Important

International Scientific Meeting on safety of Vaccine Substrates

Minutes of discussion between scientists at a FDA called conference on Vaccine Substrate safety - ie this includes on the safety of using monkey kidney cells as the growth medium for manufacturing the Sabin and Salk polio vaccine.

note that privately, between themselves, they admit to being unable to completely purify the polio vaccine - that millions of viruses, toxic particles, etc could be present.

from http://www.fda.gov/cber/minutes/0907evolv.txt.


1
U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES PUBLIC HEALTH SERVICE
FOOD AND DRUG ADMINISTRATION
CENTER FOR BIOLOGICS EVALUATION
AND RESEARCH INTERNATIONAL ASSOCIATION FOR BIOLOGICALS
NATIONAL INSTITUTE OF ALLERGY AND
INFECTIOUS DISEASES
NATIONAL VACCINE PROGRAM OFFICE WORLD HEALTH ORGANIZATION
EVOLVING SCIENTIFIC AND REGULATORY PERSPECTIVES
ON CELL SUBSTRATES FOR VACCINE DEVELOPMENT
WORKSHOP TUESDAY,
SEPTEMBER 7, 1999
The workshop took place in the Plaza Ballroom,
DoubleTree Hotel, 1750 Rockville Pike, Rockville, MD
20852 at 7:45 p.m., William Egan, Ph.D., Chair,
presiding. PRESENT:
WILLIAM EGAN, Ph.D., Chair
ELWYN GRIFFITHS, Ph.D., D.Sc., Co-Chair
REGINA RABINOVICH, M.D., Co-Chair
LEONARD HAYFLICK, Ph.D., Speaker
ANDREW LEWIS, Jr., M.D., Speaker
PHILIP KRAUSE, M.D., Speaker 2
AGENDA

Welcoming Remarks
Regina Rabinovich, M.D. . . . . . . . . . . . . 3
Institute of Allergy and Infectious Diseases, NIH
Elwyn Griffiths, Ph.D., D.Sc. . . . . . . . . . 5
WHO SESSION I - Evolution of Cell Substrates Used in the
Manufacture of Biologicals
Review of workshop purpose and goals
William Egan, Ph.D. . . . . . . . . . . . . . . 7
CBER/FDA Evolution of cell substrates used for the production
of human biologicals
Leonard Hayflick, Ph.D. . . . . . . . . . . . . 11
University of California, San Francisco
Major issues associated with cell substrates
Elwyn Griffiths, Ph.D., D.Sc. . . . . . . . . . 26
WHO
Developing an approach to evaluate the use of
neoplastic cells as vaccine substrates
Andrew Lewis, Jr., M.D. . . . . . . . . . . . . 58
CBER/FDA Conceptual and experimental approaches to address
product safety issues raised by novel cell substrates
Philip Krause, M.D. . . . . . . . . . . . . . . 73
CBER/FDA 3
1 P-R-O-C-E-E-D-I-N-G-S 2 (7:54 p.m.)
3 DR. RABINOVICH: Good evening. I would
4 like to ask those who would like to participate in
5 this evening's session to please have a seat so we can 6 get started. My name is Regina Rabinovich. I am here
7 from the Institute of Allergy and Infectious Diseases
8 from the NIH in the U.S., and on behalf of my co- 9 sponsoring agencies and colleagues, the Center for
10 Biologics Evaluation and Research, the International
11 Association for Biologicals, the National Vaccine
12 Program Office, which includes Marty Myers, who is 13 supposed to be a co-chair for this session and is
14 currently either grounded or wandering around Newark,
15 New Jersey, and the World Health Organization, I 16 welcome you to the meeting on Evolving Scientific and
17 Regulatory Perspectives on Cell Substrates for Vaccine
18 Development.
19 I have a couple of housekeeping comments. 20 I won't bother to tell you where the bathrooms are. I
21 am sure you can find those. There are two documents
22 that I think would be very useful, if you haven't 23 noticed them already, because they are key to the
24 workshop. The first is the document prepared by our
25 colleagues from the Food and Drug Administration, 4
1 which is under your tab entitled, Draft CBER document. 2 Because I think that the creation and comment upon
3 this is central to the scope of this meeting, as will
4 be elucidated further by Dr. William Egan.
5 Because the sponsoring groups as well as 6 the FDA particularly are so interested in the concepts
7 within the document and the discussion that will take
8 place, this meeting is being taped. And for that 9 reason, we must ask that speakers as well as those
10 that are asking questions or getting answers speak
11 into the microphones, and that we will work with the
12 moderators to make sure that everyone that wants to 13 ask a question, regardless of where they are in the
14 room, have a microphone available to them because it
15 is key that it be part of the written record. 16 The steering committee would like to thank
17 a couple of individuals that have worked very hard to
18 make the meeting possible. Two of our colleagues at
19 FDA, Dr. Keith Peden and Dr. Phil Krause, and Dr. John 20 Daugherty at NIAID.
21 In thinking about eight months ago about
22 this workshop, the scope and what it should cover, we 23 never in our wildest dreams considered that in
24 September of 1999 we would have gone through a summer
25 where there would be no question about how to spell 5
1 thimerosal, and that early information coming out of 2 sort of a group analysis of all clinical trial data
3 for the rotavirus vaccine would give us the first
4 inkling to the potential connection to
5 intussusception. Indeed, the issues regarding this 6 topic, vaccine safety, which never makes sense to
7 anyone, what we are talking about in terms of vaccine
8 safety, are elucidated by the kinds of thoughts and 9 strategies that will evolve from this meeting. And if
10 ever there was a connection between basic research and
11 what we do with human subjects as an outgrowth of your
12 deliberations, I think this is it. 13 So on behalf of my colleagues at the
14 National Institute of Allergy and Infectious Diseases,
15 I thank you for your participation. I would like to 16 go ahead and introduce Dr. Elwyn Griffiths from the
17 World Health Organization, who is my co-chair in my
18 role as Dr. Marty Myers, and will introduce the first
19 speaker. Thank you. 20 DR. GRIFFITHS: Can you hear? Oh, yes,
21 okay. (Speaking in Welsh.) It is the only
22 opportunity I have ever had to introduce a meeting 23 such as this in Welsh. I know there is one colleague
24 of mine, Merfyn Williams, who used to be at FDA, and
25 he is in the audience. So at least he understood that. 6
1 It is really an honor and a pleasure for 2 me to co-chair this session, the first session of this
3 important meeting with Regina here. Let me also say
4 that the World Health Organization is very pleased
5 indeed to be a co-sponsor of this workshop. Because 6 I think it considers the subject to be vitally
7 important from a global perspective, and we take that
8 view of course, for future development and production 9 of vaccines in general.
10 I see this evening's activities really as
11 a sort of an hors d'oeuvre, I guess you might say, or
12 an appetizer to the entrees that come during the next 13 two days or so. So it is an overview of where we are
14 going and where we have been. I think perhaps we
15 should, because of the time scale -- I think we should 16 move forward with the program, taking particular pity
17 on any colleagues who have just flown in from Europe,
18 because they would be suffering from some jet lag or
19 biological clock problem at the moment. So I think we 20 should move forward fairly quickly.
21 So it is a pleasure then really to call
22 upon Dr. Bill Egan, who is now the Acting Director of 23 the Office of Vaccines at the Center for Biologics
24 Research and Evaluation, CBER, who will actually give
25 us a review of the works or purpose and the goals of 7
1 this. So he will set the scene for where we are going 2 to go during the next few days. So, Bill?
3 DR. EGAN: Thank you, Elwyn. Can I have
4 the first slide, please? I too would like to thank
5 everybody who has been able to come to the meeting and 6 everybody who helped in the organization of the
7 meeting and all of the participants in the meeting.
8 This is a workshop and everyone will be working 9 together to try and address some very interesting,
10 some practical, and some very difficult questions.
11 Every now and then I think we need to
12 remind ourselves that viruses can propagate only in 13 live cells, and this of course holds true for whole
14 viral vaccines. They can only be produced in cells.
15 The choice of suitable cell substrates for the 16 manufacture of viral vaccines has over the years
17 engendered considerable discussion. The primary focus
18 in these discussions on cell substrates have been
19 safety, in particular the potential safety concerns 20 from residual cellular DNA and from adventitious viral
21 agents.
22 As history has shown, the need for concern 23 about cell substrate issues was real. We have only to
24 think back to the finding of SV40 in polio virus
25 vaccines to realize the extent of the risk that any 8
1 cell substrate may pose, and there is still great need 2 for concern.
