLLINGER:  Dr. Rentas.  Also, could you comment about the military and the consent that is used in the military for things like when they are in Iraq or Afghanistan, how that is done?  Because I'm sure there are things that are looked at in trauma and so on that might be used.
DR. RENTAS:  That is exactly what I was going to do.  Dr. Trunkey here is correct, that it was taking the military two years ago about 13 days to get the blood supply to Iraq and Afghanistan.  Fortunately that is not the case anymore.  It is taking us about six days now.
Now, you have got to remember what we have to go through.  We have facilities all over the U.S. collecting, sending to a specific location in the U.S.  Twice a week we have a chance to send these out there to another facility, and then from there it goes to Iraq and Afghanistan.  We are doing that in six days now.
If you look at the age of blood upon transfusion in Iraq and Afghanistan right now, there is not a lot going on in Iraq.  Afghanistan is where transfusions are going up every month.  It has gone down by about ten, 11 days in the last two years or so.  The average age of blood upon transfusion, because we have been able to get there faster, and because the trauma surgeons want to use the freshest blood available to these massively transfused patients, which is about five to ten percent of what they see out there.  A lot of these patients go through 50, 75, 100 units out there.
Where I take issue is, when they start calling 41- or 42-day-old blood garbage, and that makes it to the front page of the Washington Post.  Back here at Walter Reed in a place like Bethesda, that is what we use.  We try to use the oldest blood first.  The general population out there, you make a statement like that, they believe that the standard of care here is not as good as what it is in Afghanistan out there.
So we have made some big improvements in delivery of the blood supply to Iraq and Afghanistan in the last two years or so.  To be honest with you, the trauma surgeons, they swear that this works.  The data is starting to show that.  The NHLBI people are going to be conducting some trials out there.  We will see what the clinical outcomes are. 
Right now they have about a 91 percent survival rate on massively transfused patients in Iraq and Afghanistan.  That to me is outstanding.  That to me is outstanding.  You go to places like Walter Reed and you see these warriors walking around without legs or missing two or three limbs and you are saying, how is it possible that someone like that made it in the middle of Afghanistan and is here now alive? 
So I can't say enough about the care that is taking place in Iraq and Afghanistan right now.  A lot of these things that they are doing, like Dr. Vostal said, I think the evidence and the scientific evidence is still out there, and hopefully in the next three or four years we will have a better picture of this.  But that is what they are doing, and we are supporting that.
It is the same thing with the one to one to one ratio.  If you look at the numbers, there are just as many units of plasma as RBCs being transfused in theater.  Then you have the platelets and cryoprecipitate as well.  This is something that I looked at every week.  We are supporting that, because that is what they want, so we are just making that available to them out there.
DR. HOLLINGER:  Jay, did you have something to say?
DR. EPSTEIN:  I think Dr. Rentas made the point.  I will just reiterate it, that the NHLBI is in fact supporting prospective clinical studies to determine the actual value of different resuscitation regimens in severe trauma, including the ratio of components. 
I will come back to the fresh whole blood issue and also studies to look at the impact of age of storage of red cells.  Of course, we want the answers yesterday, but what has been recognized about the available information is that it didn't come from controlled studies, and for the older blood issue it is mainly retrospective with innumerable confounders.            So the truthful answer will come only from prospective controlled trials, which are being done. 
The issue of fresh whole blood is a little bit more difficult because it gets into the question of an average 48 hours to turn around testing on blood.  So the issue of using not fully tested blood comes into the picture if you want fresh blood.
We are in a dialogue with the possibility of 24-hour room temperature held blood, how appropriate it is to make components from blood collected and held in that way.  This would be controlled room terminology held whole blood.  But the missing link here is, you would need rapid tests for all of the transfusion transmitted disease markers in order to deliver equally safe blood. We understand that in the urgent situation in the military environment, tradeoffs are possible and are rational.  We know there has been use of incompletely tested blood.  Not all markers have a rapid test.  They collect a sample, they test it later, they notify if there was a positive exposure.  But really, to re-engineer the system to provide the product that you believe is preferable will take some effort.
The issue is not going unrecognized.  I appreciate the frustration.  I am aware that fresh whole blood is the history of transfusion.  Indeed, you used to have donors on call.  It goes all the way back to Philadelphia in the 1930s.  People would agree to be donors.  You had somebody who needed blood, someone would go bring the patient into the hospital.  There have been proposals to reactivate that type of model with, if you will, a prescreened donor.
So all this is recognized.  I understand that the time taken to change practice is a frustration, but there is a need to develop a sound evidence base, and that effort is ongoing.
DR. TRUNKEY:  I don't disagree with that at all.  I spent two weeks in Afghanistan just recently, and there is somewhat of a disconnect here.  When I met with the blood bank there, they told me that the average time to get blood into theater was nine to 11 days.
The other thing that impressed me is, 13 percent of all blood used there comes from blood drives on the soldiers.  The sense that I got is that there is a desire to use that blood over the blood that comes from the States that is nine to 11 days old.
A lot of this is a gangland approach to it, but they see it and they think it works.  So we should confirm that with civilian studies in the United States.  I think that we have to have a concerted effort to have a study in various blood banks across the country where there is a high propensity of trauma patients that have coagulapathy.  We have to look at this.  If it is confirmed, then I think we should give it to the people who deserve it, and that is our soldiers in theater.
DR. HOLLINGER:  It sounds like, Dr. Vostal, that the workshop was a success, and that there is still a lot of information that needs to be gathered.  It sounds like this is at least a step in the right direction.  Thank you.
We will go on to the next update.  It is an FDA guidance for industry on the use of serological tests to reduce the risk of transmission of Trypanosoma cruzi infection in whole blood and blood components intended for transfusion.  Dr. Duncan from the FDA will give this presentation.

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Agenda Item:  FDA Guidance for Industry: Use of Serological Tests to Reduce the Risk of Transmission of Trypanosoma cruzi Infection in Whole Blood and Blood Components Intended for Transfusion
DR. DUNCAN:  Good morning, and thank you for the opportunity to give this update on this newly released guidance.  You will see as we go through this that this committee has played a role in the development of this guidance all along.
There is a long background to it.  I would just like to highlight a few points along the way.  First of all, the FDA licensed the Ortho T. cruzi ELISA test system in December of 2006 as a donor screening test for blood and tissue products.  A second test, the Abbott Prism Chagas chemiluminescent immunoassay was licensed in April of this year.
The majority of blood centers voluntarily tested all donors for antibodies to T. cruzi at each donation with the licensed test in 2007, 2008 and part of 2009.  All repeatedly reactive specimens were mostly retested with an unlicensed radioimmune precipitation assay or RIPA, because no licensed supplemental test was available. 
Following consultation and development, an FDA draft guidance recommending universal testing of blood and tissue donors was released in March of 2009.  If you look carefully at the title to the current final guidance, it does not include tissue donors.  After receipt of comments and discussion and consultation, the Office of Cell Tissue and Gene Therapy of CBER decided that it was not appropriate to issue final guidance related to T. cruzi infection for tissue donors at this time.  So this document only covers blood donors. 
Shortly after release of the draft guidance at the April 2009 BPAC meeting, FDA presented potential testing strategies for T. cruzi infection in blood donors and a risk analysis for selective testing of donors.  The epidemiology of Chagas disease in the U.S., the experience with blood donor testing for T. cruzi antibodies during the approximately two years since licensure of the Ortho assay, and the experience with asking donors questions to assess their risk of Chagas disease were all discussed at that BPAC meeting.
At that time, the committee voted in favor of a selective testing strategy in which one negative test would qualify a donor for all future donations without further testing or the need to be asked questions regarding risk of newly acquired infection.  But it was recognized at that time that that kind of selective testing strategy would not intercept a tested negative donor who acquired a new infection.
So for that reason, the committee's recommendation was contingent on studies to define the incidence of new T. cruzi infections in previously screened negative donors.  Representatives of the American Red Cross and Blood Systems Incorporated volunteered at that time to pursue such a study.
So with comments to the draft guidance and incorporating the recommendations of the committee, a final guidance was published in December of 2010, just about a week ago.  That guidance document is available on the FDA website.     I am just going to highlight a skeleton outline of the contents of the document.  First of all, regarding blood donor testing and management, blood establishments should continue to ask the donor history questionnaire question, have you ever had Chagas disease at every donation.  It is our feeling that that question is another layer of safety that is completely different from and independent from our recommendation that it is not necessary to ask test negative donors any further questions, even though they will see that question on the donor history questionnaire every time they come.
Then blood establishments should test each donor one time for antibodies to T. cruzi.  Non-reactive donors may return to donate without further testing.  Each establishment should review its own records to determine the testing history of each prospective donor.
This one time testing is recommended for allogeneic and autologous donors when it is applicable to autologous, and I will expand on that a little bit later.
I will emphasize that written into the guidance is this contingency that the onetime testing depends on the outcome of studies to determine the rate of incidence of new T. cruzi infections.  If the outcome of that study should suggest that this is not a safe enough approach, the guidance will be revised. 
Continuing on donor testing and management, blood establishments should defer indefinitely and notify all donors repeatedly reactive by the licensed test.  Establishments should counsel reactive donors to seek a physician's advice.  They should explain to them the significance of possible cross reactivity and in this regard, additional medical diagnostic testing may provide useful in the counseling.  Blood establishments should only allow reentry once a supplemental test is licensed and a reentry recommendation is published. 
In terms of product management, blood establishments should quarantine or destroy or label Not for Transfusion all blood and blood components from a repeatedly reactive donor.  Products from prior collections from a repeatedly reactive donor should be retrieved, quarantined and destroyed or labeled.  The time period over which this retrieval should be done is a lookback period of ten years, indefinitely if there are electronic records, or in a previously tested donor, 12 months prior to the most recent negative T. cruzi test result.
Continuing on the question of lookback, and focusing now on recipient tracing, when identifying a donor who is repeatedly reactive by a licensed test for T. cruzi antibodies, and there is additional information indicating risk of T. cruzi infection such as a positive test result on a licensed T. cruzi supplemental test when such a test is available.  Until such a licensed supplemental test is available, geographic risk for exposure in an endemic area or in a medical diagnostic testing of the donor may provide additional information to guide which donors to do recipient tracing.  The recipient tracing involves notifying consignees to encourage notification of recipient's physician of the increased risk of T. cruzi infection. 
In terms of autologous donations, they should be tested when allogeneic use of these units is allowed, or units are shipped to centers where allogeneic use is allowed.  That is a citation from the regulations that relates to that.  Reactive autologous units may only be released with approval of the donor's physician, and all reactive units for autologous use must be labeled biohazard, which is also in the regulations.
Some more details.  Blood establishments must update their circular of information.  The circular of information is a little complex at this stage, because some of the units in the package may have been tested directly and some may not have.  We are suggesting wording such as this to be in the circular of information.  All blood has been collected from donors who have tested negative by a licensed test for antibodies to T. cruzi, either on the current donation or at least one previous donation.
There also will be need for biological deviation reports and fatality reports, and you are instructed to report any event that represents deviation from current good manufacturing practices, deviations from regulations, standards or established specifications that may affect the product's safety, purity or potency.  Also, when a transfusion involving T. cruzi is confirmed to be fatal, you must notify the FDA.
Finally, licensed blood establishments adopting the recommendations in the guidance must report the following changes to the FDA.  Revisions of the donor history questionnaire, test implementation and revision of the circular of information in their annual report. 
That is all my summary.  Are there any questions?
DR. HOLLINGER:  Questions, clarification? 
