P R O C E E D I N G S (8:30 a.m.)

Agenda Item: Administrative Remarks.

DR. FREAS: Good morning, and welcome to the 19th session of the transmissible spongiform encephalopathy advisory committee. I am Bill Freas. I am the executive secretary for today's meeting.

As announced in the Federal Register, and amended in the Federal Register, today's meeting and tomorrow's meeting are open to the public and the public is welcome to attend both days.

At this time, I would like to go around the head table and introduce the committee members to the public. Would the members please raise their hand when I call their name. I will be starting at the right side of the room at the audience's right.

In the first chair is Dr. Sue Priola, senior investigator, laboratory of persistent and viral diseases, Rocky Mountain Laboratories.

The next chair is empty right now, but it will soon be occupied by Dr. Michael Geschwind, assistant professor of neurology, University of California, San Francisco Medical Center.

The next chair is our consumer representative, Ms. Florence Kranitz, president of the CJD Foundation, Akron, Ohio.

The next chair is empty and that will be soon occupied by a new committee member, Dr. Laura Manuelidis, professor and head of neuropathology, Yale University School of Medicine.

Next we have David Gaylor, president of Gaylor Associates, Eureka Springs, Arkansas.

Next we have our industry representative, Dr. Taryn Rogalski-Salter. director of U.S. regulatory policy, Merck Research Laboratories.

Next we have Dr. Nick Hogan, associate professor of ophthalmology, University of Texas Southwestern Medical School.

Next we have Dr. Mo Salman, professor and director, animal population health institute, Colorado State University.

Around at the head of he table is our chair, Dr. Glenn Telling, associate professor, department of microbiology, immunology and molecular genetics, University of Kentucky.

Next we have Ms. Jan Hamilton, advocacy director, Hemophilia Foundation of America.

Next is another new member to this committee, Dr. Mark Powell, risk scientist, office of risk assessment and cost benefit analysis, U.S. Department of Agriculture.

Around he corner of the table we have Dr. James Lillard, associate professor of microbiology, Morehouse School of Medicine.

In the next chair we have Dr. lynn Creekmore, regional epidemiologist, AFIS veterinary services, U.S. Department of Agriculture.

Next we have Dr. James Sejvar, neuroepidemiologist, division of viral and rickettsial diseases, Centers for Disease Control and Prevention.

Next we have Mr. Val Bias, co-chairman, blood safety working group, National Hemophilia Foundation, Oakland, California.

Next we have another new member, Dr. Ronald Brookmeyer, professor, department of biostatistics, Bloomberg School of Public Health, Johns Hopkins University.

Next, Dr. Susan Leitman, chief, blood services section, department of transfusion medicine, National Institutes of Health.

Next is another new member, Dr. James Mastrianni, assistant professor of neurology, University of Chicago.

Next we have Dr. Richard Colvin, center for immunology and inflammatory diseases, Massachusetts General Hospital.

The empty chair will soon be filled by Dr. Richard Johnson, professor of neurology, Johns Hopkins University.

Dr. Bernardino Ghetti could not be with us at today's meeting. I would like to welcome everyone else and thank you for coming.

Now I would like to read the conflict of interest statement into the record. The Food and Drug Administration is convening today's meeting of the transmissible spongiform encephalopathies advisory committee under the Federal Advisory Committee Act of 1972.

All members of the committee are special government employees or regular federal employees from other agencies, and are subject to federal conflict of interest laws and regulations.

The following information on the status of the committee's compliance with the federal conflict of interest laws, including but not limited to, 18 US Code 208 and 21 US Code Section 355(n)(4) is being announced in today's meeting and will be part of a public record.

FDA has determined that members of the committee are in compliance with the federal ethics and conflict of interest laws, including but not limited to 18 US Code section 208, and 21 US Code section 355(n)(4).

Under 18 US Code 208, applicable to all government agencies, and 21 US Code 355(n)(4), applicable to certain FDA committees, congress has authorized FDA to grant waivers to special government employees who have financial conflicts when determined by the agency's need for that particular individual's services outweighs his or her potential conflict of interest, section 208, and where participation is necessary to afford essential expertise, section 355.

Members of the committee, including consultants, appointed as temporary voting members, appointed as temporary voting members, are special government employees or regular federal employees.

They have been screened for potential conflicts of interest of their own as well as those imputed to them including those of their employer, their spouse, minor child.

For the discussion topics, topic one, which is experimental clearance of transmissible spongiform encephalopathy infectivity in the plasma derived factor VIII products, and topic two, which is possible criteria for approval of donor screening tests for vCJD, these interests may include consulting, expert witness, testimony, contracts, grants, CRADAs, teaching, speaking, writing, patents and royalties and primary employment.

Today's agenda topics are considered general matters discussions. In accordance with 18 US Code Section 208(b)(3), general matters waivers have been granted to the following:

Drs. Ronald Brookmeyer, Michael Geschwind, Bernardino Ghetti, James Lillard, Laura Manuelidis, and James Mastrianni.

Previously approved waivers for Mr. Val Bias, Dr. Lynn Creekmore, Dr. Nick Hogan, Ms.Florence Kranitz, Dr. Glenn Telling, Dr. Mo Salman, are in effect for this meeting.

A copy of the written waiver statement may be obtained by submitting a written request to the agency's freedom of information office, Room 12-A-30, of the Parklawn Building.

Dr. Taryn Rogalski-Salter is serving as the industry representative acting on behalf of all related industries and is employed by Merck Laboratories. Industry representatives are not special government employees and they do not vote.

With regard to the FDA's guest speakers, the agency has determined that information provided by these speakers is essential.

The following information is made public to allow the audience to objectively evaluate any presentations and comments made by the speakers for topic one:

Dr. Lisa Ferguson is employed by USDA in Hyattsville, Maryland. Dr. Jiri Safar is associate professor, University of California, San Francisco. He has a financial interest in a company that is developing prion diagnostic products. As guest speakers, they will not participate in the committee deliberations, nor will they vote.

In addition, there are regulated industry and outside organization speakers at today's meeting making presentations.

These speakers may have financial conflicts of interest associated with their employer and with other regulated firms.

These individuals who were invited here to represent their companies, these individuals were not screened by FDA for their conflicts of interest, since they are representing regulated industry.

This conflict of interest statement will be available for review at the registration table. We would like to remind members that, if discussions involve any products or firms not already on the agenda, for which they have a personal or imputed financial interest, they need to exclude themselves from such involvement, and their exclusion will be noted for the record.

FDA encourages all meeting participants to advise the committee of any financial relationships that they may have with firms that could be affected by the committee discussions.

So ends the reading of the conflict of interest statement. Before I turn the microphone over to the chair, I would like to ask, if you have a cell phone, please put it in the silent mode, so that you won't disrupt those sitting next to you. Dr. Telling, I turn the meeting over to you.

Agenda Item: Opening Remarks.

DR. TELLING: Thank you, Bill. I would like to also welcome everybody here today. We have a full agenda so, without further ado, I think we will get on to the committee updates. The first speaker is going to be Dr. Ferguson, who will update us on U.S. and worldwide BSE.

Agenda Item: Committee Updates. US and Worldwide BSE.

DR. FERGUSON: I am just going to go through. If you have my handout, you see I have a whole bunch of slides, but I am going to rip through things pretty fast.

They are pretty straightforward slides, just to update what is happening in regard to BSE around the world, and then finally, in the United States.

Just a reminder for everybody, total cases worldwide still is greater than 189,000 cases. Again, most of those, actually greater than 96 percent, have still occurred within the United Kingdom and more than 89 percent of them occurred before 1996 and before.

So, when you still see all these numbers of cases, the vast majority of that reflects what happened in the United Kingdom in the late 1980s, early 1990s.

If you are interested in current totals, the OIE's web site actually has fairly good numbers, fairly updated numbers, about all countries that report cases.

Let's start off and talk about what has happened in the European Union. EU monitoring, since 2001, they have done very intensive surveillance, mandated by legislation.

In 2005, they have recently released their compiled report on all of the monitoring that went on in 2005, greater than 10 million tests in all the 25 member countries, in cattle.

Of those, about 1.5 million are what they call risk animals, which would be the same as our targeted population in the United States, and 8.6 million approximately are animals either 24 or 30 months old at slaughter.

Of that, 561 positive cases, 448 of those are in their risk or suspect animals, and 113 in the healthy slaughter population.

Again, in 2005, same as in 2004, both the number of cases and the overall prevalence in tested animals continues to decrease. The number of cases decreased by about 35 percent, overall prevalence by about 29 percent.

These reductions in the number of cases, also the increasing age of positive cases -- if you read their report, there is a lot of very good information about age of cases and how that progresses over the years. I didn't include that there, because that would have made me go on for far too long this morning.

Both of those do indicate success of control measures in Europe. They also provide very good details on analysis by year of birth of positive animals.

The peak of exposure actually appears pretty well defined in a few of the member states. This is the same as it was last year, which is a good indication.

France and Ireland, that peak appears to be about 1995, Germany, Belgium, Italy, The Netherlands, the peak appears to be about 1996. So, that is a good indication that perhaps -- well, it could be one of two things.

Either that is when the control measures really started to kick in or, more important, it could also be an indication of the increased surveillance that began in 2001. It is a little bit early to tell.

They are also doing significant monitoring in small ruminants, 614,000 tests in sheep and goats. Of that, 959 positives. Obviously, most of that is scrapie. They had no confirmed BSE cases in small ruminants this year.

Just to show you what has happened over the past five years within Europe, as you see, total number of positives by year, and that is decreasing every year, the same thing here in the risk or suspect animals versus healthy slaughter.

Those are very good indication that the control measures that they have in place in Europe are working and are doing the job that they are supposed to do.

Let's move to North America and a brief update here on BSE in Canada. As a reminder, Canada has been doing active surveillance, targeted surveillance, in the population where they are most likely to find disease since 1991.

As everybody knows, in 2003, they identified their first native case, in May of 2003. After that period, they significantly increased their surveillance.

So, these are just their numbers, in 2003, about 5,700 samples, two positives. One was the one that we found here in the United States in Washington State in December of 2003. So, increasing their surveillance again, with no cases in 2004, two in 2005, and five to date in 2006.

They did provide a very detailed epidemiological summary that was made public in January of this year. They are continuing that work and hopefully will have some more updates out fairly soon.

What this summary shows, it really talks about their geographic cluster theory, and the idea that the links between rendering, feed production, livestock production, tend to occur in clusters, in a fairly well defined geographic area. It is only logical, then, that that would be where the disease could cycle, would be in that type of a cluster.

What this report shows, which went through the first five cases, I believe, it does define links between most of these cases and links in this cluster.

There are also linkages in the more recent cases, again, to that same cluster and, as I mentioned earlier, hopefully they will be putting out some more updates with as good epi work in the near future.

To focus a bit on Japan, BSE was first identified in Japan in late 2001. Actually, it was September 2001. They imposed a feed ban, than, after that first diagnosis. So, the feed ban has only been in place since 2001.

Here is the number of cases. It stays about the same here until the past two years, where it has jumped up a bit.

One note about Japan. A lot of their surveillance and the testing numbers that you see have been primarily in clinically normal animals, or animals presented at slaughter.

It has only been since about 2004 that they have really increased their focus on the targeted animals or the risk population, as we would define it.

Now, let's move to the United States. As everybody knows, we have been doing active surveillance in the United States since 1990, and we are targeting the population where the disease is most likely to be diagnosed. That is the most efficient way for us to conduct a surveillance system.

The assumption is that if we can't find disease in that population, then it is even more unlikely for us to find it in the non-targeted population.

So, we can use the data that we get from that targeted sampling to extrapolate information to the broader cattle population.

Our targeted population has always been, and continues to be, those animals that have some type of a clinical abnormality that could even remotely be considered consistent with BSE.

So, these would be non-ambulatory animals, dead stock, which are animals that die for some unexplained reason, central nervous system cases, either called to our field people or on farms.

We work with veterinary diagnostic labs, public health labs, for rabies negative animals, and then we also work with our colleagues in FSIS, for those animals that are condemned on ante-mortem inspections when presented to slaughter.

Everybody knows we ran an enhanced BSE surveillance program that began in June 2004. Our initial intention was to run that for 12 to 18 months, with the goal of getting as many samples from the targeted population as we could.

We actually ran on a bit longer than the 18 months and ran through August of 2006, greater than 785,000 tests during that whole enhanced program. During that time frame, there were two positives identified during that effort.

Just to show on a monthly basis what we did in our enhanced program, as you can see, there is a little bit of a cycle.

With the population that we are sampling -- these are animals that are clinically abnormal in some way, and with the facilities where we were collecting, these are animal disposal facilities, rendering facilities, 3D, 4D, salvage slaughter plants.

Animals tend to get sicker, die, be culled in the winter. So, we always had a little peak in the winter. I just wanted to show folks, most folks don't quite understand exactly where we were sampling and why that might occur.

Let me explain a bit about OIE standards. These are the international animal health standards for BSE surveillance.

This reflects changes to those standards in May 2005. This is what we are using as our guidance for how we do surveillance from here on out.

It is a weighted point system. Previously it was a simple table that said if you had cattle population X, you need to get Y number of samples.

Now it is an interesting system where it recognizes that you are more likely to find the disease in certain subpopulations.

So, you get more points for that population where you are most likely to find the disease. We have four surveillance streams.

They are clinical suspect, which would be those animals with really pretty classic clinical signs of BSE, causality slaughter -- these would be those animals -- these are European terms, sometimes they fit with North American terms, sometimes they don't.

Casualty slaughter are those animals that are clinically abnormal. They would be condemned on antemortem inspections.

So, these are those non-ambulatory animals, could be the very weak, emaciated, thin, just some type of a subtle abnormality.

Fallen stock are dead stock, essentially, those animals that die for unknown reasons. Then, healthy slaughter is pretty self explanatory.

I don't know if you can actually read this. Hopefully you can. This shows the point system where it recognizes that you are most likely to find disease here in a clinical suspect.

So, you get 750 points for a clinical suspect between four and seven years old. That subpopulation is where you are really most likely to find disease if it is present.

You get essentially limited points for sampling in routine slaughter. So, what countries can do to use this table, you can access whatever population you like to meet the standards, and then the table says you need to get X number of points for a certain design prevalence, 300,000 points over a seven year period at a design prevalence of one in 100,000.