3 Early discussions on cell substrates
4 allowed only for the use of "normal" cells, that is,
5 cell populations that were derived from normal tissues 6 and that had undergone no subculture. And right now,
7 I am referring primarily to the 1954 decision of the
8 Armed Forces Epidemiological Board. 9 I can't read this -- it is hard to read
10 from this perspective back here. The use of only
11 normal cells has prevailed for many years. For
12 example, in 1971, the regulations of the Public Health 13 Service for the production of OPV stated that only
14 primary monkey kidney tissue cultures may be used in
15 the manufacture of polio virus vaccines. There were 16 similar regulations in place for the production of
17 inactivated polio virus vaccine, live attenuated and
18 inactivated measles vaccines, mumps vaccine, rubella
19 vaccine, and the adenovirus vaccine. 20 This was taken from the regulations in
21 1971, and it was in 1972 that the Federal Register
22 notice was placed that contemplated the use of other 23 cell lines other than these primary animal tissue
24 cells for use in viral vaccines.
25 Now our thinking and our practices with 9
1 regard to cell substrates has evolved over time. At 2 present, for example, in addition to primary animal
3 cell culture, diploid cell lines, both of human and
4 animal origin, are in routine use, as, for example,
5 the mumps, rubella, IPV vaccines. And in addition to 6 being manufactured in the human diploid cell, live IPV
7 is also produced in viro cells, a continuous cell
8 line. 9 We now wish to consider additional cell
10 substrates or additional cell substrate uses. In
11 particular, we would like to consider the use of
12 continuous cell lines for live attenuated viral 13 vaccines, and the use of other neoplastic cell lines
14 for both live attenuated and inactivated viral
15 vaccines. Indeed, the question that the Armed Forces 16 Epidemiology Board met to consider 45 years ago,
17 namely the use of tumor cells, specifically HeLa
18 cells, as a cell substrate is a question that we are
19 again raising. 20 The goals of the workshop are simple.
21 They are two. The first is to identify the various
22 concerns that are associated with using neoplastic 23 cell lines, to catalog them as completely as possible.
24 The second is to determine approaches and to identify
25 approaches to determining the level of the risk that 10
1 are associated with these concerns. In the decisions 2 that we will make with regard to the use of neoplastic
3 cells as potential substrates for viral vaccines, we
4 need to be guided by data; either data that already
5 exists in the literature or the data that we need to 6 generate. We should not be guided by intuition or by
7 opinion. If I may quote Dr. Hilleman from a
8 discussion about the use of tumor cell lines from a 9 meeting that was held in 1968, he said, "If the debate
10 has taken on an ecclesiastical or philosophical
11 overtone, it should not be unexpected, since it is an
12 area in which the definitive information for making a 13 judgment is clearly missing. In fact, I feel that the
14 greatest problem of all may lie in ascertaining
15 whether there is a problem in the first place." 16 The workshop in which we will be
17 participating over the next four days is designed to
18 see how far we have progressed and what we still need
19 to do. Thank you. 20 DR. GRIFFITHS: Thank you very much, Bill.
21 I think we have been given the task, then, for the
22 next few days, that is, to identify these concerns and 23 issues associated specifically with neoplastic cells
24 for production of vaccines. We are focusing on
25 vaccines. And to identify some approaches in 11
1 determining this risk associated with their use and 2 really how to move forward on these issues. I think
3 those are going to be our main concerns over the next
4 four days.
5 So we now move on to the second talk and 6 we are really looking at historical perspective to a
7 certain extent here, the evolution of cell substrates
8 used for the production of human biologicals. Because 9 as Bill already mentioned, they have been in use for
10 a long time and there has been a tremendous amount of
11 discussion over the years. And who better to give us
12 this talk and to take us through from the beginning of 13 the evolution of these cell substrates than someone
14 who has been involved right from the very beginning,
15 and the name is synonymous really with diploid cells, 16 Dr. Hayflick. So would he come forward and give his
17 talk on the evolution of cell substrates for the
18 production of human biologicals. Dr. Hayflick?
19 DR. HAYFLICK: Although the first cell 20 culture is generally attributable to Ross Harrison in
21 1907, it was not until 1949 that contrary to the then
22 existing dogma, which held that the polio virus could 23 replicate only in neural cells, that Enders, Weller
24 and Robbins showed that the virus could replicate in
25 cultured human fibroblasts. This set the stage in the 12
1 late 1950's for the development by Salk and his 2 colleagues of an inactivated polio vaccine prepared in
3 primary monkey kidney cells.
4 I should define primary cells, because
5 neophytes are using it to mean something entirely 6 different and are oblivious to the fact that its
7 original meaning still has a legal FDA definition. A
8 primary culture is simply a population of cells 9 derived from impact tissue that has not undergone any
10 subcultivations. After a few years of use of primary
11 monkey kidney cells, it became apparent that these
12 cells contained many unwanted viruses, some of which 13 were lethal to humans. At about this time in 1961, we
14 described the development of normal human diploid cell
15 strains that were free of contaminating viruses, and 16 we suggested that they may be useful for the
17 preparation of human virus vaccines. It took about 10
18 years before our suggestion became generally accepted
19 in the scientific community. 20 Normal human diploid cell strains have
21 several interesting properties. If I can have the
22 first slide? The properties that we described are 23 listed here. Very briefly, point number one, if
24 derived from human embryos, they undergo about 50
25 population doublings. Contrary to the belief that 13
1 this is a serious limitation, the limit is actually 2 about 50 million metric tons after 50 doublings.
3 Human diploid cells undergo a number of population
4 doublings inversely proportional to donor age. This
5 suggested to us that the finite replicative capacity 6 of cultured normal cells is an expression of aging at
7 the cell level. This notion received considerable
8 experimental support in subsequent years and is now 9 the basis for the field of cell senescence or
10 cytogerontology.
11 We also found, of course, that the cells
12 derived from this tissue had the diploid karyotype and 13 are incapable of replication in suspension culture.
14 The next point, number four, human cell strains will
15 not produce tumors when inoculated into the hamster 16 cheek pouch or even when we directly introduced them
17 into terminal human cancer patients, something that
18 was relatively easy to do in the 1960's.
19 Human diploid cell strains can be 20 cryogenically preserved. When, for example, WI38,
21 which was the first highly characterized human diploid
22 cell strain which we developed in 1962, is preserved 23 at a particular doubling level and then reconstituted,
24 the number of doublings remaining is equivalent to 50
25 minus the number of doublings spent prior to 14
1 preservation. The cells have an extraordinary memory 2 and remember at what doubling level they are
3 preserved, even after 37 years of storage in liquid
4 nitrogen. WI38 has been cryogenically stored longer
5 than any other normal human or animal cell population. 6 We also reported that human diploid cell strains had
7 the broadest virus spectrum of any cell population
8 known, and even heretofore unknown common cold viruses 9 were isolated with these cells at that time.
10 As a result of this characterization, we
11 suggested that cell populations in culture could be
12 classified into three groups: primary cultures, which 13 I have already described: cell strains, which are
14 populations that have a finite capacity to replicate
15 and don't produce tumors when inoculated into 16 experimental animals, have the karyology of the tissue
17 of origin and are anchorage-dependent; and the third
18 category, cell lines, which are populations consisting
19 of immortal cells that may produce tumors when 20 inoculated into lab animals, don't have the karyology
21 of the tissue of origin, and are usually anchorage
22 independent. 23 Our description of these three fundamental
24 cell classes in which we define normal cell strains to
25 be mortal overturned a dogma that existed from the 15
1 turn of the century. The dogma was that all cells 2 placed into culture have the capacity to replicate
3 indefinitely. And if they do die, you simply do not
4 have the proper culture conditions. We upset this 60-
5 year-old dogma by proving that normal cells do have a 6 finite capacity for replication and showed further
7 that normal cells have an intracellular counting
8 mechanism and that the replicometer is located in the 9 nucleus. Without our having proven that there exists
10 mortal normal cells, then the field of cancer research
11 rooted in the concept of immortalization of normal
12 cells clearly could not have developed. 13 Our efforts to define these classes of
14 cells were not universally accepted, and
15 terminological chaos has reigned for the last 40 16 years. A good recent example can be found in the very
17 first paragraph of the major document that you all
18 received in the package of materials for this
19 workshop. It is titled, "A Defined Risks Approach to 20 the Regulatory Assessment of the Use of Neoplastic
21 Cells as Substrates for Viral Vaccine Manufacture."
22 Reference is made to a 1954 U.S. Armed Forces 23 Epidemiological Board recommendation that "normal
24 cells, rather than cell lines established from human
25 tumors be used for the development of adenovirus 16
1 vaccines." By normal cells, this Board meant immortal 2 cell lines as I have just defined them but derived
3 from normal tissue. The cells lines referred to by
4 the Board are not normal by any criterion. In fact,
5 the normal human diploid number was not known in 1954, 6 and no normal cell had yet to be described.
