A BRIEF HISTORY OF CELL SUBSTRATES USED FOR THE PREPARATION OF HUMAN BIOLOGICALS

Leonard Hayflick, Ph.D.University of California, San Francisco

School of MedicineP.O. Box 89

The Sea Ranch, CA 95497Email:len@gene.com

Presented at the Workshop on

EVOLVING SCIENTIFIC AND REGULATORY PERSPECTIVES ON CELL SUBSTRATES FOR VACCINE DEVELOPMENT

Doubletree Hotel, Rockville, MD 20852

September 7-10, 1999

Short title: History of Vaccine Cell Substrates

Key Words: WI-38, human diploid cell strains, vaccines, cell aging, Cell substrates, viruses, cell lines, theft

Copyright 1999, Leonard Hayflick

INTRODUCTION

In this retrospective I will describe some of the events that led to the present regulations, principles and opinions that govern cell substrate usage for the preparation of human virus vaccines and other biologicals. I will also discuss the properties of several categories of cell lines with unique properties that make them superior candidates as vaccine cell substrates.

The failure to know history is evident in today's scientific literature where many papers report allegedly new observations or insights that were actually made years ago. One reason for the failure to understand the past is the current use of electronic data bases that begin in the 1970's and whose use results in the emasculation of papers published prior to that date.

The history of cell substrates used for the preparation of human biologicals reveals that much of what we were so deeply concerned about in the past is, today, considered to be either obvious or trivial. It is certain that what passionately concerns us today will, in the future, also be thought of as either obvious or trivial.

We are all a part of history and should appreciate that we cannot recall with certain knowledge all that happened in the past and why. I don't believe that history can be written without bias and what I will recount for you here will be no exception. An historian is as much a victim of his data as is a scientist. And, I suspect we all have been victimized by our data to the extent that we can sympathize with the plight of the historian. With that caveat squarely in place I shall proceed in my duel role as both historian and scientist. How, then, have today's cell

substrates become recognized as safe for the production of human biologicals?

RECOGNITION THAT THERE IS A CLASS OF NORMAL CELL POPULATIONS

Although the first in vitro cell culture was set in 1907 and is generally attributed to Ross Harrison [1], it was not until 1949 that Enders, Weller and Robbins [2] launched the modern field of vaccinology by showing that, contrary to the dogma at that time, the poliomyelitis virus could replicate in cultured non-neural cells. Prior to this finding, it was believed that the poliomyelitis virus could only replicate in neural cells and because these cells are incapable of replication in vitro, the development of a vaccine produced in cell culture would be difficult if not impossible.

Primary monkey kidney cells were chosen in the late 1950’s for the preparation of the first poliomyelitis vaccine because (1) much was known about the monkeys that were then used for research on that disease, (2) the poliomyelitis virus only replicates in primate cells and (3) the kidney is a large discrete organ that is easily accessible. Until the early 1960's the only cultured cells licensed for use in vaccine preparation were primary monkey kidney cells. Primary cells are defined as cells obtained by tissue disaggregation and used without subcultivation. This is not only the correct biological term but it is also a regulatory authority legal definition. Contrary to what is found in much of the modern cell biology literature “primary cell lines” or "primary cell strains” are non sequiturs, - a matter that will be discussed later.

After a few years of use, primary monkey kidney cells were found to contain many unwanted or indigenous viruses, one of which (the B virus) was lethal for humans. Although our research in the period 1958-1961 was not directed toward developing new virus vaccine cell substrates, our research findings serendipitously impacted on that field [3].

In 1961 we described a new class of normal human diploid cells and suggested that they may be useful for the preparation of human virus vaccines [3]. We reported that these strains have several interesting properties:

(1) If derived from human embryos, these normal fibroblast cells undergo about 50 population doublings. Contrary to the belief that this is a serious limitation for practical use, the potential cell yield from 50 population doublings is about 20 million metric tons [3].

(2) We reported that human diploid cells undergo a number of population doublings inversely proportional to donor age. This suggested to us that the finite replicative capacity of cultured normal cells is an expression of aging at the cell level. This notion received considerable experimental support in subsequent years and is now the basis for the field variously called cell aging, cell senescence or cytogerontology [4-9]. In subsequent years we provided evidence for the existence of a molecular mechanism that counts cell replications and also determined its’ nuclear location [4,7,10,11]. Our finding that normal cells have a limited lifespan governed by a replicometer is the basis for the remarkable discoveries made in the last decade that have revealed telomere attrition to be the molecular explanation for this finding and that the expression of telomerase is necessary for the immortalization of normal cells [4,7,12-16].

(3) We found that, if derived from normal tissue, cell strains have the diploid karyotype and are incapable of replication in suspension culture [3,10]. This property later was called anchorage dependence.

(4) We also reported that human cell strains do not produce tumors when inoculated into the hamster cheek pouch or even when we directly introduced them into terminal human cancer patients [3].

(5) We found that human diploid cell strains can be cryogenically preserved. When, for example WI-38, the first highly characterized and widely distributed cell strain that we developed in 1962, is preserved at a particular doubling level and then reconstituted, the number of doublings remaining is equivalent to 50 minus the number of doublings spent prior to preservation [3,10]. These cells have an extraordinary memory and "remember" at what doubling level they were preserved even after 37 years of storage in liquid nitrogen [17]. WI-38 has been cryogenically stored longer than any other normal human or animal cell population.