So, a country can then use this table and figure out what type of samples in what population they want to sample to reach those numbers of points.

So, you could sample a pretty small number of clinical suspects and reach that point value, or design prevalence, or you can sample a much higher number of routine slaughters. It recognizes that you can access both of those populations. It is just how many samples you want to get.

We did a summary of not only our enhanced surveillance program, but also what we have done for surveillance for the past seven years. That was made public in April of this year.

Just to give you kind of a graphic example of how to do this point system, these are the points that we obtained in our surveillance for the past seven years.

So, close to three million OIE points over the past seven years in the different surveillance streams. For those who ask about this healthy slaughter one, knowing that we really are not sampling healthy slaughter animals, this is a function of our data base.

Especially in some of these earlier years, our data might be somewhat limited. If we could not pull out of the data base a specific clinical sign, to assign this to one of the other surveillance streams, by default it would go into the healthy slaughter for this calculation.

So, with that summary of data, not only did we put out there just a raw data summary, we also did an estimate of BSE prevalence in the United States.

We used two methods to do this. One is the BsurvE model, which is a model developed by the Europeans with some input from our colleagues down under in New Zealand.

It looks at what we know from the epidemiology of the disease in Europe and factors in population data when animals are most likely to leave the population.

It can be used to help a country set up a surveillance system, can also be used to estimate prevalence.

We then also tweaked this model a bit and came up with what we call the Baysian birth cohort model, which incorporates what we would expect to see the effects of a feed ban, which the Bsurv doesn't show, and it also sets up some linkages between birth cohorts.

We also did several sensitivity analyses in this report, just to make sure that our assumptions weren't way off base.

The overall conclusion was that the BSE prevalence in the United States is very low, less than one infected animal per one million adult cattle.

If you are interested in most likely values, with the Bsurv model the most likely value was seven infected animals, with the BBC model the most likely value was four.

With the sensitivity analyses, those values ranged from one up to about 40, which all of those then led us to this conclusion, pretty solid, that the prevalence is less than one infected animal per one million cattle.

So, what are we doing now and where are we going from there? We have used these same methods and have moved forward into what we call ongoing surveillance.

We have been transitioning here since the end of August. What we figure is about 40,000 samples per year, again, still from this targeted population.

This will allow us to continue to monitor the status of U.S. cattle and will allow us to detect prevalence if it starts to increase.

We did this calculation, again, based on our analysis of the enhanced surveillance data and using the Bsurv model.

We first of all looked at OIE recommendations, which are at a design prevalence of one in 100,000. We wanted to make that a bit more sensitive. We wanted to stick with one per one million. So, we used the Bsurv model to estimate sample numbers and points.

Again, think of that basic premise that I described for the OIE, with a different number of points for different subpopulations.

With this, we need to get three million analytical points over a seven year time frame. When we look at what we did in enhanced, we averaged about 9.5 points per sample. So, we just divided and that gets you to about 40,000 samples per year, assuming we will get the same average points over a seven year time frame.

That is a very quick run through of both an update of what is going on in the world, what the international standards are, and where we are headed in the future. I think I have time for questions.

DR. TELLING: Yes, you do. Thank you, Dr. Ferguson. Are there any questions?

DR. GESCHWIND: Dr. Ferguson, when you were talking about the Japanese cases, could you comment about the ages of the animals?

Before 2004, I believe they were testing every cattle within a certain age range, and that they did find BSE cattle among those that would not be identified with the current methods in the United States. Can you comment on that?

DR. FERGUSON: I am not quite sure what you expect me to comment on. I think as we all know Japan, in 2001, by their regulations, required that every animal slaughtered, regardless of age, be tested for BSE.

They have recently changed that reg slightly, and it is only animals 20 months of age and older be tested at slaughter.

They did find two animals, a 21 month old and a 23 month old -- unless my memory escapes me at this point in time -- young animals, apparently normal at slaughter.

They were positive on the screening tests, negative on IHC, and then positive again with the way Japan has done the western blot.

They have put those into mice to see if there is transmission. To the best of my knowledge, those results aren't out there yet, but there is no indication that they have gotten any signs of disease in those mice.

Perhaps some of the researchers in this group can clarify that, if I am mistaken on that point. That is the situation in Japan.

DR. EPSTEIN: Lisa, my question is, does USDA have information about food chain controls in non-U.S. countries? Are we in a position to comment how adequate the food chain controls are from country to country?

DR. FERGUSON: I can speak for AFIS per se, since we are not the food safety group. That is really not part of the information that we have.

Our colleagues in FSIS, through their equivalency evaluations, work with certain countries, will have some information on essentially the red meat inspection and control, similar to what they would do in the United States. They would have that type of information. I am not sure how much further in the food chain you need to go beyond that.

MS. KRANITZ: Dr. Ferguson, in the United Kingdom and Japan they have found cases of BSE in animals that are not symptomatic. So, I would like to know why the USDA doesn't consider random sampling of healthy stock.

DR. FERGUSON: I think it is shown in that OIE table. We all recognize that you can pick up disease in animals before they begin to show clinical signs.

It all comes down to what is the purpose of your surveillance program and how do you best accomplish that purpose.

In the United States, the purpose of our surveillance program is animal health monitoring, to help us define either the presence or the absence of disease in the U.S. cattle population.

The purpose is not to identify each and every individual case of BSE that might be out there. In fact, that is an impossibility to do with current test methods that are there.

So, we have chosen the most efficient way by targeting that population where we are most likely to find disease if it is present, to give us sufficient information to help us define the status of the U.S. cattle population.

DR. MANUELIDIS: As a point of information, how many cows, adult cows, are there in the United States, so that the 459,000 that were tested is what percentage of the population versus Japan.

Then the second question is, the Japanese found more cases. Would you sort of compare a little bit or say something about the method of testing and the adequacy of the American testing method in its generality as compared to the more extensive Japanese testing and European testing.

DR. FERGUSON: Let me make sure I remember it. The first question was about adult cattle populations. We estimate adult cattle populations to be about 42 million currently.

I haven't done those numbers. I am not going to stand up here and do math in my had and divide 759,000 over 42 million. I will let you guys do that, if you so choose.

As far as the adequacy of our surveillance efforts in the United States compared to other countries, we feel very comfortable and very solid with the information that we have obtained, both over our enhanced efforts for the past two years, and all of the surveillance that we have done prior to that.

The prevalence estimates that we have done uses some very solid analytical methods, we believe, to come to the conclusions that we have.

So, we feel like the surveillance that we have done, targeted in the population that we have, is sufficient to help us define what is going on in the United States.

DR. MANUELIDIS: I am really not trying to be difficult. Perhaps you can't answer the question. I really wanted to know the specific methods that you use and how they compare to the Japanese or the Europeans.

DR. FERGUSON: Sorry, I forgot that part of the question. I assume you mean specific test methods.

DR. MANUELIDIS: Yes.

DR. FERGUSON: We are using -- actually, at this point in time, I think everybody knows we are using the Biorad test for our screening. Then, for confirmatory testing we will use both IHC and a western blot to help confirm disease.

That is essentially the same as in Europe. They have other rapid tests that are also available for use, not just Biorad, but still confirmatory testing is with IHC and/or western blot. A similar thing in Japan. They are also using Biorad, and confirmation is with IHC and western blot.

DR. HOGAN: In terms of identifying your targeted population, how are those animals being identified? Is it by government employees or by industry, and what is your sense of the compliance rate?

DR. FERGUSON: We have had very good cooperation with the industry over the past two years, actually since 1990, since we have been doing surveillance.

As everybody knows, our surveillance is not mandatory. We do have some regulatory authority to do that, but we have chosen not to exercise it at this point in time. We have gotten where we are today with cooperation with the industry.

In our enhanced program, since our goal was to get as many of these samples as we can, it has not been an issue of picking and choosing.

It is an issue of, okay, is this animal old enough to meet our target, is it greater than 30 months of age, does it meet this target, and is the sample of sufficient quality that you can test it. If you are pouring the brain out, we don't really want that.

So, that was sort of the criteria. We have had AFIs personnel collecting samples, we have had state personnel collecting samples. We have had contractors collecting samples, where we have done the initial training, set them up on our data base, and go in and cross check on them. So, it is a wide variety of folks who are collecting the samples for us.

DR. TELLING: Okay, thank you, Dr. Ferguson. If there are no further questions, I think we should move on. The second update is from Dr. Scott, who will update us on variant CJD epidemiology and transfusion transmission.

Agenda Item: vCJD Epidemiology and Transfusion Transmission.

DR. SCOTT: This is going to be a brief update of vCJD epidemiology and transfusion transmission cases. First, I am going to mention what the total number of cases is worldwide of clinical diagnosed variant CJD.

The total number of cases right now, as of August 2006, reported on the UK web site is 196. This is both deceased and diagnosed and still alive.

These are the top three. In the United Kingdom, there are 162 cases, in France 20, Republic of Ireland four, and then there are a number of other countries that have had one to two cases each reported, including the United States.

I want to point out that, in the case of Italy and many of the cases in France -- about 19 -- and the Netherlands, those patients in those particular countries had no significant travel outside of their home countries.

So, in other words, it might be speculated that these cases were acquired endogenously. Some of the other countries had travel to the United Kingdom of less than six months, such as the patient in Portugal and the patient in Spain. The point I mean to make is that not all of these cases are directly derived from visiting the United Kingdom.

The rate of variant CJD deaths has been declining in the United Kingdom over the past several years. This is the number of deaths from definite and probable variant CJD reported in the United Kingdom.

I am showing you from 2000 to the present, but remember that the first case was published in 1995 and probably developed symptoms in 1994.

I am beginning here with the peak year where there were 28 deaths from variant CJD in the United Kingdom. As you can see, while the years go by, you get a decline in the number of cases. So, in 2006, there were three deaths reported.

I want to point out that, in the United Kingdom right now, there are six patients right now still living with this disease. So, it doesn't look as if we are going to have a large number, as we did in the year 2000.

There have been three reports of transfusion transmission of variant CJD in recipients of non-leukoreduced red cell concentrates from donors who subsequently, post-donation, developed variant CJD. They were healthy at the time of donation.

Two of these cases had already been reported at the time of the last advisory committee meeting, but the third case was reported in February of 2006.

Two of these cases were recipients that developed clinical variant CJD. The donors to these patients developed their disease about 18 to 42 months after they donated blood.

The recipients developed symptoms of variant CJD six-and-a-half to eight years after receiving the transfusion from these donors.

The new case had a donor who developed clinical variant CJD 18 to 20 months after he or she donated, and the recipient developed variant CJD eight years after receiving that donation.

In addition there was one infected asymptomatic recipient of blood from a patient that developed variant CJD. This person died of an unrelated illness five years post-transfusion but, at autopsy, the variant CJD associated PRP protein was found in the spleen and lymph nodes of this person.

The other thing that makes this case unusual, besides being diagnosed when asymptomatic, is that they were heterozygous for methionine and valine at PRP codon 129.

So, this is a genotype of the prion protein that previously had not been reported in people with clinical variant CJD.

This was the first and the other two types are MM, which are all the clinical cases reported so far, and VV, and we will get to that in a minute.

To continue on the same theme, I am showing you an update or new information that has recently come out concerning the study by Hilton et al that was published in 2004.

This was a United Kingdom tissue survey where anonymized tonsil and appendix samples were taken from subjects that had undergone surgery between 1996 and 1999 in the United Kingdom.

The samples that were studied were from patients aged 20 to 29. Very interesting and important, three out of the 12,674 samples that were deemed adequate for study were positive, suggesting one in 4,225 people in this age group might actually be infected with variant CJD. All of the positive samples did come from appendices.

Now, what is new about this is that prion protein genotyping was done on two of these samples. In this first sample, there was not enough to do genotype testing, but the tissue was taken and used in a transmission study into mice, and those results are pending. We don't know what has happened to those mice just yet.

In the second subject sampled, the genotype was found to be valine homozygous. So, this was the first report of an infection in a valine homozygous person. The second was also a valine homozygous individual.

So, to summarize, variant CJD clinical cases are declining in the United Kingdom. We have had three transfusion transmission infections reported in the United Kingdom, one fairly recently.

I would just like to point out that, out of the 18 identified living recipients of blood from people who came down with variant CJD, recipients that have survived at least five years post-transfusion, now three out of 18 of these people have developed vCJD infection. Two of those are clinical and, as I showed you, one of them was preclinical or subclinical at the time of death.

This implies a fairly efficient transmission by blood. This amounts to about 17 percent. Also, we now know that all three prion protein genotypes at codon 129, the MM, the MV and the VV, are susceptible to infection.

What we don't know is whether people with this genotype ever develop clinical illness. This brings up the continued possibility that there are silent and asymptomatic infections that may never become symptomatic, but may pose a risk of iatrogenic transmission to others.

In particular, we are concerned with blood and plasma, but there are other iatrogenic transmissions to be considered as well. That is all for my update. Thank you all for your attention.

DR. TELLING: Thank you, Dr. Scott. Any questions of clarification for Dr. Scott?

DR. GESCHWIND: Dr. Scott, just regarding the Saudi Arabia case, as I recall, when that was originally presented at the academy meeting, that patient had lived in the United States for, I believe, greater than six months and, on the way to the United States had spent the night in London on the way there. Any thoughts about that, in terms of the risk of transmission?

DR. SCOTT: I think that is somewhat similar to the Japanese case, where that person spent 24 days. This has been reported so far in two different ways that I have seen.

One is the WHO report dating from June, where they state that only in France, Italy and the Netherlands did the people have no significant travel outside their home countries.

In the United Kingdom, it is reported differently, in that they looked at cumulative residence in the United Kingdom of greater than six months, how many people in the other countries had that kind of residence in the United Kingdom.

In Saudi Arabia, they report zero. So, obviously, that is a person that could have been there for a night or three months and wouldn't have been counted in the UK way of tabulating things.

I think the question does always become, was this endogenously acquired or acquired in some other country, and it probably isn't possible to answer that for certain. It may be that a single exposure to high titer BSE could infer infection.

