Workshop on Standards for Inactivation and Clearance of
Infectious Agents in the Manufacture of Plasma Derivatives
from Non-Human Source Materials for Human Injectable Use

The workshop took place in the Masur Auditorium, National Institutes of Health, Bethesda, Maryland at 8:00 a.m., Mark D. Heintzelman, Ph.D., Chair, presiding.

Present:

MARK D. HEINTZELMAN, Ph.D., Chair
JESSE GOODMAN, M.D., Speaker
JOHN S. FINLAYSON, Ph.D., Speaker
DR. PETER NEUMANN, Speaker
DR. HANNELORE WILLKOMMEN
PHILIP SNOY, DVM
THOMAS J. LYNCH, J.D., Ph.D.
KEITH HOOTS, M.D.
MR. JASON BABLAK
BARBEE WHITAKER, Ph.D.

INDEX

Welcome and Introduction

I. Regulatory Perspectives and Issues

II. Starting Materials

III. Techniques and Methods

IV. Special Interest Groups Perspective

V. Industry Experiences

Panel Discussion

PROCEEDINGS

(8:03 a.m.)

DR. HEINTZELMAN: Good morning. It's Monday morning. It's time to get going. I'd like to welcome everybody here. My name is Mark Heintzelman. I'm the chairperson for the workshop. I'll be introducing Dr. Jesse Goodman who is going to give the introduction and welcome.

We have just a very few administrative issues to discuss. I want to let you know that there is a cafeteria here. Getting there is not too hard. All you've got to do is follow the arrows and it's downstairs. Quite easy to do. I don't think they have an Dr. Atkins line, so for those of you who are pursuing such an endeavor you'll be on your own.

We got funded for this week and that's always a nice thing. President Clinton signed a continuing resolution which I think expires on Friday. So it's very happy our workshop is this week. He's making noise about not doing this again and trying to not put gas in the car, but having the car ready to go and don't start it is a real challenge and it would have destroyed our plans.

Our first speaker is Dr. Jesse Goodman. He is our Deputy Director for Medical Affairs at CBER. He's going to give you an introduction and welcome and we'll begin our workshop on Standards for Inactivation and Clearance of Infectious Agents in the Manufacture of Plasma Derivatives From Non-human Source Materials for Human Injectable Use.

Dr. Goodman?

DR. GOODMAN: Well, good morning to you hardy souls. Since I've been saying to my children for the last two hours in various stages of trying to get them to school, one missed the bus, one was still asleep when I left home, so -- I think you have a small group here, but I think in many ways that should encourage you to speak up, have a real interchange here on this subject. But I guess I'd like to start out by welcoming you to this workshop on the Inactivation and Clearance of Infectious Agents from Plasma Derivatives From Non-human Sources for Use in Humans. My background is both as an infectious disease person and a hematologist, so I'm quite familiar at least some of these products and their importance.

These are, as you know, very unusual and special products which meet special needs and they range -- they're often lifesaving products that range from antivenoms to factors for people who have multiple antibodies and as such, although at the present time they tend to have small constituencies and small amounts of use, they're critically important and lifesaving.

And as was pointed out to me, and Mark asked me to say hi here, unlike the situation with the human plasma industry and plasma derivatives, there really is no sort of safety net or set of universally adopted safety standards for this product. So that's what you're being asked to consider.

Now why in the world would one take this issue on now? And I think there are several points that I want to make about that. One is there's an expanding catalog of infectious agents of animal source which potentially contaminate products in humans. And of course, the parvovirus is an example that you're probably familiar with.

There's definitely an increasing awareness of the ability of pathogens to cross species and my area of research interest is in tick borne infections and we've worked on avian leukosis and babesiosis and both of these are obviously common infections of exactly some of the kinds of animals that the products you're interested in are made from and then used in humans. So there is an awareness of this transfer of pathogens.

I think perhaps even more important is the realization that there are contaminants in not just animal, but human biologic materials which don't cause acute and obvious disease, so you -- we tend to think we have quite a good warning system because if something is wrong we will know about it. But as the situation with retroviruses indicates, there can be real problems in source materials that may have an outcome that is only apparent many years later and may not necessarily be easy to tie to the source material.

And then finally, I understand that this

-- one of the oldest areas of sort of the plasma industry here of preparation of materials from animal plasmas also may have some room for expansion in the current biotechnological era in terms of things like development of transgenic plasmas, possibilities of making new immunoglobulins that will be used in human therapy.

So I think you'll hear an overview of these issues today and the question will be what can be done. I think first of all, the reason you're here is because we're all increasing our understanding of both the sources and the nature of these kinds of pathogens that may be in these materials and that has to increase. There's clearly a scientific need here. Again, there hasn't always been -- xenotransplantation has helped stimulate interest in these animal pathogens which may be less obvious causes of disease and we need to begin to apply modern molecular methodologies to search for pathogens that might be important.

I think one of the things that you'll want to discuss is the parallels to human plasma and the potential for incorporating pathogen inactivation steps into the routine management of these materials. Can that be done without sacrificing biologic activity? Can that be done economically? Is that something that is necessarily uniform across different products or will it most likely differ for different products?

And this should be able not only to inactivate known pathogens because, as far as I'm aware there haven't been major crises in this area that you're here to consider today. It's not just the known pathogens you want to deal with. It's the unknown pathogens. It's affording some margins of error and again, this is where there is another parallel to xenotransplantation.

So just thanks to all of you for coming and considering this issue and I hope you'll discuss it carefully and the pros and cons of the various kinds of steps that you can take and begin to move this field forward and I'd just like to thank Mark and the Office of Blood for inviting me to say hi and say that I would like to stay and listen, but I've got to run out and go talk about antibiotic resistance with the folks at CDER, so thanks very much and have a good day.

Return to Index

DR. FINLAYSON: Good morning again. As I look out here I'm afraid that the echo coming back may do away with what little hearing I have left. Nonetheless, I'm John Finlayson. I'm the Associate Director for Science of the Office of Blood Research and Review at CBER and I trust all of you are sufficiently familiar with us that we can use these three and four letter codes to represent our agencies.

Could I have the first overhead? Oh, I have the first overhead. All right. The first line there is an abstract of the title of this workshop which surely must deserve some sort of a prize for lengths of titles for workshops, but the point is I'm going to talk about plasma derivatives and try to tie this to our interest in plasma derivatives from non-human sources. I will attempt to give a historical overview, but as you will see from the next slide which I don't want just yet, the perspective that I'm going to take is not that of someone who has spent a great deal of time with plasma derivatives from

non-human sources. As a matter of fact, I suspect that my major qualification for speaking to you at the beginning of the program this morning is simply that I was the most historical person that Dr. Heintzelman found as he was wandering the halls of Building 29.

Nonetheless, I'm going to try and provide a historical overview and if I can have the next overhead. Could I have the next overhead, please? What I'm going to try and describe are as Dr. Goodman referred to, lessons learned from plasma derivatives from human source materials. Now throughout the day we're going to be talking about plasma derivatives because that's the term that we have become accustomed to, but I hope everyone is aware that the same considerations would apply if we were talking about material made from serum or whole blood or blood cells rather than plasma per se. So regard the term plasma as partially precise and partially shorthand.

However, in talking about plasma derivatives from human source materials in an attempt to give a historical overview, it is also entirely appropriate to consider the history with respect to animal plasma derivatives and there are several reasons for this. The very earliest plasma derivatives that we had were from animal sources. If I could have the next overhead?

Already in 1890, Behring and Kitasato described antitoxins made from animal blood, animal plasma, animal serum, mostly, but not exclusively equine in origin. And these antitoxins have been with us ever since. Furthermore, not only the first plasma derivatives, but the very first biological reference standard in the world was in animal preparation. If I could have the next overhead?

Paul Ehrlich in 1897 was faced with the problem of standardizing the potency measurement of, I'll say this term in German, diphtheria Heilserums, literally healing sera. Or as we said a little later, therapeutic sera. We are fortunate to have a representative from the Paul Ehrlich Institut with us today and she'll be speaking a little later on the program.

Faced with the necessity for doing these potency measurements and for standardizing the measurement process, what Ehrlich decided to do was to choose one antitoxin as the reference preparation, determine its ability to neutralize toxin and then report the potency of the other antisera in terms of comparison with this reference standard.

Now closely allied to this procedure and closely allied to the two facts that I've said, namely that the first plasma derivatives were of animal origin and the first reference standard was of animal origin is the alliance to the legislative authority for the Center for Biologics Evaluation and Research. That is to say, CBER. These antitoxins or antisera were, when they came in use, prepared locality. In other words, if they were needed in the New York City area, they were prepared in New York. If they were needed in the Washington, D.C. area, they were prepared in the Washington, D.C. area. And sometimes they worked and sometimes they didn't work.

Now in 1901 there was a serious outbreak of diphtheria in St. Louis and so immediately a program was initiated for administering diphtheria antitoxin, again, locally prepared in St. Louis, this program was begun. Tragically, in this immunization and of course it was passive immunization, ten children died not of diphtheria, but rather of tetanus. Why did this happen? This happened because the horse from which the antiserum was collected had tetanus and in the rush to immunize, collect the antiserum, immunize the human recipients, it was considered that there was not sufficient time to do safety testing.