DR. RENTAS:  Was travel to endemic countries outlined anywhere in this guidance?  For example, you test a donor once.  Now he goes to Brazil for two or three weeks and comes back.  Was there anything there, any guidance on that?
DR. DUNCAN:  There was a lot of discussion and consideration of that question.  With the experience that we have had to date of no new cases, no case of a T. cruzi positive individual having a profile that would indicate a recently acquired infection, it was felt that the probability of that occurring was low enough that there would be no recommendations for risk questions such as that.
In the presentation just following me, Dr. Susan Stramer will outline the contents and the results of the incidence study that is ongoing.  But that kind of thing is exactly what is being looked for in that study, to find out what the rate of that occurrence might be. 
DR. HOLLINGER:  This circular of information that they are being asked to do, is there also a circular of information for testing for other things, too, that goes with this, like HIV and HCV and so on?
DR. DUNCAN:  I'm not an expert on the circular of information, but I have looked at it briefly.  It is a long document that describes various aspects of the contents of the blood that is packaged.
PARTICIPANT:  There is a circular of information, and it does get reviewed by FDA.  It has been put together by AABB, ABC, ARC, Department of Defense.
Currently within the body of the circular is a statement for all of the testing that has been recommended by FDA under AABB.  Until the issuance of this guidance, testing for T. cruzi was not recommended.  But on the AABB website, there has been a statement that we have recommended to be used by centers that do tests for T. cruzi.  That statement has been for those centers that are performing universal testing.
So until the time that the printed brochure is updated, we won't move forward to replace that statement with the one that has been included now in the guidance document.
DR. HOLLINGER:  Thank you, Dr. Duncan.  Now the final update this morning is by Dr. Susan Stramer, who will give us an update on the study to further define the incidence of T. cruzi infection in the U.S. blood donor population.

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Agenda Item:  Update on the Study to Further Define the Incidence of T. cruzi in the U.S. Blood Donor Population
DR. STRAMER:  Thank you.  Dr. Rentas, your question was the perfect lead-in to what I am going to discuss today. 
Again, Blaine has provided the title.  Let me tell you what we have done in blood donor screening in the U.S. focused on the Red Cross and blood systems, or the United Blood Services' collection areas.
The Ortho T. cruzi test that was the first one that was licensed that Bob mentioned in December of 2006 is used for blood screening.  Both organizations involved in the study implemented the test with universal screening at the end of January 2007.  All donations that are repeat reactive are further tested using a research RIPA test by Quest Diagnostics.  All RIPA positive donations are considered confirmed.  Donors of repeat reactive donations are followed and tested by repeat serologic or parasyllogic tests, including hemaculture and PCR. 
Donors are also asked to respond to a detailed survey regarding risk factors.  For demographic or risk comparisons, our RIPA positive donors are defined as our cases and our RIPA negative donors are defined as our controls or our RIPA unconfirmed as controls.
Following the implementation of selective testing which both organizations moved to after a long period of universal testing, blood regions having the highest prevalence in the southern portion of the United States however did remain to test each donation every time the donor presents, or universal testing.  That was to determine donor incidence.  So we would follow repeat donors over time. 
The three areas of the Red Cross that are involved in the study are our Los Angeles region called Southern California, our collections in Texas, Arkansas, and the collections that the Red Cross tests for the Community Blood Center of Florida.  That started at the end of July last year, 2009.  Also included in the incidence protocol is UBS, United Blood Services Center, that is Central Coast in California.  They continued with universal testing while the rest of their organization went to selective testing on April 1 of this year.
At the last Blood Products Advisory Committee meeting where we discussed Chagas, I presented the 22-month experience.  Now we are up to the 43-month experience. 
Looking at the United States, the pink states give you the states that have had RIPA positives.  The numbers in the states show you the number of RIPA positives.  So we have seen RIPA positive donors across the continental United States, including a few in Puerto Rico. 
We have had a total of over 1,000 RIPA positives since testing began at the end of January through the 43-month experience that ended in August this year.  That is over 28.1 million donations screened with an overall prevalence -- this is combining the selective and universal testing -- of about one in 25,000. 
For the Red Cross, if you look at the demographics of the donors that we find, these are some of the questions and the odds ratios based on univariate analysis of what risk we sees.  Born in an endemic country is number one, lived in an endemic country, mother and maternal grandmother.  These are the demographics that we expect that are traditionally associated with T. cruzi infection.  Doing multivariate analysis, you can see that born in an endemic country still is the highest risk.
But born in the endemic country is not the only risk.  You can also acquire T. cruzi infection in the United States.  We have seen autochthonous infections and we have investigated autochthonous infections in a study with UBS Centers and CDC have tried to identify suspect autochthonous cases which are shown on this map.
This map gives you in green the states that have the vector for T. cruzi and the reservoir, and the yellow states show you only the vector.  West Virginia I guess doesn't report.  But anyway, the blue dots show you donors by state of zip code of residence that were not confirmed, and the red show you the 15 defined by our study protocol as probable cases of autochthonous infection.
Let me remind you that these were all remote.  None of these represented recent infections.
We also looked at the collective lookback experience before we went to selective testing.  ABC had done a survey that was presented, the 22-month experience.  I extended ours for the 43-month experience.  If you combine everything together, this is 253 total recipients tested, we only saw two platelet recipients that were positive, and they both received blood from a single PCR positive donor from Argentina.  This was reported by New York Blood Center at the SEERS AABB.
If you look at the sensitivities of different testing methods according to the gold standard, which is the FDA licensed test, if you were born in an endemic area, there is only about 75 percent sensitivity, because as I told you, there are autochthonous cases that occur.  Using the screening method that Rob described that is in the guidance of testing a donor once to qualify the donor as negative for future donation, that has a sensitivity of about 96 percent. 
Our 22-month experience, now looking at incidence, that changes from screening non-reactive to repeat, reactive and confirmed, when we looked at our combined experience for 22 months, we had tested about 2.5 million repeat donors, that is, with greater than two donations, and they contributed about 2.3 million person years of observation.  Again, the 22-month experience, all the intervals between donations that were tested were counted, for a mean interval of .9 years.  When we combined this with the UBS data, we now came up with instead of 2.3 million person years of observation, we had about 2.6 million person years of observation.  This also included in the case of UBS donors who were questioned pre-donation about travel and other risk factors to Latin America, and none of those showed any incidence or new infection. 
The protocol that we decided following the BPAC meeting was to continue universal testing for five years, to extend the time frames for detection of incident infection in repeat donors in regions with presumed increased travel to endemic areas, and those with the greatest limitation of autochthonous infections.  So we would combine both endemic area risk and U.S. autochthonous risk.
Universal testing remained in the highest prevalence regions based on the assumption that high prevalence regions would be the highest areas of incidence.  Other regions moved to selective testing.  So the goal was to add two million person years to the 2.6 million person years I showed you to try to approach approximately five million person years of observation.  We wanted to extend the inter-donation interval from .9 years to about two years or 1.9 years. 
It is important to define in such a study what an incident case is or a seroconverter.  It is a donor who screens repeat reactive and is RIPA positive with a period donation having a low signal and who is serologic progression on follow-up.  So it is a person that we can monitor change in, not someone who has positivity, but who is just running around the cutoff or a false positive.
If you look at our experience with selective testing and the blood systems or the UBS data are being collated, so I am just going to show you now the Red Cross data.  Since the time that we converted to selective testing, we have seen a stable reduction in testing volume for those regions that have migrated to selective testing. 
We see about a one in 42,000 prevalence in those regions in comparison -- and I changed this from what is on my handout -- one to 86,000 was our prevalence when we did universal testing for 22 months in the same regions.  So this number is of course increased, because now it is enriched for first time donors.
In the sites that remained on universal testing, the three Red Cross regions I mentioned, we had a one in 6,000 experience for prevalence when we did universal testing, and now that we have remained testing, the prevalence has dropped a little bit to one in 12,000.  Over this time we have seen no incident donors identified.  That is over a total of 4.6 million person years.  So we are rapidly approaching the five million person year mark.
We have seen eight confirmed positive repeat donors in selective testing regions.  One was a false positive donor and seven had no prior T. cruzi test result.  So none of these are considered incident cases.
In the universal testing sites we have seen 18 confirmed positive repeat donors.  Three were false positives and 15 had no prior T. cruzi test result, so again, no incidence.
Showing you the calculations for the 4.6 million person years, we have isolated all donors that had screening performed over the time period.  This was cumulative across all regions.  It included about 3.5 million donors with two or greater donations that contributed to the 4.6 million person years.  We counted the interval in days between the first and last screen to give person days divided by 365 to give person years.  Over this 4.6 million person years of observation, the mean interval was 1.33 years.  So greater than .9 which I have shown you before, not quite 1.9.
We have seen no seroconverting cases, that is zero per 100,000 person years, and the protocol specified and FDA requested that we give an upper 95 percent confidence interval to that number.  So that is 3.69 for the 4.6 million person years or .08 cases per 100,000 person years.  Or to make that a little simpler to understand, less than one case per million person years.  The criteria established for the study was an upper limit of acceptable risk of 2.4 cases per million to be comparable with other infectious disease risks, or one to two cases per year.  But again we have seen zero.
In conclusion, a selective testing strategy based on qualifying a donor by a single negative donation has a high sensitivity and has significantly reduced the amount of testing required without compromising recipient safety. 
Thank you.
DR. HOLLINGER:  Thank you, Susan.  Questions for Dr. Stramer?  Yes, Dr. Bower.
DR. BOWER:  Yes, Susan, thank you for that.  I probably missed this somewhere in the presentation, but how are the false positives -- there was the one false positive from the selective testing and the three false positives from the universal testing, how were those determined to be false positives?
DR. STRAMER:  We looked both at the index test collection, the prior donation and subsequent donation.  All of these donors were followed.  We saw no evolution of serological patterns.  That is, they were low level Ortho repeat reactive, about a one S to CO, which has a 50 percent chance if you repeat g of being negative, or 50 percent chance of continuing to be repeat reactive.
So the prior donation was just below the cutoff.  The index donation was just at the cutoff, and follow-up samples were also within the cutoff variability.  So we see no evolution.
Because the Ortho T. cruzi is a lysate test and it is the same lysate as the RIPA, in a way you enrich for not only -- not only are you able to confirm true positive infection, but you also enrich for false positivity.
DR. EPSTEIN:  Thank you, Sue, for sharing these data.  We very much appreciate the effort of Red Cross and UBS to carry on these studies so that we can get a true scientific answer.
Just a very, very small comment.  You mentioned the RIPA research test.  It is a medical diagnostic test which is done --
DR. STRAMER:  It is an LDT, yes.
DR. EPSTEIN:  Right, it is an LDT that uses a class A analite specific reagent.
DR. STRAMER:  I know.  As I was reviewing my slides this morning, I realized that is an LTD, right, home brew.
PARTICIPANT:  Just to address one of the questions about travel, in addition to the monitoring of positive donors in the U.S. who travel and asking of questions in our UBS centers for travel with no yield, we have conducted and just published a paper from Brazil.  In the three large centers in Brazil, in Sao Paulo, in Belorizonte and Recife, these are urban large hemo centers.  We monitored under the NHLBI REDS program about a quarter of a million donors for over a million person years, repeat donors, and haven't seen a single incident infection in repeat donors within Brazil.
So within most of the regions where people travel to, there is actually very little ongoing transmission of T. cruzi.  So I think this travel issue continues to be one that we are paying attention to, but doesn't seem to be accounting for any incident infections in the U.S., let alone in Brazil.