DR. COLVIN: In the case of the UK tissue survey, out of the patients that were surveyed, that 12,674, was it known if those people, for one thing, had had any blood transfusions or, secondly, if any of them had been users of any kind of plasma or plasma-derived products.

DR. SCOTT: That is a very good question. In order to get the study accomplished, they had to completely anonymize the samples. So, those people will never be identified and we don't have any information about them.

DR. TELLING: Any other questions for Dr. Scott? If not, thank you very much. Our next speaker is Dr. Williams. He is going to talk about a draft guidance for industry, an amendment. He is going to be talking about a donor deferral for transmission in France since 1980.

Agenda Item: Draft Guidance for Industry: Amendment.

DR. WILLIAMS: Good morning. I am going to present a very brief update on recent draft guidance issued by FDA pertaining to deferral of donors with a history of transfusion in France since 1980.

The current recommendations are for vCJD related donor referrals, as have been seen by this committee many times, but I wanted to runt through them quickly just for the new members of the committee.

Most of these, or all of these, are captured in guidance to industry published in January 2002. Deferrals include residents with travel of greater than or equal to three months in the United Kingdom from 1980 to 1996, residents with travel of five years or greater in Europe for the same period of time.

For donors of source plasma, this criteria applies only to France, which is considered to have five to 10 percent consumption of UK Beef, and therefore be at proportionately higher risk compared with the rest of Europe.

Combined with the presumed prion production reduction in the course of fractionation, this deferral was modified specific for plasma donors.

In addition, donors who spent greater than or equal to six months on U.S. military bases in Europe between 1980 or 1990, or 1980 to 1996 respectively for regions in the north and the south, are deferred.

This is based on importation of UK beef into U.S. commissaries, and this differed between the northern and southern bases during this period of time.

The guidance also defers donors for history of transfusion in the United Kingdom from 1980 to the present and for receipt of bovine insulin sourced in the United Kingdom after 1980.

At the meeting of this committee on October 14, 2004, the committee reviewed current FDA regulations regarding vCJD related donor eligibility.

After considerable discussion, they did not make recommendations for further FDA actions to protect the blood supply.

However, there were discussions at the meeting concerning the predictive value of donor questions that were used to exclude TSE risk and just how effective the questions were, as well as the feasibility of deferral for history of transfusion outside of the United Kingdom, but no specific recommendations were made at that meeting.

Subsequently, at the February 8, 2005 meeting of the committee, FDA brought the issue back for consideration, based largely on several recent observations at that time.

At that time there were two observed variant CJD transmissions associated with transfusion and it was recognized that two recent variant CJD cases observed in France had had a relatively large number of prior blood donations.

That is not necessarily a scientific rationale for considering the issue, but it really did raise the visibility of the fact that any potential patient could be a blood donor.

At the time, as well, there were some actions in Europe where donors were deferred for any previous transfusion.

In france, this had been in place for some time since 1998, in The Netherlands deferral was implemented for any history of transfusion in 2004, and in the United Kingdom this took place in 2005.

Also discussed at that meeting was the potential impact of any increase of donor deferral for history of transfusion.

The history of transfusion deferral for the United Kingdom had already been accomplished. The calculations for this are reflected in the transcript, but were estimated to be about two per 10,000 donors.

Computed proportionally, the estimated loss for donor who had a history of transfusion in France was figured to be 1.4 per 10,000 donors.

Also, under discussion at that meeting, was the potential for deferring any donor who had a transfusion anywhere in Europe.

When considering this excluding the United Kingdom, that would add another three per 10,000 donors. There were no data available regarding source plasma donors and their history of transfusion or travel, but simply due to the younger age group of the source plasma donor, this would be expected to be somewhat less.

In the TSEAC deliberations at that February 8 meeting, TSEAC recommended deferral of blood donors transfused in France since 1980 by a vote of 12 for, three against, and one abstention.

However, the committee did not recommend deferral of blood donors transfused elsewhere in Europe since 1980 by a vote of zero to 15 against and one abstention.

By a somewhat mixed vote, the committee also did not recommend deferral of plasma donors transfused in France. That vote was five for, seven against and seven abstention, or other European countries, with a unanimous vote of 16 against.

In issuing its draft guidance for industry, FDA is basing the guidance on the rationale of being prudent preventive measures to help prevent or reduce the risk of vCJD transmission by transfusion.

These factors are the relative likelihood of dietary BSE exposure in France - and this has been an underpinning of any of the discussions that have been held through the years about potential donor exposure.

At the time of assembling the guidance, there had been three presumptive cases of variant CJD transmission by transfusion. So, this was no longer a theoretical possibility.

There were 14 definite or probable vCJD cases observed in France. It is now observed that the variant CJD incubation period may be as long or longer than 12 years, and asymptomatic prionemia may be over three years prior to the expression of illness in an infected donor.

With respect to plasma donations, experimental studies of prion reduction and fractionated plasma are reassuring.

However, not all fractionated products have been studied and observations do not necessarily reflect the blood form of the variant CJD agent.

So, the guidance itself is published as a draft amendment to the January 2002 guidance entitled, Donor Deferral for Transfusion in France since 1980.

FDA recommends deferral of donors who have received a transfusion of blood or blood components in France since 1980.

This applies to whole blood and blood components intended for transfusion, as well as blood components intended for further manufacturing into injectable products including recovered plasma, source leukocytes and source plasma.

The implementation target for this is within six months of publication of the final guidance and the mechanism that will be used.

This amendment is published as draft, but the 2002 guidance will be updated and published in final to incorporate this as final guidance. Thank you.

DR. TELLING: Thank you, Dr. Williams. Question?

DR. HAMILTON: Has there been any consideration in the Untied States of the areas of the country that the people tend to eat brain matter and offal, consuming that? Has there been any consideration of that?

DR. WILLIAMS: There has been consideration of it. In fact, the NHLBI sponsored red study actually did a survey of consumption of brain matter and had some preliminary data on that.

I think this has been an area of consideration but I think to date the scientific evidence supporting this as a potential factor in transmission of disease hasn't been strong enough to support this as a deferral.

DR. TELLING: Thank you very much. If there are no further questions we can move on. Dr. Cai from Telecris Biotherapeutics, will discuss some critical factors influencing prion decontamination using sodium hydroxide. This is a PTTA collaborative study.

Agenda Item: Critical Factors Influencing Prion Decontamination Using Sodium Hydroxide.

DR. CAI: Good morning. Thank you very much for inviting me. On behalf of the Plasma Therapeutics Protein Association, I would like to present the results of a collaborative study designed to understand the critical factors influencing prion decontamination using sodium hydroxide.

This work was primarily done at Telecris Therapeutics. It used to be Bayer. Some of the work was done at Bioreliance.

So, we are all working together to establish a systematic, comprehensive approach to minimize the risks associated with potential SE transmission.

So, as the first line of defense, we have donor deferral and material control. As a second line of defense we have the capacity of the manufacturing processes to remove or clear prions.

In supplement to that capacity, we have cleaning and sanitization procedures. If those procedures can inactive prions, that would add additional benefit in terms of risk reduction. So, this is my focus of the talk today.

So, speaking of prion inactivation, we know that a prion is very resistant, very difficult to inactivate, using conventional viral inactivation methods, simply because, on the one hand, it is a nuclear acid base to viruses and we are dealing with protein prion infectious materials.

However, the prion has its own vulnerability, and it has its own weaknesses, because proteins can be unfolded under many circumstances.

Over the time, researchers have developed many methods to inactivate prions, including enzymatic digestions coupled with detergent treatment, and also acid or strong base treatment, or also some other chemical reactions such as titanium dioxide, photocatalytic inactivation.

Let's not forget our old friend, which is the strong base, which has been used widely in the industry to clean equipment, which includes potassium hydroxide and sodium hydroxide.

Those are readily available and inexpensive, rapid, ineffective and comparable with most of the major equipment, like stainless steel equipment, although it is not very comparable with silicon based materials. Those can be treated or disposable.

So, actually, over the time, researchers performance many studies on sodium hydroxide in terms of inactivation of prions, all the way back to the 1980s.

They used various spiking material or model prion agents for either rodents or humans, and they examined various concentrations, various temperatures, as well as various incubation times in terms of treatments. Of course, you got various results as well.

Then this is a reduction factor of the output prion titer relative to the input after specific treatments. You can see there are various reduction factors based on the conditions.

However, if you look closely, whenever there is a presence of detergents, there is a good reduction. So, please keep this in mind, and this is very relevant to our discussion.

Secondary, you can also observe that among these reduction factors, you know, most of them are quite effective.

However, not all of them give a complete inactivation. In other words, there is still residual infectivity or prions remaining after the treatments. So, the question is why.

That is quite consistent to what we have observed during the early stage of our study, the Bioreliance. In this case used scrapie brain homogenate, which contains hamster prions at one percent, and mixed with sodium hydroxide at .1 molar, incubated at 18 degrees.

The top panel is the titration of the input material. So, you can see quite significant amounts of prions gives us -- this is a half log dilution.

After the treatment you can see the majority of the signals -- the signal strength is significantly reduced. However, there is still remaining signal.

So, there are two questions here. One is, what is behind this dramatic reduction after the treatment. The second question is, what is the nature of this residual signal.

So, we set to address these questions by designing experiments that would mix the purified scrapie brain homogenate with sodium hydroxide, and incubate it with or without two percent sarkosyl, which is a detergent, after incubation at the various temperatures and the various time periods.

Then the sample is withdrawn and neutralized and treated with proteinase K in order to detect the pathogenesis conformation, as is run by electrophoresis and detected by western blot to see the signal strength of the prions.

Now, what we observed was that if you have detergents in the sample, then the residual signals can be eliminated to below the detection limit of western blot.

On the upper left panel at four degrees, you have this much of input material. In the absence of sarkosyl, after 60 minutes, we observed about three logs of reduction with a residual signal.

When the temperature is elevated, then the reduction is slightly increased. However, in the presence of sarkosyl, at 15 minutes, the reduction is increased and, at 60 minutes, the reduction is more than 4.6 logs. So, it reduced below the detection limit. At the elevated temperature, this disappearance occurs earlier.

So, we know that a detergent mainly affects the lipid composition or the disrupted structure of the lipids and detergent has very little effect on the overall structure of proteins.

So, it is highly possible that, in the sample, there are two subpopulations of prions. One is protective by lipid components and the other is protein alone.

So, this one is protective against, not accessible, by the sodium hydroxide. So, after the treatments and when you use proteinase K to probe the structure, obviously this structure is not going to be digested, resulting in a remaining signal.

Now, in the presence of detergents, the protection is removed and the entire population is vulnerable to sodium hydroxide or extreme pH. Then, when you use this probe to probe the structure, then the structure is no longer there.

Now, prions are about protein folding and miscoding. So, it can exist in a normal conformation with alpha helixes and exposed epitopes. Those epitopes can be accessed by antibodies as well as proteinases.

It can be mis-folded into this pathogenic form. So, in this case, the structure is mainly beta sheet and the epitopes, some of the epitopes, are buried and no longer accessible by the antibody or proteinases.

Then in order to inactivate this moiety we need to somehow unfold it or degrade it. So, this is the place, I think, that is for the prion activation.

So, this experiment was designed to further analyze the conformational change behind the prion inactivation or the structural change of prion proteins upon the incubation with sodium hydroxide.

In panel A, which is in the absence of detergent, and absence of proteinase K treatment, the trace buffer saline is a control.

It gives an input, the titer of input sample, and sodium hydroxide treatment you can see pretty much remaining, the signals are pretty much remaining the same, with very little reduction.

That tells us the peptide chain backbone is pretty much preserved after the treatment. However, the conformation is no longer there because in the panel B, once you use proteinase K to probe this structure, the structure is obviously greatly damaged, with a big reduction in terms of titer. However, there is a little residual signal again.

Now, in the presence of detergents, again, the protection was supposed to be removed after sodium hydroxide treatment.

There is 90 percent of the signal that was reduced in the absence of proteinase K, which tells us actually the peptide chain in this case, or the side chains of the epitope are damaged.

In the presence of both detergents and proteinase K we can see the signal disappeared completely using this assay because the structure is unfolded and it was digested by proteinase.

To further support these observations we conducted immunoprecipitation assays, tried to demonstrate that the sodium hydroxide unfolds the prions.

So, in the normal conformation this epitope is exposed to the solvent, accessible to antibody. If you have antibody beads, then you can immunoprecipitate the structure.

That is what we observed for this amount of input material in the buffer control, or preneutralized sodium hydroxide, which says that there is the same amount of solutes of the base, but it was neutralized before.

So, the sample does not experience extreme pH. So, in both cases you can see a good recovery of the signal by immunoprecipitation.

Now, it is totally opposite to the pathogenic conformation, where the epitopes are buried and you will not be able to precipitate it. In this case you see no or very low signal.

Now, treatment with sodium hydroxide, it unfolded this structure and made this epitope accessible. So, you can see the immunoprecipitation.

This is pretty much, the overall consequence, is pretty much similar to what guanidine kinase does, which is chemotropic agent, unfolds the prion, which is used as a control in this case.

So, in summary, we think the critical effects influencing decontamination using sodium hydroxides include, of course, the concentration of the agents and the presence of detergents, and temperature and time also contributes, to some extent. So, overall, the sodium hydroxide works by unfolding and degrading the structure of the prion.

So, I would like to thank people who contributed to this study, especially the PPTA collaborators. Also, the experimental work was done by Dr. Pat Bauman and her research team.

I would also like to thank contributes, former PPTA members, as well as others who contributed to this study. Thanks.

DR. TELLING: Thanks, Dr. Cai. Are there any questions?

DR. SALMAN: Can you comment on what type of media you used for the prion? What type of vehicle do you have it in?

DR. CAI: You mean the spiking material?

DR. SALMAN: Yes.

DR. CAI: That was clarified brain homogenate from masters with the 263K strain.

DR. SALMAN: Have you tried to see if there is any type of effect of the organic matter on the decontamination?

DR. CAI: We haven't specifically examined it in that respect.

DR. SALMAN: I have another question. What is the reason to decide, as far as the maximum temperature of 18 centigrade?

DR. CAI: We chose several temperatures, such as four degrees, 18 degrees. Actually, those are conservative. So, we tried to model production processes, manufacturing processes.

