Eleven

TESTING BIOLOGICAL PRODUCTS

1. HORMONES


Hormones are chemical substances produced in organs of the body, chiefly but not exclusively from the endocrine or ductless glands. They move throughout the body in the blood, regulating functions such as metabolism, growth, pregnancy and sexual development. They include insulin, thyroid hormones, and the gonadotropic, androgenic and estrogenic sex hormones among many others.

Alternatives

Tests to measure the amount of hormones in body fluids which formerly used animals have now been partly superseded by biochemical in vitro methods.

1. Instead of rabbits, mice and toads once used in great numbers in the gonadotropin pregnancy test, pregnancy may now be diagnosed by an agglutination test which eliminates the animals. (PDR, 1975, p. 2028).

2.
Instead of the rats, mice and birds formerly required to detect estrogens, progesterone, corticosteroids and 17-oxosteroids in the body fluids, a chemical assay is now the method of choice. (NRC,1971, p.134).


Insulin

Although some hormones like the steroids are produced by chemical synthesis, the most important sources of insulin and certain other hormones are the endocrine glands of animals slaughtered for meat. The insulin assay used by the Drug Bioanalysis Branch of the U.S. Food and Drug Administration is described in the U.S. Pharmacopeia XIX, p.611 (1975). Insulin manufacturers are required to follow the same method. The solutions to be tested are compared with official standard insulin: different dilutions are injected into rabbits and after one hour, and 2 1/2 hours, the effect on the rabbits' blood sugar is compared. This gives a quantitative evaluation of the potency of the test substances. I asked Dr. John Collins of the Drug Bioanalysis Branch whether the rabbits were anesthetized for this procedure and he replied that the pharmacopeia does not call for it:

"My opinion is that this is impractical for a number of reasons. Aside from the possible physiological intrusion to insulin response caused by anesthesia one must consider that practicality demands that the rabbits be used more than once, and it is certainly undesirable to expose animals to chronic anesthesia. The rabbit test is not usually hard on the animal; indeed an outpouring of epinephrin1 in the course of the assay would result in glucose elevation and possible assay invalidity. The rabbits do receive a subcutaneous injection through a sharp needle, and never appear to show any distress. If the blood sample is obtained carefully from a marginal ear vein, the animal also seems to suffer little. In my opinion, anesthesia is undesirable in this case because it is comparable to not using anesthesia for penicillin injection or venipuncture in the human." (Collins, J.,1980).

He said that the rabbit bioassay had replaced the "mouse convulsion" test in the U.S. many years ago; however, the latter is still done in England using vast numbers of mice. Even though the injected mice are given glucose to bring them back to normal as soon as the convulsion begins, a convulsion is nevertheless a required end-point of the test; thus it is an "all-or-none" procedure which does not yield quantitative information. Dr. Collins added that the Identification Test A on p.253 of the U.S. Pharmacopeia XIX, which describes an injection of test insulin into six rabbits, three of which must convulse and recover after glucose administration, is not used by the Food and Drug Administration. If it is obsolete, it's a pity the pharmacopeia continues to publish it.

A blood glucose test on mice has now appeared in Appendix 14 of the British Pharmacopeia as an alternative to the mouse convulsion test. P.A. Herman of the British Home Office Inspectorate writes:

"You will be interested to know that it has been estimated that the introduction of the blood glucose method of assay into the Addendum of the British Pharmacopeia may reduce the number of experiments in this area quite considerably. Indeed the estimate of one establishment carrying out this work is that the number of experiments it performs will be reduced by over 75%." (Herman, P.,1979).

Dr. Collins says that his laboratory is looking into this test as a potential replacement for the rabbit method.


Synthetic Insulin and Other Hormones

The chemical synthesis of bovine insulin was accomplished in the late 1960's, and in the early 1970's human insulin was synthesized by the Swiss drug firm Ciba-Geigy. The quantities produced were apparently small, so the announcement in 1978 that the San Francisco pharmaceutical firm Genentech had synthesized human insulin, using a bacterial "gene-splicing" technique which has a potential for producing significant and eventually commercial amounts, earned headlines for the company and for their collaborators at the City of Hope Medical Center at Duarte, California. A further technical advance, the bacterial production of human proinsulin (the precursor of insulin), was announced by Genentech in Mar. 1980. (Anon.,1980g).