Well, as a result of this tragedy, if I could have the next overhead, Congress passed the Biologics Control Act in 1902. This act is variously referred to as the Virus Toxin Law and the Vaccine Virus Toxin Law and other shorthand terminologies. The point is that it was the predecessor of our current day Public Health Service Act.

Now if you publish an act for the control of something, you have to give some group the authority for enforcing it. And Congress gave the authority for enforcing the Biologics Control Act to a division of the Hygienic Laboratory. By that time, 1902 the Hygienic Laboratory had moved from New York to Washington, D.C. It's worth noting that the Hygienic Laboratory was the predecessor of the National Institutes of Health and the particular division that was given authority for enforcing the Biologics Control Act was the predecessor of CBER.

Now among the classes of products mentioned in the act, you see, was therapeutic serum and was antitoxin. These animal antitoxins and analogous products still exist and are still with us. If I could have the next overhead. They have been joined by a number of other products from animal sources and I have listed here animal species from which we have currently licensed biological products and I might add that others are under development even as we speak.

If I could have the next overhead which is something if you think you've seen it before it's an indication that you are awake and oriented and paying attention, just to remind us that we're back on the track of seeing what lessons have been learned from plasma derivatives from human source materials.

Now to glean these lessons, we need to fast forward from the time of Behring and Kitasato and Paul Ehrlich and the Biologics Control Act enactment to the time of World War II. In the 60 years between the onset of World War II and the present, we truly have learned a great deal about viral clearance. If I could have the next overhead.

Much of the recently obtained information has come from such procedures as cell culture of the virus in question when Dr. Willkommen from the Paul Ehrlich Institut gives her talk, she will refer to these as relevant viruses, for example, HIV. In other words, the actual virus that we are concerned with that is inhabiting the plasma that is the source for our plasma derivatives.

Another powerful technique in recently obtained information is the use of cell culture of model viruses, for example, BVDV, bovine viral diarrhea virus has proved to be an extremely useful model virus for the hepatitis C. And if neither of these is appropriate, we now have available to us a nucleic acid testing where we can test for the genome or parts of the genome of the virus in which we are interested.

Now if I could have the next overhead, we can see by methods such as these, we can determine the quantitative reduction in the viral load. That is to say we can quantitate the viral clearance. We can get an idea of the reproduceability of that clearance by a particular manufacturing step or series of manufacturing steps or an overall manufacturing process and depending on the particular procedure that's being used to eliminate viruses, we may even be able to get information by using these approaches about the kinetics of the clearance.

However, I'm not going to talk about these things because Dr. Lynch is going to be talking about them this afternoon. So for now, let us, as a certain program back in the days of radio, if there's anyone in the audience old enough to remember the days of radio, used to say let us return to those thrilling days of yesteryear, specifically to the time of World War II and look at the next overhead.

Here's some facts about the manufacture of human plasma derivatives in the 1940s which is essentially when the whole industry began. There were no viral screening tests available to use on the source plasma. That is to say on the plasma donors. You could look at the donor's eyeballs to see if they were bright yellow. You could ask the donor if he had ever had jaundice, if you were really a forward looking blood collection center, you might even do one of the indescribably nonspecific liver function tests, but there were no specific tests available to screen for viruses that might be in the donor's blood and therefore the donor's plasma.

Moreover, the manufacturing process itself for preparing human plasma derivatives was still evolving. Next overhead, please.

So how could you tell that the product was, from a viral point of view? Safe, or conversely, that it was unsafe? And how could you tell that the manufacturing process was or was not clearing virus? Well, I think I should digress for just a moment at this point because sometimes we become very taken with our modern status and self-importance to say that even back in the 1940s and 1950s people were aware of the procedures that we have available to us today. That is to say, to culture a virus and to harvest that virus, spike it into the plasma and see where it went during the purification process.

There were only two major problems in the 1940s and early 1950s with this approach. And that is one, since virtually nothing was known about the biology of the viruses that were in human plasma and could infect potentially recipients of plasma derivatives, there was no way of knowing whether these viruses that could be cultured and harvested were or were not good models for the viruses that you were interested in. So the best that they could do was to use a variety of these viruses with different physical and biological characteristics.

The second problem was that when such procedures were carried out in the early 1940s with a fractionation procedure which was a distant precursor of the way that most human plasma derivatives are made today, what we found was that the viruses that were used as tracers showed up in all fractions harvested. So even though there may have been some quantitative reduction in the viral load, it forced people to use other procedures for determining whether the material was virally safe and whether the process being used for manufacture had cleared virus.

So if we take a look at the next overhead we'll see some of these other approaches. Well, one of the useful, I would say, intermediate approaches has been the use of animal models. But you see I put there parenthetically, eventually, because in the 1940s and the early 1950s these animal models did not exist. These models which are primarily primate models began to evolve at the very end of the 1960s and continued to develop through the middle of the 1980s.

However, one approach that was available from the earliest time was the use of epidemiological studies. Sometimes these epidemiological studies consisted of following the patient populations, that is, the recipients of a particular plasma derivative simply to see whether there was disease development. On some occasions there was investigation of adverse events and these too provided useful information.

The last thing that you see on the list there is studies with human volunteers. I would like to spend a little time on this for several reasons. First, because these studies were done it the late 1940s and the early 1950s, and there has been such a long lapse of time between then and now, these studies are not well known to many of today's investigators. And the other reason is that these are studies that obviously could never be done again, so it is worth seeing what information was taken away from them.

If we look at the next overhead, here is an experiment which studied the effect of ten hour heating on hepatitis. Now the hepatitis that people were talking about in these studies, these studies were reported in 1948 and done a number of years earlier, was that this hepatitis was a so-called homologous serum hepatitis which today we know to be hepatitis B. If you will look down in the footnote down here and let me see if I can make this work, you'll see "icterogenic" pooled plasma. In those days, pooled plasma was a licensed product and you have probably all seen the posters showing the wounded serviceman lying on the beachhead and the medic there with the inverted rifle with the bayonet stuck in the sand and he's infusing this reconstituted plasma as part of the casualty resuscitation procedure. Well, it was known that pooled plasma carried the risk of transmitting so-called homologous serum hepatitis and in some occasions there would be pools, lots of this plasma which seemed to be particularly capable of transmitting hepatitis and these were designated "icterogenic" pools. So in this particular experiment, 10 milliliters of an "icterogenic" pooled plasma was mixed with 40 mls of 25 percent human albumin which was the only way that albumin was formulated in those days and 10 milliliters of this mixture was after the treatments, which I'll come to in just a minute, was injected into human volunteers. You see that 10 milliliters of such a mixture would be equivalent to 2 milliliters of the plasma and therefore, presumably would transmit, have the potential for transmitting the infectivity in that plasma, those 2 milliliters of plasma plus any infectivity that might be present in the albumin itself.

The first treatment that this underwent, Group A, was nothing, simply to make the mixture and put it in the refrigerator. The second was to heat the mixture for 10 hours at 60 degrees Celsius. Now anyone who has ever tried to heat human plasma or serum at 60 degrees Celsius knows that you start to coagulate it or turn it into gelatin very quickly. So being able to do an experiment like this was dependent on finding stabilizers that would allow albumin to be heated for 10 hours at 60 degrees Celsius and in fact, to a certain extent, if diluted properly would allow whole plasma to be heated. So heating, you see, for 10 hours at 60 degrees Celsius in the presence of stabilizers or for 10 hours at 64 degrees Celsius in the presence of a somewhat different mix of stabilizers eliminated the transmission of hepatitis and thus seemed to have been a very effective method for clearing virus. Now I'm not going to elaborate on this because let's take a look at the next overhead because the obvious question was well, suppose you took the "icterogenic" pool of plasma and simply fractionated it to prepare albumin. What would be the infectivity of the resulting product? And as you can see from Group A here, this albumin again, prepared as a 25 percent solution, just like the clinical preparation, but undergoing no heating, did not transmit hepatitis.

Now recall that two or probably even one milliliter of the "icterogenic" plasma when injected into human volunteers would infect at least half of them with hepatitis. Here we're injecting three milliliters and we're injecting a 25 percent solution which depending on how you want to do the calculations, amounts to at least 18 milliliters of the starting plasma and there is no evidence of hepatitis. When that albumin was heated and the same dose was given by the same route, again, no hepatitis.

When a much larger dose was given, something that is like a clinical dose or maybe twice a clinical dose that might be given by the route that the clinical dose would be administered to one icteric and one non-icteric case of hepatitis was found. On the other hand, when this albumin was heated, no hepatitis.

Now let's go back and take a look at this line here. One hundred milliliters of 25 percent albumin, again, depending on how you want to do the calculations amounts to at least 625 milliliters of the starting plasma. This is plasma of which one or two milliliters would be expected to infect half of the recipients. And so the message here is that simply the purification process to obtain the albumin in a purer form and albumin in those days was prepared to a purity of at least 97 percent, simply the purification procedure in the absence of the heating was capable of the great reduction in the viral burden. And seeing that there could be virus still remaining, this was eliminated by the heating procedure which is consistent with the information that we saw on the previous overhead.