During manufacturing, the cleaning procedures vary depending on manufacturer, such as whether it is upstream, is it downstream, whether it is -- the condition of the equipment.

So, there are hundreds of standard operation procedures for each manufacturer to define the specific procedures for cleaning.

All those procedures are validated based on a validation package, you know, according to common practice, where you use many measurements to determine how much residual protein is remaining.

Often we use total carbon measurements. So, if the total carbon measure is under a certain level, then you are confident there is no residual, or small amounts of residuals, remaining.

In that regard, back to your question, it is an organic compound and those effects are probably very limited.

DR. MANUELIDIS: I would like to sort of clarify something here, to make sure that in the rest of the meeting this is clarified by the speakers.

The question is, did you ever inoculate any of this material to see how infectious it was? You are making an assumption about inactivation of abnormal prion protein and infectivity, which other types of studies -- there are many numerous studies including heat inactivation, guanidinium, et cetera, where the correlation is not there.

So, I would really like to know, did you do any infectivity studies? I think it can be misleading to sort of say that we have inactivated this as a titer. Titer usually refers to infectious titer as measured biologically.

DR. CAI: We did do infectivity study, although the data is not shown here. We observed quite good correlation between the inactivation measured by western blot as well as hamster bioassay.

In addition, previous studies also demonstrated, as listed here -- a lot of them were done by using a bioassay, for example, infectivity as it was labeled as a green star here. Many of those cases are done by bioassay. So, they have quite good correlation.

DR. SEJVAR: Just to clarify, the conditions that you have been describing would be compatible with real life experience, in other words, actual decontamination of, say, surgical instruments, et cetera.

DR. CAI: Actually, there is some subtle difference between surgical instruments and the manufacturing processes for plasma products.

A surgical instrument is in direct contact with central neural systems and often has a much, much higher degree of protein binding to those instruments.

The manufacturing equipment, on the opposite side, has -- it is rigorously cleaned, of course, but if you think about if there is any donation got into the manufacturing side, the infectious titers could be very, very low.

So, it is kind of a different scenario but the general approach should be applicable to both. A lot of the surgical instruments are also treated with sodium hydroxide.

DR. SEJVAR: But you are talking about specifically plasma products, et cetera?

DR. CAI: Right.

DR. MASTRIANNI: In your experiments you used sarkosyl at two percent. I am wondering if you did a titration curve to see if there was a dose response that correlated with increasing levels of detergent to show a decrease in signal of western blot.

DR. CAI: That is a good point. Although we didn't titrate it, we did a spot test. You do need a certain amount, one percent, two percent, but if you go down to .1 percent, the effect will be reduced.

DR. COLVIN: As opposed to the indirect method of looking at the structure of the prion protein through either proteinase K susceptibility or through the western blot through antibody affinity, did you look at any direct measures of conformational change, such as using circular dichroism, NMR, spectroscopy, something that would show there has been a change, or even differential centrifugation of the products?

DR. CAI: Limited by our methods, we were unable to use a lot of physical means. We did these studies primarily in the BSL-2 lab in our setting, which is set up for pathogen research. So, we don't have extensive physical characterization testing available.

DR. GESCHWIND: Related to those last two questions, one is, there is clearly a difference in inactivating human CJD as in animal, as shown in the Peretz paper, Journal of Virology that came out recently, where human prions were found to be 100,000 times more difficult to inactivate than in animals, hamster 263 prions.

I think that is an important point to consider, that the only effective way of testing really should be -- I think this is an important point for the committee to consider for the next two days, is that really the human prions are going to be different than animal prions and we have to realize that.

Then the second issue is, is there any equipment in the processing for the plasma that would be exposed to metal, as clearly there is a difference between brain homogenate and testing in which they have looked at small pieces of metal put into the brain.

So, brain homogenate has always been easier to inactivate than the steel rod method. I am just wondering if there is any possible exposure to metal in particular during any of the processing.

DR. CAI: Yes, the first question about Dr. David Peretz' paper, I guess using acid in combination with detergent to inactivate prions. So, they compared between hamster and human materials and there was a big difference.

I would like to point out in that case it is weak acid at a pH 3, 4. In our case, sodium hydroxide or potassium hydroxide is a strong base. It is a strong electrolyte. So, they are very, very different in their nature.

In addition, the previous studies demonstrated by almost 10 groups using the sodium hydroxide treatments with the absence of detergents, they consistently demonstrated significant removal or reduction using various strains of material, including rodents and humans.

So, the second question is whether the manufacturing process has metal components in the equipment. The answer is yes. Of course we use a lot of metal equipment, including stainless steel, all of that.

Several groups, including Dr. Safar's group, they use metal instruments into rodents to detect the prion infection. That is a very good approach and gives us a better understanding.

The study we did, you know, they were designed to address in general those conformational changes, and to understand the significance of a residual signal, how to remove residual signal. Those studies should be complementary, I think.

DR. TELLING: Thank you. We need to move on. The final speaker in this update section is Dr. Safar from the University of California, San Francisco, who will be talking about human prions clearance in plasma lipoproteins.

Agenda Item: Human Prions: Clearance and Plasma Lipoproteins.

DR. SAFAR: First of all, I would like to thank the committee for this opportunity to present some new data that we think are very relevant to the task of this committee.

I think that fundamental issues facing the prion research, I have selected those that I think are very relevant for this meeting and for the present agenda.

I think that the three in the square are interrelated. The pathogenesis of the prion diseases is important to understand and to plan the most effective therapeutics which would halt the prion formation and remove existing prions.

The condition for that is to have a very sensitive presymptomatic diagnostic test. If we would initiate any therapy late in the symptomatic stage of disease, there is very little hope for the recovery. The situation is very similar to Alzheimer's disease.

This is a table which I put in. It is very complex, but I think that it is really important to realize the progress of the field in the last few years.

There were originally described two entities related to the prion diseases, normal PRP, cellular form of the PRP protein and the resistant form of PRP, PRP^SC, which is infectious.

We found that there are very similar species, which actually in many cases is dominating disease, we called protease sensitive form of PRP^SC.

They have different conformations. The PRPC has exposed most of the epitopes against monoclonal antibodies, where PRP^SC, both S and R forms, those epitopes -19 and -125 are already buried.

The secondary structure of PRPCs is 40 percent helix. RPRP^SC is 40 percent beta helix. We don't know the conformation of the PRP^SC.

Quaternary structure of PRP^SC is a monomer PRP^SC or oligomers, and $PRP^SC can polymerize into analoid-like rust.

Standard PK destroys both PRPC and SPRP^SC, but leaves behind a proteolytic fragment of PRP^SC which we call PRP22-30 by molecular weight.

Current PK, which we found very simply, hydrolyzes selectively PRPC and leaves behind a proteolytic fragment which is typical for SPRP^SC. So, that is the first really direct evidence that this comes separately from what the RPRP^SC comes from.

Another way to separate PRPC and PRP^SC, both S and R, are polyoxometalate, polyoxometalate precipitation. Non-denaturing detergents are solubilizing PRPC, they have mixed effect on the SPRP^SC, they don't solubilize RPRP^SC.

Infectivity, normal protein is non-infectious, of course, and the RPRP^SC is infectious. Levels during infection, there is no change, no up-regulation of PRPC. SPRP^SC and RPRP^SC are in equilibrium, which is typical for different prion strains.

In RML infected mouse, the clearance half time for the SPRP^SC and RPRP^SC is 1.5 days. I showed this slide because I think it is becoming increasingly important.

There is a large percentage of the sheep scrapie which carries selectively the SPRP^SC forms, and wouldn't be detected without the availability to detect this PRP^SC. We saw it in Norway cases and more and more cases in Europe.

There is a growing number of human CJD cases, and they were presented last week in San Francisco by Luigi Gambatti(?) from his CJD surveillance collection.

He estimates that it may be up to 14 or 15 persons which display selectively SPRP^SC and practically no detected RPRP^SC proteins.

So, this is becoming very important for two reasons. First, practical detection and identification of the prion disease. Second, in a theoretical sense, how is it related to the disease and how important is it in pathogenesis.

The direct PRP protein with proteases, we designed 10 years ago the protocol which avoids proteinase K. It is called conformation dependent immunoassay, and it recognizes antibodies which are exposed in PRPC and hidden in PRP^SC. This is the beta helix of PRP^SC, helix A and C, which are still remaining there.

If you test simultaneously one sample which is native and the second of which is denatured after denaturation with sodium hydrochloride, you compare the signal.

If you don't see any increase in the signal, you know that they are PRCP proteins, or if there is a very small increase, you can account for it by establishing for the size of cattle.

If there is an increase in the signal after denaturation, you know that you have a certain percentage of PRP^SC in the original sample, which had hidden epitopes. that is a quantitative parameter indicating the presence of PRP^SC protein.

We weren't very happy with the sensitivity. So, we are looking for the compounds which would selectively precipitate PRP^SC and leave behind PRPC. One of those compounds was the keggin structure of polyoxometalate, where the phosphate is in, and they are coordinating the constant oxide of the hyderons around.

There is a misconception that it is some small cell. The polyoxometalates are actually very large. The monomer of PRP structure, monomer of PRPC is about 1.8 nanometers. The polyoxometalate in this case, kaggin structure, is about one nanometer large.

So, those are very large compounds which can be synthesized in a way which modulates either size, shape or charge.

We found out that some of the polyoxometalates are efficient in aggregating PRP^SC, the S and R forms, and large polyoxometalates actually have a positive effect. They dissociate PRP^SC proteins.

This dual effect is still not understood exactly at the molecular basis. It is definitely related to the charge and size polyoxometalate.

So, kaggin structure, small polyoxometalate cyclates facilitate prion formation and decrease cell growth from prions, and the large polyoxometalate have the opposite effect. They dissociate PRP C protein and they make smaller complexes.

Our studies of SVRPLC proteins, the protease sensitive form, was initiated with generating biogenic systems to regulate the PRPC level of expression.

So, we could shut down PRPC and look what happens to PRPC proteins, both the S and the PRP^SC. The first surprise came following the incubation time.

When you shut down PRPC expression and then follow the incubation time of the animals, those which express downregulated PRPC from 100 percent to about five percent of residual expression, extended their incubation time by about three-fold.

So, that was a really amazing result. We didn't expect it because we were afraid that the small leak we have in the background would inevitably lead to very small changes in incubation time.

When we measure the PRPC protein prions, we found that the PRPC has a half time of about 18 hours, PRP^SC has a half time of about 36 hours, measured by both CBI and western blots.

So, that was really an amazing finding, indicating that this is a very powerful mechanism in the brain, physiological mechanism, which is able to clear prions in one and a half day, and practically 50 percent of already formed prions.

It is apparently related also to the strain. When we compare CO1V, an animal strain, CO1V, which has a slightly extended incubation time, it cleared about twice as more slowly. Also, the accumulation was slower.

So, there is an interrelationship between the stability of the prions for prion strains, incubation time, the accumulation rate and clearance rate. Those functions are strain specific.

If you look at the pathology of the animals where the PRPC was likely to be expressed during the incubation time of prions, we see large deposits of PRP^SC proteins.

If you look at animals which were inoculated and then, after 98 days, which is about two thirds, we shut down PRPC, we see how clean those brains are.

There are only some deposits in the corpus callosum in the white matter. Most of the cellular areas are completely clean. Other deposits are around the vessels.

This slide, I think, is really optimistic. It has got a therapeutic approach. If we would be able to down-regulate PRPC, you would effectively cure the disease because the brain has a very powerful clearance mechanism for clearance.

Additionally, the therapeutic level, we know now that it is possible that the prions are continuously synthesized at the low level and that the brain has -- that they have some physiological function in the brain, and that the brain has, at the same time, a very strong clearance mechanism, which is how you get rid of them.

So, where do prions go? We know from other experiments, when you inoculate directly prions into brain, 99 percent are lost within the first 24 hours.

So, there is massive outflux of prions from the brain, and obviously the target in this case has got to be the first circulating blot and cerebral spinal fluid.

There is no question that there is infectivity in the blood, and there are many studies indicating them. The issue in this case is which compartment of blood it is. Is it out of blood or plasma or both.

So, we established a system where we looked really blindly in both plasma and white blood cell compartments. White blood cells are sorted by facs, flow cell activated cell sorting, and by myelin Bs, and we have tested up to now granulocytes, monocytes, CD4, T cells, B cells, circulating dendritic cells from different animals and also from CJD patients.

The results are -- the results really increased in plasma. We started to supply them with polyoxometalates. Polyoxometalates have not only very specific precipitation capability of prions, but they also precipitate lipoproteins. They have a still not fully understood affinity to the lipoproteins.

So, we decided to test where the prions would go in the human plasma by using polyoxometalate fractionation. By increasing concentration of polyoxometalates, you can supply plasma into the LDL particles, lipoprotein particles, immunoglobulins, HDL and other plasma components.

When we spike the plasma with prions from brain, sporadic CJD prions -- so, this is a homologous system, plasma, human plasma, and sporadic CJD and one case of CJD.

We found that all the prions, by western blots, and by CDI, were fractionated or precipitated into the VLDL or LDL fraction of the lipoproteins.

It is not actually so surprising. The PRP, prion PRP has a very high affinity for cholesterol. It is very difficult to separate them.

So, the lipoprotein particles that are about 60 percent of cholesterol and phospholipids, and about 30 percent hydrophilic proteins. So, the fact that they have affinity for each other is not very surprising.

What came up as a surprise was the level of the affinity. When we tested in our affinity assay, the binding of the sporadic CJD prions to the lipoproteins coated on the late, the mid-points which was the indication of the affinity constants, they were in the low picomolar range, between 30 to 100 picomoles.

That was a real surprise. The second surprise was the selectivity. When we coated the plate with HDL, there was practically no specific binding, no cooperative binding.

So, despite a similarity in the lipid content between LDL and HDL, there was a big difference in the affinity for the LDL, a preferential binding for the LDL.

The second surprise came from electron microscopy when we purified the sCJD protein from the brain and then incubated them with the VLDL and LDL, or HDL.

We saw decoration of the human neurons only with LDL. We didn't see any decoration with HDL. If you compare the signal of our best monoclonal antibodies, and decoration with LDL gold, we see how few dots we actually got on those prion neurons. On the other hand, we have a massive accumulation of the LDL on the human prions.