7 This confusion led Maurice Hilleman to
8 prepare a live adenovirus vaccine in the Henley 9 intestine cell line, which was thought to have been
10 derived from normal human tissue, but was in fact a
11 grossly abnormal cell line later identified as HeLa.
12 The six human recipients of this vaccine fortunately 13 did not suffer any ill effects after 25 years of
14 follow-up. It was not until 1961 that we showed how
15 to make normal human diploid cell strains that normal 16 cells became available.
17 The advantages of these cells are shown on
18 the next slide. In 1961, we suggested that these
19 normal diploid cells have definite advantages over 20 primary cell populations. I won't go into detail on
21 each of these items, but simply give you some details
22 on two of them, the first and the last actually. In 23 respect to latent viruses, primary adult monkey kidney
24 cells, as we all know now, harbor quite a number of
25 serologically distinct latent viruses, several of 17
1 which were demonstrably lethal for humans. The B 2 virus and the Marburg agent both were lethal for 23
3 people after accidental percutaneous inoculation of
4 handlers of monkeys and their primary kidney cultures.
5 SV40, as was mentioned earlier, known to produce 6 tumors in rodents and with the unknown potential to do
7 so with humans was also an early contaminant of
8 primary monkey kidney cells. SV40 was also capable of 9 transforming normal human cell strains into cancer
10 cell lines in vitro. SV40 was in fact inadvertently
11 administered to several million recipients of early
12 lots of inactivated and attenuated polio virus 13 vaccines. Latent viruses have never been found in
14 WI38. To this day, no evidence exists for an
15 endogenous human oncogenic agent, latent virus, 16 transforming principle, slow virus or endogenous
17 intact retro in any human diploid cell derived human
18 virus vaccine.
19 I won't discuss cost of cell procurement, 20 available cells, cell storage. These are all fairly
21 obvious to most of us by now. But the last point I
22 think is worth emphasizing, and that is that the 23 essential point that we tried to make was that a
24 diploid cell strain was more attractive than a primary
25 cell population in its ability to be thoroughly tested 18
1 before use. In a word, standardization. Thus, the 2 concept of cell standardization was first introduced.
3 Once standardization is achieved, cryogenic storage
4 permits the acceptable strain to be held indefinitely
5 for subsequent use. It is not possible, practical or 6 economic for primary cells to be similarly handled.
7 The argument holds equally well now for
8 cell lines that all of you now take as perfectly 9 obvious. But it was not so obvious from 1962 to 1972.
10 In the late 1950's and early 1960's, cell lines were
11 generally believed to be a forbidden candidate for the
12 production of human biologicals because they shared 13 properties with cancer cells. Our suggestion that
14 human diploid cells could provide a safe and superior
15 substrate for the production of human virus vaccines 16 was met with considerable resistance for the next
17 decade. The major reasons for this resistance were
18 the belief that hypothetical cancer viruses might be
19 present and that spontaneous transformation might 20 occur resulting in the presence of human cancer cells.
21 Latent viruses were such a problem with primary monkey
22 kidney cells that a worldwide moratorium on the 23 licensing of all polio virus vaccines was called in
24 1967 because of death and illnesses that occurred in
25 monkey kidney workers and vaccine manufacturing 19
1 facilities in Germany and in Yugoslavia. The 2 responsible virus, of course, was the Marburg agent.
3 The arguments against the use of human
4 diploid cells for the preparation of human virus
5 vaccines were used as the basis for the philosophy of 6 the Division of Biologics standards, the precursor of
7 what is today CBER, that "the devil you know is better
8 than the devil you don't know." 9 In 1961, together with Dr. Stanley
10 Plotkin, we prepared the first human biological
11 produced in a human diploid cell. It was an oral
12 polio vaccine which we showed to be both safe and 13 efficacious. By 1963, 7,000 people received a similar
14 vaccine produced in WI38 with no safety problems.
15 Many of you use the concept of the master working cell 16 bank in the production of biologicals, but few know of
17 its origins. In 1963, I suggested the concept of the
18 two-tiered system of master and working cell banks for
19 WI38, although these terms were not used. The terms 20 I used were master and working cell seeds. I based
21 this reasoning and the terms on the identical way in
22 which virus seeds were then utilized. The first 23 publication of these procedures was made at the
24 landmark Opatia, Yugoslavia conference in 1963.
25 By the 1980's, when heteroploid and mortal 20
1 cell lines like CHO lines were used for the 2 manufacture of human biologicals, the procedures for
3 characterizing and handling WI38 were lifted over for
4 use with cell lines with very few changes even to this
5 day. In the mid-1960's, several WI38 users expressed 6 difficulties growing the cells that were traceable to
7 variations in the medium reagents used by various
8 laboratories. As a consequence of this, we developed 9 a practical standardized cell culture powdered media
10 in kilogram lots, that unlike wet media was easily
11 transported worldwide and used by many labs
12 simultaneously and for which only two variables 13 existed, water and serum. As a result of our
14 development of powdered media, the WI38 growth problem
15 was quickly resolved and the use of powdered media has 16 now become commonplace throughout the world.
17 In the years following our development of
18 WI38 in 1962, as a matter of national pride other
19 countries produced their own strains. The Medical 20 Research Council in the UK produced MRC5. In 1970,
21 the Japanese made TIG1 and the Chinese made 2BS, et
22 cetera. Today, most of the world's virus vaccines are 23 made on WI38 or similar strains. It has been used to
24 provide more individual doses of a biological than any
25 other cell substrate ever used. This slide following 21
1 gives you a summary of or a rough idea of the extent 2 of the use of these populations.
3 I am frankly astonished to observe that
4 after 10 years of debate on the risks of moving from
5 frequently contaminated primary monkey kidney cells to 6 virus free normal human cells that the time required
7 to make the final leap to abnormal cell lines for
8 producing human biologicals occurred in the 1980's in 9 a matter of weeks. Today, the molecular mechanism for
10 our finding that normal cells have a replicometer has
11 been substantially described as a result of a
12 fascinating story that time constraints prevent me 13 from describing. The explanation is briefly described
14 on the next slide and consists, as most of you here
15 know by now I am sure, of the shortening of telomeres 16 in normal cells each time DNA replicates. And for
17 those cells that are immortal, they escape the
18 inevitability of telomere shortening by switching on
19 an enzyme called telomerase that adds on the 20 nucleotides at each round of DNA replication, thus
21 maintaining the telomere length constant.
22 The remaining question, as I just 23 described, in respect to telomere shortening was the
24 discovery of this enzyme called telomerase, an enzyme
25 that contains an RNA template and a catalytic moiety, 22
1 among other components. This is the only enzyme that 2 contains a reverse transcriptase and is part of normal
3 cell biology. 90 percent of cancer cells express the
4 enzyme. Only normal stem cell populations do so.
5 As for the use of abnormal and mortal cell 6 lines for the production of human vaccines, that has
7 already been done. The Vero cell line derived from
8 the kidney of an African green monkey kidney has been 9 licensed in France, Belgium, the United States and
10 Thailand for the production of both live and
11 attenuated polio and rabies vaccines. There are now
12 more than 20 million vaccinees. The Chinese have made 13 a Japanese encephalitis vaccine in the abnormal cell
14 line, BHK21. Recombinant vaccines have been licensed
15 for hepatitis B grown in a CHO cell line, and phase 16 III trials are currently underway for an HIV vaccine
17 in these same cells.
18 There are other immortal cell lines that
19 bear consideration as substrates for the preparation 20 of human virus vaccines. About two years ago, I
21 initiated a culture of fibroblasts from a skin biopsy
22 that I took from my knee. This culture was the first 23 to be immortalized by transpection with human
24 telomerase reverse transcriptase. Three other human
25 cell strains were similarly treated and these results 23
1 were published in Science in January of 1998. These 2 immortalized normal cell populations have now
3 undergone well over 400 population doublings. This
4 opens up enormous opportunities, not only to
5 immortalize WI38, but many other normal cell types for 6 applications like -- for exploitation actually as
7 vaccine substrates among many other potential uses.
8 Another category of immortal cell lines 9 that merits consideration are those transformed by
10 agents other than viruses. SUSM1 is a cell line
11 derived from normal human lung that we transformed
12 with a chemical carcinogen. KMSD6 is also an immortal 13 cell line derived from normal human fibroblasts and
14 transformed by exposure to cobalt 60 gamma radiation.
15 I might add that contrary to the belief of some, early 16 passage WI38 does exist in ample quantities, and
17 anyone wishing to obtain these cells should feel free
18 to contact me.