Genentech had previously been associated with the synthesis of the brain hormone somatostatin, and in the summer of 1979 they had achieved the synthesis (almost simultaneously with another team at the University of California, San Francisco) of human growth. hormone - all through the use of recombinant DNA technology, or gene splicing. The major producer of insulin from animal sources, Eli Lilly, is also in the race to make synthetic insulin in commercial quantities. With 1,300,000 American users of insulin, the financial stakes are high.

Although the coming of synthetic insulin is a big step along the road toward the elimination of animal in favor of chemical testing, it should be mentioned that no attempt to synthesize the protamine modifier of insulin has been reported. Protamine is an amino acid-rich substance obtained from fish sperm (now mostly imported from Japan); it makes insulin longlasting and is prescribed for some diabetics. Animals are needed to determine the biological reaction curve owing to the variables from the differences in lots of this insulin modifier. However, there is an alternative: an extended insulin zinc suspension known as "Ultralente." This is as long-lasting as protamine insulin but has a modifer which is inorganic and thus does not require the animal assay.


Hoped-for Phasing-out of Testing with Animals

As for insulin itself, and the expected replacement of the beef or pork hormone with a product derived from human genes by DNA recombinant technique, Dr. Collins makes the following encouraging comment: "If the product is as good as we all hope, it is quite possible that a few chemical methods such as electrophoresis or chromatography can suffice to identify it, and animals will not be needed." (Collins, J.,1980).

And if the product were always made in exactly the same manner2, each batch would be identical and animal testing for potency and efficacy would also be superfluous.


2. VACCINES AND ANTITOXINS

This section has to do with the viruses, minute living parasites which inhabit living cells; with bacteria, and the toxins which some of these produce; and with the body's weapons against them: antibodies and antitoxins. These defenses are mobilized in answer to a call to arms, and this is sounded when information comes from a surface or coat protein carried by the microorganisms in the form of a code. Certain specialized body cells, especially the lymphocytes, are able to "read" and react to this code, which consists of a particular sequence of protein molecules formed on the surface of the microorganism out of the DNA - the genetic material of living things. This surface protein, when it provokes a cell to produce antibodies and antitoxins, is called an antigen.

When on the warpath, we can imagine that these microorganisms, like placard-carrying militants, display codes spelling out messages such as "Death to the cells!" This may result in an orderly counterattack by the lymphocytes and other members of the immunological defense system. Or it may panic the cells into an overreaction, like the body's response to gram-negative bacteria which display lipopolysaccharide in their walls and which our tissues read, in Lewis Thomas's words,

"...as the very worst of bad news. When we sense lipopolysaccharide, we are likely to turn on every defense at our disposal; we will bomb, defoliate, blockade, seal off, and destroy all the tissues in the area .... Pyrogen is released from the leukocytes, adding fever to hemorrhage, necrosis and shock. It is a shambles." (Thomas, L.,1974,p.92).

These overreactions are part, and sometimes the most uncomfortable and dangerous part, of what we call "disease."

However, we have learned how to use the milder forms of immunological defense to our advantage by actually cultivating the virus, then "taming" it either by weakening (attenuating) or killing (inactivating) it and thus producing a vaccine. The attenuated virus, taken by mouth, multiplies in the intestinal tract: the inactivated virus does not multiply, so enough has to be given by a series of injections. Both kinds of vaccine stimulate sufficient antibodies to protect against the next invasion of a virulent virus, but in their attenuated or inactivated form do not themselves produce the damaging effects of the disease. Bacterial toxins can also be cultivated - from the bacteria grown in the laboratory. The toxin is treated with formalin to render it harmless, but it retains the surface antigen and can stimulate the production of antitoxin if injected into an animal, such as a horse.