(6) We also reported that human diploid cell strains have the broadest human virus spectrum of any cell population known [3]. Even heretofore unknown viruses were discovered using these cells [18].

CLASS DISTINCTIONS

As a result of this characterization we suggested in 1961 that all cultured animal and human cells be classified into three groups [3]:

Primary cell cultures

Cell populations derived from animal tissue and that have undergone no subcultivations.

Cell strains

Cell populations that have a finite capacity to replicate, do not produce tumors when inoculated into experimental animals, have the karyology of the tissue of origin and are anchorage dependent.

Cell lines

Cell populations consisting of immortal cells, may produce tumors when inoculated into laboratory animals, do not have the karyology of the tissue of origin and are usually anchorage independent.

Our observation that cell strains are mortal overthrew a dogma that had prevailed in the cell culture field since the turn of the century. It had been believed that all cultured cells have the capacity for unlimited replication. The fact that this was never observed was thought to be attributable to the failure to understand what in vitro conditions were necessary to reveal the innate ability for all cells to reveal their alleged capacity for unlimited replication.

The distinguishing characteristics that define these three classes of cells suggested to us that cell population immortality was a fundamental criterion for distinguishing normal from cancer cells [3]. This was the basis for the distinction that we made between the phenomena of aging, which occurs in normal cells, and cancer that is usually a property of immortal cells [3]. The field of research on cell immortalization then emerged after we described this fundamental distinction.

WHAT'S IN A NAME?

Our definitions were not universally accepted nor has anyone succeeded in bringing order out of the past and present terminological chaos. Because of this, today the scientific literature in this field is virtually unintelligible. I will use the terms "cell line" and "cell strain" as I have defined them above in an effort to avoid the chaos and because there is historical precedent for doing so [1,19].

I will give one illustration of the seriousness of this problem by asking: What is a Chinese hamster ovary (CHO) cell culture?

There are at least three possible answers. A CHO cell could be a primary culture, a cell strain or a cell line as defined above. The term "CHO cell" is used in the literature to mean any one of these three fundamentally different cell populations. This results in enormous opportunities for misunderstandings and confusion. But, the matter of clarity of expression doesn't end here.

We suggested in 1961 that the phenomenon by which a cell strain acquires the properties of a cell line be called an "alteration" [3]. We suggested this because the term transformation had a specific meaning in microbiology and we believed that the term should be reserved in the event that transformation, similar to pneumococcal transformation, would some day be discovered to occur in eukaryotic cells. Our suggestion was ignored and, as predicted, transformation similar to pneumococcal transformation ultimately was described. Thus, a new term had to be invented for a phenomenon that already had a recognized name in microbiology. Now, what is called transformation in bacteriology is called transfection in cell biology.

Today, even the word transformation is rarely used as originally defined. And, that original meaning is vital because it defines one of the most important areas in all of cell biology, - the acquisition by a population of normal cells of the properties of abnormal or cancer cells. This is the basis for a substantial part of the worlds’ biological research yet there is no agreement on terminology or even on the proper classes of cells to be used in order to study the phenomenon.

The word transformation is so abused that the only way possible to know what it means is to ask its user who often defines it vaguely. If you substitute the word "interesting" for the word transformation in the worlds' scientific literature today, nothing substantive would be lost.

Two timely examples of terminological chaos and the misinformation that becomes perpetuated can be found in the very first paragraph of the document prepared for the participants in this workshop and entitled, “A Defined Risks Approach to the Regulatory Assessment of the Use of Neoplastic Cells as Substrates for Viral Vaccine Manufacture” and in the summary of this workshop published in Science [20]. Reference is made in both places to a 1954 U.S. Armed Forces Epidemiological Board recommendation that “normal cells rather than cell lines established from human tumors be used for the development of adenovirus vaccines.”

By “normal cells” the board meant spontaneously derived immortal cell lines derived from normal tissue as I have defined cell lines above. The cell lines referred to by the Board were not normal by any criteria. What the board meant were allegedly spontaneously transformed cell lines derived from normal tissue. All of these cultures were found to be contaminated with the HeLa cell line (derived from a human cervical carcinoma) several years later. Indeed, the correct normal human diploid number was not known in 1954 and a normal human cell population was not described until we did so, seven years later, in 1961 [3].

GENESIS

The first cell line used for the preparation of a human virus vaccine was made in the 1950's for the preparation of an experimental adenovirus vaccine. It was not until the early 1980's that the first cell lines became licensed for use in the preparation of human biologicals. The CHO cell line was used for the manufacture of tissue plasminogen activator in the United States and the Vero cell line for an inactivated poliomyelitis vaccine in France [21].

As stated above, the U.S. Armed Forces Epidemiological Board ruled in 1954 against the use of HeLa cells for the production of an adenovirus vaccine for use in the military. They would, however, accept "normal" cells for that purpose [22,23]. The Board meant abnormal, immortal cell lines

spontaneously derived from normal cells. In 1959 Dr. Maurice Hilleman, believing that he was using an authorized "normal" human cell line called Henle intestine (a cell line thought to have been derived from the spontaneous transformation of normal human intestine cells), prepared a live trivalent adenovirus vaccine in these cells and administered it to 6 adult volunteers.