19 Thus, there exists several methods for 20 transforming well-characterized normal human and
21 animal cell populations to immortal cell lines using
22 at least three proven methods. What should not be 23 overlooked, however, in our zeal to condemn any
24 immortal abnormal cell line from consideration as a
25 human virus vaccine substrate is the fact that 24
1 downstream processing has reached such a level of 2 efficiency that the preparation of a human virus
3 vaccine containing only the purified moieties capable
4 of eliciting an immune response is possible now.
5 Affinity technology in particular is clearly capable 6 of meeting this exacting goal. Nevertheless, as we
7 all know, economic considerations will surely guide
8 most of these decisions. Thank you very much. 9 DR. GRIFFITHS: Thank you very much
10 indeed. We do have time for one or two questions if
11 people want to start the ball rolling and ask Dr.
12 Hayflick any questions. I don't see any immediate -- 13 oh okay. Sorry, I can't see your name from here. Can
14 you introduce yourself? Is there a microphone?
15 PARTICIPANT: I am Jim Cook from Chicago. 16 What all types of animals have WI38's been inoculated
17 into for tumorigenicity testing besides the hamster
18 cheek pouch?
19 DR. HAYFLICK: Oh my. 20 PARTICIPANT: I mean, have they been put
21 into newts. Have they been put into other kinds of
22 immunocompromised animals? 23 DR. HAYFLICK: All immune compromised lab
24 animals -- mice, rats --
25 PARTICIPANT: What about things like the 25
1 odd observation that Balb C3T3's when hooked onto 2 plastic disks will make tumors in newts? Has anything
3 unusual like that been done with WI38's?
4 DR. HAYFLICK: Yes. And to the best of my
5 knowledge, there has been no tumor formation. There 6 have been occasional reports of nodules where the
7 cells will replicate to a point where they reach a
8 macroscopic size. But usually what happens is that 9 they fail to grow beyond that state.
10 DR. GRIFFITHS: Johannes?
11 PARTICIPANT: Johannes Loewer. You
12 mentioned that the human diploid cell lines have a 13 very broad host range for many different viruses. Is
14 it known whether neoplastic cells have even a broader
15 host range or is it more or less similar to diploid 16 cells?
17 DR. HAYFLICK: You are talking about cell
18 -- immortal cell lines?
19 PARTICIPANT: Yes, immortalized cell lines 20 or neoplastic cell lines.
21 DR. HAYFLICK: From human material?
22 PARTICIPANT: Yes. 23 DR. HAYFLICK: To the best of my
24 knowledge, the human diploid cell strains have a far
25 broader virus range than the immortal cell 26
1 populations. Most or many of the rhinoviruses were 2 first discovered on WI38 and later on MRC5. But the
3 fact that they could ultimately be adapted to growth
4 in immortal cell populations was also revealed.
5 However, I think it is still fair to say that normal 6 human diploid cells are the most sensitive cell
7 population to most of the viruses that are now well
8 characterized. 9 DR. GRIFFITHS: Any other points that we
10 should take? Because we are right on time, so we are
11 really doing quite well this evening. If not, then
12 thank you very much indeed. 13 DR. HAYFLICK: Sure.
14 DR. RABINOVICH: Our next speaker is Dr.
15 Elwyn Griffiths from the World Health Organization, 16 who has also worked in this field for -- I won't even
17 venture to guess how many years. He is going to talk
18 about major issues associated with cell substrates.
19 DR. GRIFFITHS: Thank you very much. I 20 must say I was very impressed when I got this badge
21 actually from the organizers. I mean, I don't know if
22 they get a prize for the number of flags that is on 23 it. It is very impressive. I mean, I was only asked
24 to do this particular talk and stand in just a couple
25 of weeks ago. I think it should have been Dr. 27
1 Petricciani who would have done this and done it far 2 better than I am sure. But in standing in, perhaps I
3 should have stood in for the patron committee or
4 something just to have another badge on here.
5 Anyway, for this evening to a certain 6 extent this talk really is something of a trailer to
7 the more detailed discussion we are going to have over
8 the next few days, and really an extension of the 9 points already touched upon by Dr. Hayflick.
10 The way I thought I would like to do it is
11 to take a different stance in the beginning. Before
12 dealing with the major issues which really revolve 13 around these different cell substrates, to start with
14 a reminder. And I think I would like this reminder to
15 be borne in mind throughout the meeting that what we 16 are dealing with has great benefits for mankind. I
17 mean, the benefits of the vaccines produced on cell
18 substrates. There has been enormous success in cell
19 substrates for producing vaccines. These particular 20 vaccines have had a tremendous influence on human
21 health.
22 I would just want to pick up on polio, for 23 example, because that is something which the WHO
24 clearly has a -- and everybody else, I guess -- has a
25 major interest in. The last case of polio in the 28
1 whole of the Americas, that is PAHO, was in 1991. In 2 the Western Pacific region, it was in March of 1997.
3 The world is moving towards global eradication of
4 polio in roughly about -- it is expected in 2000. I
5 am not sure if 2000 is realistic -- 2000, 2002 or 2003 6 or so. You can see the difference from -- the top one
7 is 1988 really to 1998. There are patches left in
8 1998, and this is gradually getting smaller and 9 smaller.
10 So essentially, global eradication of
11 polio, which will have enormous benefits for mankind,
12 has been achieved primarily through live polio 13 vaccines. These vaccines are produced on a number of
14 cell lines -- primary monkey kidney cells, diploid
15 cells, and also in vero cells. I think it is true to 16 say on a global perspective that the majority of the
17 producers are producing on primary monkey kidney
18 cells.
19 Now having said that, I just wanted to 20 have that borne in mind. When we deal with the
21 issues, we really do have some major benefits from
22 these products. So it really is how much of a 23 risk/benefit equation which we have to deal with here
24 and not forget the actual benefits by focusing too
25 much on the issues. That isn't to say the issue is 29
1 not important. 2 The major issues associated with the use
3 of cell substrates really relate to safety. I mean,
4 that is our main problem here. This is something
5 which has been really extensively debated over the 6 last 40 years or so, with the acceptance of cells for
7 production really being a hot topic as they come up
8 from time to time -- primary cells, diploid cells and 9 continuous cell lines, as Dr. Hayflick already
10 mentioned. The perceived risks being attributed to
11 contaminants coming from the cells. The main ones of
12 interest really being viral contamination, DNA, and 13 transforming proteins.
14 The whole of these issues have been
15 discussed -- I wouldn't say ad nauseam, but they have 16 been extensively discussed over -- what is it, from
17 1967 -- over 30 years or so. And what is interesting
18 is when you look back over this, this is a selection
19 of meetings. I am sure there are many, many more 20 meetings. You see, I am only just taking this from
21 1967. It is the same players essentially or the same
22 cast as we have for this meeting. We have the FDA. 23 The IABS features strongly in this, the WHO, the NIH.
24 So they are all there really right from the very
25 beginning. 30
1 The main point here is that some of the 2 issues -- I mean, each meeting will have a special hot
3 topic, if you like. In 1967, it was diploid cells.
4 Then in 1978, that meeting was organized essentially
5 to see whether we could move forward with the use of 6 continuous cell lines, primarily because namalva cells
7 have already been produced by the Wellcome Institute
8 for producing interferon. And I suppose the 1978 Lake 9 Placid conference gave us a cautious yes to the use of
10 continuous cell lines for the production of
11 biologicals. And I am broadening this now not just
12 for vaccines, but for the production of biologicals. 13 But there was still a lot of "yes, but" at
14 that meeting. I wasn't there, and I am sure that John
15 and other people who would have been there would say 16 the same thing. It seems that it was, yes, we can go
17 forward, but, but. And certainly not in the USA, I
18 guess. That was the sort of general feeling at that
19 time. It was really the meeting in 1986, the WHO 20 meeting, a study group in Geneva, which really allowed
21 the field to move forward in so far as use of
22 continuous cell lines were concerned for the 23 production of biologicals.
24 At that time, there was an agreement from
25 this particular study group that continuous cell lines 31
1 could be used for the production of biologicals 2 provided that the level of residual DNA or residual
3 host cell DNA was kept below 100 picograms of DNA per
4 dose. That is the origin of the magic figure which has
5 been used for the last 10 years or so. And this 6 really allowed -- this decision really allowed the
7 field to move forward in the expansion of the
8 biotechnology industry to produce recombinant DNA 9 biologicals in continuous cell lines. This has been
10 the situation for the last 10 years or so or more now.
11 At the last meeting of the WHO, which was
12 1996, a WHO and NIABS consultation at Merieux 13 Fondation in Annecy, that is the time where the field
14 was reviewed, if you like. Where are we after 10
15 years or so of using this figure of 100 picograms? 16 Incidentally, that figure of 100 picograms was used as
17 the guidance for all the national regulatory
18 authorities. The FDA went a little bit further and
19 went down to 50 at one time, less than 50 picograms. 20 Industry was pushing toward the limited detection.