Sue, one thing, just glancing back at the incidence protocol that we submitted to FDA, it has two denominators.  One of them is accruing five million person years, which we are very close to.  The other was to accrue up to five years of follow-up of donors from the very beginning of implementation of screening.  I think the reality is that although we have saved some resources in terms of testing, we actually have not saved money in terms of the reagents, because the companies have more than doubled the price of the assays, and have offset what we hope would be a cost effectiveness gain through conversion to selective testing, one-time testing.
So the sooner we can discontinue these repeat donor testing, ongoing incidence monitoring, potentially the better for the industry.  I would hope that FDA would consider that target of five million person years as acceptable in terms of completion of the study.
DR. HOLLINGER:  Sounds reasonable.
DR. NAKHASI:  Hira Nakhasi, FDA.  Sue, I just wanted to follow up on Willy's question.  These false positives, they come from (foreign accent) the first time.  Then when you followed up to -- how long did you follow?
DR. STRAMER:  Variable times of follow-up.  Some donors had four subsequent samples.
DR. NAKHASI:  And each time when they tested they were repeat reactives?
DR. STRAMER:  Sometimes they were, sometimes they weren't.
DR. NAKHASI:  And not accounted for by the RIPA.
DR. STRAMER:  Sometimes they were, sometimes they weren't.  This is typical stable false positivity.
DR. NAKHASI:  I understand.  You had the cutoff and the gray zone. 
DR. STRAMER:  Yes.
DR. NAKHASI:  Thank you.
DR. HOLLINGER:  We are going to take a break until 9:30.  We will start again at 9:30 on a review of the research programs of the laboratories in hemostasis and plasma derivatives.
(Brief recess.)

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Agenda Item:  Review of the Research Programs in the Laboratories of Hemostasis and Plasma Derivatives, Division of Hematology, OBRR
DR. HOLLINGER:  As we indicated, this is going to be a review of the research programs in the laboratories of hemostasis and plasma derivatives.  We have several talks regarding that as overviews, with some places in the second half for some questions and answers.
Let's start first with the overview of CBER research by Dr. Dr. Wilson.

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Agenda Item:  Overview of CBER Research
DR. WILSON:  Good morning, thank you.  I am going to start with just a brief review, give you an overview about the Center.
Our mission if you are not already familiar, is to insure the safety, purity, potency and effectiveness of biological products, including vaccines.  Obviously we will be talking today on the focus of your committee, blood and blood products, cells, tissues and gene therapies for prevention, diagnosis and treatment of human disease conditions or injury.
The vision is to use innovative technology to advance the public health.  We do that in order to protect and improve public health and individual health in the U.S., and where feasible we also try to engage globally with our international partners, facilitate development, approval and access to safe and effective products and promising new technologies, and to strengthen our Center as a preeminent regulatory organization for biologics.
You may know that we regulate a variety of complex products.  I know you are familiar with blood, blood components and derivatives.  We also regulate vaccines, both preventative and therapeutic, allogeneic products, novel medicines like cell and gene therapies, that includes things like xenotransplantation, and also human tissues and a variety of related devices.
Our research is seen as critical for us to fulfill our regulatory mission.  This diagram is meant to illustrate how we view the role of research.
As you know, most of the products we regulate are going to be driven by a public health need.  That will generate either a novel product or it may also result in an application of perhaps a novel technology to existing products that are already licensed.  But those new things may raise regulatory challenges, and there may be scientific gaps in addressing those regulatory questions that come from these new issues. 
That is where research can provide an important role, both in terms of discovery, developing new tools, providing reference materials, and from that we can also make more science-based regulatory decisions, help inform our development of policy.  As we have better guidance for sponsors, we will have improved data and be able to make a better informed decision as it comes to evaluating benefit and risk.  From that, hopefully the end product will be a licensed product that is both safe and effective and will have a positive impact on the public health.
The organization of our Center is shown here in tissue culture plastic ware.  In the middle is the immediate Office of the Director where deputy and associates reside.  We have seven offices.  The three petri dishes support all of the offices, Office of Management, Communication, Outreach and Development and Compliance, Biologics, Quality. 
The four epindorph tubes are our four offices where we have some component of research.  Biostatistics and Epidemiology is obviously desk-based research, but they have an active research component, and then Office of Cellular Tissues and Gene Therapies, Vaccines Research and Review and Blood Research and Review, which we will be talking about today.
Our research facilities.  We provide some core support in terms of a biotechnology core facility which provides an array of technologies to support the research going on in the Center.  We have some limited core support in things like flow cytometry and confocal microscopy as well.  We have a state of the art vivarium with procedure rooms and the ability to support research using both rodents and non-human primates, and with BSL-2 capacity so that we can support our work with infectious agents.  We also have on site access to BSL-3 laboratories and animal BSL-3 laboratories.
We have an array of scientific expertise within the Center, showcasing a number of novel technologies like NMR, mass spectrometry, flow cytometry and high throughput sequencing.  We have a lot of microbiology, as you can imagine, with parasitology, bacteriology and virology, a lot of immunology, biochemistry and molecular biology, cell and developmental biology.  So it is a very interdisciplinary Center, which provides a great opportunity for cross-disciplinary collaborations. 
Our research scientists are what are called researcher regulators, which means that they spend about up to half of their time doing review activities.  The type of review that these individuals do includes review of submissions to the Center, regulatory submissions, going out on inspections, writing guidance documents, participating in advisory committees like this one, organizing workshops and so on. 
The outcome of this integration of the research and the review responsibilities in one individual means that we can apply our research resources in a manner that is timely and use the expertise in a wise manner to address our regulatory issues. 
We manage our research program through an iterative process which involves evaluation of our in-house and horizon scanning of regulatory and public health needs.  From that we derive a set of priorities for our research programs, both at the Center level and within each office, and the research programs then are designed -- they are PI driven, but they are intended to address priority areas of research.  A critical part of our management is the external review and input that we receive through our site visit reviews of each laboratory every four years, and then evaluation at the advisory committee level. 
Our research priorities for FY 10.  We are in the process of revising these for FY 11.  Our aim, to insure the safety, efficacy and availability of biologic products and use and development of appropriate regulatory pathways through development and evaluation or methods, reagents and standards, evaluation, development and integration of novel scientific technologies and preclinical models for use in product regulation, including the three R's approach, to reduce, refine and replace use of animals, facilitate development of new biologic products for control of high priority public health threats, including pan flu.  You hear a lot about emerging infectious diseases and agents of bioterrorism.  Then in the biostatistics and epidemiology side, we have improving clinical trial design, evaluation, including things like adaptive design approaches, enhancing risk management, assessment and communication sciences, and developing improved analytical tools to access large medical databases to look at active population-based safety surveillance.  This is part of a larger FDA-wide sentinel initiative.
As I mentioned, we do cyclic review of every principal investigator every four years.  The site visit is the external aspect of that, where we do peer review by outside scientific experts in the same field.  Then we also have an internal review that goes through our promotion, conversion and evaluation committee.
In addition to the four-year review, we have an annual review of our research program, where we have a research reporting database where on an annual basis each PI provides a progress report of accomplishments, future plans, budget requests.  It also collects information on presentations and publications, other relevant output, sometimes guidance documents or workshop advisory committees may also be relevant to the research program.
That information is then reviewed at a variety of levels, at the lab chief level, division level and office level, and also at the Center level.  It is looked at for both the relevance to the stated priorities, the productivity of the research program, and the quality and feasibility of the proposed plans.  Then based on that evaluation the funding is allocated accordingly.
The site visit report that you will be looking at today is a draft report that was generated from a subcommittee of this advisory committee.  In this case we will be talking about two separate site visits that were done.  It is your responsibility to evaluate that report and choose whether or not it is acceptable in its current form or needs to be changed.  Once it is approved by the full advisory committee, that final report can then be used in a variety of ways.  It becomes a part of a package for internal peer review by our Promotion Conversion Evaluation Committee.    The PIs take the scientific recommendations as far as direction or other suggestions very seriously to improve their own research program, and management also takes these recommendations very seriously as it impacts resource allocation. 
Finally, I just want to thank the site visit reviewers who came for these two site visits and the advisory committee, your input to finalize this report is very important.    These external reviews really are critical for us to make sure our research programs are fulfilling our regulatory mission.
Thank you.  Any questions? 
DR. HOLLINGER:  Thank you.  The next talk is an overview of the  Office of Blood Research and Review by Dr. Atreya.

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Agenda Item:  Overview of the Office of Blood Research and Review
DR. ATREYA:  Good morning, everybody.  What I will do today is, I will briefly provide you an overview of our mission and related research that is going on in Office of Blood Research and Review.
Briefly, the office structure and staff, I will go through that.  As you all know, Dr. Epstein is our Office Director, and then we have our Deputy Director, Ginette Michaud, sitting there.  Then we have four associate directors for different activities.  One is the Associate Director for Regulatory Affairs, that is Alan Williams, and the Associate Director for Medical -- who is acting now, Basil Golding sitting there in the audience.  He is also the division director for one of the divisions, which I am going to show you on the bottom side.  Then finally, Associate Director for Research, and then we have another Associate Director for Communications and Policy, Jennifer Scharpf.  Most of you know her through her e-mails.
We have three divisions in our office.  Two are research oriented and then the third one is review.  The first one is the Division of Imaging and Transfusion Transmitted Diseases.  Dr. Hira Nakhasi is the Director and then Paul Meade is the Deputy Director.  Then we have the Division of Hematology, which Dr. Golding is going to elaborate a little bit more its activities in his overview.  He is the Director for the division and the Deputy Director is Dr. Fauci.  Then we have the third division which is Division of Blood Applications.  The division director is Richard Barry and the Deputy Director is Cheryl Kotchkin. 
So this is the structure we have in our office.
The office responsibilities are based on the products we regulate.  The three divisions have three different roles to play here.  The Division of Imaging and Transfusion Transmitted Diseases mostly deal with the blood donor screening tests for infectious diseases and also retroviral diagnostics. 
The Division of Hematology has several products.  There are plasma derived products, for example, like IGIV and coagulation products, and blood and blood component collection devices.  Then we also have bacterial detection devices and then also plasma expanders, including hemoglobin based and carrier solutions.
The third division is the Division of Blood Applications.  It has the blood and plasma licenses responsibility and then blood establishment software, and also blood grouping and HLA reagents.       So under FDA's Critical Path Initiative which was initiated in 2004, March, the Critical Path Initiative is a national strategy for transforming the way FDA regulated products are evaluated, developed, manufactured and used. 
There are six priority topics, which are better evaluation tools, streamlining clinical trials, harnessing bioinformatics and moving manufacturing into the 21st century and developing approaches to address public health needs, also, the specific at-risk populations, that is, pediatrics.
These are the variety of topics under which FDA's different centers work up on their own goals. 
We at the Division for Research, an important goal is to support the critical path of the FDA for product development.  What we mean by that is, we try to focus on scientific questions critical to effective regulation.  Then we focus our concentration on areas where our unique role as regulators is most contributory.  Also we have a provision of an infrastructure for investigation of product limitations and failures.  Overall our goal is to facilitate the progress towards goals and promise of 21st century medicine.  The examples are a proteomic and genomic approach and then applications of nanotechnology.
We do have research programs and we have a way of managing them.  When we call in short is the managed research programs in our office.  The way we approach this is that we identify scientific needs and then evaluate available expertise and resources within the Center and within the Office, and the feasibility of success of those programs.  Then we look at the public health significance and expected outcomes of those programs.     Then based on these, our Office currently has six high priority research areas.  These areas were started in 2007, I think in the August 2007 BPAC, and then we are trying every four years to update and refine these as we go by.  The current ones are all equal priority, even though a number of them one to six, they are all equal priorities for the Office.