No untoward effects attributable to the cell substrate have been seen in these vaccinees after 25 years of follow up. It later developed that the Henle intestine cell line used was, in fact, HeLa.

A second, somewhat similar incident, occurred when a cell population also later shown to be HeLa was used for the preparation of an inactivated poliovaccine administered to 144 people in 1959 [24]. There has been no published follow up report.

ADVANTAGES OF HUMAN DIPLOID CELL STRAINS (HDCS) OVER PRIMARY CELLS

FOR VACCINE PREPARATION

Prior to our work, the only cell culture substrate licensed for use in the production of human biologicals was primary monkey kidney cells.

In 1962 in order to demonstrate the safety and efficacy of a vaccine produced in a normal human diploid cell strain, we prepared the first vaccine produced in a passaged cell population using normal human diploid cells. This poliomyelitis vaccine was shown to be both safe and efficacious [25]. We suggested that HDCS have definite advantages over primary cells for vaccine production [3,25-28].

1. Latent Viruses

Primary adult monkey kidney cells, then used for poliomyelitis vaccine production, harbored about 20 serologically distinct latent viruses, several of which were lethal for humans. The B virus and the Marburg agent both killed 23 people after accidental percutaneous inoculation of handlers of monkeys and their primary kidney cultures [26].

S.V.40, known to produce tumors in rodents, and with unknown potential to do so in humans, was also an early contaminant of primary monkey kidney cells [26]. S.V. 40, as was later learned, was capable of transforming normal human cell strains into cell lines in vitro. S.V. 40 became the first virus shown to be capable of transforming a cultured normal human cell into a cancer cell [29]. It should not be difficult to appreciate the level of concern engendered when this virus was found to have been inadvertently administered to several million recipients as a contaminant of early lots of inactivated and attenuated poliomyelitis vaccines [26].

Latent viruses have never been found in HDCS intended for the manufacture of human virus vaccines. To this day no evidence exists for an endogenous human oncogenic agent, latent virus, transforming principle, slow virus or endogenous retrovirus in any HDCS derived human virus vaccine. Although it is theoretically possible that entities like these might be found, a contaminated HDCS strain would be discovered and discarded well before use, unlike a primary culture.

2. Cost of cell procurement

A continuous supply of primary tissue is far more costly than is the use of one highly characterized, cryogenically stored, HDCS for the production of a human biological.

3. Available cells

For primary cultures the number of cells available from a single tissue source is small for the production of one vaccine lot but is virtually unlimited for a HDCS. Twenty million metric tons of cells are the potential yield from a single HDCS with a 50 population doubling potential [3]. Successive vaccine lot production in cells derived from a single tissue source is practical with a HDCS but is not possible with primary cultures. Thus, the belief that a HDCS, like WI-38 with a 50 population doubling potential, is a less practical substrate than an immortal cell line is spurious. Hundreds of millions of vaccine doses have been produced worldwide in WI-38 since 1962 and there is sufficient remaining in my repository and those of others to last for several more decades. A summary of the proceedings of this workshop published in Science in which it is written that it is "more efficient" to use immortal cells continues to perpetuate the myth of the unavailability of adequate amounts of a HDCS [20]. After 37 years of distribution I am still prepared to supply anyone with WI-38 at very early population doubling levels.

4. Cell storage

Cryogenic storage of primary cells is less efficient as their recovery rates are inferior to those of HDCS.

5. Tumorigenicity in man

Several HDCS were tested for tumorigenicity directly in human volunteers [3,25], and then used for the production of vaccines [25]. One HDCS so tested could provide virtually unlimited quantities of several vaccines thus minimizing the frequency of such studies. This is impractical or potentially dangerous to do with primary animal cells. The inoculation of living cells into human volunteers was commonplace in the 1950's and 1960's.

6. Endangered species

The continued capture of wild monkeys and the losses sustained during their transportation, quarantine, and subsequent use, presented a clear threat to their existence as a species.

7. Standardization

The essential point that makes a diploid cell strain more attractive than a primary cell population is the ability to thoroughly test a diploid cell strain before use. In a word the advantage is standardization. Once achieved, cryogenic storage permits the acceptable strain to be held indefinitely for subsequent use. It is not possible, practical, or economic for primary cells to be similarly handled. This argument holds equally well today for cell lines.

For the first time the concepts of cell characterization and standardization became introduced into the field of cell substrate usage.

EARLY ATTITUDES TOWARD THE USE OF HDCS FOR HUMAN VIRUS VACCINE MANUFACTURE

Prior to our suggestion that HDCS be used for vaccine manufacture [3], only two classes of cultured cells were recognized and, therefore, were the only candidates for substrates to be used for the manufacture of human biologicals. These were primary cell populations and cell lines.

Cell lines were generally believed to be a forbidden candidate for the production of human biologicals because they shared properties with cancer cells.

One of the earliest published statements made opposing the use of cell lines was that of Westwood et al. in 1957 [30]:

"It is the fear of malignancy more than any other single factor which precludes the cell lines at present available from use in the production of virus vaccines. The risk lies in the possibility of inducing malignant changes in the cells of the human subject by the introduction of an as yet hypothetical virus or non-living transforming principle analogous to that inducing change of type in the pneumococcus."