21 And it is interesting to see the European
22 Pharmacopoeia has a limit for recombinant hepatitis B 23 vaccine produced in continuous cell lines of 20
24 picograms per dose, which is very low and very
25 stringent. This meeting in 1996 was to review that, 32
1 and things have changed a little since then and I will 2 come to that later on.
3 The present meeting, of course, really
4 focuses on cell substrate for vaccine production, not
5 for biologicals in general. I think there is this 6 distinction which I would like to make later on. We
7 are now moving to potential use of overtly neoplastic
8 cells for vaccine production, and I think this is a 9 very timely meeting.
10 The sort of landmarks -- and I am really
11 going over a little bit the ground which has already
12 been covered by Dr. Hayflick. These were sort of, I 13 think, the sort of landmarks as you might say. The
14 gradual overcoming of the hurdles -- the use of
15 primary cells for vaccine production in polio really 16 exemplifies that in the 1950's. In the 1970's,
17 diploid cells were accepted for vaccine production,
18 but not without problems as we have already heard.
19 Then in the 1980's, after the WHO meeting, continuous 20 cell lines then became acceptable for vaccine
21 production -- namalva cells for interferon, vero cells
22 and so on -- hybridomas for monoclonal antibodies 23 before then of course, and recombinant DNA
24 derivatives.
25 Now the progress during this period of the 33
1 1960's to the 1990's has been really on a number of 2 fronts. I think it is fair to say -- I can say this
3 because the WHO isn't really a regulatory agency. But
4 I think one can now say that regulatory decisions are
5 made more on a scientific basis and not on opinions as 6 such. And the progress that has been made in
7 scientific knowledge really over this period is
8 enormous. We have to remember that. I just put a few 9 up here to flag them -- new diagnostic procedures,
10 let's say, such as PCR; better understanding of
11 molecular mechanisms of pathogenicity; and new
12 concepts, the concept of validation has been 13 introduced.
14 So we are now in a position -- it is
15 perhaps unfair to say that the regulatory decisions 16 can be made more on a scientific basis. We have got
17 more scientific basis to make the decisions. I think
18 that is perhaps the main point to make there. We can
19 make decisions, better informed decisions, and I think 20 this is an important part of the gradual move to
21 acceptance of different cell substrates.
22 Part of this process as well has been co- 23 defining, if you look -- perhaps co-define is too
24 strict. Having regulations and guidelines and
25 recommendations for production and quality control of 34
1 these products using cell substrates has also been a 2 major development since that time, both on a national
3 basis, on a regional basis, such as in the European
4 Union and the European Commission, and the WHO, of
5 course, on a global perspective. And I think what we 6 are doing is really learning how to manage the risks
7 to take advantage of the benefits. And I think that
8 is the important -- what we want to do to take the 9 field forward.
10 Very briefly then I will go through just
11 to remind ourselves and really to reiterate what Dr.
12 Hayflick has said. The primary cells -- I mean, I 13 will just mention the disadvantages here in the sense
14 that many people tend to use them because they tend to
15 be easy to prepare and so on. But they really do have 16 this major disadvantage of microbial contamination and
17 especially of viruses, of course.
18 Can we go to diploid, the next one, and
19 just go through these very quickly. Again, and it has 20 already been eluded to, the great advantage of using
21 diploid cells is that they can be well characterized
22 and standardized. And so production then becomes based 23 on well characterized cell banks, the master cell bank
24 and a working cell bank. And this is true also for
25 the continuous cell line. 35
1 But they all have their advantages and 2 disadvantages. I just want to focus on the
3 advantages. An infinite life span for the continuous
4 cell lines, so you can keep them going forever I guess
5 really. And again, production on well characterized 6 and standardized cells for an indefinite time basis
7 really.
8 Now when you look at the disadvantages 9 amongst these cell lines, the primary cells had the
10 problem with the contamination. But the main
11 disadvantage of the continuous cell line is that many
12 do express endogenous viruses and there has always 13 been this concern over tumorigenic potential, should
14 we say, associated with cellular DNA.
15 The main three risks then with these 16 different cell lines for producing biologicals are
17 contaminating viruses, and we must include here, the
18 TSEs, the transmissible spongiform encephalitis
19 agents, whatever that may be in the end, residual host 20 cell DNA, and growth-promoting proteins. And as I
21 said, the trick really is how to manage these. And by
22 developing procedures, we can actually move forward 23 onto guidelines and so on.
24 In fact, there are three principles
25 essentially really to production of biologicals. And 36
1 these are really the basis for moving forward on all 2 these cell lines. Control of the starting material,
3 such as the baseline data, characterization of the
4 host cell, and so on, if you are able to do that
5 depending on what sort of cell line you're using. 6 Then you control the manufacturing process itself,
7 this is for adventitious agents and so on, and also
8 control of the final product. And that includes tests 9 performed during development. Because not all tests
10 will be carried out on a routine lot-to-lot basis.
11 There will be some testing on development. And then
12 there is a subset of those tests, which will be 13 carried out on a routine basis. These are the lot-to-
14 lot releases.
15 Now historically, assuring the quality and 16 safety of what you might call traditional biologicals
17 has been what I call disaster-led. And you can make
18 a nice list of all the problems we have had over the
19 years and you find that control procedures are really 20 established or altered following some major mishap,
21 let's say. For example, Creutzfeldt Jakob disease
22 from the bottom there in recipients of pituitary- 23 derived growth hormone. I mean, that meant that we
24 weren't now going to use the pituitary-derived growth
25 hormone and move over to recombinant. So it is these 37
1 sorts of issues which really trigger the traditional. 2 But if you look at the situation which I
3 call applied to biotechnology products -- I mean novel
4 biotechnology products. I mean, it is all
5 biotechnology I guess really, but I am thinking of 6 recombinant DNA, monoclonal antibodies, cell cultures
7 and so on. The approach has been somewhat different.
8 Guidelines have been established early-on in the 9 development of the new field to try and pre-empt any
10 disaster. I mean, the idea here is that we think of
11 what the problems might be and try to make sure that
12 we don't land ourselves in these sort of situations. 13 And developing guidelines and points to consider,
14 whatever you might want to call them -- and the
15 different countries and different agencies call them 16 different things -- I think these have been
17 instrumental in establishing safety and quality of
18 these newer type of biologicals, the biotech products
19 if you like. Because they are by far the best 20 characterized and best purified biological medicines
21 in clinical use.
22 You see during this period in the 1970's 23 and 1980's, guidelines on production and control or
24 you might call them points to consider have been
25 derived by many agencies. The main ones being the CBER 38
1 FDA points to consider, Japan has guidelines, the 2 European CPMP biotech working group, those are the
3 European Commission guidelines, and WHO. Now what is
4 interesting is that the majority of these guidelines
5 do have the benefit of mutual consultation during 6 their development. If you look at them in some
7 detail, you find that essentially they all say the
8 same thing, but they are actually viewing it from a 9 slightly different perspective with slightly different
10 words. The ICH guidelines, for example, on cell
11 substrates, that actually deals with more of the
12 characterization of the cell lines and so on. So we 13 find that these different type of guidelines do all
14 have the same thrust, but they take it slightly in
15 different perspectives. But they all say essentially 16 the same thing.
17 Now as far as WHO is concerned, the first
18 requirements for cell cultures for production of
19 biologicals was the 1959 requirements for primary 20 monkey kidney cell production of polio vaccine. Now
21 all the other major agencies -- FDA and in Europe and
22 the individual national states -- would also have 23 their requirements as well. But WHO is the embodiment
24 of a global situation. And using the primary cells,
25 monkey kidney cells, for polio vaccine really did lead 39
1 to some confidence in developing other primary cell 2 lines for producing a range of vaccines. And these
3 are still in use. We mustn't forget that. The
4 primary cell is still a major source -- for example,
5 the chick embryo cells for measles vaccine and mumps 6 vaccine as well. So the development of these at this
7 point was a sort of major move forward.
8 Now I am not going to try and sort of 9 compare and contrast the different guidelines, because
10 I don't think that is particularly helpful. But what
11 I will try and do is just to scan briefly through the
12 guidelines and points to consider which have been 13 developed in relation to the cell cultures. These,
14 again, reflect considerable discussion. I mean, I have
15 just put the WHO ones. The first one there was the one 16 for oral OPV, and these are updating. As time goes on,
17 there is always a need for updating because some
18 technology changes and things move forward.