Now I mention to you that the procedure for purification, that is the manufacturing process itself was still evolving at this time. The method for manufacturing this albumin was a fractionation procedure which was called Cohn Method 6. That group that worked out these procedures under the leadership of Professor Edwin Cohn at Harvard Medical School continued to develop methods and finally, eventually got up to Method 12. In Method 12, one prepared, among other fractions, what was called SPPS, Stable Plasma Protein Solution, which was made up as a five percent protein solution and as you can see when this was administered, hepatitis indeed was transmitted. This was a less pure preparation of albumin. It was rich in albumin, but only about 69 percent of the total protein was albumin. Nonetheless, despite this impurity and the fact that it could transmit hepatitis when it was heated for 10 hours at 60 degrees, again, the hepatitis transmission did not occur.

Well, you can ask, is this a real result? In other words, I just got through telling you that there were no specific viral tests available in those days so how did people decide whether or not there really was transmission of hepatitis? Well, first thing one would look for was jaundice and obviously if there was jaundice, the chances were very, very high that hepatitis had been transmitted.

If there were not jaundice, one did all of the liver function tests that one could get one's hands on, looking for serum bilirubin, bromsufalein test, the thymol turbidity test and other tests that were in the armamentarium of the investigative physicians at that time. However, in I think testimony to the vision and face of the investigator who led the carrying out of these studies, namely, Dr. Roderick Murray, he bled these recipients of these products serially, obtained the serum, froze an array and kept the records on the faith that some day there would be specific serological tests for homologous serum hepatitis. And indeed, when 15 or 20 years later Murray and a different co-worker thawed out these samples, coded them, tested them under code, to make a long story short, the recipients who were said to have had hepatitis had hepatitis B and those who were said not to have had hepatitis, didn't have hepatitis B.

All right, let us move from albumin and ask what about other plasma derivatives? Consider the product that today is called immune globulin. Its major constituent is what we call today IgG. In the 1940s and 1950s, there were no effective stabilizing conditions to permit the heating of IgG or immune globulin and therefore it wasn't heated. Furthermore, there were no other known effective viral clearance techniques and so the use, obviously, were not employed either. Nonetheless, as I indicated, the methods for purification, that is, the actual manufacture of the product was still under development and so we can take a look at the next overhead to see a comparison here.

Here we have the infectivity of immune globulin made from, that is fractionated from a pool of "icterogenic" plasma. Here we have it fractionated by Method 6 of Cohn and Method 9 of Oncley which, in fact, is the way that most of the immune globulin for intramuscular administration is still made today. And we can see here that a 2 milliliter dose of 16 percent protein solution and I might add parenthetically that this is very much like what is used today, 16.5 plus or minus 1.5 percent protein is the concentration of immunoglobulin that is manufactured and used clinically today. When 2 milliliters of this was administered to 10 recipients, no hepatitis was found and again, these recipients were bled serially and their sera tested again 20 years later and the results confirmed. Sixteen percent solution, 2 milliliter dose amounts to at least 32 milliliters of plasma and recall that the starting plasma, 1 or 2 milliliters would be expected to infect about half of the recipients.

Now, let's go to this elegant method, Method 12. When I say elegant, that is not irony. From a physico chemical point of view this was a truly elegant method. The only problem was when that immunoglobulin was injected, 5 out of 5 of the recipients got hepatitis. Now you may say well, yes, but it wasn't a fair trial because you really were studying the route of administration here. That very well may be, but despite that this experiment spelled the death knell of Method 12 for anything other than a laboratory method for purification of plasma proteins. But it is legitimate to ask was this result, namely no hepatitis from the immune globulin prepared from "icterogenic" plasma by the method that, as I say, is still used today was this a real result or was one simply lucky or was one simply skimming off somehow the tip of an iceberg?

So on the next overhead, we see some of the follow up of recipients of immune globulin. Here we have a study that was carried out and reported during World War II. Eight hundred sixty-nine recipients of immune globulin evidenced no jaundice. Admittedly, a crude measure, but better than nothing.

In 1952, remember, we were still a little time away from the development of polio vaccine, so the only medicament that was available for prophylaxis for poliomyelitis was so-called poliomyelitis immune globulin, a preparation of IgG from people who had recovered from polio and 2,800 recipients of this prophylaxis were followed and again, no jaundice was seen.

Also, in 1952, we were fighting a war in Korea and so immunoglobulin was being given as prophylaxis for what was then called infectious hepatitis or as we call it today hepatitis A and so 1,977 recipients of this prophylaxis were followed and these were followed both by looking for evidence of jaundice and by liver function tests and again, no product related hepatitis was seen.

Now I would say that the take home message at this point is it seems that immune globulin, despite the fact that it undergoes no deliberate viral inactivation steps, seems to be safe, but the reason for the safety is not clear. Now an incident that took place in the 1970s which in the interest of time I will not describe, this incident and the follow-up thereof suggested that the presence of some antibody, that is to say, antibody to the hepatitis B surface antigen or anti HBS, some antibody in the product itself was important for neutralizing any hepatitis B virus that might have escaped detection and might have found its way all the way through the fractionation process.

Furthermore, in the 1980s and the 1990s, there were numerous occasions to perform very intensive follow up of immune globulin recipients both with respect to transmission of hepatitis and with respect to transmission of HIV which had reared its ugly head by that time. Some of these follow ups took place in the context of clinical trials. Some of them took place in the wake of reports of adverse events. Some of them took place in the wake of rumors. For example, in the 1980s, word got out that one recipient of RHOD immune globulin, RhoGAM and as you are aware RhoGAM is a trade name and I am using it advisedly here, that one recipient of RhoGAM had developed HIV infection. You can imagine that this lit up the switchboard both at the Ortho Corporation and at the FDA. And so an immediate intensive follow up took place involving both of those organizations and the CDC. It proved that eventually to have been strictly a rumor. The recipient had a number of other modes of becoming infected, but on this occasion there was very wide follow up recipients of not only this product, but other immune globulins.

Along in the early 1980s, we also had intravenous immune globulins developed and licensed and the recipients of these were followed in the context of clinical trials as well as post-marketing surveillance. There was no evidence ever of transmission of HIV or hepatitis B virus. There were, however, some rare transmissions of hepatitis C virus including one set of episodes of transmission of hepatitis C virus by a U.S.-licensed immune globulin intravenous.

In view of this situation, FDA requested that all manufacturers of immune globulins, be they for intramuscular use or for intravenous use have validated viral clearance steps in their manufacturing process.

Now, to continue tracing the evolution of plasma derivatives we should ask what other major class of products evolved? And the answer is clotting factors. Now if we look at the early stages of plasma derivative development on the next overhead, we see that in their early stages of development albumin seemed to be safe from the viewpoint of transmission of viruses and we felt that we had a pretty good idea why this was so, that is, the purification process lowered the viral burden and the, by that time mandatory 10 hour, 60 degree Celsius heating was effective in inactivating viruses.

In the case of the immune globulins by contrast, they also seemed to be quite safe, but the reason was not clear. And again, I emphasize in the early days there were no deliberate viral inactivation steps that were possible and therefore none was carried out.

When some decades later, clotting factors or sometimes they're called clotting factor concentrates became available it was known that these were risky products. In fact, they were called high risk products. Nonetheless, the benefit risk ratio was so high that it was deemed appropriate to use them. It was deemed appropriate by the FDA and the predecessor control organization. It was deemed appropriate by the manufacturers. It was deemed appropriate by the physicians and most importantly, it was deemed appropriate by the patients because these were truly life saving products.

Now I might say parenthetically at this point in the discussion, mainly because there's no other appropriate place to say it, some products that did not have such a high benefit risk ratio were simply taken off the market. For example, human thrombin was delicensed as a therapeutic product in the 1950s. It was shown that it transmitted hepatitis and there was an alternative product, namely bovine thrombin available. Human fibrinogen was taken off the market in the 1970s. It also was found to transmit hepatitis and as information accumulated about its clinical use, it was found that its clinical benefit was very, very low.

Now, anti-hemophiliac factor was first licensed in 1966 and since then there have been numerous developments. Of course, the one that immediately leaps to mind is the tragic transmission of HIV to hemophiliacs who were receiving such preparations. But let us look at the next overhead and we'll see some of the progress in clotting factors since 1966.

First, there's been the introduction of specific screening tests for the plasma and for the donors. Now bear in mind that with the exception of the syphilis test, all tests for infectious diseases to which the plasma of plasma donors and blood of blood donors is subjected had been introduced since 1966 and 100 percent of the tests that we do for viral markers have been introduced since 1966, so all of this is within the time frame that is the history of clotting factors.

Second, there has been the introduction of deliberate viral inactivation steps. The first of these was introduced in 1983 and they became universal by 1985. You see below here, I have indicated discovery of methods for stabilization depending on the particular method that was used for viral inactivation or viral clearance. Sometimes the introduction of a particular method was dependent on the discovery of a method for stabilizing the clotting factor so in fact it could be subjected to this procedure. Bear in mind that one of the major impediments to obtaining purified clotting factors in the first place was that compared with proteins such as albumin, they were much less stable, simply from a protein point of view.

And then finally we had over this time period since 1966 advanced purification procedures, procedures which were developed to obtain a purer protein, that is a higher specific activity, clotting factor, but which in fact, could be validated and very often shown to have a great deal of viral clearance capacity.