One is the novel methods of pathogen reduction and inactivation in blood and blood products.  Then the multiplex platforms and high sensitivity methods for pathogen detection, including variance of EIDs and bioterrorism agents, the development of infectious agent panels for assay standardization and standards and reagents for product lot release testing, development and evaluation of proteomics and genomics based biomarkers for efficacy, quality, toxicity and consistency of blood components, their products and their analogs.  The fifth one is development of predictive models including animal models for evaluation of blood components, blood derivatives and their analogs, and then also to study pathogenesis of blood-borne emerging infectious disease agents.  The last but not least one is development of methods to evaluate efficacy of immunoglobulins of pandemic and bioterrorism importance.
How we manage this research program.  I will just give an overview.  At our office we have 26 investigator initiated programs located in seven laboratories, three in the DETTD division, 12 PIs, and then in the DH there are 14 PIs and then there are four labs.  These labs are located in three different buildings, what we call the 29 Complex on the NIH campus.
The subject expertise include microbiology, which includes viruses, retroviruses, parasites, bacteria and -- then cell biology, toxicology, immunology, biochemistry, physiology and hematology.
The way we do the budget process, Dr. Wilson has already given a brief introduction to that.  But each office, how we do that is, individual programs in each division are evaluated annually for public health impact, mission relevance, feasibility and productivity.  Then based on a form that we internally developed, by the division directors in consultation with me and the office director, each program receives a score that translates into some operating dollars for each program.  Then once this process is approved by the office director, funds will be distributed to each PI.  This is the way we operate.
In conclusion, I think I gave the impression to you that research is highly integrated and is integral to the mission of OBRR and CBER.  OBRR facilitates product development and is aligned with FDA critical path research.  The next round at the division level, Dr. Golding is going to provide his overview.
Any questions I will answer.
DR. HOLLINGER:  Thank you, Dr. Atreya.  We will move to the next overview, the Division of Hematology Research Program by Dr. Golding.

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Agenda Item:  Overview of the Division of Hematology Research Program
DR. GOLDING:  Good morning.  I welcome this opportunity to resent the division and the two labs that are being reviewed at this public meeting.
I am going to have two presentations.  The first presentation will be an overview of the division and the second presentation will be an overview of the Laboratory of Hemostasis, including the regulatory and research work.
The Division of Hematology consists of five branches if I am counting right.  From left to right, it is the four branches that are involved in research, the Biochemistry and Vascular Biology Branch, Cellular Hematology, Hemostasis and Plasma Derivatives.  You are going to hear later from Dr. Scott about the Plasma Derivatives Branch.  I am going to present in my second presentation the Hemostasis Branch, which are being reviewed today.  There is also a Clinical Review Branch which consists entirely of regulators.  These other branches perform both review and research, and these are the number of principal investigators in each branch.
I am not going to go over this because Karen already mentioned the CBER mission.  The Division of Hematology in terms of its scope of regulation and research, I think I will be able to convince you that the scope in the division is very broad, very diverse and very complex.  The research helps solve regulatory problems using the critical path and serves to enhance the expertise of scientific investigators who have review responsibility for these products.
Scientific evaluation of biologic products derived from blood include those isolated from blood or plasma, and analogous materials derived from recombinant DNA and transgenic technology.
Clinical indications.  This is not a comprehensive list, but they include a wide variety of clinical conditions, including prophylaxis and treatment of bleeding disorders, trauma shock, infectious diseases, immunological deficiencies, replacement therapy and congenital or acquired plasma protein deficiencies, counterterrorism agents and pandemic preparedness, and also we review volume expanders.
The regulatory products and process.  The whole spectrum of applications that come to the FDA come through our division.  These include biologics, drugs and devices.  The products themselves are diverse complex proteins and also carbohydrate polymers for volume expansion.  We also regulate all the blood cell components. 
Our regulatory decisions are based on scientific data showing safety, efficacy and purity and the decision making process includes internal review, meeting with manufacturers, consultations with other parts of the agency, consultation also with special government experts, presentations to advisory committees and workshops.
To continue to talk about the regulatory process.  I have already talked about the scientific review, but in addition to the scientific review, laboratory investigation is sometimes needed.  You will see some examples of this in my presentation and Dr. Scott's presentation. 
We had been traditionally and still are involved in lot release, doing testing at the lab level.  This is being transferred to another part of CBER, to the Office of Compliance.  We are working very closely with the laboratory in the Office of Compliance to transfer our expertise to that laboratory. 
We also are involved in standard development for lot release of different products.  These standards are often distributed as CBER and/or international standards. We work with other international regulatory agencies to develop these standards.
We are also involved in surveillance.  This involves inspections, adverse event reports and product failures. 
This is an average number from a couple of years ago, just go give you a flavor of the workload.  We get on average nine original BLAs per year, a large number of BLA supplements because we have a large number of products that are already licensed.  These are device reviews.  We get NDAs.  These are drug applications, drug application supplements, abbreviated drug applications and supplements, and original INDs.  The bulk of the work is the original INDs and the original BLAs, and we also have a considerable number of meetings with sponsors on a yearly basis.
In addition to all of that, we are involved in policy and guidance document development, harmonization with other countries.  We now have regular meetings with the European regulators.  We have liaison meetings with other government agencies, regulatory agencies of foreign governments, international bodies, consumer groups, nonprofit organizations such as IVF and NHF and with industry.  Our communications besides the usual letters to sponsors and meetings involve placing things on the FDA website, especially safety notification and dear health care letters.
Getting into the research arena, related to the types of products that we regulate, we have research in the area of protein therapeutics, looking at structure-function relationships and new tools for characterization of proteins, including mass spec, using aptimers, phage display libraries, and lately we now have two crystallographers in the group, and we are involved in looking at crystal structure.
We also look at immunogenecity, which is a major concern with protein therapeutics.  You will see some more details about this in terms of in silico characterization using aptimers to look at structures and developing knockout/knock-in animal models to look at immunogenecity, so these will be more predictive.
Part of the research looks at the polymorphism of protein therapeutics using bioinformatics.  This is to optimize expression and function of these proteins.  We also develop new assays.  You will be hearing a little bit about the new thrombin generation assay that has been developed, and assays looking at van Willebrand Factor, alpha-1 proteinase inhibitor and C1 esterase inhibitors.  We also have a group working on nanomaterials.
Continuing the research.  We have research in the area of infectious diseases.  This is mainly looking at immunoglobulins, so for the pandemic flu and counterterrorism.  We have a research group that has made major progress in identifying neutralizing antibodies against HCV epitopes and understanding the interplay between the neutralizing and the interfering antibodies.
We have a major research effort in oxygen carrying compounds, including developing animal models to test these compound and to develop biomarkers for measuring the efficacy.  This also relates to research on red cells, plasma and platelets, looking at storage lesions.  This came up in a previous discussion today, looking at pathogen reduction, how this may affect these components, and looking at lyophilized products.
The research budget.  They come from different sources.  There is the operating budget, which each division gets funds from CBER's center office.  It goes through our office and then is divided among our divisions.  The additional budget can be obtained through internal and external FDA sources.  This is the internal Office of Women's Health, there is also funds that are available from NIH intramural programs, and there is also the DoD, the DARPA program, critical path, which is part of now an FDA program.
Just to give you some idea of what the average PI can hope to get as an annual budget, it is somewhere between $50,000 to $100,000 as an operating budget and from the outside also an additional $50,000 to $100,000.
The budget allocation that we have developed within our office is based on certain research elements.  Each PI's work is judged and scored based on mission relevance, public health impact, productivity and feasibility. 
Just to give a little bit more breakdown of the resources, an average principal investigator has at least one but maybe two or three fulltime employees.  Depending on his own budget, his or her own budget, may be able to hire additional contract employees.  I mentioned what the budget is.  The space is rather limited.  Each PI in our division has both a regulatory and research responsibility.  Most of the PIs are spending at least 50 percent on regulatory work.
The research output consists of published papers, about 2.5 per year per PI.  Guidance documents for the division, about two per year, and workshops for the division, about two per year.
Thank you for your attention.  I will answer any questions.  If not, I will go on to the next presentation.
DR. DEMETRIADES:  Dr. Golding, thank you very much for this update.  Can you give us some more information about the studies on storage lesions?  Are these studies done in elective surgery or trauma patients, because they are very different groups of patients.
DR. GOLDING:  Just to try and give you the background, we are not involved directly in clinical trials.  We don't have the resources to be involved in clinical trials.  What we are doing mainly is research of the products themselves.  What we have developed in the lab are animal models that can be used to look at delivery of oxygen for example or hemoglobin based oxygen carriers, and look at the tissues and see what kind of lesions you get in the tissues; are these products safe and effective, in other words, they are not inducing some damage to the tissues, and are they effective, are they delivering oxygen.
We are trying to develop animal models and biomarkers that can be used in the type of studies that I think others will do, which is to take young blood and old blood and use it in surgery.  So the level of our research is more related to looking at the products and developing animal models and biomarkers.
DR. DEMETRIADES:  Thank you.
DR. KEY:  I would like to congratulate Dr. Golding on the trauma workshop last week, and particularly the emphasis on bleeding as an end point for clinical trials.  Trauma might be the front end of the wedge, but assessing bleeding as a clinical end point is increasingly important as new hemostatics come along for not only major trauma, but also bleeding disorders, reversal of anticoagulation, et cetera.  We really haven't got this worked out.
So I hope there will be other workshops that maybe will spin off that with emphasis on other areas where bleeding as an end point needs to be figured out.
DR. GOLDING:  Thank you for that comment.  I think we will consider additional workshops in the future related to the types of issues that you are bringing up.
DR. RENTAS:  Are you moving to the White Oak facility here?  If you are, is that going to have any effect on the space, the way you go about doing things on a day to day basis? 
DR. GOLDING:  We have been moving to the White Oak facility for about five years, at least.  We are being told that the move will actually take place probably towards the end of 2012, early 2013.
I'm not the expert in this area, but we are going to move.  We did spend a lot of time with Carolyn and others in developing plans so that we would have adequate animal facilities, lab space, office space and so on.  As we talk, the CBER grows and the space becomes too small, but we are going to get there eventually.

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Agenda Item:  Overview of the Laboratory of Hemostasis
DR. GOLDING:  I am putting the hat on now of the branch.  One of the branches being reviewed today is the Laboratory of Hemostasis. 
The organization of the Laboratory of Hemostasis.  The acting lab chief is Tim Lee, who couldn't be here today.  The PIs that are being reviewed in the site visit are Drs. Chava Kimchi-Sarfaty, Zuben Sauna, Andrey Sarafanov and Mikhail Ovanesov.
I would point out that a few years ago, not very long ago, we did not have any basic research being done in the Laboratory of Hemostasis.  We were doing some standard and assay development, but it is only in the last few years that we were able to recruit these people.  I hope I can convince you that they are doing high quality work, and it serves the mission of the FDA.
These are the players that I just mentioned.  In addition to the PIs, we do have fulltime regulatory reviewers, and this lists them here.  Natalya Ananyeva, Roman Drews, Don Lebel, who just retired, Tim Lee, Ze Peng and Laura Wood.
Just to give you a flavor of the type of products that are regulated in the Laboratory of Hemostasis.  Obviously coagulation factors, Factors VIII and IX.  These are both human plasma derived and recombinant.  Factor VIII/von Willebrand Factor Complex, these are plasma derived, and fibrinogen concentrates.  Then there are bypassing products.  These are used in cases where patients develop inhibitors to Factor VIII and IX, so they are Factor VIII inhibitory bypass agents.  Then there is recombinant Factor VII which is also used as a bypassing product and is indicated for that use.