In 1962 several NIH scientists met to address the issue of cell substrates for the production of human virus vaccines [31]. This meeting, which was closed to outsiders, published several conclusions that ignored our work and we published a refutation [32]. For example, the NIH group embraced the dogma, disproved by us, that "...continuously cultured cells eventually develop characteristics suggestive of malignant change" [31]. In respect to our suggestion that HDCS be used for vaccine production they argued that this was dangerous because "...the resultant cell populations are heterogeneous, which means that they are not precisely characterized genetically or otherwise and may be subject to random fluctuations in properties"[31].

The NIH group ignored the fact that the same criticisms apply to primary cells and they further ignored the extensive published karyological analyses performed on HDCS and their inoculation into humans that demonstrated their genetic normalcy unequivocally [3,25]. Although this committee was unable to distinguish between cell lines and cell strains they were, in fact, the first group to recommend the use of cell lines for the production of human virus vaccines [15].

The belief that cell lines shared properties with cancer cells was widespread and no alternative to primary cultures was available until our report in 1961 [3].

Our suggestion that human diploid cells could provide a safe and superior substrate for the production of human virus vaccines was met with enormous resistance for the next decade. The major reasons for this resistance was the belief that hypothetical viruses might be present or that spontaneous transformation might occur resulting in the presence of human cancer cells. Yet, no one has ever found a latent virus in any HDCS that might be a candidate for vaccine production although that possibility can never be entirely eliminated. The argument that there may exist an unknown virus for which no test exists is tenable for any cell population. Yet, latent viruses were commonly found in primary monkey kidney cells but never found in WI-38.

Latent viruses were such a problem with primary monkey kidney cells that a worldwide moratorium on the licensing of all poliovirus vaccines was called in 1967 because of deaths and illnesses that occurred in monkey kidney workers in vaccine manufacturing facilities in Germany and Yugoslavia. The Marburg virus was the etiological agent.

The theoretical possibility that human cells might harbor a human cancer virus gained further support because of the belief that if human cancer viruses existed, they would probably be specific for humans, consequently the use of human cells would increase the risk of spread. Yet, at that time, the only known mammalian oncogenic viruses were DNA viruses and all of these were oncogenic, not in the indigenous species, but in foreign species (e.g. S.V.40). Thus, the use of human cells for the production of human biologicals would be safer than the cells of other species because, as we pointed out, all the then known oncogenic viruses were oncogenic only in species in which the virus was never found in nature.

It was also suggested that even if HDCS were demonstrably normal the possibility remained that a small number of cells could subsequently transform and become part of the pool of cells used for production. Although this is an extremely rare event, there are sufficient safeguards in the testing procedures to recognize the occurrence well before vaccine release. The same criticism could be made of primary cells but this was ignored and no

determination of karyological normalcy or tests to detect transformation in primary monkey kidney cells are required even to this day! Both are still required for HDCS.

THE PRIMARY PROBLEM

It is a curious footnote to this history that no quantitative definition for a primary cell culture exists. Legal primary cultures, sparsely inoculated with cells, will undergo many population doublings before confluency is reached and the culture used for vaccine production. Indeed, the prudent manufacturer will encourage this because more primary cultures can be produced in this way from a given amount of expensive primary monkey kidney tissue. In so doing the original cell inoculum will still be legal because no subcultivations have been made but the population will have undergone many population doublings before use as a vaccine substrate. In fact, the number of doublings that a legal primary cell culture can undergo may be equivalent to, or even greater than, the number of population doublings that a HDCS like WI-38 would undergo at the time that it is used for vaccine production! Cell strains, primary cell cultures and cell lines are all multigeneration populations [33].

ENTER THE DEVIL

The arguments used against HDCS for the manufacture of human virus vaccines supported the belief by the Division of Biologics Standards (the precursor of CYBER) that “The devil you know is better than the devil you don't know." Yet, every study that was ever conducted showed that HDCS were safer for the production of human biologicals than were the then used primary monkey kidney cells. Even the introduction of live S.V. 40 virus into millions of vaccinees and the deaths and illnesses that occurred in monkey kidney workers from B virus and the Marburg agent were not persuasive.

In 1961, we prepared the first biological produced in a HDCS and showed that it was safe and efficacious [25]. This was a trivalent oral poliomyelitis vaccine. By 1963, 7000 people received poliovirus vaccine produced in WI-38 with no untoward effects attributable to the cell substrate [34,35].

EFFORTS TO EDUCATE THE SCIENTIFIC COMMUNITY ON THE ADVANTAGES OF USING HDCS FOR THE PRODUCTION OF HUMAN BIOLOGICALS

In 1963 it became clear to advocates of the use of human diploid cell strains as substrates for human vaccines that a program of developmental research and education should be mounted in order to establish minimum requirements for their use and mount an effort to overcome the resistance to their use.

Under the sponsorship of the Permanent Section on

Microbiological Standardization of the International Association of Biological Societies, the Cell Culture Committee (CCC), was born. In the next eight years this committee was to play a significant role in the subsequent worldwide licensing of human biologicals produced in WI-38 and similar strains. The CCC sponsored several important conferences where research results using HDCS were presented [36].