The common component to all the VLDL, LDL, IDL, and not HDL, is the apoprotein B. The other apo-C, E and so on, are exchangeable, but they are not present in LDL.

So, we decided to test specifically the apoprotein B, purify for protein B. The affinity was only about four to 10-fold lower than the affinity of the original LDL.

So, in conclusion, the PRP SC protein, both the S and R forms, have a very high affinity for lipoproteins containing apoprotein B, or apoprotein B itself.

The binding is conformationally specific. If you compare the affinity constants of the alpha helical PRP versus random cold PRP, versus native PRP SC protein in prion neurons, hey go in that sequence.

So, alpha helical PRP doesn't have practically any binding, random coil higher, and then followed by the native prions.

The stoichiometry is also is also different between recombinant PRP and the native prions, where we see the binding ratio about three, we see only one to one ratio for recombinant PRP in the random coil conformation.

The LDL suffers from a misconception. Most of the people have the impression that LDL is cholesterol. That is actually not true.

It is about 30 percent protein, which is called apoprotein B. It has a molecular weight about 550 kilodaltons. It has 4,536 amino acids.

It composes about 30 percent of the weight of the LDL particle. The rest is cholesterol and phospholipids in the monolayer, and the apoprotein B, which is hydrophobic alpha beta sheets and alpha helices, are basically wrapped around the particle and presents no specific fusion with the cell.

If there wouldn't be apoprotein B, we would die from atherosclerosis of age two or three, probably. So, it is a very important mechanism which, through the LDL receptor domain, directs the LDL particles to the cells, which express the LDL receptor and, if they meet, influence of the cholesterol. So, it is a very important regulatory mechanism.

Is it conformation specific also from the other side? In the prions, when we test the different prions from CJD, sporadic CJD, CN hamster(?), scrapie, RML, we saw a totally different binding curve, indicating different stoichiometries and different affinity constants.

So, it is not only conformation specific for PRP, for human PRP, but it is also able somehow to discriminate between PRP, different prion strains.

So, human LDL and apo-B binding with AP CJD prions, it is conformation specific. It has a very low affinity constant down to 30 picomolars.

The PRP affinity for binding is present in 19 to 31. The order of the binding goes from the beta HPRP to the denatured PRP to the alpha helical PRP.

The different stoichiometries, three to one for native prions, one to one for recombinant PRP. The lipids of LDL are not essential for the binding. Glycolipids and glycosylation are not essential for the binding in PRP. LDL and APO-B binding to denatured PRP is sequence specific.

So, did we look into sporadic CJD cases. I think that the first step before that, we actually realized that first we have to validate our assay.

We have to show that we have a -- that we can truly detect PRP^SC protein and, second, that we can truly measure quantitatively PRP^SC protein, and correlate it with the prion infected.

So, in this study, which was actually initiated with Glenn Telling, and whose transgenics he generated in San Francisco, we inoculated three different cases of sporadic CJD in the end point titration experiment in different transgenics to determine end point titers.

At the same time we made homogenase from the brain and tested by CDI, the dilution curve, in parallel. When you see the correlation, there is a very clear overlap.

It shows one important difference. The 50 percent transmission rate indicating one infectious unit per ml, at that level, CDI has a reading skill of about 20,000, which is about 1,000-fold over the capability of the CDI assay. So, in effect, the CDI is more sensitive than the bioassay in transgenics.

So, one more question was correlating the established procedures of immunohistochemistry and pathology with the infectivity and with the CDI.

So, we blindly tested PRP C protein in those different forms of prion diseases and in 18 different anatomical areas in eight sporadic CJD cases.

We could detect the RPRP^SC protein everywhere. In contrast, the immunohistochemistry and localization profiles in many areas the sensitivity of both was not exceeding 20 percent, or was even lower than that.

So, one conclusion. First, the testing has to be in a diagnostic aspect. It has to be in different anatomical areas.

Second, the CJCDI shows absolute diagnostic sensitivity and specificity in all of those anatomical areas.

The second important finding was related to the SPRPSE. When we looked at the concentrations of RPRP^SC versus SPRPSE, in all frontal and white matter areas we tested, there was more SPRPSE protein over the RPRP^SC protein. The RPRP^SC protein actually formed about five to 10 percent of the total.

The next presentation is going to be humoral. This is just the first data showing the CDI on the VLDL fraction and plasma of the CJD cases.

When we tested total PRP concentrations in 21 donors and 20 sporadic CJD cases, we didn't find any difference in total PRP or in the SPRP and the PRP^SC protein. It is below the threshold.

When we separated VLDL, there was a significant difference in the sense of more total PRP protein in the VLDL from sporadic CJD cases, and most of the total PRP increase was RPRP^SC protein, actually.

So, what are the conclusions? I think the apoB containing lipoproteins are strong candidates for carriers of sporadic CJD prions in human plasma, and I will talk about it as the diagnostic implications emerge.

Binding of apoB containing lipoproteins to native sporadic CJD prions is conformationally specific with Kilodaltons down to 30 picomolars.

The existence of highly stable lipoproteins prion complex in plasma suggests that it may be that the lipoproteins have a role in the clearance of prions from the brain and other tissues.

Both conformational specificity and high affinity will lead to the development of new assays for prions.

The data on low affinity prion ligands suggest that lipoprotein binding may impact the infectivity of sporadic CJD prions.

Conformational specificity of the apoB binding may lead to the new ways of differentiating human prion strains, including variant CJD.

Plasma lipoproteins provide, in my opinion, highly specific ligands for prion removal from plasma. Thank you.

DR. TELLING: Thanks, Dr. Safar. We have time for maybe two quick questions before the break, if there are any?

DR. MASTRIANNI: I have got so many, but I can talk to you later. One just generalized question. Why does LDL bind scrapie better than PRPC? Do you have a conformational model for that? Maybe I missed it.

DR. SAFAR: We don't have a really good conformation model of apoB. ApoB is almost as difficult to study structurally as PRP^SC. It is very large, very hydrophobic, and there are no three-D structures.

The model I showed is a computerized approximation of the CD infrared spectroscopy of the entire EM. So, it is a very large protein.

On the other hand, there are many tools in molecular biology, including fragments of apoB and LDL, which will allow us to determine which domains specifically, in the first approximation, are responsible for the binding and we are studying it now.

DR. MANUELIDIS: If I understand it, you are saying that if you take out the LDL fraction of plasma, you would lose a lot of the infectivity in prions. Is that correct, and is that true?

DR. SAFAR: We think that practically all the PRP^SC from the CJD brains spiked into the plasma co-precipitated with LDL and VLDL, yes. We haven't measured the infectivity.

So, to your question, we measured PRP^SC protein, which we correlate with the CDI, which we correlate with viruses.

So, we are very sure that what we detected is infectious PRP^SC protein. Formally, we haven't done a bioassay yet.

DR. TELLING: Thanks, Dr. Safar. I would like to keep this on track, which we more or less are. I would like to take a break and reconvene at 10:30.

[Brief recess.]

Agenda Item: Topic One: Experimental Clearance of TSE in Plasma-Derived FVIII Products.

DR. TELLING: We are going to move on to topic one, experimental clearances of transmissible spongiform encephalopathy infectivity in plasma derived factor VIII products.

We are going to start out with subtopic A, BSE clearance studies of factor VIII, study methods and clearance levels, and a presentation by Dr. Scott from the FDA.

Agenda Item: TSE Clearance studies for pdFVIII.

DR. SCOTT: Mine is the first of two presentations. The second will be given by Dr. Thomas Kreil, representing the Plasma Protein Therapeutics Association.

We are going to have somewhat different presentations. What I would like to do is outline some of the challenges in TSE clearance studies, and to introduce the committee to some discussion questions that we would very much like your scientific input on.

Our TSE safety concerns are that, theoretically, plasma derivatives might transmit variant CJD or other TSE agents, since we certainly know that blood can do this and that plasma of infected animals is also infectious.

Any such risk is probably very low, based on the fact that no cases of variant CJD have been reported worldwide in any recipients of plasma derivative, including in the United Kingdom where vCJD risk is greatest.

However, we seek to assure the safety of plasma derivatives, especially plasma derived factor VIII, against the risk of transmission of TSEs.

The clearance of TSE agents in manufacturing of plasma derived factor VIII and other plasma derivatives has a major impact on estimated risk. In a minute I will go through what I mean by clearance and clearance studies.

In a risk assessment, a draft risk assessment that we published on the internet after the TSE advisory committee meeting in 2005 for plasma derived factor VIII, that risk assessment had a sensitivity analysis, which gives you an idea which inputs to the risk assessment most affect the output or the level of risk.

Indeed, the clearance of TSE agents during the manufacturing process is one of the major things that did impact the ultimate estimated risk to recipients.

However, standardized methods for studying TSE clearance in products have not been defined,in part because there are a great number of challenges associated with standardizing these methods, since we don't know everything there is to know about the TSE agents in blood.

We seek your advice about whether standardized methods and assessment criteria are feasible now, and if they are appropriate for determining TSE clearance in the manufacturing processes for plasma derived factor VIII products.

In particular, these are your three items for discussion, the feasibility and scientific value of adopting standardized methods to assess TSE clearance in manufacturing of plasma derived factor VIII products, and whether a minimum TSE agent reduction factor might reasonably serve as an appropriate standard for demonstrating vCJD safety of plasma derived factor VIII products.

If there is a minimum level that might reasonably serve as such, what action should FDA consider if only lower levels of clearance can be demonstrated for a given factor VIII product.

I am going to go into a little bit of the previous history of TSE clearance studies and FDA's involvement in those.

Some of the members here today might remember that we discussed TSE clearance studies with you in February of 2003.

Since then, we have engaged in case by case review of the following types of information on TSE clearance. So , this is information submitted by manufacturers officially to FDA, requesting a labeling claim based on specific data that they had generated.

These studies include a rationale for the animal model selected and a rationale for the spiking preparation. I will get into some of these details in a minute, characterization of the spiking agent, demonstration of accurately scaled down processes, robust and reproducible experiments, well characterized assays for TSE infectivity.

These submissions will contain estimated logs of TSE clearance by the processing steps that were studied, and also will demonstrate or describe mass balance, that is, accounting for all the input infectivity in the output samples that are assayed.

There should also be a demonstration that mechanically similar clearance steps are or are not additive in the process, and an accounting for conditioning of infectivity.

By conditioning, what I mean is, a prior step in manufacturing might affect the physical state of the TSE agent and, in turn, impact the amount of clearance that can be effected downstream in the actual clearance step that is being studied. Again, I will go into this in more detail in just a minute.

Since that time, four labeling claims have been approved. These are for Carimune NF and panglobulin NF. These are immune globulin products, and these are the reduction factors and these were the steps that were studied, precipitations and nanofiltration for these two reduction factors of 7.2 and 4.4.

Gamunex, another immune globulin product is where a combination of cloth and depth filtration were studied and the clearance level obtained was an average of 6.6. Thrombate III, precipitations were studied with a reduction factor of 6.0.

Now, you notice there aren't any plasma derived factor VIII products here. That doesn't signify whether we are evaluating submissions or not, but it does tell you that no such studies have been approved for labeling claims.

Why are we particularly concerned about plasma derived factor VIII? Well, we are concerned, of course, about all plasma derivatives, but cryoprecipitation is the first in manufacturing of plasma derived factor VIII.

There are many other steps that may follow as there is increasing purification to make the product. Dr. Kreil will be talking about those more.

This is just a schematic of Cohn-Onclay blood plasma fractionation process. What you can see is the cryoprecipitate which becomes plasma derived factor VIII comes off very early in the scheme.

So, one of the reasons to be concerned is that there is not much opportunity for clearance like there might be for albumen, which undergoes a series of sequential precipitations to be purified, and immune globulins where the case is similar.

Many people have looked at experimental clearances, either PRP^TSE or infectivity by cryoprecipitation, and this is just some of the references that have reported this.

You can see here that the log 10 reduction factor is really one log or less in all of these studies, and whether a bioassay or a binding assay was used of a surrogate marker for infectivity.

So, these are the major challenges that we face in standardizing these studies or even understanding how best to do these studies.

I am going to talk about four things: the exogenous or spiking experiments, endogenous experiments and their relevance and feasibility, the TSE strain and animal model that is used, and output measures of infectivity reduction -- bioassays, which are infectivity assays in animals, and in vitro assays such as the western blot and the conformation dependent immunoassay, which depends upon antibody binding.

This is a very simple schematic of how an exogenous TSE clearance study might be done. The example I have given you here is the cryoprecipitation steps.

So, here you would have plasma. You would spike a preparation typically from brain of an infected animal into this plasma, and then that would undergo the manufacturing step where you get cryoprecipitate and cryopoor plasma supernatant.

The infectivity would be measured here and here and compared to the amounts that you put in at the beginning to determine a reduction factor.

So, for right now, I am going to be talking more about spiking experiments. There are a number of studies that have been done this way.

The reason that spiking experiments are done with brain homogenate is that it tends to have a very high infectivity level. So, you can achieve or demonstrate a wide range of clearance values. It is practical.

The form of the infectious agent, a number of these have been used, including membrane associated forms, brain homogenate that is just centrifuged to clarify, ultra centrifuged preparations including microsomal preparations and coevally like domains.

People have also used detergent solubilized hemogenates from brain. There are also membrane free infectious materials that have been used.

Again, they are derived from brain. These would be fibrole preparations, but also more purified preparations of PRP^TSE .

In general, they can be somewhat insoluble and they are felt, perhaps, not to be the best representatives of blood infectivity, which is believed to be more soluble.

Here I am just going to show you some examples of what we know about the spiking form of the agent. This is by Bey et al, published in Biologicals.

This is just to demonstrate that the form of the spike impacts clearance by precipitation. What you see here is the manufacturing process, cryoprecipitation, two different alcohol precipitations, and glycine precipitation.

These are the logs of PRP^TSE reduction that were measured with respect to the supernatant. This is microsomal spikes. You can see that, for cryoprecipitation and glycine precipitation you have a fairly low clearance.

If you look at a more purified PRP scrapie spike preparation, you get somewhat higher levels of clearance, here two, three and four.