Other hemostatic agents that we regulate are thrombin.  These come as bovine, human, which is plasma derived, and recombinant human.  We also regulate fibrin sealants.  These are examples of different fibrin sealants.  And anti-coagulant proteins such as protein C and antithrombin III.   This was the first FDA approved transgenic protein.
The regulatory responsibilities of the lab include pre-submittal support, which is reviewer briefing materials for meetings, meeting with sponsors, preparation of summaries of these meetings.  We review applications for investigational products, marketing of new products and changes in manufacturing or indications for licensed products.
I mentioned that we are involved in lot release.  The Laboratory of Hemostasis has been particularly involved in doing lot release.  It was this laboratory that worked out the problems with one of the products, Refacta, and was able to reconcile the differences between the different assays that were out there, the chromogenic assay and the one stage clotting assay. 
We are also involved in the inspection of manufacturing facilities.  This involves compliance and enforcement actions, review of biological product deviation reports, assessment of risk and response, developing policy and guidance, and this relates to product safety and efficacy, patient and physician information, and current good manufacturing practices.
This is just a list of new products licensed by this branch in the last few years.  You have a list here of Ceprotin, Recothrom, Evithrom which is human thrombin, Xyntha which is human Factor VIII, Artiss, which is a fibron sealant, RiaSTAP, which is a human fibrinogen, Atryn which is anti-thrombin III, Wilate which is a vWF/Factor VIII complex, TachoSil, a fibrin sealant patch.
Over these few years, this branch has reviewed 400 BLA supplements, 100 annual reports, 100 other miscellaneous submissions, 1600 lots tested for release, 50 original INDs and the associated amendments.  They participated in facility inspections on site and by phone.  Some of these inspections are in Europe.  They have also been involved in international calibration studies for reference standards.
These are some of the reference standards that we have been involved with in this lab.  I am only listing the ones where we have participated in looking at panels and doing lab work in collaboration with other international regulatory agencies.  So this is a Factor VIII standard, a thrombin standard, Factor IX standard, a newer Factor VIII standard and a vWF concentrate standard.
These are anticipated products.  You can Google and find all of these products.  I'm not revealing anything that is not in the public domain.  We anticipate newer products coming to us such as recombinant porcine Factor VIII, PEGylated coagulation factors, Fc fusion factors linked to coagulation factors to increase the half life, proteins for rare bleeding disorders such as Factor XIII and Factor XI and Factor X, and rADAMTS13 and recombinant von Willebrand factor. 
In addition to anticipating all these newer products in the coming years, we also anticipate an increased workload because of the passage of the health care bill.
Now we are going to the Laboratory of Hemostasis research activities.  First of all, I am only going to give you a glimpse of these -- in the time constraints allowed, I am only going to give you a glimpse of the research, starting out with Dr. Kimchi-Sarfaty.  Her heading is towards more effective treatment of blood clotting disorders, pharmacogenomic studies of ADAMTS13 and related proteins.
In terms of FDA mission relevance, why is this relevant?  A new draft guidance for pharmacogenomics of products regulated by FDA has been published.  Dr. Kimchi was part of the working group to develop this guidance.  This guidance is a new concept paper, guidance on clinical pharmacogenomics, and discusses the following issues.  FDA's current view on whether or not clinical pharmacogenomics studies should be performed as part of licensure studies, general strategies for using pharmacogenomic information in drug development, design of clinical pharmacogenomic studies, incorporation of pharmacogenomic information in the drug label.  However, currently there are no regulations or guidelines vis-a-vis the choice of sequence in the development of a recombinant therapeutic protein.
In her research I am just going to be focusing on one of her projects related to single nucleotide polymorphisms.  What is a single nucleotide polymorphism?  The DNA has these triplets which form amino acids.  You can change one of the nucleotides and change the amino acid that it accounts for, but in this example given over here, you are changing a triplet, and it accounts for the same leucine amino acid.  This is called a synonymous single nucleotide polymorphism.  When there is a change in sequence it is called a non-synonymous single nucleotide polymorphism.
Polymorphisms are defined as a DNA variation in which the sequence is present in at least one percent of the population and is the most common type of genetic variation, and may occur in every 100 to 300 bases.
What is the potential effect of these polymorphisms, single nucleotide polymorphisms on protein structure and function?  With the non-synonymous, where you are getting a change in primary sequence, it is very easy to understand that this could affect very broad aspects of protein expression, conformation and secretion.    However, regarding synonymous mutations, which does not affect amino acid sequence, how can these affect the actual protein?  Work from Dr. Kimchi and others has shown that depending on the nucleotide sequence you can get differences in messenger RNA structure and stability, and that the codon usage can affect the kinetics of translation.  Also the nucleotide sequence can affect alternative splicing.  She has published already examples where synonymous mutations do lead to differences in expression and protein conformation.
The aims of this part of her research is to understand the consequence of synonymous and non-synonymous mutations and SNPs on the development of recombinant therapeutic proteins, and to develop tools to predict the consequences of changes made to wild type sequence during manufacture of recombinant therapeutic proteins.
This is a complicated slide.  What she has done here is taken the ADAMTS13 protein.  These are the different domains.  Using databases she has found single nucleotide polymorphisms across the protein.  Then she studied this in her laboratory.  Some of these are synonymous and some are non-synonymous.  The bolded ones are synonymous.  So for example going from a proline to a proline.  The non-synonymous one are going from for example an arginine to a lysine.
So the question is, how do these nucleotide differences affect the protein.  She has performed in silico, in other words, computational studies of 12 SNPs in ADAMTS13 located in various domains of the protein.  Based on the in silico studies, concluded that there could be changes both in secondary structures on the protein, related primarily to beta sheets and alpha helices.  Codon usage could be different and could cause changes in conformation.  The nucleotide changes could also change messenger RNA structure and stability.
The question then was, how did this correlate with in vitro studies.  All of these 12 SNPs from ADAMTS13 were cloned and studied, using real time PCR, flow cytometry, FRETS, which is a functional assay looking at cleavage of vWF, trypsin digestion and western blotting.  Based on these studies, she was able to conclude that both the synonymous and the non-synonymous mutations affect protein expression, conformation and/or function.
So regarding the 12 -- I am not going to give you the details -- regarding the 12 SNPs, some of them, five of them, were not different from wild type, but in seven of them including synonymous SNPs, she was able to find examples of changes in expression, changes in conformation of the protein, and changes in function of the protein. 
This is obviously important in trying to determine which clone to use in making a recombinant product.
Here we have a case study.  This is regarding Factor IX, in other words, hemophilia B.  This is a mild form of hemophilia in which there is a synonymous mutation in position 107 in the Factor IX gene.  It was found in five unrelated Swedish hemophilia B patients.  Dr. Kimchi performed in silico and in vitro assays and the conclusion was that the computational analysis suggested that the synonymous mutation was critical for protein folding.  It could change transition rate and rhythm, therefore affecting the protein's conformation.  And experimental analyses demonstrated a significant change in conformation between this mutation and wild type Factor IX.
Future studies will focus on developing new assays that can differentiate between different molecular forms of ADAMTS13, von Willebrand factor, Factor VIII and Factor IX, based on increased sensitivity, characterized ADAMTS13 SNPs in healthy TTP individuals without ADAMTS13 and getting into trouble with thrombosis, and correlate the in vitro analysis with in vivo characterization.  Also, to characterize additional Factor IX SNPs and mutations using in silico and in vitro analyses, and study wild type versus the mutated Factor IX in hemophilia B knockdown mice.
I have still got a few more to go through. 
This is Dr. Zuben Sauna's work.  He is looking at the immunogenicity of protein therapeutics.  This is obviously a very important mission related research and involves all protein therapeutics that we are concerned about in immunogenicity.
The importance of the project to the FDA.  The FDA has been regulating biotechnology derived protein products since the 1980s.  More than 200 biopharmaceutical proteins have now been approved.  There is widespread use of these proteins.  It has been demonstrated that all biologicals can elicit antibody responses.  Inhibitory antibodies compromise efficacy and also can cause cross react with endogenous factors to cause serious toxicity.  Even non-inhibitory antibodies can complicate interpretation of toxicity, PK data and PD data.
The specific aim is to develop computational and in vitro methods for the preclinical identification of T-cell epitopes on Factor VIII protein, determine the role of sequence mismatch between the endogenous, albeit non-functional, and infused Factor VIII in eliciting immunogenicity.  So the presumption is that you are tolerant to your endogenous, but then when you get a wild type which is different from the endogenous, you might break tolerance and get antibodies.
Pharmacogenics and the development of inhibitory antibodies to Factor VIII, evaluate the genetic variability in the endogenous Factor VIII gene with respect to polymorphisms is a risk factor for immunogenicity, and evaluate HLA restriction of patients and the consequence of sequence mismatch, and as a risk factor for immunogenicity.  So clearly the ability to develop an immune response is based not only on the Factor VIII mismatch, but on the ability of MHC class two proteins to bind to the mismatched peptides and to engender an immune response.
The accomplishments of this research.  Computational predictions of peptide binding to specific MHC proteins using 56 Factor VIII peptides correlated well with in vitro binding measurements, using purified recombinant MHC plus two alleles with a very low P value.  Historical data showed that some missense mutations in the Factor VIII gene are associated with inhibitory antibodies, while others are not.  Missense mutation results in a localized mismatch between the sequence of the endogenous and infused Factor VIII.  That is, peptides from this region are foreign or seen as foreign. 
An immunogenicity score was devised by Dr. Sauna's lab, and discriminates between mutations that do and do not elicit inhibitory antibodies with a very resectable P value.
A recent report suggests that African-Americans develop inhibitory antibodies at higher frequencies due to the greater diversity in Factor VIII gene polymorphisms in these patients.  Peptides that span SNPs associated with inhibitory antibodies bind with higher affinity to MHC alleles, commonly found in African-Americans.
Demonstration that cells and tissue samples from hemophilia A patients with the intron 22 version synthesized the entire Factor VIII polypeptide chain.  However, the primary sequence is contained in two polypeptide chains.  The intron 22 inversion represents about 50 percent of severe hemophilia patients. The immunological implication of our finding is that these patients, despite a large gene alteration, may be tolerized to the Factor VIII protein.  That is because even though they have this inversion, there is some intracellular expression of the truncated polypeptide chains and some of these individuals may be tolerant to the Factor VIII.
The conclusions from these studies are that a pharmacogenetic approach based on individual patients is necessary for the accurate prediction of immunogenicity.  This is because of the MHC differences between individuals and because of polymorphisms that may be different between individuals.  The extent and position of sequence mismatch between the endogenous and infused Factor VIII due to disease causing mutations in SNPs, and the HLA restriction of the patient are important risk factors for immunogenicity.
Future plans, to develop a hierarchical decision making tool for individualized immunogenicity risk assessment for HLA patients using clinical samples to test the hypotheses; develop strategies to reduce the disproportionate frequency of adverse alloimmune events in vulnerable populations, and determine whether principles developed in Factor VIII for a personalized assessment of immunogenicity risk can be applied to therapeutic proteins in general.
The third of the four PIs, Mikhail Ovanesov.  He has been studying the regulation of blood coagulation kinetics.  This is a public health issue.  Existing clinical assays of blood coagulation have poor predictive value and sensitivity.  Coagulation testing can be improved to reflect physiological conditions.
The research approach was to develop and optimize novel assays that measure global hemostatic function, and particularly investigate potency and thrombogenicity of Factor VIIa, Factor IXa and Factor XIa.
The regulatory contribution would be for product evaluation, and enhancing the assurance of efficacy, safety and quality of coagulation products.