Now again, I am not going to discuss all of these items here because Dr. Lynch is going to talk about them this afternoon. I mean I certainly hope Dr. Lynch can live up to this advance billing that I'm giving him.

What I am going to do is to give a summary of some results of epidemiological follow up, much of which was, in fact, most of which was obtained in the setting of clinical trials of hemophiliacs who received antihemophilic factor and if you look at the next overhead we can see that information that was gleaned over a number of years. Now, I should say that if we went back in time before that we would see that those earlier studies on human recipients were preceeded by studies with animal models and in fact, virtually of them were with the chimpanzee model. Nonetheless, because the denominators in those studies were considerably smaller than those that we have here, I think we can look directly at the results with human recipients.

Now also bear in mind that this all took place after 1985 and that means that the plasma, the donors of the plasma that was used to prepare those materials were being screened for markers of hepatitis B and HIV and furthermore all of these products were subjected to one or more deliberate viral clearance processes. After this point then screening for markers of hepatitis C came in as well. Products A, B, C and D are simply different U.S. licensed antihemophilic factor products. A prime is not a U.S. licensed product, but was made in manner similar to the method used to make product A and was licensed in a different country.

Suffice it to say without belaboring the denominators that you see that all of the numerators are zero. This is follow up of recipients of antihemophilic factor, that is to say Factor VII concentrate. Factor IX safety data was mostly published later. These studies that I have selected here for reported in 1993 in a review by two employees of CBER, Drs. Bill Fricke and Dr. Mary Ann Lamb. But subsequently information on Factor IX concentrates became available as well with the same results so that we can say since 1987 there have been no, zero, transmissions of hepatitis B virus, hepatitis C virus or HIV by U.S. licensed clotting factors and there was only a brief episode in 1995 of the transmission of hepatitis A by clotting factor made by one firm.

So what do you say about these effective, I would even go so far as to say proven approaches to viral safety that have evolved in the decades since human plasma derivatives came into the picture?

Let's take a look at the last overhead and I think the message is that the combined use of screened plasma, that is to say screened plasma donors, validated purification steps and by that I mean not only validated from the manufacturing point of view, but purification steps to prepare a purer product also validated for their viral clearance capacity, validated deliberate viral clearance steps and certainly not to be forgotten adherence to current good manufacturing practice. This combi approach has served us very well, so I think that the lessons are that not only has this combi approach served us well in the field of plasma derivatives, but to use a word that the computer people like very much, this approach seems to be exportable and in particular, it should be exportable in whole or in part to plasma derivatives made from non-human source materials.

Thank you.

(Applause.)

Return to Index

DR. HEINTZELMAN: My name is Mark Heintzelman. And I'll be speaking regarding the regulatory requirements for plasma derivatives. As soon as we can get the projector to come up. Our computers now are now very high tech and very safe and the one I have in particular has so many layers of passwords and security codes on it that if this takes more than three minutes this could take forever. So hopefully we'll be moving along quickly very soon.

I would like to thank Dr. Finlayson for that overview. I feel that he is eminently qualified to educate myself, in particular. He's been a mentor of mine since my career here at CBER and I always benefit greatly from listening to him.

His comment about the length of the title is very true and you have to remember that when you have a last name as long as Heintzelman, you tend to see length differently than many people and I happen to notice shortness and brevity much more readily.

Something to point out not generally noted is I've tried to avoid as much as possible the use of red and green in these slides for people who are red/green color blind. Projections like this can drive you crazy. I happen to know from personal experience. So they may lack luster, but I can read them for a change.

My name is Mark Heintzelman. I work with the Division of Blood Applications in the Office of Blood Research and Review, Center for Biologics. My talk is concerning the regulatory requirements for plasma derivatives in the United States.

Page down, please. The title is Standards for Inactivation and Clearance of Infectious Agents in the Manufacture of Plasma Derivatives from Non-Human Source Materials for Human Injectable Use. Long, but for a reason because there are a number of animal derived products that get manufactured into a variety of final applications and we wanted to try to make this so that when you read the title you would at least recognize that we're not talking about in vitro diagnostics or a variety of other products.

Next slide. I will discuss the regulatory requirements for plasma derivatives that pertain to pathogen reduction and try and review them at all stages from pre-IND through post marketing because while there are a number of products that are licensed that are made from plasma derivatives, there are -- and we have many manufacturers who know the regulations, many manufacturers and consultants here, who know the regulations incredibly well. We are hoping to address some of these issues to people that were newcomers to the field also, so there may be a minor amount of review for those of you with a considerable amount of experience.

Which products? Well, specifically we're talking about plasma derivatives, regulated by the Center for Biologics Evaluation and Research within the Office of Blood Research and Review, not those regulated by the Office of Therapeutics and not those regulated by the Office of Vaccines. Though we may share the same concerns, we may in the long run end up in the same place for those products, but we're here to talk about blood and blood products.

Of course the issues that are pertinent are zoonosis and safety. When considering this product line, it is important to compare the two steps in the manufacture of human plasma derivatives. Setting standards for pathogen reduction in animal derived products should be no less rigorous. I think Dr. Finlayson has done a wonderful job of showing how our base of information has come from human success stories in restricting and reducing viral and pathogen contamination.

Examples of infection that can be quickly recognized when sourced from human plasma or serum do to their rapid rate of infection are well known to many of us. For products manufactured from animal plasma or serum, the infection rate can be much more gradual as is suspected say in the course of BSE or for an opportunistic pathogen of animal origin in aggressive infection with high morbidity and mortality is also possible. So we see the gamut on both sides of its ability to demonstrate itself epidemiologically.

We're going to discuss now, and as I said I would review the regulations. I realize that reading the regulations can be the greatest cure for insomnia known to mankind and I will try to keep it from falling within that purview, but I will review the regulatory pathway to eventual licensure for these products, trying to point out at appropriate intervals where these pathogen reduction and removal or inactivation schemes can be gleaned from the guidance and the documentation that we have.

First opportunity to discuss this issue is at a pre-IND meeting. Certainly a formal meeting, typically conducted with a sponsor prior to submission of the IND. Prior to filing an IND we encourage that you meet and discuss source materials and pathogen reduction concerns with CBER when you have a product that may have within it this liability. This is a great opportunity to lay the groundwork.

At this point in time a really good recommendation to a manufacturer is to ask them what is your intended use statement to be? If your intended use is clearly defined at the pre-IND stage, you will certainly find that is a much more direct path to the final testing of your hypothesis in accomplishing the Phase III pivotal trial, rather than deciding what your intended use statement will be after completion of the Phase III pivotal trial. So it's really a good first question to ask.

Of course we're now faced with changing technologies and changing technologies bring new species into production and new concerns and the discussion that we have today will be certainly based upon the five or six species that Dr. Finlayson pointed out as being a manufacturing species for these products. I'll mention Dr. Snoy's talk in a while. He will cover these animal issues and requirements in detail.

As everyone knows the pathway to licensure should begin with pre-clinical data, Phase I, Phase II and Phase III testing within the IND. These regulations are found in Title 21 Code of Federal Regulations, Section 312.

Another good opportunity that presents itself as the IND progresses is at the pre-Phase III meeting. Typically, will have met with the sponsor prior to the filing of the IND. Generally, there are a number of conferences and calls, sometimes even meetings required during Phase I and II, but before you get into Phase III it's highly recommended that you meet and discuss with CBER in detail the plans to make sure that you have consensus as to where you're going. So at this opportunity is also a very good opportunity for discussion, to discuss and agree on the pivotal trial and the validation requirements for the product. These would include pathogen reduction and pathogen inactivation standards.

After having completed your Phase III, you'll be considering submitting your license application and a pre-licensing meeting is essential. Here, we find final agreement for pathogen reduction can be identified, now that you're going to be scaling up and begin talking about providing final large volume of your product. Scale of manufacturing and appropriate validation requirements are identified. If you will be going from pilot to scale we have a number of guidance documents that concern themselves with those requirements, but there are instances where scale up does dramatically affect the production modality. And can require a new look at viral or pathogen reduction inactivation standards.

The licensing requirements, of course, are found in the Code of Regulations, Title 21, Section 314.

We'll find as we go through this talk and as John began to point out very concisely when he reviewed the Cohn and Oncley fractionation steps and methods that many manufacturing steps will have pathogen reduction capability. The value of those steps should be identified and quantified and not just looked at as serendipitous.

Additional specific steps may be required to be incorporated into the manufacturing process as you proceed to consider pathogen reduction and inactivation.

These typically are seen as steps such as solvent detergent treatment and heat inactivation. Dr. Lynch, who did make it will be here and discuss these steps in detail.

Now I'd like to begin with a very brief quick overview of some opportunities to discuss pathogen inactivation at the IND stage up through pre-license. Now the manufacturer has met and discussed in detail with CBER these requirements and we have some documentation that's available to you to help get through the filling out of the form 356H and to eventually obtain licensure.

A document that is very pertinent to this issue is our CMC guidance. This is the chemistry and manufacturing and controls and establishment description information for human plasma derived biological products, animal plasma or serum derived products which was issued and finalized in February of 1999. This document, we always have to say this, this document represents FDA's current thinking on the content and format of the chemistry and manufacturing controls and establishment description information for human plasma derived biological products, animal plasma or serum derived products. Current thinking is current thinking, subject to change and modification as technology and time advances.