So, spike has a major impact on the amount of clearance that you measure, which means it is very important to choose, if you will, not necessarily the worst case spike, but perhaps the one that might be most representative of infectivity in blood.

There is also the impact of conditioning. This is an example of conditioning, where detergent treatment of the infectious preparations diminishes its clearance by nanofiltration. This was published in 2001 by Tateishi.

The feed solution is a starting solution. So, this brain homogenate was treated with detergent or without detergent, and these are the titers of infectivity that they got.

By the way, these experiments were done by bioassay. So, all of these output measures are amount of infectivity by bioassay.

So, this is what you begin with, the detergent and non-detergent treated material. That was put through a 35 nanometer filter.

What you can see here is that material, brain homogenate, that was not treated with detergent had a very nice clearance by nanofiltration, 35 nanometers.

When detergent was added -- I think this is sarcofil three percent, you get a much lower level of clearance.

So, what does that mean? That means that if you have a detergent step, that that might impact the filterability of the agent at the end.

What you can also see is that, at lower pore size filters -- that is, 15 and 10 nanometers -- you do get good filtration in either case.

What this means for TSE clearance studies is that you really need to consider the upstream steps that might impact the agent.

You could get, in a sense, a result that might not really reflect, if you only looked at nanofiltration and not the upstream steps before, he impact on the agent, it means that you might over-estimate the amount of clearance, for example, by nanofiltration.

This is one of the complexities of doing these kinds of studies and one of the challenges faced by industry.

Another example of conditioning is PRP^TSE clearance by membrane filtration and depth filtration. This was shown in a paper by Van Holten, to increase in the presence of alcohol.

It appears, from his data, that the alcohol the used and the concentration of alcohol caused aggregation of PRP^TSE , which obviously influenced how well it was filtered by fairly large pore sized membranes and depth filtration. It was clearly filtered much better in the presence of alcohol.

I am going to briefly review the endogenous infection model. In these models, plasma is taken from a TSE infected animal.

This is very low titer material, somewhere on the order of a couple of IDs per ml to maybe 50 or 60, depending on the animal model used.

It undergoes the manufacturing step again, and you measure the amount of infectivity in the supernatant in this case, or the precipitate.

The endogenous TSE clearance studies have relevance to blood infectivity. I would point out, though, that the comparison of results from endogenous and exogenous infectivity studies suggest similar reductions for some precipitations, but the number of comparative studies is extremely limited.

However, endogenous infectivity is probably the most relevant to infectivity that we would find in human plasma.

The characteristics of endogenous infectivity are thought to be a fairly small size of the infectious particle, difficulty in sedimenting in its native form -- and by sedimenting I mean by high speed centrifugation. It is probably poorly aggregated and it may be lipid or plasma protein associated.

The relevance to human blood is highly likely, but you can only demonstrate limited clearance because the starting infectivity is so low.

That means that a large number of donor and assay animals have to be used to compensate for these low titers. So, in other words, if you have two infectious doses per ml, but you assay 100 mls of material, you will likely find this infectivity.

Just to give you an idea, the volume injectable intracerebrally to titrate this is only about 20 microliters -- sorry, .02 mls at 20 microliters per ml, or about 50 microliters per hamster. For 100 mls of plasma, to completely titrate it, you need 5,000 mice or 2,000 hamsters.

What is done in real life, I don't know if anybody has ever done quite this many, but they will do a portion of their output sample and look at infectivity and calculate essentially how much they could have missed if they get a zero.

Large animal models, in theory, might be nice to study. We know that sheep can have natural infection with scrapie and can be experimentally infected with BSE.

We further know that the blood of these infectious animals is infectious to recipient sheep. There are experimental logistical hurdles in doing these kinds of studies

. Among those are herd management, and the fact that there would be very limited locations where you would be allowed to have a scrapie herd or a sheep herd infected with BSE. Furthermore, these would have to be very carefully segregated away from the control animals.

These sheep also have very long incubation times. So, you would have to wait even more years than you would have to wait for a hamster or mouse study and they will be limited in availability.

Of course, the logistics of scale down would be different because you would have much larger volumes to work with, but you would still have to use a pilot laboratory to simulate the manufacturing process. Probably new or different pilot laboratories would have to be set up to study clearance in large animals.

In terms of TSE model selection, there are many papers that show that TSEs differ in their resistance to inactivation but, to date, clearance of TSEs in plasma products has only been demonstrated by partitioning studies.

The reason for that is that the inactivation methods that have been used for TSE infectivity are very harsh methods and you would destroy the proteins that you are trying to isolate and purify for people to use by any of these methods.

There are very few direct strain comparisons in TSE clearance study plasma derivatives, but alcohol precipitations were looked at by Stenlin(?) and the group at Telecris. They found similar clearance levels using western blot for BSE, CJD and vCJD spiked samples.

Alcohol precipitations, at least in theory, could be influenced if strain related differences exist in aggregation properties of the infectious agents.

This is a theoretical concern, but it might also be a real concern, and we don't have the data to know whether or not that is the case in these particular scenarios.

So far, strain differences for partitioning clearance experiments have not been demonstrated. You can see how limited in number the studies are.

What kind of assays should be used for TSE agents in clearance studies? Bioassay is usually done by limiting dilution titration into susceptible rodents and, as you have already heard today, PRP^TSE is felt by many to be a good surrogate marker for infectivity, and this is usually measured by western blot or conformation dependent immunoassay, as you have heard.

There is a rationale for retaining bioassay use, because although binding assays detect PRP^TSE , they are examples of infectivity without detectable PRP^TSE .

I should qualify that by saying very often this has been PRPres, that is, PRP^TSE as assayed by its resistance to proteinase K.

There are also examples of PRP^TSE occurrence without infectivity and also I would note that conditioning that I have shown you before might differentially affect binding versus infectivity.

There is a paper by Silvera and his group suggesting that, at least from brain homogenase, a certain size of prion particle seems to be associated with greater infectivity, and that larger aggregates and smaller aggregates are less associated.

So, even in the context of the protein only hypothesis there are some caveats that one would have to consider in terms of using PRP^TSE binding solely as a surrogate for infectivity.

Furthermore, binding assays currently are not as sensitive as bioassays. We have just heard, however, that for the conformation dependent immunoassay, this may be otherwise, and we look forward to additional data in that respect.

The limit of detection for binding assay, more typically, is two to three logs of infectivity. So, you can't demonstrate as wide a range of occurrence when you are using, in general, these kinds of assay.

An additional challenge in TSE clearance studies is their interpretation, how much clearance is significance. Well, we are going to ask the committee to discuss that, but I can give you something to work with.

In viral validation or viral clearance studies that are done for all plasma derived products in the United States, it is typically demonstrated for effective viral clearance that there are at least two to three logs greater clearance than the maximum potential absolute amount of virus present. When I say the amount of virus present I mean the amount of virus that would be expected in infected plasma.

This added margin of safety is probably important, because we don't know in every case how much virus might be in infected plasma. We have a very good ballpark estimate based on what is reported.

Furthermore, manufacturing itself has its -- is not entirely robust. You might not get exactly the same value each time you do a manufacturing step because of slight changes in parameter.

Again, this margin of safety, this addition of more logs on top of what you think absolutely has to be removed, is probably a good idea.

Now we come to TSE clearance. If TSE infectivity is present in a unit of plasma, how much might there be. Well, if you take an 800 ml plasma unit -- this would be the top amount you might expect, and multiply that by the potential infectivity in it -- and we really don't know exactly if these numbers are right -- so, these are estimates based on other studies in the literature from animals.

So, you multiply by this range, two to 30. What you get is 1,600 to 24,000 range of infectious doses possibly expected in this infected plasma.

That works out to 3.2 to 4.4 log 10 total infectious units. Actual infectivity might be less than this due to the blood brain barrier and due to host susceptibility, but this gives you some numbers to start with and to think about.

I am going to introduce the questions, but Dr. Kreil will be following up with additional thoughts about TSE clearance studies and more detailed information about where industry has been studying clearance in plasma derived factor VIII.

We are asking you to comment on the feasibility and scientific value of adopting standardized exogenous or spiking study methods to assess TSE clearance in manufacturing of plasma derived factor VIII.

We would like you to comment on your thoughts on optimal spiking material and its preparation from the standpoint of relevance to blood infectivity, the selection of TSE strain and animal models.

TSE immunoassays for PRP^TSE versus bioassays for infectivity, the use of these as output measures, and identification of manufacturing processes that might alter TSE agent properties.

We would also like for you to comment on the feasibility and scientific value of adopting standardized endogenous study methods to assess TSE clearance in plasma derived factor VIII.

We would also like you to discuss whether a minimum TSE agent reduction factor demonstrated using an exogenous spiking model in scaled down manufacturing experiments, like the ones I have described, might reasonably serve as an appropriate standard for demonstrating TSE safety of the products.

Considering the outcomes to that discussion in question two, in cases where only lower levels of clearance can be demonstrated for plasma derived factor VIII products, what should we consider:

Labeling that would differentiate the lower clearance products from other products with sufficient TSE clearance;

Recommending addition of TSE clearance steps to the manufacturing method; Performance of TTSE clearance experiments using endogenous infectivity models, or any other actions.

I will leave that with you and give the podium over to Dr. Kreil. Thank you very much.

DR. TELLING: Are there any questions for clarification for Dr. Scott at this time?

MR. BIAS: Dr. Scott, is there a reason that there haven't been any experiments done using human blood of vCJD victims?

DR. SCOTT: That is a very good question. I think that if this was easily available, they definitely would have been done.

I think by the time the patients come to their clinical disease, the ability and the ethical constraints on collecting a lot of blood or plasma from them has been limited.

In the United Kingdom, they are particularly careful to assure that patients have a choice and that their families have a choice.

That is what has caused the limitation. It is not obviously the patient's fault. There aren't very many patients to begin with, but there aren't very many people with this disease at any given time that are in a situation where they might be able to give a large amount of blood or plasma.

i know Dr. Minor is in the audience and I wouldn't want to just call on anybody in the audience at random, but either Dr. Asher or Dr. Minor might have more insight on the availability or the potential availability of variant CJD blood. I guess I will let Dr. Asher, because he is on duty here.

DR. ASHER: I hope that we will hear some thoughts on the issue tomorrow from Dr. Minor who is here, and Dr. Turner who we expect to arrive this evening.

The problem has been this. With sporadic and familial CJD, infectivity has not convincingly been demonstrated in the blood.

So, if you collected it, either by epidemiological look back studies -- and the American Red Cross' study is really now quite extensive -- and a very limited number of studies done at the NIH transfusing whole blood into chimpanzees, none of whom ever became ill with Creutzfeldt Jakob disease.

So, the blood from the forms of Creutzfeldt Jakob disease generally available in the United States, the hypothesis that there is enough infectivity present to be detected at all with any of these assays has not been demonstrated.

With variant Creutzfeldt Jakob disease, as Dot pointed out, the number of patients available has been very small.

In the two cases in the United States, I believe that Dr. Gambetti has a small amount of blood, and I know the Canadian case there is a small amount of plasma available, but nothing approaching what would be needed for the kinds of studies that we have been talking about.

I am afraid at the moment we are stuck with blood from endogenous infectivity. We are stuck with blood from animal sources.

DR. GESCHWIND: So, at UCSF we have actually shown that it is pretty feasible to get large volumes of blood from patients with CJD.

We have -- Jiri Safar probably can give you the fact numbers, but probably we have over 50 patients in whom we have gotten 200 to 400 mls of blood.

So, bring in patients from around the country and at certain points when we have funding we have been sending out a nurse to get 200 mls of blood from patients with CJD, and we have been collecting it every two to three months from patients during the course of their disease, depending upon -- we do very strict safety tests that are more conservative than for the Red Cross blood donations, prior to doing this.

So, it is feasible, particularly in patients whom we have diagnosed earlier in the disease course, and in patients who have a slower course.

DR. SCOTT: I think that Dr. Minor also has a comment maybe about the variant CJD cases.

DR. MINOR: Well, I am very jealous of the comment that has just been made. I have discussed this extensively with the people at the CJD surveillance unit in Edinburgh, and they won't touch it.

They basically say that the ethical concerns are such that they will not take a unit from people who have variant CJD, no matter who wants it.

I will be talking a little bit about human samples tomorrow in the diagnostic presentation, and the availability of human samples is absolutely tiny, relevant human samples, like within the United Kingdom, is absolutely zero.

There has also been a recent introduction of a thing called the human tissues act, which means that if you don't do it right, you get sent to prison. That has actually been a major inhibitory effect on actually trying to get these kinds of samples.

I am actually very impressed by the fact that you can get those kinds of volumes around. If we could get those kinds of volumes, I think I would put them into diagnostics rather than into plasma fractionation, frankly.

DR. TELLING: What about blood from BSE infected cattle?

DR. MINOR: This is like experimental infections you are talking about or what?

DR. TELLING: Either experimentally infected -- well, presumably that would be the most convenient source.

DR. MINOR: Again, I will talk about some of that stuff tomorrow. There is a study which is going on with Ferna(?) Huston on sheep, blood transfusion, where I think this is actually a kind of interesting animal model for this.

The idea is that the sheep will be infected by mouth by BSE or whatever, and then blood will be taken from them and transfused into other sheep which are negative.

If you can actually keep a sample of the blood which is transfused, and you can also follow the blood samples from the transfused sheep -- I don't now how confusing I am making this sound -- you can start talking about when the diagnostic tests become positive and when they become negative.

You can also in theory, I guess, use those kinds of materials for fractionating plasma proteins. I think the wrinkle to that is it is not clear to me that plasma proteins fractionate from sheep plasma in exactly the same way as they fractionate from human plasma.

So, there may be a doubt about even the relevance of the model. I am sure that can actually be done. I am not sure that has been done, but I think it can be.

DR. TELLING: Any further questions?

DR. SAFAR: This is more a comment or offer. I think that starting with Jim Mastrianni, who is at the table, and followed by Michael Geschwind, it was a very difficult and challenging project, the logistics and technical issues and the protocol issues.

With the help of NIH, with Michael Nunn and many other people who cooperated, I think that all of those logistical issues -- and that is an answer to Phil Minor more than anybody else -- it can be overcome.

It took time and it was really difficult, but I think that it is feasible to collect a significant amount, two ml, 200 ml, at a session from CJD patients, either variant CJD or sporadic CJD.