This gives a time line, a seven-year plan.  This is the assay development, assay application.  He is focusing on the thrombin generation test, plot growth assay, and coagulation in mouse models using deep tissue imaging.
The development of the thrombin generation test to facilitate the characterization and regulation of plasma derived products was funded by FDA CBER Modernization Science.  This is another FDA sponsored funding source.  The research goals: optimization of the assay for hemophilia treatments and thrombogenicity, comparative analysis of commercially available and in house thrombin generation test variants, and application to outstanding regulatory issues, for example, comparing products with known controversies in potency assessment such as Factor VIII and Factor VIIa variants.
Now we have a case study.  Dr. Ovanesov was merrily proceeding with his research, and then we got a whole series of adverse event reports regarding immune globulins.  Dr. Scott, who regulates immune globulins, went to Dr. Ovanesov and said, hey, Mikhail, do you want to study this?  Maybe you can figure out what is going on here.
So what Mikhail did I think was quite remarkable.  Octagam is the product.  It has been in the public domain, so I can talk about it.  This is an immunoglobulin intravenous.  In the last year there was a high incidence of thrombotic adverse events, mainly arterial, strokes and myocardial infarctions.  Testing indicated a possible root cause was Factor XIa from previous literature and from the type of clinical presentation.
As a result of the adverse events in TGT testing performed by Dr. Ovanesov, or partly as a result of that, Octapharma voluntarily withdrew the product from the U.S. market in September of 2010.
What did he find?  I am going to let the video microscopy show you what he found.  What he is doing here, he is taking the implicated lots, non-implicated lots and a negative control.  This is going to repeat multiple times, so if you miss it the first time you can see it the second or third time. 
I'm not speaking fast enough, but this is 40 minutes of time lapse photography.  It starts out with a thrombogenic surface and then he starts adding IGIV and you see this clot formation developing.  A little bit of clot forms with the non-implicated lots, which is also not surprising.  We think there may be very low levels of Factor XIa in these products.  But there is no clot developing in the control.  So this is a dramatic presentation of the fact that these lots did contain some coagulant activity.
The ongoing efforts of his research team, distribution of this assay protocol.  What I showed you was video microscopy.  I am not showing you the results of the thrombin generation assay because of constraints of time, but this assay that he has developed with the protocol has been distributed among all the major IGIV manufacturers so that they can screen plasma derived products for thrombogenetic contaminants.  We have also been collaborating with other regulatory agencies and the industry, and we hope to develop standards together with other regulatory agencies.
The relevance of the project to the FDA mission.  Deficiency in Factor VIII results in coagulation disorders.  The disease is treated by infusions of Factor VIII products regulated by the FDA.  Currently new Factor VIII products with prolonged half time in the circulation are under development.  Regulation of these products requires understanding of the structure of Factor VIII in relation to the mechanism of its clearance from the circulation.
That concludes Dr. Ovanesov's work.  He considers that assays that he is developing will help better characterize all the products and the products that we anticipate to come to us in the future.
The fourth project.  This is Dr. Sarafanov's project.  What we are talking about here are the LRP and LDLR receptors.  These are receptors in the liver mainly that catabolize various ligands, including coagulation factors such as Factor VIII.
These receptors are evolutionarily related and similar in structure.  They express in the liver and serve a function to catabolize various ligands, plasma lipoproteins, et cetera.  They cooperate in clearance of Factor VIII.  Deficiency in both receptors results in prolongation of the half life of Factor VIII by five times in a mouse model.  On this basis, it is highly likely that there are molecular interactions between Factor VIII and these two receptors.
What he wants to do is to map the binding site on Factor VIII that binds to these receptors, and also map the binding site on the receptor that binds to Factor VIII.  The selection of Factor VIII amino acid residues will be done by scanning mutagenesis, expressions of fragments of Factor VIII and of the receptor will be looked at.  He will perform binding assays for Factor VIII variants in receptor fragments and will test receptor mediated internalization of Factor VIII variants in cell culture and also test the clearance of these Factor VIII variants in mouse models to see if changing the binding site alters the half life of the protein.  The critical thing is to determine the critical residues of Factor VIII for binding to these receptors.
This is a general overview of what he is going to do.  This is the Factor VIII structure.  Here is the light change in the A3 domain, the biding site is thought to be in the A3 domain, so to perform scanning mutagenesis to decide which part of the A3 domain binds to the receptor. 
In a complementary fashion, he will look at the receptor in the liver, the LDLR receptor, and use two complement type repeat regions from different parts of the ligand part of the receptor to determine which fragment binds to Factor VIII.  It can then perform experiments to change either the LDLR or the Factor VIII to see what effect that has on Factor VIII pharmacokinetics.
The future plans.  The generation of Factor VIII having a decreased affinity for LDLR and LRP yet needs to retain functional activity and not have increased immunogenicity.  The improved Factor VIII would have a longer half life in the circulation, thus requiring less frequent infusions into the hemophilia A patients, and reduced cost for the treatment.
Thank you.  Oh, wait a minute, I've got one more slide.  I like to end happy.
DR. HOLLINGER:  Thank you, Dr. Golding.  Questions in regards to the Hemostasis Laboratory that was presented here?
MS. BAKER:  Not a question, but a comment.  I wish to commend you and your staff for their tremendous efforts on behalf of the patients and the physicians and all the caregivers.  It is absolutely unrecognized, the level of intelligence, creativity, the persistence that you do under very restricted resources, and I wish to commend you.
DR. GOLDING:  Thank you.  I think the credit goes entirely to some of the people that you see in the audience.
DR. RENTAS:  I was going to wait until the last presentation, but since somebody took the lead here, let me go ahead and equal that on behalf of the members of the armed forces. 
You may not realize what you are doing out there to keep the safety of the blood supply that is collected in theater safer, what I do and a lot of us do.  That is because of the amount of work that you do over here.
Let me just give you an example.  The hepatitis C virus rapid test that you just approved this year is already on its way to Afghanistan.  So anything that is collected out there will be tested with this FDA license.  Yes, it is a clinical diagnostic rapid test, but there is nothing else available out there.
So we encourage you to keep on working with us.  At the same time on behalf of the armed forces, we thank you for the work that you are doing out there.
DR. GOLDING:  Thank you, much appreciated.
DR. HOLLINGER:  Thank you for the comment.  Anyone else?  We will move then to the overview of the Laboratory of Plasma Derivatives.  Dr. Scott will give us this overview.

Table of Contents

Agenda Item:  Overview of the Laboratory of Plasma Derivatives
DR. SCOTT:  Good morning.  I am going to try to give you a snapshot of what we do.  I am going to skip a few things for the sake of time, and so that you have a chance to ask questions.
Our lab is the Laboratory of Plasma Derivatives.  We have a mission statement, which is to meet the public health needs for safe and effective products by performing high quality research that directly impacts the safety, effectiveness and availability of plasma derivatives.
This is our group.  It is rather large, but you will notice there are four principal investigators.  Some of the people in our group do regulation only, but most of them contribute to the research in one way or another.  I wanted to have their names out there, because they are the people who are responsible for everything that gets done.
We regulate ten immune globulin products for a multiplicity of indications, as well as specific immune globulins for various pre and post exposure, prophylaxis and treatment of mainly viral diseases.  Also, infant botulism and prevention of newborn hemolytic disease.
We also regulate all the anti-venoms and antitoxins.  These are orphan products, but they tend to be very important if you are one of the people that was bitten by a snake or exposed to botulism, for example.  We regulated the anti-thymocyte globulins, which are made in animals for treatment of transplant rejection.  This is a product that I will be talking about quite a bit today, the alpha-1 proteinase inhibitor product.  Right now it is indicated for treatment of emphysema and in alpha-1 proteinase inhibitor deficiency, but there are many other types of applications that have been proposed.
I am going to follow the format that Dr. Golding followed, to tell you that we do a lot of other things that may not be directly considered basic or more fundamental research on our products, but things that are experiment for our products to remain safe and potent.  That is, developing standards, reagents and methods for lot release testing.
Dr. Yu in particular has typically led this effort.  She and her group has many years in development of international standards and test methods that will increase the safety of plasma derivatives.  Among these are standards for nucleic acid testing for all plasma derivatives or for source and recovered plasma that goes into plasma derivatives, to assure safety against certain types of transfusion transmitted viral infections.
What is ongoing now.  This is a summary review.  We are currently working to test a new CBER potency standard for immune globulins.  This is necessitated by the regulations, which say that we will supply such a standard.  That is called Lot 177.  We are in the middle of starting a collaborative study with industry and other partners to establish the potency against polio and measles. 
Development and testing of a new international B19 virus.  That is standard for plasma testing.  Quite a few of these have already been developed by us and by others.  There is still a little more work to do.
We recently took the co-lead in characterizing the first international A1PI standard, and we do a lot of research on potency testing and safety correlates.  Some of this research that I am going to tell you about has to do with tests that we could develop to make the products -- to demonstrate potency better for our products or to create products with better potency.
We also develop predictive models for preclinical product evaluation of safety and efficacy.  I will be talking about most of these except for the influenza project, where we are looking for more rapid test methods for neutralizing antibodies so that plasma can be screened for these antibodies in hopes of making an influenza immune globulin.
We also evaluate the efficacy and safety of immune globulins for pandemic and counter terror responses.  This includes the animal model evaluation of vaccine immune globulin products and other therapies, evaluation of animal models for other counterterrorism related immune globulins.  Right now it is a matter of public record, there are anthrax immune globulins and botulinum immune globulins that are under investigational study.
The research framework that we have, and the standards and testing that we have developed or helped to develop give us a framework to scientifically address issues that come up.  I know that Dr. Golding just mentioned Dr. Ovanesov's ability to figure out what was wrong with those lots of Octagam that were implicated in thrombotic adverse events.  Similar things are happening all the time.  We are able to have products in the lab and to test those and to perform research on them to provide insights into usually potential adverse events.
So for example, in the past four years we have evaluated immune globulin products that were reported to cause clinically relevant hemolysis in patients.  Dr. Marszal has identified biochemical changes in alpha-1 PI products, detected those and analyzed them, and that led to changes in product manufacture to eliminate some of these biochemical changes from the natural protein. 
Dr. Golding has already talked about the evaluation of thrombosis associated IGIV product lots.  We are heavily involved in the manufacturing root cause evaluation for that problem.  But what he has done very much informs what we are able to do.
Dr. Yew identified B19 virus transmission by a coagulation factor.  This has led to increased screening and enhanced safety of our products with respect to nucleic acid testing.  I will be talking about Dr. Golding's project to identify bacterial constituent contaminants in products.
In preparing for the future, some of the things that we would like to do generally is address the lack of predictive animal models for immunogenicity, particularly using alpha-1 proteinase inhibitor as a model.  There are a number of things we could do to perhaps improve IGIV safety, efficacy and potency, and these include enhancing neutralizing potency for specific immune globulins, characterizing aggregates and their impact on potency, safety and immunogenicity.  Large protein molecules have a tendency to aggregate in solution, and this is a problem not only for our products but for many products across CBER.  But we don't really know very much about how they form and exactly what their structures are.
We want to facilitate development of potency assays that are most relevant to immune globulin use for primary immune deficiency patients.  We are very interested in a platform for rapid collection and testing of plasma so as to manufacture specific immune globulins that can be used in pandemic or bioterrorism scenarios.  We are also interested in developing preclinical models for inhaled alpha-1 proteinase inhibitor to look at efficacy and immunogenicity and toxicity.