19 I have already mentioned the meeting of 20 the study group in 1986. It was published in 1987,
21 which allowed the field to move forward, and that was
22 the acceptability of cell substrate for production of 23 biologicals. And then in 1987 as well, there was the
24 continuous cell line for the production of
25 biologicals. The FDA, the ICH, the CPMP also have 40
1 their guidance documents as well specifically on cell 2 substrates.
3 Now I just want to say something about the
4 situation of 1998 as far as the WHO cell substrate
5 document is concerned. This document actually 6 reflects considerable international consultation. I
7 think this is probably the latest one or major
8 international one. It was published last year, but the 9 actual meeting -- there was a meeting in 1996 with
10 WHO, IBS, and we had the benefit of input from the
11 ICH, which went into great detail about how we should
12 control the quality of the biologicals produced on 13 primary, diploid and continuous cell lines. It covers
14 the whole three for production of viral vaccines and
15 the biologicals. It is not restricted to viral 16 vaccines. And it is interesting that this document is
17 very clear in that it encourages the move away from
18 primary cells to cell substrates which can be
19 generated from well characterized cell lines. As we 20 have already mentioned -- Dr. Hayflick mentioned this
21 idea of the well characterized cell line goes back a
22 long time, probably to the meeting -- I think there 23 was a meeting in Zagreb where this was developed of
24 having the master cell bank and so on.
25 Now in this particular document, there has 41
1 been a major change in certain parts of the way we 2 handle the control of some of these cell lines. There
3 is, for example, for well characterized diploid cells,
4 a relaxation of the karyology and tumorigenicity
5 testing and so on. But I just want to touch on one 6 particular point which is important for the next part
7 of the issues. And that is related to the DNA and the
8 viruses. The main risks -- in this document, we still 9 see that the main risks still are for the continuous
10 cell lines residual DNA and maybe growth promoting
11 proteins, and I will come to that in a moment, and for
12 all cell lines, contaminating viruses. 13 Now as far as the DNA is concerned, there
14 has been a major departure in the previous
15 recommendation regarding residual DNA. And this is 16 really due to a reevaluation of the situation and new
17 data over this decade from 1986. But also new
18 technologies have appeared on the scene -- gene
19 therapy and DNA vaccines, which actually are involving 20 injection or administration of in the case of DNA
21 vaccines quite large quantities of DNA. So one has to
22 be consistent here. Either DNA is really a big 23 problem or it isn't. Or is it the type of DNA? And
24 this is the issue.
25 If you look at the 1986 summary, I guess 42
1 really, the cause of concern for that time was 2 transmission of oncogenes. And the 1986 consultation
3 provided this way forward by saying that if you can
4 get your DNA down to less than 100 picograms per dose,
5 then there is no realistic risk, and this is what 6 everybody went forward on. The perceived problem was
7 really not DNA post-op, but the specific sequences of
8 DNA which may be encoding oncogenes and the possible 9 insertion of endogenous or host cell protooncogenes or
10 inactivation of suppressor genes by the DNA. The DNA
11 itself was largely ignored, I think, and that is were
12 the move forward is on DNA vaccines. We can go forward 13 there because although there is a risk, the acceptance
14 of this minimum risk is something which has been
15 actively discussed and debated for the last two or 16 three years.
17 So the figure now is that we now think --
18 by we, the current opinion. I think this is
19 everybody. As I say, everybody has partaken into 20 these general discussions here. They are based on
21 current state of knowledge. The suggestion is that
22 DNA from continuous cell lines can be considered -- 23 the DNA itself now -- as really a cellular contaminant
24 rather than itself as a significant risk factor
25 requiring removal to extremely low levels which needed 43
1 validation and testing of each lot and so on. And the 2 relaxation now is that we think that a figure of 10
3 nanograms of residual DNA from a continuous cell line
4 will apply. And this applies primarily, of course, to
5 purified recombinant DNA biologicals. 6 This is something which I want to touch on
7 here. What we are talking about is removal of DNA to
8 reasonably low levels. But the guidelines do make the 9 point that residual DNA from continuous cell line may
10 pose a higher risk if it might include infectious
11 retroviral or provirion sequences, and then we may
12 well still have to go down to lower levels. Now this 13 is something I am sure that is going to come up in
14 this meeting. What is the actual level which is
15 applicable to vaccines. Can you actually go to that 16 lower level.
17 I don't want to say much about the growth
18 of multi-proteins. These are sort of more or less
19 dismissed, I suppose, in the WHO guidelines. Growth 20 factors that are produced by cells, of course, the
21 risk is very limited. Yes, they do affect cell
22 growth. It is usually transient and reversible. They 23 don't replicate and many are inactivated in vivo. So
24 the general thrust of the argument there was that they
25 are not such a problem as we originally thought. 44
1 Now that might be different today, but the 2 viruses remain the major and real concern for all cell
3 types. The test for viral contamination really is a
4 major part of cell bank characterization. In the
5 guidelines, in the WHO ones, it covers the primary 6 cells here. Now, here we are taking cells directly
7 from animals. Sometimes in the wild animals for polio
8 -- from monkeys for polio. And quite clearly now, we 9 have to move into a situation if you are going to use
10 animals that they must be healthy animals subject to
11 veterinary and laboratory monitoring, very closed
12 colonies wherever possible, not from the wild if 13 possible. And animals, if they do come in from the
14 wild, then they really have to be quarantined and
15 appropriately monitored in the time before you 16 actually take the cells for preparation of vaccines.
17 And serological screening of donor animals for
18 relevant pathogens.
19 Now you might say what is a relevant 20 pathogen. Well that really is changing as time goes
21 on, of course. The problem with doing this for a
22 primary cell is that you have to do it for every 23 vaccine batch. I mean, it is not the case of doing it
24 once. You need to test the cells vigorously. It is
25 an awful lot of work. And of course you can only test 45
1 for a certain number of known viruses. You can have 2 a sort of catch-all, but there are mishaps. As we
3 know, SV40 was missed, and this is an issue that comes
4 back to haunt us as time goes on.
5 With a cell bank, we can actually do this 6 testing, extensive testing for exogenous and
7 endogenous agents, once only. You can do it and then
8 you can make sure that this is the master cell and 9 then you move on to the working cell bank. The
10 further testing really is on the working cell bank or
11 production cells really to detecting common
12 adventitious agents, and that could be sort of a 13 catch-all. It is not such a major issue.
14 It is interesting that the working group,
15 the WHO study group, in 1986 valued the well 16 characterized cell banks so much that it recommended
17 the establishment of a well characterized cell line
18 that would be available to national control
19 authorities and manufacturers of biologicals globally. 20 And, in fact, the WHO master cell bank for vero cells
21 was established following the recommendation by this
22 group. It comes from African green monkeys and it was 23 selected -- the vero cell was apparently selected
24 because of the immediate prospect for improving the
25 quality and quantity of several vaccines. Now it has 46
1 been used but perhaps not used as widely as was 2 thought at the time.
3 Now the strategy for preventing viral
4 contamination then is basically what all the documents
5 go through. Tests on starting materials -- not just 6 the cells. We have to remember that some of the
7 agents will come in from the reagents -- the serum and
8 so on. We have to be very aware of excipients as 9 well.
10 Evaluation of production processes. That
11 is the ability to remove or inactivate potential viral
12 contaminants. That is your validation essentially. 13 This is the concept where validation has come in. And
14 then tests on final product or appropriate
15 intermediates. And essentially what people think is 16 that it is not a single approach. One approach isn't
17 sufficient. It is the combination of approaches which
18 gives you this degree of security.
19 Now if you look at the WHO guideline, and 20 I think this is true of most of the points to consider
21 in the guidelines, they are really focusing on biotech
22 products, that is, highly purified recombinant DNA 23 products essentially. Now if you look down at the
24 bottom, the purified biotechnology product -- you can
25 have a very robust virus clearance system that you can 47
1 check for validation of virus clearance downstream and 2 so on. So even if you are -- I mean, I don't think
3 there are any biotech products produced or recombinant
4 DNA products produced in primary cells, but even if
5 you were doing it that way, you could be sure that -- 6 you could actually be sure that everything was going
7 to be cleared up by the system. When you come to virus
8 vaccines, you have much less purification, and the 9 possibility of virus/virus interaction during
10 production needs to be borne in mind, and this is an
11 issue which will come up later in the meeting.
12 So virus vaccines, though, are a different 13 ballpark really from the general biologicals, which
14 everybody -- you know, most of these guidelines are
15 focusing on clearance of DNA and clearance of viruses, 16 looking at the characterization of cells. When you
17 move to virus vaccines, there is less you can do.
18 Now we mustn't think, of course, that we
19 know everything at this stage. I mentioned that we 20 have had enormous progress in the last 30 years or so.