So, I think that this is one of the issues which should be discussed tomorrow in more detail, how to organize such a collection and how such a repository should be handled, funded and organized.

DR. TELLING: With the obvious caveat that sporadic and variant CJD may differ radically in their biological properties with respect to infectivity in blood.

DR. SAFAR: Absolutely, yes.

DR. MANUELIDIS: I would like to make one comment about sort of the definitive comment that David made about sporadic CJD.

It was shown in guinea pigs in 1978 that the blood is infectious and the spleen is infectious. It only makes sense, really, that it would go to spleen if the sporadic CJD, the agent itself, went through blood.

The second thing is that there were two studies that were published in Lancet, one by our group and one by Tateishi's group, showing that human blood actually transmitted as well.

Now, the Japanese group might have a slightly different variant of their CJD because of the different geographic region.

I think that probably the amount of infectivity is much lower than it is in vCJD, but it is likely to be there, from everything we know about these infections and the fact that spleen is infectious.

DR. ASHER: We agree that in blood of patients with sporadic CJD, infectivity is likely to be there, but the amount would certainly have to be smaller.

Over 100 patients who received blood transfusions from donors subsequently confirmed as having CJD followed for more than five years by the American Red Cross, none of them came down with CJD, whereas a very small number of recipients of blood components in the United Kingdom -- 18 -- got presented, three have already come down. So, it is clearly a very different situation.

Now, I suggested for variant CJD that cadaver blood for some purposes would be satisfactory, but the UK authorities, apparently found that idea distasteful. You can get more than a liter of blood from a cadaver, and privacy rights end at death.

DR. TELLING: Thank you. I think we had better move on to the second presentation. Dr. Kreil, industry TSE clearance studies for factor VIII.

Agenda Item: Industry TSE Clearance Studies.

DR. KREIL: Good morning, ladies and gentlemen. On behalf of the pathogen safety steering committee of the Plasma Products Therapeutics Association -- that is the group that I am going to talk on behalf of today -- I would like to thank you for giving us the opportunity to comment on some of the aspects that Dr. Scott has raised in her presentation just a little while ago.

This is just to remind you basically of our constituency, worldwide manufacturers of plasma derivatives. Specifically the considerations here focus on one class of products. Those are the plasma derived factor VIII products.

The manufacturer, just to give you a schematic of that, actually commences very early onwards from the plasma. This is thawed up to just a little above freezing temperature, at which point, in plasma, there is a precipitate that forms, the so-called cryoprecipitate.

By centrifuging these, precipitates can be removed from plasma and that way you basically split up plasma into two fractions, one being the cryosupernatant here, for the production of certain coagulation factors, but then for also the classical Cohn products, Igs and albumen.

Then the cryoprecipitate, where factor VIII products are manufactured, historically they have been turned into high purity. That is meant to say that, beyond factor VIII, they may contain albumen factor in addition.

This is the principle of how TSE or, for that matter, all virus or prion clearance studies are being performed.

So, you have a very large scale manufacturing process. Typically we are talking thousands of liters. Obviously, you cannot work with pathogens at that level for GNP considerations to start with.

So, what is done is, we are scaling down these processes into a scale that we can work with these processes in what we call pathogen safe laboratories.

There we can work with biosafety level agents, and what we do basically is, we run these processes at a very small scale, typically upstream with a little less pure intermediate, a little larger in volume, and then running through one of these purification steps.

What you get is typically a smaller volume intermediate of higher purity. Now at the laboratory scale, what we can do is, we can add upstream, for the purposes of today's discussion, prions but, again, we are doing the same thing with all sorts of viruses also.

Then, after this is added, you go through this manufacturing process at the laboratory scale. Then you can determine the input of prion activity or prion surrogate markers and the output.

Then, by comparing that, and taking into consideration the volumes before and after, you can actually derive what is the so-called reduction factor, so you can understand what reduction of prions for that purpose is achieved by that manufacturing process.

A very important point obviously is, this needs to be exactly like this. Otherwise, the numbers that we obtain are not meaningful for the large scale production of biological medicinal products.

That is why a lot of time is spent, once you have established the so-called downscale, into validating that down scale.

Really what we mean by validating that down scale is that we are going to validate that this downscale is equivalent to the large scale manufacturing process, because this, again, is the fundamental principle under which we can derive knowledge about the large scale processes, from doing these small scale experiments.

So, what can be done to make sure that the information that we derive at the small scale is meaningful? Well, first, the intermediate that we use for running these small scales is directly derived from our manufacturing facilities, or is somewhat specialized materials and we get them maybe from a pilot scale.

In other words, this is regular intermediate that would be manufactured into commercial product. Now, for this product, a number of different parameters can be assessed, not only for the input, which is equivalent anyway by means of its origins, but also, after doing the small scale purification, you can determine whether the amount of protein concentration or activities, for the purification that supposedly should occur in the large scale, does also occur in your small scale.

Further, a number of process parameters can be monitored and, as you can see from the numerous examples on this slide, these do vary depending on the step that we investigate.

If it is a precipitation step, then obviously the concentration of the precipitating agent, the time that the intermediate is stirred with this agent, the temperature would be important.

Then, for example, calling for other steps, then things like pH, conductivity, ionic strength or contact time with the resident would be more important.

Again, this is sort of the prerequisite under which we have to operate. So long as you can't demonstrate that there is a perfect equivalent of the small scale with the larger scale, all further information that you derive would be meaningless for the large scale.

Now, for prion clearance studies specifically Dr. Scott has pointed out already that there are a number of choices that one needs to make before going into a prion study.

There is first the choice of a spiking agent from which organism we want to derive that. Secondly there is a number of different possibilities for particular spikes for the initial spike preparation.

Initially people have used the organ that does contain typically the highest levels of prion levels -- the brain -- and have more or less purified that as a spiking material.

So, brain homogenous has certainly been the first material used, and then different purification forms off that, such as microsomal fractions, detergents treated or sonicated intermediates of that.

Finally, there is a choice of which assay you want to use for a readout of your prion clearance study. That would be either in vivo, which is cost intensive, resource intensive, and you will have to wait a long time to get a result or, alternatively, in the in vitro assay such as the western blot or the CDI.

Now, it is important to understand that, for these prion quantifications, there are a lot of controls also put around this such as, for example, physical reactions are quality control.

There are good laboratory practices applied and, where it is not possible to become certified for the application of good laboratory practices for such studies, and that, as I said, is not possible in all geography. Then at least the principles are being followed.

Then finally, the preparation of the spike materials and how the assays are performed, when an assay is acceptable or not.

All of this is writ down in what we call standard operating procedures so that there is a lot of control being put around the reproducibility of these assays.

Obviously also, such as with every good assay, controls are being put in place, such as a positive control, negative controls, controls for interference of the matrix with the performance of the assay, et cetera.

So, I guess we can say that, by putting in place all of these controls, we can certainly guarantee that the assay is suitable for the purposes of a prion reduction study.

The agency has asked the question whether further standardization or a validation of such assays would be useful, and we would like to make a number of arguments why we believe that that would be useful.

First, I have to mention that, during these manufacturing processes, it can be observed that the initial spike is being conditioned through the process, such that actually you would want to use different materials for investigating different manufacturing processes.

If, for example, you had a solvent detergent treatment upstream from the process that you want to investigate, then any prion that would have been present in plasma, that would have come down to the step that you investigated, would have gone through that contact with solvent detergent.

So, it might be a chance to consider using a spike that has been detergent treated. That would reflect, probably most adequately, whatever was upstream in the process.

Also, investigating the potentially additive effect of sequential steps will require you, for example, to do a run without subsequent spiking. So, again, standardizing how exactly you need to do that experiment is going to make the interpretation difficult and will limit your ability to demonstrate removal.

That is why we would argue that it is more important to rely on good expert judgement first, and then obviously also justification of that judgement on a case by case basis.

Another useful example, we believe, to look at this -- and again, Dr. Scott has shared with you the very same example that I will bring up here -- it has been shown that there can be very substantial removal here by log steps, for example, using a filter with a nominal core size of 35 nanometers.

In the presence of detergent during the preparation of the prion spike, however, that removal becomes less significant, and on the smaller core sized filters, we have been able to remove the spike.

Now, further data that are available and yet unpublished have shown that if you use more drastic detergent treatment and sonication of the prion spike material, then in reality you can get the material even through a 15 core sized filter, with virtually no removal at all.

So, one might argue that that would then be a worst case and such a spiking material should be used for the evaluation of nanofilters.

So, our belief is that these conditions are certainly very important in trying to understand the elementary nature of the infectious unit for prions, but I think these should be seen as experimental conditions.

During manufacturing, we do certainly not add these high levels of detergents, and certainly we do not sonicate our intermediate.

Therefore, should any prion agent be present in plasma, then it would not be sonicated detergent treated to the degree that has been used in this more experimental set up, to understand better the nature of the agent.

That is why we believe that the reduction capacity for nanofiltration has been widely demonstrated under more relevant conditions for manufacturing.

There are some recent, I would like to call it, advances in science that would suggest that maybe we should be using different spiking materials to the ones we have used so far.

A very recent piece of evidence has come from an Italian laboratory where it was demonstrated that starting from brain homogenate at a titer of roughly 10⁸ infectious units per ml, after a very high spin, you can actually device material in the supernatant here, and that supernatant still has a very high level of infectivity, yet very little or even no PRPres demonstrated by, for example, western blot.

That paper, in the conclusion this should suggested that there should be a suitable spiking material to use in validation.

Now, some of the limitations there already are, that you would only be able to do in vivo assays because if there is no PRPres even in the starting material, then that would not be a good readout for a reduction study.

Another complication, however, is that this 10⁵, while still a reasonable titer, represents only one-thousandth of the original input. So, it is a tiny little minority of the original PRP agent.

Now, that we have seen already in an earlier study, where endogenously present infectivity has been fractionated using a less drastic centrifugation here.

Even for endogenous or, if you will, the relevant former sensitivity, you can see that with centrifugation you can pellet quite a bit of that infectivity.

So, the question becomes, if you are looking at these infectivities, are you interested in the majority of the infectivity or do you want to investigate a tiny little minority that, in behavior, may not at all reflect what would be present even in an endogenous infectivity situation.

Another piece of recent information, a very elegant paper has been published recently that I would like to discuss because it seems to have pertinence to the conduct of reduction studies.

It would be an experimental model, a transgenic mouse model, where these might express PRP without the GPI anchor.

Now, if these mice are infected with prions, then they do not develop a classical pathology of scrapie. What is interesting is that they have very high levels of infectivity circulating in their blood.

One might argue that that would be a high titered blood spike so something rather usable for validation studies. Certainly this has been suggested to be the case by the authors.

I guess one argument that I would like to convey to you is that this PRP protein is devoid of the GPI anchor and, therefore, this truncated version is of unclear relevance to the pathophysiologically relevant prion agent that w are concerned with, should it really be demonstrated to occur in plasma.

I would argue, if we investigated the removal of this truncated form, then those results might, because of the similar or dissimilar nature of the agent, tell us something about the true agent or might not tell us something.

In summary, we feel that certainly through the validation of equivalence between large scale and down scale, the controls we put around all the prion spiked materials and also the controls that we put around these prion assays, prion clearance studies as we have performed them up until now certainly have generated meaningful information.

We feel that, therefore, further standardization would, in fact, inhibit process specific investigations more than anything else.

We feel that we should more rely on expert input, obviously providing the adequate justifications. Further, given the enormous advances that science comes up with at a very rapid pace -- the two papers that I have just shared with you have actually been published in the last two months only -- would also prevent using novel approaches that might allow us to investigate more meaningful processes, and I think would really discourage, more than anything else novel approaches.

As Dr. Scott has mentioned, we are now going to share with you a summary of different prion clearance studies that have been performed throughout the industry on specifically plasma derived factor VIII products.

Before showing that to you, I would like to make a number of qualifiers. You need to keep in mind that not all of these products are manufactured using the same manufacturing process.

This also results in different clinical usability, such as some of these products contain non-relevant factors in addition to factor VIII and therefore cannot be seen as typical or just another factor VIII product, if you will.

Also, for the reduction factors that you are going to see for the overall clearing, it is not necessarily so that all the manufacturing steps have been investigated.

So, a lower clearance factor may just mean that not all of the steps have been investigated. Should that be done, the numbers could be much different.

Also, we would like to point out that, for the products that have been licensed in the United States -- and that is mentioned in the footnote of the slides -- these data have been shared in more comprehensive fashion with the agency.

We would also like to point out that, at this point, there are a number of research studies going on. The results we will await and will provide further results.

So, this is the first one. We have taken a look at two manufacturing processes that have been investigated, one being purification with a monoclonal antibody column, and then there is another ion exchange chromatography column.

This is since the 263K strain of scrapie adapted to hamsters have been used, with an infectivity assay. So, bioassay was used for generating the numbers here. You can see a total log reduction of roughly eight logs was demonstrated.

Here is another product, also licensed in the Untied States. Here four different manufacturing processes have been investigated -- actually three, I apologize. That is a PG precipitation here, another affinity chromatography step here, and then a final precipitation plus final filtration.

What has been used here are two different preparations of spike, one being a microsomal preparation here and a detergent 3-D preparation here. Here, the same detergent 3D preparation with the brain homogenate as a complement, and again here, the microsomal and the detergent preparation.

Two independent runs have been performed first by preparation and the mean reduction factors you can see down here. The product overall has a demonstrated safety margin of greater than nine logs of prion.

Another product here has investigated two combinations of steps, one being a sequence of precipitation procedures, and the other a sequence of chromatography events.

There have been, in this instance here, two spike preparations used, here one, and two independent ones, first by preparation, have resulted in these reduction factors here. It should be pointed out that this product is not licensed in the United States.

Another product from the same company, again not licensed in the United States, again, sequential procedures have been investigated here.

Here the spike preparation is mentioned here, in a single run, again using the western blot in vitro assay, if you will, with a cumulative roughly six log reduction for these products.

Company D, that product is also licensed in the United States. Again, two different spike preparations have been used, a purified PRP^SC or microsomes.

It has been assayed with the CDI assay and two runs were performed per spike preparation, resulting in these mean log reduction factors here.