So that is the broad future outlook.  There are many other things.  What I am now going to do is give you a snapshot of what people are doing in the lab.  This is in your site visit notebooks, and there are a lot of other things there, but I just wanted to highlight a few of the projects that are ongoing.
The first I am going to talk about is improving treatment for progressive vaccinia, which is a formerly lethal complication of the smallpox vaccine seen in immune compromised people.  In the setting of a smallpox outbreak, and I know that we don't seem as worried about that now as we used to be, but nevertheless it is still considered a category A threat.
Immune compromised people are likely to be exposed to the vaccinia either through inadvertent vaccination or through contact exposure.  This has actually happened since military recruits received this vaccination.  That is where we have sometimes seen the complication.  It is not used routinely anywhere else other than for lab workers.  They can get it by contact exposure with someone who has been vaccinated as well.
The treatment and post exposure prophylaxis for progressive vaccinia are not well defined.  There still is a high mortality rate.  If patients don't perish from this, they do require a lot of intensive support until they get better.
We thought it would be useful to develop and characterize an animal model to study the pathogenesis and treatment of progressive vaccinia.  That kind of a model could be used to assess new therapies and current products, and it would help us understand the mechanism of potency of vaccine immune globulin, which could lead to improvements of products and improved potency assays for product release.
We looked at the severe combined immunodeficiency mouse as a model.  We scarified these mice at the base of the tail, and they developed lesions similar to the human lesions, with a similar clinical course, a slow progression and eventual lethality if untreated.  This is responsive to vaccinia immune globulin, because early treatment eliminates infection, that is, treatment beginning two days after exposure to the virus, and vaccinia immune globulin in combination with topical cidofavir can result in long term survival, even if treatment is delayed for seven days after infection. 
Our conclusions were that the SCID progressive vaccinia model simulates many features of human progressive vaccinia, and it could be useful for testing combined treatments and new antibody treatments.  You can't test these in people because there simply aren't enough cases, fortunately.
The studies do also support the likelihood that early post exposure prophylaxis might prevent progressive vaccinia in susceptible hosts.  We have ongoing studies to identify the mechanisms of action of the vaccinia immune globulin, particularly with respect to the FC portion of the molecule.  We have also begun funding animal model studies of maternal fetal vaccinia complications.
I am going to switch gears almost completely.  Dr. Zhang is proposed for a promotion to principal investigator.  I am going to tell you about one of his very striking findings that I think could have a big long term impact on how hepatitis C virus immunity is understood, and hopefully on vaccine development as well as plasma derivative development. 
A large number of people, 170 million worldwide, are infected with HCV, including four million in the U.S.  HCV associated cirrhosis is the leading indication for liver transplantation in the U.S. 
However, the public health problem is, we have no vaccine licensed. There is no immune globulin product licensed for prophylaxis or for treatment.  This is in contrast for example to hepatitis B virus.  And treatment for this disease is only about 50 percent effective with the current antiviral regimens.
So to address this problem, Dr. Zhang took the strategy that he wanted to identify the molecular and structural characteristics of epitopes that are critical for HCV neutralization.  He came up with an unexpected finding of what we call interfering antibodies.  I am going to explain this as briefly as I can.  This is probably the most complicated slide out of the group of slides I have, but I show it to give you an idea of what is happening.
He studied the E2 protein.  This protein is known to have neutralizing epitopes, but the exact nature of those epitopes has not been well understood.  He took a region of the E2 protein and he made overlapping peptides.  Then he affinity purified a polyclonal hepatitis C immune globulin intravenous preparation using each of these peptides.
What he ended up with was four preparations, one that contained antibodies to this whole A peptide, preparation to the B, to the C and to the D.  Then he took these preparations and he tested them for their reactivity to the epitope they were absorbed against, and also to these other regions.
What he found was not exactly what would have been predicted.  For example, he looked at the ability of the D antibodies to bind the D epitope and sure enough, they did.  They also bound as you would expect the A epitope here.
But when he looked at antibodies to this entire region, they did not bind here.  Why?  His hypothesis was that some of the antibodies to this portion of the A peptide were interfering with biding to antibodies of this portion.  Why does that become important?  That becomes important because the antibodies to the D epitope are the ones that are able to neutralize HCV in vitro in an infectivity assay.  A antibodies don't do it, control IGIV doesn't do it.  This is 100 percent infection, essentially. But if you add A antibodies to D antibodies, what happens is, you get rid of this D neutralizing activity. 
So based on structural predictions concerning this epitope, his hypothesis is that D antibodies find a neutralizing epitope in the E2 protein, A antibodies find a non-neutralizing epitope, but these two epitopes are conformationally very close together.  What happens if you have a mixture of these antibodies is that the A antibody which has pretty high affinity binds here, and it blocks binding of the neutralizing antibody.
That isn't the only evidence that he has for this.  He has shown in many different ways that this is likely to be the case.  For example, he depleted the interfering antibodies and increased the HCV IGIV neutralizing activity by doing this.  He also depleted interfering antibodies in infected chimpanzee sera, and that revealed cross-genotype neutralizing antibodies that had formerly been blocked.
He also has evidence that interfering antibodies are found at higher levels in chronic HCV patients and in people who recovered from HCV.  He has evidence from sera samples from one chronic HCV patient before and after infection that interfering antibodies in that patient arose prior to neutralizing antibodies.
What does this mean?  It means that this kind of immune globulin preparation might be greatly improved by removing interfering antibodies and enriching neutralizing antibodies.  It also means that you can figure out how to design epitope based binding assays to measure HCV neutralizing antibodies in sera for clinical trials, and HCV IGIV products.  But also with respect to vaccine design, and he has an active collaboration with Steve Feinstone at CBER to look at this, vaccine design might be improved if you could develop antigens that will provide neutralizing but not interfering antibody responses.
Oddly enough, Dr. Golding has been up here.  I would like to invite him up here to talk again, but it would probably take too much time.  He is part of our immunology group in the Lab of Plasma Derivatives.  I am going to be presenting a snapshot of his work as well.  That is the use of toll-like receptor assays to detect and identify microbial contaminants in biological products.
His goal was to develop a panel of toll-like receptor expressing cell lines.  These should be able to detect the presence of microbial components in biological products.  We can go into how do those get there, but the point is that they do get there.  Although these types of products are aseptic except for live vaccines, they still may contain microbial components that can be immune stimulating and cause adverse effects in people. 
This is a case study where he used these cell lines to determine what was the likely contaminant that caused adverse events in patients receiving an IND product.  Adverse events were described.  These were things such as fevers and chills and an intolerance of this recombinant product. 
We received a panel of blinded product samples labeled A, B, C and D.  Two of those samples were made from two different manufacturing methods for this product, and both have passed lot release testing, including testing for endotoxin, total residual DNA assay and host cell protein ELISA.
One of those samples was a positive control and one was a negative control, and he tested these against the cell lines with various TLR ligands.  He found that sample B activated cell lines expressing toll-like receptor five. The ligand for toll-like receptor five is flagellin, which happens to be an E.coli protein.  He did in fact show that this sample contained flagellin by western blot, and that was confirmed by mass spec.
So what this demonstrated is that there truly was and is a practical application for this kind of a panel, where you have the ability now to test products for contaminants that might cause adverse events. 
So this is what I am going to present about his work, but there is a fair amount of other work also included that is relevant to this in the site visit report.
Dr. Marszal has proposed to become a staff scientist.  I am going to talk a little bit about her work, which involves the biochemistry and biophysics of protein aggregation, particularly looking at the alpha-1 proteinase inhibitor, which has a tendency to aggregate in vivo in people with a certain variant of this protein, as well as in vitro, in products.
These protein aggregates in general are not well characterized.  That isn't just for alpha-1 PI, but for many protein aggregates that can occur in our products.  But structural and biochemical understanding of alpha-1 PI polymers is critical to enable development of therapies for alpha-1 PI ZZ variant associated liver disease.  So this liver disease is a result of aggregates of this abnormal alpha-1 proteinase inhibitor that form and deleteriously impact liver function.
But it is also important to understand aggregation of the normal alpha-1 proteinase molecule to improve product stability.  To address these scientific questions, she investigated the basis of alpha-1 PI polymerization in the structure of those polymers in vitro and in vivo. 
This is ongoing work.  I am going to show you just two slides that illustrate more or less what we are up against and what she has been able to do so far.
First of all, this is HPLC from three of our licensed alpha-1 PI products.  What you can see here is that they will have different aggregate profiles, and they all have aggregates.  You can also see this by isoelectric focusing, where we are looking at normal plasma alpha-1 PI.  These are three of our products that are licensed, and they have different aggregate profiles and charges.  You can also see that the alpha-1 PI itself has some differences. I am not going to go into that, but we know what these differences are now.
She also looked at alpha-1 PI products by dynamic light scattering.  Even for these two products you get a different hydrodynamic radius, which also implies some aggregate formation and different aggregate formation in quantities.
So she developed some models in vitro of alpha-1 PI polymerization.  She did this by denaturing and renaturing alpha-1 PI.  This is an artificial system, but it becomes important when you want to develop and study models, because some of these may be relevant to how polymers form in vitro and in vivo.  Basically, that by denaturing under different conditions, high temperature, low pH, or use of guanidinium isothiocyanate, you get different polymerization patterns of alpha-1 proteinase inhibitor.
When you refold these, yu also get certain polymerization patterns.  She has been able to characterize these polymers by a number of methods.  What this has resulted in without going into the details is a lot of evidence that alpha-1 proteinase inhibitors can not only polymerize in a head to head model which has previously been described, but also that you can have polymerization at the tail of alpha-1 PI.
This is important, because it means now you can form a chain of polymers.  Now you have a way to do that.  You are not just forming dimers.  In fact, this is what can be seen by atomic force microscopy.
This slide is half of the size of what it used to be, but there is still a lot on here.  I do want to say a sufficient amount about her work.  She was able therefore to propose a new model of polymerization of alpha-1 PI.  This had a direct impact on selection of formulation of buffer for a specific product, so as to avoid those tail to tail polymer forms.
She is working on continued investigation of alpha-1 PI polymer structure using atomic force microscopy, and this will help to establish if there are more than one polymer types in alpha-1 PI deficient patients.  I can already tell you from looking at her AFM that there are.  So it makes this even a more complex type of disease to tackle in the liver. 
She proposes to investigate whether polymers formed in different tissues have the same structure.  She can also now link the physiological relevance of model polymers to that of the polymers that form in PIZZ transgenic mice and in people.  She has ex vivo samples of liver tissue from PIZZ patients.
There is always the possibility in the future, this method or combined methods for polymer characterization could be used for personalized treatment, and that methods used to characterize polymers might include other things to determine their surface structure.
What I think we really need to get at is how these polymers are forming in vivo, so as to be able to develop molecules that will prevent that polymer formation.  This is another important method that might enable us to do this.
She also has undertaken a large collaborative study as the lead of protein aggregates in alpha-1 PI.  I am not going to talk about that more here, but there is extended cross center and cross industry interest in this project.
Very briefly, I am going to talk about another alpha-1 PI project.  This is addressing a different public health issue.  That is very simply that intravenous alpha-1 PI is approved to treat emphysema in patients with alpha-1 PI deficiency.  It makes sense.  They don't have enough alpha-1 PI, they have emphysema, this should work.  But the original licensure was based in 1987 on achievement of serum alpha-1 PI levels, and long term clinical studies are very challenging to accomplish, because the progression of emphysema is quite slow, and there aren't that many patients with this deficiency to study.
However, we do know based on the literature and based on data that has been presented publicly that even treated alpha-1 PI deficient patients may continue to have declining pulmonary function and exacerbations of their disease.  So maybe there is something that can be done to improve treatments for these patients.