ORIGIN OF THE MASTER AND WORKING CELL BANK CONCEPT

The concept of the well-characterized and standardized master cell bank, working cell bank, and the first "Minimum Requirements" emerged from a small CCC sub-committee meeting consisting of Dr. Frank Perkins, Dr. Drago Ikic, Dr. Hilary Koprowski and myself at the landmark Opatija, Yugoslavia Conference in 1963 [37].

It was at this conference that I suggested the concept of the two tier system of master and working cell banks for WI-38 although those terms were not used. The terms used were master and working cell seeds. This reasoning, and the terms used, was based on the identical terms and ways in which virus seeds were then utilized. That is, from an approved original virus seed, an aliquoted master pool was prepared by a manufacturer from which was derived the working virus seed stock. The same principles of virus characterization, permitted rounds of virus replication, and other concepts were lifted directly from this procedure and modified for use with WI-38 and it's precursor WI-26. The original publication of these procedures was made in the 1963 Opatija Conference Proceedings [38].

In the 1980's when heteroploid, immortal, cell lines like CHO were used for the manufacture of human biologicals our procedures for characterizing and banking WI-38 were lifted over for use with these cell lines with few changes and they are used to this day.

POWDERED MEDIA

In the mid 1960's several WI-38 users outside of the United States expressed difficulties growing the cells that were traceable to variations in the medium reagents being used by various laboratories. As a consequence of this we developed a practical standardized cell culture powdered media [39] in kilogram lots that, unlike wet media, was easily transported worldwide, used by many laboratories simultaneously, and for which only two variables existed, - water and serum. I recall transporting powdered media equivalent to hundreds of liters in my brief case to the former Soviet Union, Czechoslovakia and Yugoslavia. As a result of our development of powdered media the WI-38 growth problem was quickly resolved and the use of powdered media has now become commonplace throughout the world.

THE BANDWAGON STRIKES UP

In 1967, through the efforts of Dr. Drago Ikic, Director of the Institute of Immunology in Zagreb, Yugoslavia (now Croatia) the world's first attenuated poliomyelitis vaccine produced in WI-38 was licensed for use by the Yugoslavian control authorities.

By 1968 over one million people world-wide had received vaccines produced in WI-38 both orally and parenterally against such diseases as poliomyelitis, adenovirus types 4 and 7, measles, rabies, vaccinia, varicella, and arboviruses [33]. All of these vaccines were reported to be safe and efficacious.

In 1970 an attenuated polio vaccine manufactured by Pfizer Laboratories, Ltd. in WI-38 was licensed in the United Kingdom. A similar vaccine, produced by Merieux in France was licensed in 1971 and another, produced in the USSR, was also licensed.

In 1972 Pfizer Laboratories was licensed to sell their WI-38 produced poliomyelitis vaccine in the United States exactly ten years after our original proposal was made.

At about this time the U.S. control authority, now convinced that HDCS had some merit as substrates for the manufacture of human biologicals, did an about face and let several contracts to develop diploid cell strains from rabbits and monkeys. This was done in an effort to directly compete with our work. The NIH was only partially successful in developing a monkey cell strain that was rarely used.

After I produced the first widely distributed normal human diploid cell strain WI-38 in 1962, others followed our published procedures and produced their own strains. New HDCS can be produced at will and, as a matter of national pride, many countries have made their own strains. For example, in 1970 the Medical Research Council in London produced a HDCS called MRC-5 [41].

Other examples include TIG-1 produced in Japan and 2 BS produced in China [42].

Today there are many licensed virus vaccines produced in WI-38 or similar strains (TABLE 1).

INSERT TABLE 1 HERE

WI-38 and MRC-5 derived human virus vaccines now have a 25 year record of safety after extensive and ongoing pharmacovigilance. The products from HDCS have been administered to more humans than have the products from any other cell substrate. No unwanted side effects have ever been found to be traceable to a HDCS substrate.

Dr. David McGrath, director of the Virology Division of the World Health Organization wrote in 1991, that HDCS are “without any evidence of any adverse reaction attributable to the substrate, or any report of the detection of a virus contaminant, either infectious, latent or oncogenic." McGrath further said "..the two well characterized cell lines (sic) WI-38 and MRC-5 may perhaps be regarded as truly safe substrates" (44).

HOW THEFT BECAME NATIONAL POLICY

In 1975 an incident involving WI-38 occurred that was to have a profound effect on biologists rights to their intellectual property and on the establishment of the biotechnology industry. That incident involved the ownership of WI-38 specifically, but generically it involved title to any self-reproducing system.

When we first described the human diploid cells in 1961 efforts were made by the Wistar Institute to patent them. Other than roses and fruit trees, the U.S. patent laws at that time had no provision for patenting living things.