I'm going to review a number of areas within the document where are steps taken or steps are identified that can serve to address the issues of pathogen reduction and inactivation. First of all, we find in the general information section two definitions a statement about virus clearance. The number of principles may be used to demonstrated expected removal or inactivation of infectious virus. That's a very nice way of saying that CBER is open to technological advances. It recognizes that there are standards that are out there, such as solvent detergent and heat treatment, but new, novel creative methods that render a product safer without adulterating its activity are always being sought after and would readily be considered during manufacturing.

The manufacturing scheme may include steps which are intended to specifically address removal and steps which specifically address inactivation. This was the first time that I was able to encounter specific notification that we consider these issues to be separate and distinct even though they may result in the same end product where we are looking at removal and inactivation. Removal serendipitously may be through the fractionation process and intentional steps added in for inactivation.

Under Part 1 of the CMC section within the introduction, going from the general information to the introduction, we find the starting materials for human plasma derived products are known to be capable of transmitting infectious disease and many of the infectious agents of primary concern have been identified. There's nothing surprising here.

It goes unsaid, but it's not included within the document that for animal plasma derived products a different set of agents is of concern, but no less concern than for human plasma.

Part 2 within the biological substance product component of the document, C, methods of manufacturing and packaging within the manufacturing methods. It says (1) starting materials. Materials used in the processing and collection of the biological substance should be fully described. Such a description could include any endogenous pathogens within the species that are being used for production.

1(a). For purchased raw materials, representative certificates of analysis from the supplier or the manufacturer's own acceptance testing results should be submitted. It's typically interpreted in to mean that that would include identification of any potential pathogens.

(b). The tests and specifications for materials of animal source that may potentially be contaminated with adventitious agents, for example, bovine spongiform encephalopathy for fetal bovine serum and viruses and products of human and animal origin should be fully described. Here we find a direct notification that we would like to have information regarding any potentially contaminating viruses identified at this point. And it should not be just construed to be limited only to viruses. Any pathogens would be appropriate to identify.

Information or certification supporting the freedom of reagents from adventitious agents should be included in the submission. That goes unsaid. In-depth discussion regarding the quality of the animals used in production will be discussed by Snoy shortly. I will not pursue information at this time regarding the species and the pathogens of concern, but continue on with the regulatory pathway for these products and their relationship to the reduction standards that we will discuss, hopefully, when we get to the discussion panel, leaving the information for the specifics regarding animal serums and production with Dr. Snoy.

Under process controls within the CMC guidance document there's validation data should be provided for a number of processes.

A description of the validation studies which identify and establish acceptable limits for critical parameters to be used and in process controls, to assure the success of routine production. Reference can be made to flow charts and diagrams. Certainly critical areas to determine appropriate levels for would be in pathogen levels during the processing.

Validation studies for the purification process or a description of the validation of the purification process to demonstrate adequate removal of extraneous substances such as chemicals used in purification, column contaminants, endotoxin, antibiotics, residual plasma proteins, nonviable particulates and viruses should be provided. Yet another notification that we are looking for this information for these license applications.

Within microbiology is an unusual twist to this, but a description of the validation studies for any processes used for an activation of waste for release into the environment should be provided. If you're going to be releasing waste into the environment as a result of your manufacturing process and that waste is contaminated with animal pathogens, that too should be identified and corrected. So it's a little bit out of the manufacturing stream within the final product, but still within the concept, overall, of pathogen reduction and removal.

Within specific analytical methods 1(b) is the statement lot release protocols including specification, ranges of representative lots of the product should be provided. Specifications may include, but are not limited to biochemical purity which may, for example, include PCR testing of the final product to look for pathogen DNA or RNA, safety, which I'll discuss later, but safety is clearly one of the regulations we have that directly addresses the issues associated with pathogen reduction; appearance, pH, residual moisture, excipients may or may not be, endotoxins and sterility.

Under (f), specifications, analytical methods, excipients; (b) refined for noncompendial excipients, tests and specifications should be described. For novel excipients, the preparation, characterization and controls should be described. As technology continues to move forward, novel, the statement here for novel excipients leaves wide open manufacturing techniques that will undoubtedly include derivatives from animal, serum and plasma and the need again to consequently identify those pathogens that may be removed or inactivated throughout the process.

For inactive ingredients of human or animal origin, you need to provide certification or results of testing or other procedures demonstrating their freedom from adventitious agents. So direct correlate to these excipients and their possible contamination with adventitious agents.

An impurities profile needs to be provided. A discussion of the impurities profile with supporting analytical data should be provided. But certainly within an impurities profile for anyone whose product may contain zoonotic organisms we would want to see it addressed fully at this time. As you can see, we begin to build a huge foundation upon which these issues are addressed and found throughout the regulations.

It's an understatement to say, please be sure to consult the CBER listing of guidelines, policy statements and points to consider as you go through your license submission. Within the document, the CMC document for plasma derivatives, at the back is a complete listing of the guidelines, points to consider and policy statements that are referenced throughout it. And there are a number of opportunities and many of these separate documents to find again specific references to pathogen reduction requirements found throughout each one of the individual steps. I didn't list them all because there's a huge number and they're constantly being updated. These are all available on the web.

Also, and within the CMC document, you'll find the international conference on harmonization guidelines mentioned for specific issues and those are the rules that we are following also.

Now we've, in a very cursory overview considered IND, the opportunities during the IND to discuss pathogen removal or inactivation, talked about important documentation that is requested throughout the licensure process. Let's look at licensure and post-marketing and those regulations to see where once again we find specific mentions of steps that would help to render these products safer.

Under 600.3 in the definition section, (p) the word safety means the relative freedom from harmful effect to the persons affected directly or indirectly by a product when prudently administered, taking into consideration the character of the product in relation to the condition of the recipient at the time. It's not a direct mention here of pathogen reduction, but certainly coming down with hepatitis, HIV, West Nile Fever or virus infection or any of these other pathogens that are out there would be a direct step back to our regulations where we have very strong statutory authorization.

Again, in the definitions section, purity means relative freedom from extraneous matter in the finished product, whether or not harmful to the recipients or deleterious to the product. Impurity here can be taken to mean that whether the animal pathogens that may be found in the products made from animal sera or plasma are infecting human beings and showing disease is not important. The fact that we can find them means that he product is not pure and the regulatory authorization is quite clear on that matter. So again, we find good statutory authorization for requiring removal of these products or products that don't contain them here in the CFR.

Under 610.13, purity, products shall be free of extraneous material, except that with is unavoidable in the manufacturing process described in the approved license. How you interpret unavoidable becomes a very big issue.

Now what I've done is I've gone through and I picked some of the additional standards for products that are licensed. You may have noticed that our CFR is kind of on the Atkins Diet itself and has lost considerable weight in the last five to ten years and there are a number of products that are not found there any longer, but some of the regulations are still there and I looked through the CFR to try to find specific instances where even though this is for a human, where pathogen reduction and/or inactivation is mentioned so that it's clear that the stance that CBER takes is very much so directed towards that goal. And here for human albumin, albumin human, excuse me, under 630.80, under source material, the source material of albumin human shall be blood, plasma, serum or placentas from human donors determined at the time of donation to have been free from disease causing causative agents that are destroyed or removed by the processing method. So we can start with the material that may have some contamination with pathogen in it, but the regulation identifies that those need to be destroyed or removed during manufacturing.

Under 640.81, processing for albumin human, heat treatment is noted. As Dr. Finlayson pointed out with the original identification that when the value of heat treatment was first come upon, in the regs we find heat treatment, heating of the final containers of albumin human shall be in within 24 hours after completion of filling.

Heat treatment shall be conducted so that the solution is heated for not less than 10 or more than 11 hours at an attained temperature of 60 degrees centigrade. Heat treatment obviously as was seen in those earlier experiments is an effective method for reducing hepatitis within the recipients.

Under 640.90, plasma protein fraction human, we see similar information provided. Not too surprising. Source material. The source material of plasma protein fraction human shall be blood, plasma or serum from human donors determined at the time of donation to have been free from disease causative agents that are not destroyed or removed by the processing method as determined by a medical history of the donor and from such physical examination and clinical tests as may appear necessary for each donor at the time the blood was obtained. So specific mention again that your starting source material has to be well identified.

Again within the plasma protein fraction, (e), we find heat treatment. Heating of the final containers of plasma protein fraction human shall begin within 24 hours after completion of filling. Heat treatment shall be conducted so that the solution is heated for not less than 10 or more than 11 hours and at attained temperature of 60 degrees C.

The next product line that is included in this is 640.100, immunoglobulin human. Source material. The source of immunoglobulin human shall be blood, plasma or serum from human donors determined at the time of donation to have been free of causative agents of diseases that are not destroyed or removed by the processing methods as determined by the donor's history and from such physical examination and clinical tests as appear necessary for each donor at the time the blood was obtained. So this is an early recognition that the donor as the source for these products will always be of question and the manufacturing process needs to be stepped up to assure that the products come through safely.

Within manufacture, 640.102, manufacture of immune globulin human, sterilization and heating. The final product shall be sterilized promptly after solution. The statement, clearly such sterilization would be a good inactivation of any final contaminants that might be found.