A further product that is licensed in the United States, here is the sequence of events, if you will, when one goal has been investigated. So, sequential steps.

This has been done with two different spike preparations, and one run per spike preparation was performed with a mean reduction factor of 3.7 to 3.8.

Finally, this is the last product, again not licensed in the United States. Here, again, another sequence of steps has been investigated with a single spike material, brain homogenate, and that resulted in a 3.5 log reduction.

So, summarizing all of these already summarized data, I would like to point out that we feel that manufacturing processes for plasma derived factor VIII products do remove prions, to varying degrees.

The individual reduction factors that we had on the summary slides really depend on, first, the specific manufacturing process. That is also resulting in different product quality, if you will. Secondly, obviously these numbers depend on the number of steps that have been investigated. The more steps investigated, the higher the numbers.

Finally, to some degree, on the experimental design. Using in vivo assays, for example, allows you with a higher dynamic range to demonstrate larger reduction factors. So, there may just be larger reduction factors inherent to the assay system that you use.

In summary also we feel that, in terms of the safety margins of these products, it is important to point out that the level of risk at this point remains unknown, the specific level of risks, but very likely the level of risk is low.

There is not any evidence for the transmission of prion diseases by plasma derived factor VIII products, andthat despite the very high level of pharmaco vigilance, I would like to mention the multiplication exercise that the United Kingdom has gone through.

Patients, where it is known that their product has been derived from also the contributions of latent bearing(?) CJD donors have been notified of their presumably increased risk, and these people are being closely monitored.

Epidemiologically, I think it is also important to point out, as Dr. Scott has also mentioned, that the exposure is low and the exposure seems to be getting lower still.

There is, as I did hopefully convince you, a reduction of prion agents by all the plasma derived factor VIII manufacturing processes that I have shown to you.

Therefore, we feel that the quantification of reduction versus an unknown certainly low level of risk is an open equation at this point, really.

In conclusion, we would like to say that, given the unsubstantiated level of risk associated with plasma derived factor VIII, we feel that this is not a rational basis for implementing further measures, because it needs to be kept in mind that any additional steps that might be implemented might also adversely impact the product characteristics, starting with clinical safety, but then with additional manufacturing steps, also typically yields suffer and, therefore, availability may be affected.

I can say on behalf of industry that certainly we continue to be committed to research. We have done these studies on a voluntary basis and, as I said, further studies are currently being conducted and results will be made available. Thank you very much.

DR. TELLING: Thank you, Dr. Kreil, for that perspective. Are there any questions at this point, or comments?

DR. HOGAN: Tom, I can assume, then, that the products, after these additional steps, are all biologically active and have been evaluated for safety?

DR. KREIL: The steps that I have summarized for you this morning are the steps that are being conducted during the manufacture of commercially licensed product.

So, all of these products are clinically usable because they wouldn't have received a license otherwise. It is just that history has shown that, whenever manufacturing processes are changed, such that, for example, greater virus reduction is afforded, that typically that results in a reduced yield of these product, and clinical usefulness of the product then needs to be reestablished by clinical trials.

DR. HAMILTON: What concern is there about the exceedingly long incubation period for CJF and variant CJD, and also could you speak about leukoreduction effectiveness?

DR. KREIL: Well, regarding the long incubation period of variant CJD, this is one of the aspects that still will not allow us to come up with a final judgement, I guess. It is one of the uncertainties. We just need to wait for further advancement and understanding of these diseases.

This is why we don't say that there is categorically no risk, because I think at this point we cannot say this.

In leukoreduction, leukoreduction has been very elegantly investigated for the reduction also, prion activity.

As far as published data, leukoreduction has actually been shown to just result in a marginal reduction of prion infectivity.

There is no further research going on to enhance this leukoreduction to provide the filter with an added prion removal capacity but, to my knowledge, these devices are not yet available.

DR. HAMILTON: Could you compare this rarity or lack of -- this low incidence in this situation to the low incidence that was supposed in the early 1980s with HIV?

DR. KREIL: That is a very difficult comparison, I think. Certainly with HIV, very quickly, during the early 1980s it was realized that there was a blood transmissible agent there.

While the virus was not known at that point, research did quickly establish its presence, and actually the virus was present at very high levels, as we know.

To compare this with prion agents where, despite intensive research, the demonstration of presence in plasma has not been successful, I think would be very different.

I mean, certainly the levels of risk are very different. It has been mentioned today that, out of 18 potential opportunities for transfusion transmission of variant CJD, three have resulted in a transmission, which is very different from viral diseases as we know them.

There you would be looking at a 100 percent transmission likelihood, if we took the figures of blood and transfused it into a recipient. I think this comparison would not be appropriate.

DR. BROOKMEYER: could you comment some on the reproducibility of some of those reduction factors of the data that you showed?

Some of the data, it looked like there was only one independent run or one or two independent runs. If you could just comment on the variability and also on how much you think those reduction factors depend upon the input and how much is actually being spiked in. What are the sources of error or variability in those reduction factors?

DR. KREIL: I was trying to point out that obviously we do everything possible to control these experiments well.

This is what scientists do. At the end of the day we like to have information that is meaningful. We are not trying to make up numbers, if you will.

Regrading the reproducibility, you are right that some of these experiments have been informed with an N of one, but others have been performed with numbers of repeats.

I can tell you that these repeats have been very reproducible. So, I certainly do believe that the studies as I have told you have been controlled adequately to ensure that the numbers are, first, reflective of what occurs in large scale and, secondly, have been adequately controlled so that the numbers are meaningful.

Now, all the limitations, all the caveats that Dr. Scott pointed out are acknowledged. I mean, we haven't seen the agents occur in plasma. Therefore, we don't know exactly what it would look like in plasma, should it occur there.

So, all the spiking materials that we are currently using are models. Therefore, the specific number with the agent, would we have it, as it occurred in plasma, would it occur there, might look slightly different.

I guess one important point to mention is, all these numbers are numbers on a log scale. So, if a number is 3.1 or 3.3 quite frankly, for all practical purposes, the same thing.

In a log scale, again, there are potencies of 10 that you are measuring against. So, minor differences that might occur during experimental set ups would be insubstantial versus the reduction factors that we have seen.

DR. TELLING: If there are no further questions, thank you, Dr.Kreil. Next on the agenda is the open public hearing. Bill, would you let us know who is registered for the open public hearing?

Agenda Item: Open Public Hearing.

DR. FREAS: As part of the advisory committee procedure, we hold open public hearings so that members of the public can address the committee on issues pending before the committee.

Mr. Chairman, at this time, I have received four requests to speak in the open public hearing sessions, one request for this morning, two for this afternoon's sessions, and one for tomorrow.

I would like to invite the speaker for this morning, Dave Cavenaugh, government relations for the Committee of Ten Thousand, up to the podium.

While he is coming to the podium, Mr. Chairman, I would like you to read the open public hearing statement required for the meeting.

DR. TELLING: Both the Food and Drug Administration and the public believe in a transparent process for information gathering and decision making.

To assure such transparency at the open public hearing session of the advisory committee meeting, FDA believes that it is important to understand the context of an individual's presentation.

For this reason, FDA encourages you, the open public hearing speaker, at the beginning of your written or oral statement, to advise the committee of any financial relationship that you may have with any company or any group that is likely to be impacted by the topic of this meeting.

For example, the financial information may include the company's or a group's payment of your travel, lodging or other expenses in connection with your attendance at the meeting.

Likewise, FDA encourages you, at the beginning of your statement, to advise the committee if you do not have such financial relationships.

If you choose not to address this issue or the financial relationships at the beginning of your statement, it will not preclude you from speaking. So, Dr. Cavenaugh?

Agenda Item: Statement by Dave Cavenaugh.

DR. CAVENAUGH: My name is Dave Cavenaugh. I am government relations staff for the Committee of Ten Thousand.

I think as many of you may have heard in one place or another, our president, Cora Dubin, likes the expression coined in the last year or so of having an arm in the game.

All during this discussion I have been sitting here and thinking we have all these studies that show that prions can be reduced in factor.

We don't have a lot of information that they are being reduced in factor. That factor that is going out now, the factor that is going into people's arms, is still the same factor that it has been.

Today's discussion of prion reduction through fractionation brings little comfort to persons with hemophilia in the United Kingdom.

Different interpretations of the findings of science show the United States and the United Kingdom to be going down very different roads on this subject.

As long ago as 1999, when the FDA first announced that screening for classical CJD was no longer needed, the agency began distinctly identifying the greater perceived safety of plasma products to whole blood:

"...experimental studies in animal models for CJD suggested that manufacturing procedures used for plasma derivatives could lower the amount of infectious material present in plasma derivatives compared with whatever levels could be present in blood."

In 2002, FDA moved further down this road:

"...we recommend that you defer donors of whole blood and blood components intended for transfusion. source leukocytes and recovered plasma, but not donors of source plasma, who have resided in Europe for a cumulative period of five years or more, between 1980 and the present."

This exemption of plasma occurred when the whole blood geographic donor ban was first being expanded beyond the United Kingdom.

Since that time, there has been no retraction from this position by the agency. Thousands of units have been collected throughout Europe, pooled and fractionated to make factor VIII, IVIG, albumen and other products, which of course by now have all been consumed, largely by Americans.

It was in 2003 and 2004 that true cases of vCJD transmission by blood were reported in the literature. From that point on, all of the language of prior government and industry regarding theoretical risk of transmission became obsolete.

The United Kingdom, following a different time line regarding discovery of the dangers of variant CJD in blood, learned of the contamination of plasma pools years earlier, and declared the recipients of products from those pools to be at great risk.

The now famous 2004 letter from the Ministry of Health to 4,000 homes of persons with hemophilia instructed them not to donate blood, organs or tissue, and to inform medical, surgical and dental providers, so that disposable instrument can be arranged for in advance of any procedures.

Two years have passed since the risk communication exercise in the United Kingdom. The stigma it brought to every family with hemophilia is somewhat dulled now, although it was unprecedented and disruptive for weeks at the time.

So, which is it? Are we at great risk or is there no risk, or rather undetectable risk, which is not the same thing?

CBER advisory committees are often asked to decide on issues for which there is inadequate data to make sound judgement.

COTT has watched this country's response to TSEs unfold, from the USDA's denial that there is any problem to the untracked venison eaters in areas of US CWD outbreaks.

We ask that you do not give ground. Do not expand the exemption from geographic donor bans which plasma collection now enjoys. We further ask that you retract altogether this dangerous exemption of source plasma from geographic donor bans. Thank you.

DR. TELLING: Thank you very much. Are there any questions or comments? Is there anyone else in the audience who would like to address the committee at this time?

Okay, I thank everybody. We can adjourn until 1:00 o'clock for some lunch.

[Whereupon, at 11:50 a.m., the meeting was recessed, to reconvene at 1:00 p.m., that same day.

A F T E R N O O N S E S S I O N (1:05 p.m.)

Agenda Item: Open Committee Discussion.

DR. FREAS: Before we begin the afternoon session, Mr. Cavenaugh has asked to address the committee for a brief announcement.

MR. CAVENAUGH: I would like to correct some possible misinterpretation of what I said before, just the part about the dissimilar geographic donor ban between whole blood and plasma in Europe creating a lot of blood collected in Europe from countries that don't have the ban and brought here from processing. That doesn't occur, and I know that.

People who live there for some time, who are American, live over there six months or more, in many of the countries to which we now have bans, come back here and give plasma, and that is basically exposed to European risk factors. The other is definitely correct.

DR. TELLING: Thank you. Dr. Epstein?

DR. EPSTEIN: Let me just state it in my own words. I agree with what Dr. Cavenaugh just said. The committee needs to understand that plasma for fractionation into U.S. licensed products has never been sourced outside of the United States.

DR. TELLING: Are there any other comments? If not, thank you. So, the next item on the agenda is open committee discussion. Dr. Scott, would you like to rephrase the questions for us?

DR. SCOTT: Would you like me to go through all of them or just one by one?

DR. TELLING: I think one by one for now.

DR. SCOTT: So, the first question is, we are asking you to comment on the feasibility and scientific value of adopting standardized exogenous or spiking experiment study methods, to assess TSE clearance in manufacturing of plasma derived factor VIII.

We are asking you to consider and to comment on what is the optimal spiking material and its preparation from the standpoint of relevance to blood infectivity.

Second, whether you feel there is any particular preference for TSE strain or animal model in these types of studies.

Whether immunoassays for PRP TSE -- well we would like for you to comment about the use of immunoassays for PRP TSE in the context of these clearance studies, as compared with bioassays for infectivity.

Lastly, identification of manufacturing processes that might alter TSE agent properties. We would like for you also to comment on that based on what you heard and perhaps other things that you know about the agents.

DR. TELLING: Thank you. So, before we get an answer, let's discuss the question as it stands. I would like to make it open to the committee for discussion right now. Does anybody have any comments or additional questions?

DR. SALMAN: This is a point for a clarification. I thought we had discussed in the previous sessions what FDA has done with the risk assessment.

To my knowledge, the risk assessment led to the conclusion that there is almost like very low risk or negligible risk related to the factor VIII. Maybe somebody from FDA can clarify that.

DR. TELLING: Could somebody from FDA comment and clarify on that?

DR. EPSTEIN: Thank you very much. Actually, in the October 2005 TSE advisory committee meeting, we discussed the model that FDA would apply to estimating the vCJD risk from U.S. manufactured factor VIII.

We didn't actually show an output of that model. We do intend to bring to a forthcoming meeting of the committee the results of that assessment.

Generally speaking, looking at the input parameters, we think the risk will be lower than for products made in the United Kingdom, but we have not actually brought forward yet and output of the risk assessment for U.S. licensed factor VIII.

DR. SALMAN: Thank you for the clarification. I think now that you remind me, you are right. Would you think it is better to try to do the risk assessment before we decide about the protocol for how you assess the clearance of the plasma?

DR. EPSTEIN: I don't know that that is a question that just one person should answer. We do think that the risk assessment is highly informative.

The main message, though, is that the factors that most affect the assessed risk are the clearance, the prevalence in the donor pool, and the product usage in the patient community.

Far and away the largest variable really is the clearance. That is why we felt we need an antecedent discussion about just how well do we understand clearance and how concerned should we be about the absence of sta