In 1962, because of the enormous demand for WI-38 starter cultures mainly from NIH grantees, the NIH provided us with a contract to distribute the cells. Many original ampoules were given free to commercial cell culture manufacturers, pharmaceutical companies, and repositories in Europe, and in the U.S.S.R. In the decade 1962-1972, WI-38 starter cultures were sold by many cell culture manufacturers and grossed, conservatively, well over 25 million dollars. No value was placed on the additional hundreds of thousands of WI-38 cultures used by vaccine manufacturers or cultures sold by third and fourth parties during that decade. Today, the sales by manufacturers of WI-38 itself or of products produced in it have yielded billions of dollars in revenue. Other than reimbursement for the costs of distribution, no funds ever reverted to me, the U.S. government or to the Wistar Institute where I developed WI-38.

The attitude during the 1960's was that academic biologists had no intellectual property rights nor should title be vested in them or their institutions for their development of new or unique life forms. Commercial organizations in the US and elsewhere in the world could, and did, freely obtain WI-38 starter cultures from me and sold or used their progeny for enormous profits. Yet, the academic institutions, the U.S. government, and even the scientists who actually developed WI-38 were forbidden from benefiting. I felt that there was incredible irony and injustice in this. I took the position that a biologist, like physicists, chemists and software developers do have intellectual property rights even though their work might be supported directly, indirectly or partly with federal funds.

The NIH took the position that I had no rights. In order to resolve this question I asked the NIH to send a lawyer to discuss the matter. Instead, they sent an accountant who, when presented with this unique legal problem and with the biology of self-replication, concluded that I had stolen government property and sold it for personal gain. In 1976 I challenged this absurdity by

filing a lawsuit against the Department of Health, Education and Welfare, the NIH and the FDA in which I claimed title to WI-38. During the time that this litigation was underway two crucial events occurred.

First, in 1978, the patent laws dramatically changed in the United States. The Supreme Court ruled that microorganisms and cell populations that were then becoming the basis for the emerging biotechnology industry could be patented. Biologists did, after all, have intellectual property rights. Second, in the 1970's and later, cell and microbial cultures developed with full or partial federal support were simply taken by researchers from an academic institution to form a new biotechnology company. There was tacit acceptance of this practice by the biologists themselves and by the NIH.

To do exactly that two years earlier in 1976, however, would be to have had yourself characterized by the NIH and FDA as having engaged in the theft of government property as they had accused me [45].

Of the many ironies that occurred during this saga none is more amusing than after a decade of futile efforts to have WI-38 accepted by the United States Control Authorities (the Division of Biologics Standardization and its successor the FDA), those same public servants eventually wanted WI-38 so desperately that they gained access to my laboratory in my absence and confiscated all of my frozen ampoules of WI-38! They then characterized WI-38 as "A national resource."

I am pleased to say and that after six years of litigation, the United States Justice Department, the FDA and the NIH, realizing that they held a losing position came to me in 1981 with an offer for an out-of-court settlement. The settlement provided that most of the confiscated ampoules of WI-38 would be returned to me and that all funds realized from the distribution of WI-38 after my contract ended belonged to me with interest. The Justice Department, the NIH and the FDA also agreed that WI-38 could be sold by anyone including its inventor, and finally, that title to WI-38 was not vested exclusively with the government as they had insisted for ten years

[46,47]. Accordingly, all copies of the accountants false accusations would hence forth be accompanied by a copy of the out of court settlement. The funds returned to me in this settlement, plus much more, were all consumed in paying the costs of the litigation.

Many believe that had we not prevailed, the present accepted practice of vesting intellectual property rights in the inventor of new life forms, developed in whole or in part with federal funds, would be quite different. Title to hybridoma populations, unique cell populations, animals, plasmids, gene products and life forms expressing new recombinant DNA properties is now vested in the institutions and/or the scientists who developed them regardless of federal support.

Several attorneys representing scientists and new biotechnology companies who claimed title to self-duplicating systems in the late 1970's intended to provide amicus briefs in our support if our case had ever gone to trial. If we had lost our suit, it is probable that the assignment of patent and intellectual property rights for new life forms would be much different than it is today. As a result of our lawsuit, most of the biological community has now completely reversed its' earlier belief that anything discovered in their NIH supported laboratories belonged exclusively to the government. That position, if held today, would be considered to be absurd, but it was widely held by most biologists in the 1960's and 1970's.

Our position received its greatest support from President Reagan when he told federal agency heads in 1983 "..all businesses should be able to retain patent rights on inventions made in the course of government funded Research and Development work.. The administration takes the view that giving the private sector clear title to patents on inventions developed under federal contracts and grants will lead to more rapid commercialization of new products and processes and combat the slump in US productivity and competitiveness [48]."

Today, many scientists and their institutions legitimately profit from the sale of self-duplicating materials that they have developed with or without federal support. I applaud this. The attitude of biologists the NIH and the FDA today is exactly the reverse of what I experienced in 1976(38).

Today, if you do not hold a patent on a cell population, plasmid, gene sequence, animal or microorganism or you are not involved with a company that exploits such materials you are a failure in cell biology or microbiology. For a more detailed discussion of these events see [46,47,50-52].

In Marcus Tullius Cicero's orations against Cataline he put it best when he said, "O tempora! O mores!" For those of you who have forgotten your Latin, - a loose translation would be, - Oh, how much our mores change with time!

FROM CELL STRAINS TO CELL LINES

After the use of human diploid cell strains for the production of human biologicals became commonplace in the 1970's, the final step in the evolution of cell substrate use occurred in the mid-1980's. That is, the sanctioning of cell lines for use as substrates for the production of human biologicals. This became necessary much sooner than many of us had expected because most of the important products emanating from biotechnology laboratories can only be made in mammalian cell lines.