So many manufacturing steps designed to provide a high level of protection to these products will help forestall a disaster. The threat of emerging infectious diseases requires a constant watch for new risks which will pose new threats to products made from animal sources. We should not just assume that because we have such a tremendous safety level with the products in that there's been no real outbreaks of problems from animal-derived products as I mentioned here, that that's how the present and the future will continue to take us. Having a level of assurance that these products are treated effectively so that pathogen inactivation and pathogen reduction are identified and prevent any future catastrophes that may occur as a result of emerging infectious disease is critical for us to consider. It's the purpose of the workshop today.

I have a case study that I want to discuss in a moment that is just an overview of where we missed it with human and the threat of after having missed it with human and preventing that from occurring with animal is incredibly important. I believe that a proactive position is a far better one than a retrospective explanation. And in these days there is a lot of explaining that goes on at all levels. As a matter of fact, on several hills I can think of and we would like to very much consider that we can be more proactive in our requirements for safety for these products.

I have a very brief case study that I wanted to point out regarding hepatitis C virus and contamination that occurred not too long ago in products of human source and final use. What I've done here is I've simply looked at CBER's position as events continue to unfold and discussed steps that CBER took in a regulatory fashion and left out many of the specifics regarding manufacturers and product lines because my talk is to consider the regulatory requirements for these products and I believe that this shows in a fairly straight forward example how we have gone forward and addressed issues when things have gone wrong and this is what we're trying to prevent.

On January 8, 1992, CBER wrote a letter, wrote to all U.S. licensed manufacturers of plasma derivatives in an effort to facilitate the implementation of new procedures for inactivation of infectious agents in plasma derivatives. These were, of course, from human source or whole blood and recovered plasma.

Subsequently, in January and February of 1992, CBER wrote to all manufacturers that were not licensed, but had pending license applications for plasma derivatives and those that had IND applications in as well with similar, within the same text.

On May 23, 1994, a letter was sent to all U.S. licensed manufacturers and all manufacturers with pending license applications for human immunoglobulin preparations. The letter acknowledged that various manufacturers of immunoglobulin for intravenous use -- oh boy, excuse me. The letter acknowledged that various manufacturers of immunoglobulins for intravenous use were at various stages of progress, i.e., some had introduced virus inactivation removal steps. Others had violated virus inactivation and removal steps.

Part of the manufacturing process in some of the clinical trials with products made by incorporating viral inactivation steps. CBER was not aware of the status of progress with regard to comparable work involving intramuscular immunoglobulin and specific immunoglobulins for intramuscular use. CBER requested that recipients of the letter reply with plans for progress in this area. Okay, that was an example of a proactive step taken by the Center.

On December 27, 1994, OBRR wrote to the appropriate license manufacturers informing them of OBRR's intent to begin HCV RNA testing in all human immunoglobulin products that had not undergone one or more validated viral inactivation/removal steps.

So you can see that there have been times where CBER has moved forward directly setting the level of safety at a technologically achievable levels through PCR testing to increase the safety profile of products. A well validated pathogen reduction scheme could have prevented the transmission of hepatitis C in these products and many other pathogens from plasma derivatives.

That's the extent of my discussion. Thank you.

(Applause.)

Return to Index

DR. NEUMANN: Good morning. I'm from the Bureau of Biologics and Radiopharmaceuticals for Health Canada, I guess we're considered the CBER equivalent. And if the first slide goes up, now this is in contravention to all the rules and regulations regarding what makes a good slide, but I'm not responsible for the title. I can blame that on Mark.

Furthermore, it's good to be speaking fairly early on because anything that I don't cover I can say will be covered by Tom Lynch later on in the afternoon or Dr. Willkommen and after my talk it's nice to have some backup.

I would like to say that I think you'll find actually a handout of my slides in your package. To keep people awake I think you'll find that was the penultimate version and there's a few spelling mistakes and other changes that might have to be made that will be on the slides here.

What I've done is taken the -- I like the word current thinking of the Bureau of Biologics with respect to plasma-derived products and essentially drawn parallels to it for what our thinking would be on animal derived products.

Now on the draft paper, next slide, if you can read that, guidance in the the manufacture of plasma derived products, human plasma derived products and this is what essentially the bureau uses and as an internal guide to reviewers in order to insure consistency of applications in front of us from manufacturers of plasma derived products. In that guide, you can see on the next three slides covers the table of contents. Some of these will be covered in my subsequent slides and I think if you'll look at the next slide as well, these cover essentially, some of these, I must say were cribbed, not entirely but derived from some of the ICH guidance documents on federation of biotech products derived from cell lines. Some of them were CPMP guidelines. Some of them were EMA. Some of them were also the FDA guidance or industry documents so in typical Canadian fashion these tend to be a hybrid of earlier regulatory guidance documents.

Now the next slide essentially describes what we're looking at today and this is -- you have a manufacturer here and this is an animal derived product, the sacrificial dog in this case and the manufacturer is, I think you can even see here he seems to have a smile on his face, but he's probably in the business for profit. I mean that somewhat cynically actually. And this is essentially the discussion of our product today. We have an animal derived product being used in human and physician oversight of the undoubtedly, in this case, adverse reactions that's likely to occur.

Next, please. Now one way of evaluating the risks of animal derived products would be looking at in decreasing risk order would be those animal diseases for which there's evidence of transmission and human disease. There's all sorts of known zoonotic diseases, pox viruses of bovine and other origins, rabies, menangle virus, swine flu, equine infectious

-- equine encephalitis, hendra virus and of course, more recently BSE and vCJD. This list could go on forever. I think we are discovering anybody that subscribes to ProMed has seen that almost every day new viruses are emerging which may have some animal and human pathogen and I think we're looking at things like West Nile Virus and so on.

So these would be the things of first consideration. Secondly, those for which there is animal disease but no evidence of transmission or disease in humans. We're looking at things like porcine parvovirus for which there's no evidence of either transmission or infection as evidenced by seroconversion. Equine infectious anemia, there's -- it doesn't appear to be infectious to humans. Louping ill, foot and mouth disease virus, pseudorabies, there are a host and a huge range of animal viruses for which there are no human infections associated.

Next. Third level of risk would be those for which there is animal disease and the theoretical transmission of risk to humans and this might be things like other prion diseases, scrapies, ruminant TSEs. The only ruminant TSE we're aware of at the moment, obviously, is BSE and variant CJD and the other ruminants that have been identified as having TSEs, they're not likely to be used as a source for human plasma and last, but not least, there's no animal disease and questionable evidence of transmission, but there's no human disease shown yet. PERVs, there have been possible seroconversion, but even this is a little bit questionable and as Dr. Weiss two and a half years ago pointed out that under certain conditions PERVs could be transmitted to human cells in vitro.

Now what this doesn't take into account, of course, and this is almost on a case by case basis, what the benefit risk of any of these particular animal derived products are. Despite the theoretical impossible risk of animal virus transmission to humans, one still has to look at whether or not these are critical life saving drugs and that's another factor to be looked at.

Next slide, please. Now what I've done here is on the left hand side taken note of our guidance documents, those things which we consider important for reducing risks of human diseases from human derived plasma. One of the things we look at, of course, is the prevalence of relevant infectious disease compared to Canadian and U.S. sources. If we were receiving plasma from non-North American sources we would want to see that the relevant infectious diseases, if there happens to be endemic diseases in some other area, those would be taken into consideration and a parallel with animals is that for bovine sources, we're looking for BSE countries of origin and whether or not there is any consideration or not, but free of menangle virus, for instance, if that happens to be a consideration; ruminant TSEs if there is to be another ruminant used other than bovines.

For donor selection, well, we look for equivalency of the donor history and risk assessment criteria compared to Canadian and U.S. practices. In animals, one might very well look for a specific pathogen free herds or flocks. Donor animals could be retested prior to successive leads. These are for animals who are not sacrificed or evidence of relevant vaccination, if one has concern about rabies transmission then animals would be expected to be vaccinated against rabies or they happen to be a rabies-free country. This is something that may be considered, are there surveillance programs for slaughterhouse operations in which the local agricultural regulatory agencies may require oversight or perhaps an on-going program looking for viral diseases in the herds from which these plasma products are derived.

Next please. Another thing we're looking at is test kit comparability. We're looking at the sensitivity taking into account, strain variation of viruses and the regulatory oversight of the manufacturer of the kits. For animal source material, one could identify commercial test use if such exists and a regulatory oversight for their manufacturer or if there are no commercial kits available, then the reference procedure is used. An awful lot of these screening tests are in-house methods and they would have to be very well validated or reference to other referenced literature sources.

Another thing we would look at for plasma derived, human plasma derived are procedures associated with reactive test results such as donor referrals, re-entry algorithms, trace back, look back procedures and quarantine procedures. Some of these things may not be and cannot be applicable to animal source material.

Now another thing we look at, doing a history assessment, written and oral questionnaires. Now what we might be looking at for animal source material is animal health history which is on-going veterinary assessment of a flock or herd and if you have a Dr. Doolittle available, then they could be asking animal risk questions. This is the original Dr. Doolittle. I think it was Rex Harrison, not some other actor.

Donor testing, since these tests have been known to transmit diseases, all these screen tests have come into account and for animal source material you'd look for disease free status and test as appropriate for species, for instance, nucleic acid testing for porcine parvovirus.