We need to better understand the effect of alpha-1 PI in lung tissue, in addition to elastase inhibition.  This may inform the dose around alpha-1 PI therapy. 
What Dr. Reed showed in the lab is that alpha-1 proteinase inhibitor which is seen in this column is expressed in normal adult lungs and also in lungs of people with various conditions, in this case RSV lower respiratory tract infection and cystic fibrosis. The expression of alpha-1 PI appears to be largely confined to macrophages.  This is staining for CD68, a macrophage marker.
Lung macrophages are derived from recruited monocytes from the bloodstream.  This is staining for alpha-1 PI in monocytes.  Very simplistically, they undergo in situ differentiation either to homeostatic, relatively anti-inflammatory wound healing and cleanup type of macrophage, or into in the setting of an acute infection a pro-inflammatory macrophage which is highly susceptible to apoptosis.  It recruits and encourages adaptive immune responses, and it has its own anti-microbial effects. 
Where does alpha-1 PI stand in all this?  We used two model cell lines initially, and we have also used some human elutriated monocytes to look at the differentiation of monocytes into macrophages and to look at their expression of alpha-1 proteinase inhibitor. 
What we found was that alpha-1 proteinase inhibitor expression is inversely related to markers of inflammation in these cell lines.  Without going through this, the cool colors are the anti-inflammatory, so it is the macrophages, and the hot colors are the pro-inflammatory sorts.  In terms of alpha-1 PI expression, you are looking at unstimulated differentiated or differentiated macrophages with the addition of a toll ligand. 
The U937 and 28SC cell lines secrete a lot of alpha-1 PI when stimulated and even unstimulated in this case.  The pro-inflammatory macrophages, which make a lot of TNF alpha and also IL-1 beta, don't make any alpha-1 proteinase inhibitor.  We were not able to induce this easily in these cells at all, or in anything close to these amounts.
The research hypothesis that we are now undertaking to investigate is that alpha-1 PI regulates macrophage function.  There are a number of studies that do describe other functions of alpha-1 proteinase inhibitor, but these are tissues other than lung. 
One of the things that alpha-1 PI can do is scavenge nitric oxide, which inhibits activating signal amplification.  It may be able to inhibit inflammasome activation or apoptosis, definitely apoptosis.  It is involves hepcidin, which inhibits iron utilization.  We are looking at this in our cell lines.  We plan to confirm any positive findings in primary monocytes and macrophages.
We hypothesize that recruitment and maturation of lung macrophages is disregulated in alpha-1 PI deficient patients.  This is one of the things that contribute to the ongoing lung damage and disease that they have subsequent to infection and their eventual pulmonary decline.            It is also possible as a corollary that increasing alpha-1 proteinase inhibitor expression in patients with normal expression could modify macrophage responses to injury and infectious insult.  So the implications are optimization of delivery of alpha-1 PI to the lungs if that is needed.  In other words, even though a lot is given intravenously, do they actually need more, and is enough getting on site to exert this anti-inflammatory effect.
Also, if it turns out that they do have a function in the lung, potency assays could be -- these alpha-1 PI expressing pulmonary macrophages have function related alpha-1 PI in the lungs.  Potency assays can be developed to reflect additional alpha-1 PI activities.
Overall, we think this will improve understanding of pathogenesis of alpha-1 PI deficiency.  This pathway may be useful as a target to ameliorate other lung diseases. 
I want to thank everybody, but particularly my group for the breadth of their knowledge and creativity and their very hard work, and their dedication to the mission of improving products to improve the lives and health of patients.  I also want to thank our management for their support and the site visit committee for their very hard work in looking at all these diverse projects and evaluating them, and to thank you for your attention to what is a large number of projects presented in a short period of time.
Thank you.
DR. HOLLINGER:  Thank you, Dr. Scott.  Questions?
DR. TRUNKEY:  Yes.  How far along is Dr. Zhang in getting phase 1 trials with the hypothesis neutralizing antibodies.
DR. SCOTT:  Well, we don't perform these trials.
DR. TRUNKEY:  I know that.  How can you fast track that?
DR. SCOTT:  It is very important.
DR. TRUNKEY:  You nicely demonstrated a forward progress in dealing with hep C.  We need this badly in the clinical area.
DR. SCOTT:  I could let Dr. Zhang address this if he would like to.  But if he feels too reticent at the moment, I would say that we are doing what we can to promote intelligent and informed evaluation and design of vaccines in an HCV immune globulin.
One of the things that he has done to encourage that obviously is to publish all of his work pretty much as rapidly as possible.  He has a number of collaborations with people in the area, particularly at NIH and at CBER, with respect to both vaccine design and to further understanding of these interfering and neutralizing antibodies.  He has already done these depletion experiments.
But I think what you are asking me is who is developing this product and are they acting on what you have seen here.  I have a hard time to answer that and stay in what I am allowed to say publicly.
DR. HOLLINGER:  Dr. Zhang, do you want to see if you can answer?
DR. ZHANG:  This is the first stage of the study.  Lots more work needs to be done.  Lots of mechanisms we still are in progress of studying.  Obviously the implication is very important, so we will try to see if we can have a good plan and to see if we can make progress on the clinical application part.  Obviously it is a very important area.
DR. HOLLINGER:  Can you pass the immune globulin through a column with the FHL for a non-neutralizing antibody on the column and take it out very simply?  That would be a very rapid way of doing this.
Secondly, do you know what the concentration of these non-neutralizing antibodies is in patients who have been treated and cured of their hepatitis C, and still have antibodies around?
DR. ZHANG:  The first question.  There is a possibility.  We did some preliminary study.  We could remove the non-interfering antibody.  Non-interfering antibody is very high affinity and very high level in the chronic patients.  We studied single patient and we studied vaccine and showed we could able to remove the interfering antibody.
In terms of the second question, we still do not know what is the clinical relevance of this interfering antibody.  The question is, because there are lots of interfering antibodies in the patient, we should sort out what is the interfering antibody, what is the mechanism first, and to study more population of patients and get a conclusion on it.
DR. HOLLINGER:  Fortunately with the new direct antiviral therapy, the care rates have gone up to maybe 65, 70 percent or so, which is really pretty good. 
DR. ZHANG:  That is the one of the genotypes.  There is about 12 or ten genotypes in HCV.  All the other genotypes, that is not a response to the interfering combination therapy or retroviral therapy.  So this is the way we have to address it.
DR. KEY:  It might be a question for either your or Dr. Golding.  In the way the protein therapeutics are going, maybe this is a futuristic question, but do you see a time when we may need to improve upon nature and use less immunogenic products, bioengineer less immunogenic products?  We see in hemophilia patients who develop inhibitors, this is the major complication now of clotting factor concentrates, and it applies in some of these other deficiency states and so on and so forth.
So as we get to recognize better the particular epitopes and the immunology, do you see that as something that is going to be mandated even in the future in terms of reducing immunogenicity of products?  Perhaps it is a little bit theoretical now.
DR. SCOTT:  It seems to make a lot of sense.  I think one of the problems is that when you are looking at immunogenicity, you tend to only find out how immunogenic something is when you put it into people.
One of the things that we are interested in is developing more humanized animal models for immunogenicity that might be able to assist in that prediction as well as undertaking whatever the structural and sequence and immunodominance information that exists out there, and using it to intelligently design recombinant products.
DR. GOLDING:  Can I just add a couple of points?  In my presentation, this was Dr. Sauna's work, there is a lot of information based on computational methods and databases that you can identify in any protein using different software and algorithms, which sequences would be immunogenic.  That has to be coupled with MHC class two, to which the peptides bind. Then there are other software algorithms for antibody induction.
So theoretically what you are asking, I think there is already a lot of information out there that can help you predict whether a protein is going to be immunogenic in a certain population.  Also by inference you can manipulate the protein. Obviously you have to manipulate it in a way that it removes immunogenicity but that retains function.
DR. HOLLINGER:  Dr. Luban, sorry for not introducing you as a new member of this committee for today's discussion.  Could you tell us also where you are from and so on, and then go ahead and ask your question?
DR. LUBAN:  Sure.  Naomi Luban.  I am from Children's Hospital and George Washington University, and I am here to assist the committee in the review of two of the laboratories.
But I have a specific question for you.  That is in regards to biovigilance for IGIV as it particularly relates to homolysis in children.  Do you foresee any organized way of collecting data on this, in view of the fact that most of the children who receive IGIV receive very high doses, two grams per kid, usually over two or sometimes even days for Kawasaki's, for example. 
In the pediatric hematology community, we recognize that either low grade or a flavorant hemolysis is occurring in some of these but not all of these children.  Yet there really hasn't been a prospective organized approach to identifying which kids are at higher risk.
DR. SCOTT:  Dr. Yu is here.  In case she wants to say something, she is invited.  But you bring up a very important issue.  In our recent workup of some adult cases of hemolysis, the products themselves met their lot release specifications for anti-A, anti-B and anti-roD.  For anti-roD it is quite low, for anti-A it is a certain titer relative to the standard. 
However, the establishment of those specifications I'm sure was based on some epidemiological work that has been done.  However, the dose that the patients were receiving was not precisely taken into account.  I think it is possible, even if you have an acceptable lot release, that you are still going to have product that if you give enough, you are giving a high enough dose of hemolysins, it is going to cause hemolysis in those patients.
So one thing that we would like to address in the future is whether or not the specifications actually need to be lower or need to be lower in certain instances where those high doses are given.  IGIV as you know was originally given and dosed for immune deficiency, and that is usually around 400 to 600 mgs per kg.  Whereas for Kawasaki and for ITP and for CIDP, you will have higher doses, and certainly for a lot of the off label uses that is the case.
So it is a very important problem.  I would say next to the thrombosis problem, those two still are things that we all need to work on hard to enhance patient safety.
In terms of biovigilance that is ongoing, that is not something that we are tasked or able to do.  I think if we could have more biovigilance it would be helpful.  I wonder if I could ask you whether there is any kind of consortium or organization that might be willing or able to undertake at least some more biovigilance than we are able to accomplish.  We only receive voluntary adverse event reports.
DR. LUBAN:  We can talk about that maybe off line.
DR. SCOTT:  Okay.  Dr. Yu has something to say.  She is the one who has done all the work, by the way.
DR. YU:  This is Megan Yu.  As Dr. Scott mentioned we have over the years have anti-A, anti-B, anti-D standards, so we have set maximum specification.  Now because they tend to use large dose, we may have to lower the ratio.
We also have an internal discussion about developing some IGIV having a lower titer of anti-A, anti-B and anti-D for such clinical use and so forth.  I just wanted to comment about that.
DR. SCOTT:  The other thing that we are very often missing is, we get these reports of hemolysis, but post hoc the patient hasn't been worked up for the hemolysins.  So we sometimes see eluate results, and sometimes they are pretty uninformative.  But many, many times we don't see that.  So we don't know if it is a combination of antibodies or if there are a few rare antibodies that are sometimes causing it.  We don't have a good feel for what proportion of cases are due to which kinds of antibodies.

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Agenda Item:  Open Public Hearing
DR. HOLLINGER:  Thank you, Dr. Scott.  I think we at this point would move into the open public hearing.  There are no prior requests to address the committee at this time, so I am going to put it open.  Is there anyone here in the audience during this open public hearing who would like to speak? 
Not seeing anybody, we are going to go into a closed committee discussion.  So we will ask that all members of the public leave, and only those FDA members who are going to be part of -- only the authorized FDA staff are in attendance, as well as the committee.
So we will take about a ten-minute break while we get this taken care of.
(Whereupon, the open session adjourned.)

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