21 We must be prepared for surprises. Two recent events
22 I think have raised awareness of the challenge there 23 is in dealing with viral contamination of cell lines
24 and vaccines which are used in their preparations and
25 the consequences. One was the detection of very low 48
1 levels of reverse transcriptase in chicken cell- 2 derived vaccines using this newly developed assay --
3 we call it PERT assay. This is from -- like measles
4 vaccine is an example and mumps is another example.
5 All the vaccines produced in eggs will contain very, 6 very low levels of RTAs. And this, of course, when it
7 first was discovered raised a lot of alarm bells.
8 People were thinking, yes, there must be retroviruses 9 in here and so on. And this really led to quite a lot
10 of activity trying to show where this activity was
11 coming from. Is it real retrovirus or what do we have
12 there? What is the problem? Or do we just have some 13 non-specific activity which really looks like reverse
14 transcriptase? And much work -- and WHO was very much
15 interested in this, of course, because if national 16 control authorities of one country ban a vaccine
17 because it is considered to be potentially unsafe,
18 this has ripples throughout the whole of the vaccine
19 community. I mean, what is not safe for one set of 20 kids in one country must be unsafe for other kids of
21 course.
22 So there was a lot of work to try and make 23 sure that this activity -- where did it come from and
24 so on. And manufacturers and the national control
25 authorities were very active here. It has now been 49
1 shown to be particle associated, but extensive studies 2 have shown no transmission or productive infection,
3 and that of course is good news. But as we will hear
4 later on in the meeting, what about the potential for
5 interaction between these particles and some of the 6 viral vaccines -- the actual virus which is used for
7 producing the vaccine during growth. We need to be
8 sure -- I am thinking of pseudotypes and so on here. 9 This is an issue which we need to address in this
10 meeting.
11 The other surprise was the detection of
12 SV40 genome in rare human tumors. This is something 13 which has come back to haunt us after 30 years or so.
14 I am sure you all know that SV40 was a contaminant of
15 some of the early batches of primary rhesus monkey 16 kidney cells used to produce polio vaccines. This is
17 no surprise. During the 1950's, these were actually
18 used in a large number of people -- in the millions.
19 There was follow-up with that to see whether they 20 actually caused any problems, and nothing much
21 materialized. And then suddenly about three or four
22 years ago, the SV40 sequences were picked up in 23 various rare human tumors. That raised the issue of
24 was the vaccine -- was the polio vaccine made in
25 primary kidney cells actually still transmitting SV40 50
1 or SV40 sequences. Because right in the beginning 2 when SV40 was discovered, measures were introduced
3 very quickly by national regulatory authorities to
4 exclude SV40 from polio vaccines. For example, I am
5 thinking here of the WHO. The 1959 requirements had 6 to be modified and they were updated and the
7 regulations in all the major national authorities were
8 also changed to exclude SV40. And for more than 30 9 years then, polio vaccine made in primary monkey
10 kidney cells have been shown to be free of SV40. Now
11 by shown to be free, it was shown to be free by
12 technology of the 1960's, I guess. I mean, it is old 13 technology. And the question which everybody was
14 rather anxious about was were we actually missing
15 something here? And then the application of new 16 highly sensitive PCR techniques for detecting SV40
17 genome was then introduced to see whether batches of
18 vaccines were actually carrying any SV40 sequences.
19 This is work carried out by CBER and also by NIBSC, 20 and we are looking back on historical samples as being
21 quite clear that the methods introduced in the 1960's
22 were effectively excluding SV40. 23 Now if I can sort of come back to where I
24 started from in the beginning in relation to polio,
25 the polio eradication is going to be with us in about 51
1 two or three years, and this is really an end-game and 2 how do we move forward here. Once polio has been
3 eradicated, transmission of wild polio would be
4 stopped. And the global certification of polio
5 eradication will come in around 2005, I guess. But 6 there will be continued use of the OPV expected for
7 maybe 5 or 10 years. It is unclear. The end-game here
8 is unclear really and how we move forward. But WHO is 9 looking towards cessation of immunization with OPV
10 around 2005 or 2010. USA will be moving to IPV in
11 2000 -- in January of the year 2000. So the question
12 is do we need to do anything about the vaccine, the 13 OPV. And we need now, because -- I think now as we
14 move to eradication, I think we do need to be
15 absolutely sure of the vaccine and introduce an 16 additional level of security. This is provided by
17 ensuring that SV40 sequences are absent from polio
18 virus seed. Because during the actual survey of the
19 vaccines, one seed I think from a manufacture was 20 found to be positive. The seed was positive. There
21 was no actual live SV40 there. There were some SV40
22 sequences there. It is considered that it is much 23 better to have the seed free of SV40. And we are now
24 actually updating the original requirements -- this
25 will come up this year -- making sure that all seed 52
1 will be tested for absence of SV40 sequences. The 2 primary monkey kidney cells for production will now
3 have to come, I think, from closed, intensely
4 monitored colonies of animals and not from the wild.
5 As time goes on, of course, new viruses 6 are discovered and new problems arise. The foamy
7 virus has been identified as one that we should be
8 really sure is absent from these vaccines. So 9 updating is something which we are moving forward
10 towards in this end-game for on the polio.
11 What is the next step then? What are the
12 challenges for the future? The use of neoplastic 13 cells or novel cell lines for vaccine production I
14 think is really a challenge. And I think one could
15 say as with any new technology, new sets of safety 16 issues are generated for consideration. Not just by
17 regulatory agents but by industry and regulatory
18 agencies together. I mean, everybody is on a learning
19 curve here. It is not one or the other dictating. 20 Everybody is together here. We need to consider
21 again, I think, some of the issues of residual DNA.
22 Is it oncogenic? What is the issue there? Is there 23 an infectious DNA in relation to what sorts of cell
24 line you've got and what is in the cell? It is really
25 timely to review and assess the risks in light of a 53
1 much better understanding of the molecular mechanisms 2 of geo-originicity and of viral/viral interaction. As
3 I mentioned, I think we will touch on that during the
4 meeting.
5 What I think we have to do is to come to 6 some realistic and scientifically sound decisions
7 concerning the use of these substrates. Now, a point
8 which I from sort of a WHO and a global perspective 9 think is very important -- I think science and
10 commerce in biologicals -- I mean, vaccines are made
11 in one country and travel the world. They are not
12 necessarily just used in one country. It really is an 13 international situation. And so too are the public
14 health questions which the use of these products
15 raise. So if there is an issue in relation to a 16 problem in one country, it is a global issue. And the
17 international dimension of the discussion of these
18 issues is vital. And I am glad that we do have an
19 international group here, and I think it is important 20 that WHO is part of this and we are very pleased to be
21 co-sponsoring this meeting. Thank you very much.
22 DR. RABINOVICH: Are there any questions 23 specifically for Dr. Griffiths? Dr. Rubin?
24 PARTICIPANT: I was not quite clear on
25 what you were saying about the presence of 54
1 retroviruses in chick embryo cell cultures. Whether 2 you thought that in fact it was present or not. And
3 the reason I am asking is we had a lot of experience
4 with chicken leukosis viruses in chick embryo cells
5 beginning back in 1960. And the thing about them is 6 they are not easy to detect because they don't produce
7 any pathogenic effect and they have to be detected in
8 indirect means. The indirect means in those days when 9 they were first found by us, at least, was
10 interference with RAS sarcoma virus preventing. And
11 then much later on came -- at least 10 years later
12 came reverse transcriptase. The thing about these 13 leukemia viruses or leukosis viruses, these
14 retroviruses, other than the really pathogenic ones,
15 is that they are highly species specific for one 16 thing. So chicken viruses in general, with some
17 exceptions, will not infect other species. So if we go
18 up the scale, I think, to the mouse leukemia viruses,
19 I think it is generally true of them also that they 20 are not cytopathic in culture. They can't be detected.
21 And probably they are of no concern to other species
22 unless they are very closely related. The concern I 23 would have of vaccines made in higher species, monkeys
24 or humans, is that probably there are -- or not
25 probably, there may be some stealth viruses like these 55
1 that don't produce any obvious effect and that we 2 don't even know about their presence to even detect
3 them in animals. Let's say the chicken virus requires
4 a flock of chickens that is not infected with them
5 that has to be infected at a very early age and you 6 have to practically wait for half a lifetime of the
7 animal to produce any statistical effect among them.
8 So I wonder if there is any concern about these points 9 with the retroviruses.
10 DR. GRIFFITHS: Can I just clarify the
11 point about the ALV, the avian leukosis virus.
12 PARTICIPANT: Yes. 13 DR. GRIFFITHS: The vaccine production
14 stipulates that the eggs are free from the -- the
15 chickens are free from the avian leukosis virus. 16 PARTICIPANT: Yes.
17 DR. GRIFFITHS: So the reverse
18 transcriptase activity wasn't -- it wasn't expected.