The chief objections to using cell lines as a substrate for production of human biologicals were the same objections made by those who first opposed the use of human diploid cell strains. That is, the possibility that cell line cells may contain a latent virus or transforming agent. In addition to this, all cell lines, by definition, are karyologically abnormal and some produce tumors or are malignant when inoculated into an immunologically privileged site or into immunosuppressed laboratory animals.

Nevertheless, these objections were overcome by utilizing cell lines that, although neoplastic, are not malignant when tested in animals and by validating the manufacturing process such that the likelihood of biologically active DNA sequences or unwanted proteins surviving from the cell substrate is not possible.

It is astonishing to observe that after ten years of debate on the risks of moving from vaccines produced in dangerous primary monkey kidney cells to their production in human diploid cell strains (which unlike cell lines are absolutely normal), the time required to make the final leap from normal cell strains to abnormal cell lines occurred in the early 1980's literally in a matter of months, if not weeks. Why? I doubt that this occurred because we were less safety conscious, or that we were weary of debating. I would prefer to believe that most of us then had read Santayana, - could remember the past, - and were determined not to repeat it.

PATHS TO THE FUTURE

There are several ways in which most of the real and theoretical problems associated with the use of cell lines for the production of human virus vaccines can be resolved. Regrettably, few of these possibilities were adequately addressed at this workshop. In the following paragraphs I will briefly mention some of these approaches.

Downstream Processing

A striking omission from this workshop is the absence of a session on the science of downstream processing. The recovery of purified biological products from complex bulk starting material has, in the past twenty years, reached such levels of sophistication that one must consider the application of these technologies to achieve the isolation of pure vaccines products of

any description and without contamination with any unwanted protein, DNA, or RNA moiety.

For example, the utilization of various affinity technologies could resolve many of the concerns expressed at this conference with putative indigenous viral contamination from cell substrates. The downstream processing technologies that either alone, or in combination, that have the potential to produce a pure vaccine products include such techniques as antibody affinity columns, protein chromatofocusing, aqueous two phase partitioning systems, affinity ligand engineering, high performance tangential flow filtration, isoelectric focusing, fluidized bed adsorbtion, microwave technology for virus inactivation, continuous annular chromatography, affinity chromatography, size exclusion chromatography, chromatographic ion exchange gels, gel permeation chromatography, expanded bed chromatography, electrochemically modulated ion exchange chromatography, utilization of protein specific ligand affinity adsorbtion, affinity chromatography, expanded bed and packed bed adsorbtion, linear gradient elution, etc.

The evolution of modern downstream processing technology for manufacture of human biologicals and the means for validating the presence of only the pure desired product has evolved to the point where any cell substrate now can be considered to be safe.

Vero Cells as a Precedent for the Safety of a Cell Line Vaccine Substrate

The Vero cell line was established from the kidney of an African Green Monkey (Cercopithecus aethiops) in 1962 by Y. Yasamura and Y. Kawakita at the Chiba University in Japan [53]. This cell line has been used for many years for the production of licensed vaccines that include oral attenuated poliovaccine (OPV), inoculated killed poliovaccine (IPV) and rabies vaccine. The control authorities of France, Belgium, and Thailand license these vaccines. More than 350,000 persons have been inoculated with the rabies vaccine with "... a very low level of clinical reactions..." [21].

More than 20 million doses of IPV have been administered to children without side effects [21].

The properties of the Vero cell line have been thoroughly studied and have met the criteria of several control authorities for use as a substrate for the preparation of pediatric vaccines [21,54]. Healthy children represent the most vulnerable group of vaccinees for the administration of an inoculated biological yet several control authorities have sanctioned the use of the Vero cell line for this purpose. One can only conclude that reluctance to use Vero or any other cell line with similar properties for the preparation of human vaccines is indefensible.

Immortal Normal Human and Animal Cells

The discovery of telomerase as an immortalizing enzyme and proof that transfection with hTERT (human telomerase reverse transcriptase) can immortalize normal human cells with full retention of their normal properties has now opened the way for the immortalization of any replicating normal cell for use as a vaccine cell substrate [55]. By employing the strategy of producing a well characterized master and working cell bank with hTERT immortalized normal cells, it is now possible to circumvent virtually all of the concerns that have been expressed about the use of abnormal, immortal cell lines for the preparation of human vaccines.

Normal Human Cells Immortalized with Chemical Carcinogens or Ionizing Radiation

A second category of cell populations that merits consideration as a substrate for human virus vaccine preparation are cell lines that have been immortalized by exposure of normal human cells to other than viruses or parts of a virus genome. In 1978 Namba et al produced an immortal cell line by exposing a normal human fetal diploid cell strain to multiple exposures of 4NQO [56]. In 1981 we produced the immortal cell line SUSM-1 by exposing normal human fetal fibroblasts to multiple treatments of gamma radiation from a Co 60 source [57]. Kakunaga described the transformation of a normal

adult human diploid cell strain to an immortal cell line using 4NQO and MNNG [58]. Normal human endometrial cells have also been transformed to a cell line with MNNG [59]. There are other examples [60,61]. These cell populations have many of the desirable properties of both cell strains and cell lines in respect to characteristics that favor their use for human virus vaccine production.