Next. For human source material, we're looking at post donation information and this is information exchanged between collection sites and manufacturing, if it's found that the donor didn't meet health criteria, develops disease or risks, have been identified, and subsequently found positive for viral markers for which they were originally found negative. And the assessment of PDIs and you would defer the donors and retrieve plasma units.

Considerations for animal source material may be that the herd be monitored for known diseases, seroconversion. If the disease had been identified in a herd, one could retrieve plasma of other animals in the herd. If donor animal is subject to rebleeds, then that animal would be restricted or eliminated from further donation and plasma which hasn't already been pooled could be retrieved.

I won't be the first and probably not the last person to say that size matters. Limiting pool size would reduce the window period collection or risks including the risk of including units contaminated with an agent for which screening can't be done. Similar considerations could be made of animal source material, a lot of it depending on the number of -- the type and material being produced. If this is a material that's a large volume material, that's likely to be used only once or twice during a patient's lifetime, that would have a different profile than those products for which there's on-going therapy is required such as hemophiliacs require weekly or biweekly infusions. For each of these human derived sorts, upper limits should be established of each product taking into account the number of lots and number of units in the pools for specific product to which the users are exposed, the infectious disease risks associated with the products and if they're added as stabilizers they should be ideally derived from the same pool as the product. Here we're looking at albumins almost exclusively.

Nucleic acid testing of pools. There should be validated methods of suitable sensitivity for different genotypes and the specificity must be supported by documentation to reduce risk of hepatitis C. Each assay line used must include controls expressed with reference to international standards. For animal source testing, not testing of pools for appropriate viruses depending on the species, for viruses for which screening tests are not sufficiently sensitive. For instance, PPV could be tested for pigs. Or not testing when the validated inactivation removal processes have not been demonstrated. Again, if there has been some risk associated with animal derived plasma, then indeed one could develop a NAT test to reduce the raw plasma as a source of contaminating material.

Next, please. The quarantine of plasma units. Now this is being widely used in the ABRA industries in North America. This is a period of time to allow for the retrieval of units prior to pooling, based on subsequent positive results of donor testing or post donation information. This is possible for animals subsequently bled for plasma and it could be possible for diseases identified in the herds. You could retrieve units from other animals. Now this is a "could" not a "should" but this is something for consideration, that if there was a quarantine period allowed, one would be able to retrieve plasma units from those animals which are being held in quarantine, plasma units in quarantine if subsequent disease is identified in the source herd.

Next. A lot of these are going to be covered by Tom Lynch. Following activation of removal procedures, this specific step must be introduced if the removal of a virus is a major factor in the safety of the product or if the manufacturing process itself doesn't remove infectivity. And similar considerations can be given to animal source material. Heat treatment which has been described quite well, for albumin, if it's used as a stabilizer can also protect the virus from inactivation. Therefore, worse case scenario consideration should be given in which case high titered spiking experiments should be used in which albumin itself is a very good stabilizer of virus and I think this same consideration would have to be taken into account for animals. Animal albumins and other stable products through which they're being used as a stabilizer, the same considerations can be taken into account.

Now animal albumins aren't typically used as stabilizers in animal products so maybe this is not a consideration here.

Next. Solvent detergents. This has frequently been described for human derived plasma as a cassette. I think the New York Blood Center has described it as such and an in-process solution should be free of aggregates particularly when you're considering this, that might harbor virus. Therefore, maybe filtration before treatment can remove some of these aggregates. Inside these aggregates could be viruses that you're well-protected from the effects of solvent detergent. And for animal sources, again, we know the toxicity and effective range of solvents and detergents to be used for human derived plasma. For animals, known animal viruses, such as PERVs, solvent detergent would very likely inactivate these kind of viruses and a whole host of unknown envelope viruses waiting to be discovered. I think in some cases maybe the unknown, if one isn't looking for them, you're not going to find them and to some extent the use of solvent detergent will be a way of proactively looking at -- treating animal source plasma so that you don't have to wait to find when the next zoonosis will be found in humans.

Next slide. Viral filters are being widely used now and they're now even being used in recombinant products and recombinant products just to remove risks of, in the most case, murine viruses which for the most part haven't been shown to cause any disease, but these manufacturers are using viral filters, along with solvent detergent treatment and coagulation factors. However, if you're using viral filters sometimes the filters themselves can affect yields. Perhaps there might be an activation of coagulation factors and obviously it's essential that filter integrity tests be done in process control and scale down comparisons with production scale.

For animal source material, its broad usage with human derived processes and it's possibly, a lot of these filters are already validated for a host of animal diseases and in some cases it would be a relatively innocuous and easy step to introduce. For human immunoglobulins, low pH, usually a pH of less than 4 inactives certain viruses, depending on time, temperature and the composition of solution. And this may also be applied to certain animal immunoglobulins.

Next. Now I'm appropriating the use of the words "relevant viruses" and "model viruses" here from some of the CPMP documents and they do seem appropriate, so I didn't invent a word of my own. The relevant viruses are either identified viruses that pose risk and for which spiking studies can be done. Model viruses are those for which infectious spiking studies cannot be done. For instance, if a virus cannot be grown in vitro such as hepatitis B or hepatitis C. And for animal sources, we'd be looking at spiking studies would be done according to the potential risks to humans. That doesn't tell you very much, but again, on a case by case basis, one would have to look into these.

And on the next slide there's a table showing you relevant and model viruses for human plasma derived products: HIV, it is a relevant virus for both HIV 1 and 2; hepatitis B. Manufacturers frequently use pseudorabies viruses, other envelope DNA viruses and perhaps along with pseudorabies manufacturers have used a host of herpes viruses and there really is no practical system for hepatitis B validation using in vivo models. I have yet to see people using duck hepatitis virus. Actually, I've seen one submission that's used that. You do go through a lot of ducks. Hepatitis C virus, BVDV, sindbis has been used. BVDV is particularly a more relevant model and BVDV strain should be used that has a high physical chemical resistance. For B-19, an appropriate model would be porcine parvovirus. It seems to be the most closely related model to B-19. Hepatitis A is a relevant virus for coagulation factor studies. You can grow hepatitis A and consideration should be paid to possible interfering antibodies, if you're looking at immunoglobulin preparations and the immunoglobulin preparation itself should be free of anti-hepatitis A antibodies. And prions, not much can be said about them and the models that people have been using, scrapie models and so on, may or may not be appropriate for the prion disease of consideration.

Next, please. Now these may be relevant in model viruses for animal plasma derived products. And all of down here is a list, an array of viruses or virus families with a representative species of virus which have an array of genomes, envelope, non-enveloped and resistance to pH and chemicals and different shapes. And again, prion diseases, there may be various hosts that could harbor these and has high resistance to pH. The thing that could be said about prion diseases is there may be some evidence of partitioning of prions, at least it has been shown with the plasma derived albumins, for instance, which have been shown to decrease prion load, at least if one is using a scrapie model by about four logs.

Next, please. Now the conduct of viral spiking experiments, I think a lot of the work has been done for us. The ICH technical requirements for registration, etcetera, and these are for biotech products. And some of the considerations for the spiking experiments have already been dealt with in that document. Essentially reduction is the sum of the individual factors. Less than one log is not considered significant. Steps with four log reduction are generally considered significant for package insert claims. This is above and beyond those serendipitous fractionation steps which must be used in the manufacture, but coincidentally do remove viruses. And considerations could be given for animal source material and the conduct of spiking experiments. As I said, the work has been done for you.

Next. In the conduct of viral spiking experiments, there are specific precautions that are outlined in that ICH document. Things like avoiding aggregation with high titered preparations. The dilution effect on the spike of stabilizers. A few years ago we received submissions in which in the same submission they demonstrated that a difference of 10 percent on the stabilizer used would make a remarkable difference on the degree of viral inactivation and yet, the dilution of the spike and their spiking experiments haven't taken that into account. When you have a 10 percent spike, you obviously have a 10 percent reduction in the stabilizers that are being used in the product and that has to be accounted for.

And again, steady scale versus production scale, all of the parameters that one measures, all the end process controls and things that ones looks at at a production scale must be mimicked perfectly in the study scale.

Next. Further limitations, the tissue culture virus that's in a production step may be different than the native virus. People may very well be using laboratory strains of virus in their spiking experiments and sometimes these get passage to some degree and they may no longer reflect what wild type viruses exist and this is another consideration to take into account, that the viruses used in these spiking experiments must from time to time be

re-passaged from wild type viruses that one might expect to contaminate a product. And the reduction values of identical procedures should not be included unless they're justified. If you have a column fractionation step and it requires a specific type of column, two subsequent steps cannot be pooled together and considered two separate reduction steps.