19 There are very low levels of RTAs. I mean, it has 20 been tested by the ordinary methodologies -- the sort
21 of standard methods. It was only when you went down
22 to these very low levels. And the origin is now known. 23 Where it comes from is endogenous viruses, retros.
24 And they do form particles, but as I said they are
25 non-infectious particles. But on a global basis, 56
1 yellow fever for example -- the yellow fever vaccine 2 -- this is some sort of a risk/benefit which the WHO
3 has to sort of wrestle with which a national authority
4 has the benefit of not necessarily -- like the USA,
5 for example, doesn't have to. It can make a decision 6 right away. But, for example, WHO requirements --
7 they will be changing, but they have allowed
8 production in ALV positive eggs because you cannot get 9 ALV-free flocks in many -- in places where you are
10 actually making yellow fever vaccine, and it was
11 considered that the lack of yellow fever vaccine was
12 much more of a risk than the presence of the ALV. 13 Nevertheless, Western Europe and USA making yellow
14 fever vaccine is all in ALV-free -- from eggs from
15 ALV-free chickens. And that will now be introduced 16 into WHO requirements as well, I think, in the future
17 because these countries which were producing in ALV-
18 positives can now actually get the eggs free.
19 PARTICIPANT: Okay. 20 DR. RABINOVICH: One last question?
21 PARTICIPANT: I was really going to
22 comment on actually the last comment from Dr. Rubin. 23 And reiterate your point that the sequence that appear
24 to be responsible for the RTA activity appear to be
25 the AEV gene and not the ALV gene. 57
1 DR. GRIFFITHS: That is right. Yes. 2 PARTICIPANT: But actually both the AEV
3 and ALV genomes can actually undergo recombination.
4 One of the subgroups of ALV is actually based on the
5 AEV envelope gene sequence. But I really wanted to 6 challenge the assumption that we should not be
7 concerned about the species specificity of the
8 retroviruses. In fact, you can induce tumors quite 9 readily in certain species quite diverse from any
10 species with RAS sarcoma virus that contains the ALV
11 envelope gene in subgroups like Subgroup D. So it is,
12 I don't think, a correct assumption to state that 13 viruses like the avian retroviruses are not of
14 concern. They could be.
15 DR. GRIFFITHS: I think David's point -- 16 did you introduce yourself David? That is David Onions
17 for the record.
18 DR. RABINOVICH: Thank you.
19 DR. GRIFFITHS: But I think the point made 20 is very important that although we assume that some of
21 these things are innocuous, I think it is much better
22 if you can get rid of them to have them out of the 23 way, and I think this is the point really. Because
24 there are a lot of interactions going on and there is
25 a lot of new information coming through, which I am 58
1 sure will be discussed over the next few days. 2 DR. RABINOVICH: Thank you, Dr. Griffiths.
3 I would like to introduce our next speaker, who every
4 single presenter and panel chair at this conference
5 knows well because he has been contacted individually 6 by him. It is Dr. Andrew Lewis from the Center of
7 Biologics and Review at the FDA.
8 DR. LEWIS: Thank you, Regina. I think 9 pestering is a better word perhaps.
10 DR. RABINOVICH: Persistence, leadership.
11 DR. LEWIS: We have had a lot of
12 conversations with everybody. As you've heard from 13 Dr. Hayflick and Dr. Griffiths, until the end of the
14 1980's, the use of all types of neoplastic cells for
15 the production of biologicals was controversial. With 16 the development of the defined 100 picogram limits of
17 residual DNA, the development of the concept of viral
18 clearance, and the World Health Organization's
19 acceptance in 1987 of interferons and monoclonal 20 antibodies that were produced in tumor cells, the
21 issues regarding the use of neoplastic cells for the
22 production of purified biological products were 23 resolved. However, the use of neoplastic cells as
24 substrates for live virus vaccines continue to be
25 controversial. 59
1 A number of factors are motivating the 2 need to reconsider using neoplastic cells for vaccine
3 development, and I have listed these factors on the
4 next slide. These factors include the development of
5 the whole virus or traditional vaccines to HIV, 6 bioengineering approaches to viral attenuation to
7 vaccine development, the rapid development of vaccines
8 to emerging viruses such as the H5N1 and the H9N2 9 influenza viruses, significant progress in
10 understanding carcinogenesis and in detecting
11 adventitious agents, and finally the very successful
12 experience with the highly purified biologicals that 13 were derived from neoplastic cells.
14 Now conflicting with the need to use
15 neoplastic cells as vaccine substrates are the 16 regulatory concerns over the possible risks associated
17 with vaccine manufacturers in these cell types. I
18 think it is no secret that issues regarding vaccine
19 safety receive a very high level of public attention. 20 It is also no secret that public confidence in vaccine
21 safety is a critical component for the success of
22 immunization programs that are important to the public 23 health.
24 The scientific and regulatory challenge is
25 to develop the concepts and the technologies that can 60
1 be used to assess the risks perceived to be associated 2 with neoplastic cell substrates in a manner which
3 sustains public confidence in vaccine safety. To
4 achieve this goal, we at CBER believe that it is
5 essential that an approach be developed which can be 6 used to objectively and critically assess using state
7 of the art technology those issues associated with the
8 use of neoplastic cells as substrates for vaccine 9 manufacture.
10 The purpose of my talk tonight is to
11 outline an approach that CBER is proposing to
12 establish an objective and systematic means of 13 managing the regulatory concerns that are associated
14 with the development of vaccines in neoplastic cells.
15 To begin the discussion of this approach, I would like 16 to define exactly what we mean by the term neoplastic
17 cells.
18 For my talk, the term neoplastic cells
19 refers to immortalized cells that are derived from 20 either tumors or from transformation of instant tissue
21 culture, and these cells can either be tumorigenic or
22 non-tumorigenic when injected into animals. 23 The approach that CBER has followed to
24 consider neoplastic cells as possible vaccine
25 substrates is based on the approach worked out in the 61
1 1980's to evaluate the use of continuous cell lines to 2 manufacture biologicals. Now as shown in the next
3 slide, this approach consisted of five components.
4 These components included identifying the issues,
5 developing theoretical and experimental models to 6 evaluate each issue, validating the models for issue-
7 associated risk evaluation, developing criteria to
8 evaluate the levels of risk, and finally discussing 9 these issues and the approaches in public meetings,
10 which we are doing over the next several days.
11 The process of developing the CBER
12 approach began with organizing and presenting a 13 discussion of the use of neoplastic cells as vaccine
14 substrates before CBER's Vaccines and Related
15 Biological Products Advisory Committee in November of 16 1998. Following this meeting, CBER drafted a proposal
17 which evolved from the Advisory Committee
18 presentations. This proposal represents the first
19 attempt in CBER to formally address the regulatory 20 issues associated with the use of neoplastic cells as
21 substrates for vaccine development.
22 Perhaps I should also point out that the 23 draft proposal which has been distributed in your
24 meeting package, as Dr. Rabinovich mentioned in the
25 introductory remarks, is not -- and I will emphasis 62
1 the word not -- an official, approved FDA document 2 offering guidance on the use of neoplastic cell
3 substrates. The issues and concerns that were
4 presented to the Advisory Committee were developed in
5 greater detail in this CBER draft proposal, and these 6 issues and concerns were use to develop the agenda for
7 this meeting. And I am going to review them for you in
8 the next series of slides. 9 Now in this series of slides, I have
10 attempted to organize the issues in a common format.
11 The title of the slide represents the issue of
12 concern. The first bullet represents the cell 13 substrates that are involved. The second bullet
14 illustrates the issues that are responsible for the
15 concern. And the third bullet is a very brief summary 16 of data that documents why the concern or why the
17 issue is in fact a concern.
18 Now concern 1 represents tumor cell
19 contamination. With regard to the presence of tumor 20 cells as contaminants, at first glance this concern
21 appears to be trivial, as viable cell substrates are
22 almost always or always removed from viral vaccines. 23 However, if </pre></body></html>

 FAIR USE NOTICE  
  This site may contain copyrighted material the use of which has not always been specifically authorized by the copyright owner - or on which copyright has expired. We are making such material available in our efforts to advance understanding of environmental, political, human rights, health, economic, democracy, scientific, and social justice issues, etc. We believe this constitutes a 'fair use' of any such copyrighted material as provided for in section 107 of the US Copyright Law. In accordance with Title 17 U.S.C. Section 107, the material on this site is distributed without profit to those who have expressed a prior interest in receiving the included information for research and educational purposes. For more information go to: http://www.law.cornell.edu/uscode/17/107.shtml. If you wish to use copyrighted material from this site for purposes of your own that go beyond 'fair use', you must obtain permission from the copyright owner.