These cell lines are immortal, can grow in suspension culture, have reduced serum requirements and can be produced from normal human cells previously determined to be free of contaminating viruses including retroviruses. Although they are heteroploid there is no reason to believe that this property would be undesirable especially because heteroploid cells are presently used to produce many licensed human biologicals and heteroploid Vero cells have been used to produce human virus vaccines that have proven to be safe and efficacious in millions of vaccinees.

SLAYING THE LAST DRAGON

There are now several opportunities to produce safe and efficacious vaccines in cell lines. The use of the Vero cell line for the production of several vaccines, including IPV safely inoculated into more than 20 million children, has set the precedent. Nevertheless, there are other cell lines derived from normal human cells that offer other possibilities. These include cell lines produced from well characterized normal human cells of almost any differentiated type and derived either by transfection with hTERT or treatment with chemical carcinogens or gamma radiation. It is clear that we have arrived at a time when the use of non-viral methods for immortalizing well characterized normal human cells in combination with sophisticated downstream processing provides opportunities for the use of cell lines for the production of safe vaccines and biologicals.

The dragon that for ten years prevented the United States from moving from uncharacterized primary monkey kidney cells to licensed vaccines produced in a well characterized normal human diploid cell strain was finally slain in

1972. Ten years later, in 1982, the second American dragon that stood in the way of licensing a biological produced in a cell line, other than a vaccine, was also slain when a license was issued for tissue plasminogen activator produced in a CHO cell line. The third dragon species was slain in Europe in the 1980's when Vero cells were used for licensed pediatric vaccines. Yet, members of this third species of dragons still roam the American countryside. It is time to annihilate this third American dragon that has been preventing us from using cell lines for the production of virus vaccines.

Curiously, all three dragon species were first eradicated in foreign countries but members of the third dragon species are still at large in the world's greatest super power.

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TABLE 1

ADMINISTRATION OF HUMAN VIRUS VACCINES PREPARED ON WI-38 OR MRC-5 ________________________________________________________________________

VACCINE MANUFACTURER DOSES

ADMINISISTERED

LIVE:

Adenovirus Wyeth Millions

Measles Institute of Immunology Millions

Zagreb Croatia

Mumps Swiss Serum and Vaccine Millions

Institute

Oral Poliovirus Pfizer England Millions

(OPV)

Rubella Merck >100 million

Pasteur Mérieux Connaught

Smith Kline Beecham

Swiss Serum and Vaccine

Institute

Varicella Merck >2 million

Pasteur Mérieux Connaught (1995-1996)

Smith Kline Beecham

Inactivated:

Hepatitis A Merck Millions

Pasteur Mérieux Connaught

Smith Kline Beecham

Poliovirus (IPV) Pasteur Mérieux Connaught 6.3 million

(1991-1995)

Rabies Pasteur Mérieux Connaught 7.2 million

(1985-1994)

__________________________________________________________________________

Data adapted from M. A. Fletcher L. Hessel and S. A. Plotkin.[43]

October 20, 1999

Ms. Alison Roth

IQ Solutions Inc.

11300 Rockville Pike

Suite 801

Rockville, MD 20852

Dear Ms. Roth,

Enclosed is the hard copy and disk of my paper presented at the workshop on Evolving Scientific and Regulatory Perspectives on Cell Substrates for Vaccine Development, Doubletree Hotel, Rockville, MD 20852, September 7-10, 1999.

Please note that I wish to have an opportunity to see galleys of this paper prior to publication and, most importantly, the galleys must be accompanied by the original of this paper, or a copy of this paper, with all of the printer's markings and text changes indicated.

This makes proof reading much easier.

Sincerely yours,

Leonard Hayflick, Ph. D.

Professor

The reference 49 is correct as it appears in your Fax to me with the exception of the large space between "Advancement" and "of."

The following are corrected responses to questions appearing after paper entitled:

"A BRIEF HISTORY OF CELL SUBSTRATES USED FOR THE PREPARATION OF HUMAN BIOLOGICALS" BY Leonard Hayflick, Ph.D.

Change first response to James Cook to read:

My WI-38 cell strain is the most rigorously tested human virus vaccine cell substrate in the world. Not only has WI-38 been inoculated into all classes of immunosuppressed animals, including nude mice, but they have also been inoculated into terminal human cancer patients where, unlike HeLa cell controls, no growth occurred. Finally, as revealed in Table 1 above, the many major vaccines produced in WI-38 and MRC-5 (which was developed ten years later) have been administered to almost one billion people with no record of a single adverse effect attributable to the cell substrate. There is no human virus vaccine cell substrate with a record like that.

Change second response to Johannes Loewer to read:

No transformed immortal cell line derived from normal or neoplastic human or animal tissue supports the range of human viruses that can be grown in human diploid cell strains like WI-38. In fact, many previously unknown viruses, like rhinoviruses, were first discovered using my human diploid cell strains.

PLEASE NOTE: I named WI-38 and that is its correct designation. It should be corrected from WI38s in the query by James Cook (where it is misspelled) to WI-38s.