Next. Specific points to consider, for instance, for immunoglobulins, unknown and envelope viruses. Before steps were introduced, there was instances of hepatitis C transmission. You're looking at these particular products. You're looking at a very large volume, but low frequency and I think these kind of considerations have to be taken into account of what your product is, how it's used and what the lifetime risk to the recipient may be. For coagulation factors, we know that hepatitis A and B-19 risks have been associated and both of which are highly resistant to inactivation. Again, we are looking at -- I shouldn't say we, manufacturers are looking at ways of reducing hepatitis A and B-19 risks by introducing PCR technology to reduce the burden of the raw material. I think we've all learned that anticipating that there will be sufficient neutralizing antibodies in these materials, particularly for immunoglobulins, that both hepatitis A and B-19 have been shown to have such high titers that there is not sufficient neutralizing antibodies in any of the pools. There has been cases of B-19 in which it was assumed that there would be sufficient neutralizing antibody, but B-19 is one of those bugs when a donor happens to be viremic, they have titers of about 10 to the fourteenth and with that kind of viral load, practically no degree of neutralizing pooled sera could possibly neutralize that much virus.

And again albumin, it has an excellent safety record and there's been some evidence of prion partitioning. We have seen some studies from manufacturers where there appears to be at least a four log reduction due to partitioning of prions in the albumin fraction.

Next, please. Now this tends to be my thinking. If it can be done, do it. I think we shouldn't be waiting for something to happen, particularly when there are cassettes, if you will, of known procedures for viral inactivation and they can be introduced into animal derived products without further reduction or loss of yield from these products and that manufacturers should be looking at ways of reducing either known or unknown risks with respect to animal derived proteins.

Thank you.

(Applause.)

CHAIRMAN HEINTZELMAN: Well, we're scheduled for a break now. We're a little ahead of schedule. That's good. Maybe we'll leave a little early. Why don't we take a 15 or 20 minute break, does 20 minutes sound okay? Twenty minutes gets us back at 10:30 and we'll reconvene with the European Union perspective. Thank you.

(Whereupon, the proceedings went off the record at 10:10 a.m. and went back on the record at 10:36 a.m.)

CHAIRMAN HEINTZELMAN: We'll reconvene, please, and get ready for our next speaker.

(Pause.)

Return to Index

DR. WILLKOMMEN: Ladies and gentlemen, it's a pleasure for me to continue now with the European perspectives and I have heard already this morning the position of the Food and Drug Administration, from the Canadian people and I must say we have not so many differences. I can stop here already. Okay?

(Laughter.)

DR. WILLKOMMEN: But I want to speak, of course, and I have thought that it would be fine or it would be interesting or maybe interesting for you to compare or to demonstrate to you the European requirements of life safety testing of many titered products derived from human or animal sources.

I'm sorry, I forgot to introduce myself. My name is Hannelore Willkommen. I am from the Paul Ehrlich Institute in Germany. It is a national authority for sera and vaccines and this institute is very much responsible for the development of national guidelines in our field and is very much also into development of European guidelines.

So I want to speak about this and I hope I can give you some interesting information. At the beginning I want to summarize, I want to give you an overview about the guidelines which are in place. You know, the European Union consists of 15 countries at the moment and we have a high need of guidelines in order to summarize our position, to find a common position in many aspects.

This is the background or this is the reason why we have a lot of guidelines in place. So these are the guidelines and I want to go through only very quickly. I want to mention these guidelines which cover these products derived from human or animal material.

First, these are the guidelines for plasma derivatives. This was revised in September 1996 and it is now a new version of this guideline is in place. And here, you see the source of the guidelines, if you go on home page of the European Agency, you can find all these guidelines and can read them.

So this guideline said how to test the source material, how to -- this guideline says also what's the capacity of the manufacturing process for the removal and inactivation of viruses. What does the figure have to be for the result.

The second guideline here, note for guidance on virus validation studies, this guideline says how to perform virus validation studies. And I think it's -- I'm quite glad about this guideline and I will come back later on a little bit on it.

So this is a guideline which you also know about. It is an ICH guideline, saying something about the quality and biosafety, especially about biotechnology products. And I have it here on the list because this guideline is applicable also for monoclonal antibodies which are derived from mouse ascites and so it is also animal and is a material used for the manufacturing derived from animal materials.

So next is a guideline for guidance on minimizing the risk of transmitting animal spongiform encephalopathies agents via immunosera products. This guideline was finalized in this year and there's also a newer version of an older guideline, but I don't want to come back on this one. I think it is -- you understand, it is another issue.

So we also have a guideline which was developed already. It started to develop in 1996 and -- sorry, in 1993, and it was finished in 1995. It is a guideline about the use of transgenic animals in the manufacture of biologic immunosera products for human use and we think that this guideline is already a little bit old and should be revised in some parts.

And then we have a new draft guideline and I must say it is at the moment the draft or the suggestion from our Institute. We discussed it already in the biotech working party, but it is not finished from the discussion in the biotech working party. It is not finished and so it is a draft and maybe it more or less demonstrates opinion of our institute.

And it is a guideline about the production quality control of animal immunoglobulins and immune sera for human use. We think that especially for these kind of products we need some regulation and need also some regulations for Europe. At the moment, these kinds of products are on the market on the basis of a nationalized sense. There are no products in place already which has a European license.

So as a general approach, biosafety means the absence of infectious viruses and we are speaking or I am speaking only about viruses at the moment. I don't speak about the prions.

This means that the source material should be tested or it should be controlled. The manufacturing process should have a high capacity for removal inactivation of viruses and in some cases it may be useful also to test intermediate products or to test the final product.

This is a general approach and we think that this approach is also applicable for this kind of product derived from animal material.

Let me go now through the different guidelines and show you the differences in the regulation or the state of regulation. I want to mention also what should be changed or what is under discussion at the moment.

These are the guidelines, ICH guideline here. It's a number of European -- and it is a guideline which covers the most of the monoclonal antibodies and the most ICH source material. You see, it is required to have close colonies and these colonies have to be tested for many, many viruses and it is very accepted that these testing is necessary and tests have been developed which are relatively easy to perform and you have no discussion about the need to test such a lot of different viruses. It is good, I think, I mention it because it is a starting point for our discussions.

With regards to the requirements on the capacity of the manufacturing process, we have an expression in the guideline that the manufacturing process should be substantially higher than the lab contamination in the source material. Very often we have contamination with retroviral particles and so in this case it should be substantially higher. It is not clearly defined. Here, it is to be considered on a case by case basis.

Testing of the final product is only in some cases required, only if the source material contains the viral contaminants and then it is limited on some lots only.

So what is expressed in the draft that I want to remind you? It is, at the moment, our draft, draft for animal immunosera and immunoglobulins. We know that it is a little bit difficult and it is not realized in each case that animals are held in closed herds, but we think that it should be at least well monitored herds. If you are thinking about larger animals that is nearly impossible for the manufacturing. They say they can't hold the animals in closed herds.

At the moment we have products on the market in Germany which came from rabbit, goat, sheep and horses. So we think that these herds have to be tested on the freedom of infectious agents and at the moment there are no requirements, no advice from industry what they have to test and we think that it should -- virus lists should be developed and should be given to the consideration of the Ministry and also of the control authorities. I will come back on this point later.

So there are no specific requirements at the moment for the capacity of the manufacturing process to remove inactive viruses for performing virus validation studies. This guideline is applicable and it is a guideline which is also applicable for the blood products.

We have to consider in the case of these products, we have to consider not only species specific viruses, very often the products need to be absorbed in human material, it is so at least in the state of anti-T cell sera. And if it is the case, we have all to consider the presence or we have to control the absence of human viruses and for all the steps of this manufacturing validation process. We have to consider human viruses too.

The final product is over here only required in specified cases, if it is not possible to arrive at the contamination of the source material.

So what is with human products? The idea today, you know, we have the development of the donors. We have a very -- we have a lot of regulations for the selection of donors and the testing for the absence of viruses. You see normally it is tested for HIV, HBV, HCV, and in Europe the HCV-RNA testing for plasma pools is introduced since July of this year. All manufacturers have to perform these testing and the pools have to be free of HCV-RNA.

The capacity of the manufacturing process should be very high. We have a special guideline for it. So testing of this capacity has to be performed according to these validation guidelines.

If I summarize the requirements in some words, then I can say it is required a high affectivity for the manufacturing process, in most cases, two effective steps which compliment each other in the amount of action required.

The testing of the final product as in each case is not sufficient in order to demonstrate the safety of the product and it is so because of the statistical reasons or because of the statistical limitations, but the safety -- we think the safety has to be demonstrated by other measures.

In some cases, can it be useful? As an example, if you look at the contamination with parvovirus B-19, it is very informative to test the final product. So but it is not the general framework or it is not normally required.

So products derived from transgenic animals, I mentioned already that we have in all the guideline here and the guidelines is sufficient we think with regard to the source materials, with recommendation to the source materials. It is required, of course, that animals shall be held in closed colonies. It is required that animals have to

-- or the colony has to be tested or it has to be controlled in the absence of specified viruses. But the guideline gave only some examples of viruses which should be considered. There are no specific requirements for the capacity of the manufacturing process, but it is, of course, expressed that the process should be effective in the removal or inactivation of viruses and it is mentioned too that mycoplasma should be considered because if not as a source material of these products, mycoplasma can go to high titers in this material.

And again, there are no specific requirements for the testing of the final product. So now I want to make some remarks to the source material testing. If you compare the animal material with the human material we can say okay, the human material is a high risk material. It doesn't work. Yes, it's a high risk material. You know the contamination is chemical. It's pathogenic for humans.

In the case of animals, you don't know exactly what the risk level is. We know that animals can also have virus infect