Moderating the Rigors of Tetanus Testing

If the disease is actually present, however, there may not be time to wait for antibodies and antitoxin to mobilize; in that case the alternative is to obtain them from a person who has had the disease and in whose serum they are still circulating. Anti-tetanus serum may be obtained in this manner. As D.H. Smyth points out, the antibodies prepared from human blood require "less safety testing than blood from a different species, and the tests for the normal hazards of human blood, syphilis and hepatitis, do not require live animals."

Tetanus toxoid production, on the other hand, involves the painful participation of guinea pigs under U.S. Dept. of Agriculture regulations. The virulent toxin has to be tested in control animals: "For a satisfactory test, the controls must die with clinical signs of tetanus: ... increased muscle tonus, curvature of the spine,...generalized spastic paralysis...." (US Laws,Stats.,etc., l979, CFR,Title 9).

But Smyth notes that the (British) National Institute "or Biological Standards and Control does not require that mice on whom the still active toxin is tested have to be observed until death (an agonizing one owing to the tetanic cramps). A smaller dose which merely causes a paralysis of the hind leg is sufficient for the test. (Smyth, D.,1975, p.83).

Production and Assay of Vaccines

The production and assay (testing for potency and safety) of vaccines consume the lives of many animals, often in painful ways. In the past, many more were sacrificed. In the early years of polio vaccine, in the late 1950's, 200,000 rhesus macaques a year were being killed for that purpose in the U.S. In the early 1950's, thousands of dogs were destroyed yearly in the production of distemper vaccine. Table 1 lists the vaccines for different diseases and the animals from which they were, or in some cases still are, derived. Most of them were grown on primary kidney tissue cultures, "primary" meaning that the cells are taken directly from the kidney of the monkey, who is killed in the process.

In recent years there has been a great reduction in the use of primary cultures in production, thus sparing many animals. This is because all the vaccines listed in Table 1, and others as well, can be grown on embryonated duck or chicken eggs, or on a human diploid cultured cell strain WI-38.


Table 1: Vaccines Grown and Assayed on Primary Cultures from Killed Animals
"P": production; "A": assay. Left: Disease vaccines; Top: Animals used in production or assay.


The last named was developed at Wistar Institute by Dr. Leonard Hayflick. (Hayflick, L.,1961). The WI-38 strain was obtained from embryonic lung tissue and, unlike a primary cell culture, can be propagated for many generations before dying. There is no risk that the cells might harbor a virus from the animal donor, nor do they undergo cancerous transformation. They can be produced in large quantities and can be preserved by freezing.

A rabies vaccine has been grown on the WI-38 strain, which unlike the presently used vaccines, is painless and is free from the complication of postvaccinial encephalomyelitis. It has been successfully tested on over 1000 people since 1975, and now awaits FDA licensing. (Anon.,1980e).

Table 2 demonstrates the saving of animals which is now possible through the use of alternative production or assay material. Of course these alternatives were developed, not for humane reasons, but because they were more economical and safer. Probably they are not fully utilized even now. Much painful testing of the vaccines on animals is still required by government agencies, although assay methods using cell cultures are available. A plaque-test is said to be four times as sensitive as one using mice (Tint, H.,1974), and instead of the old way of testing Yellow Fever virus by injecting it into the mouse's brain and observing the incidence of paralysis, the World Health Organization now recommends a technique employing plaque formation in a monkey kidney cell culture. (Whitaker, A.,1977, p.28).


Table 2: Vaccine Grown and Assayed by Methods Which Avoid Animal Killing

"P": production; "A": assay. Left: Disease vaccines; Top: Substances used in production or assay. References: 1. Hayflick, L. 1970; 2. US Laws, Stats. etc., 1978, CFR, Title 21, Sec. 170-299, 3. Russell, W., 1959, p. 83; 4. Keele, C., 1962, p. 311; 5. Tint, H., 1974; 6. PDR, 1978, p. 98.


The Suffering of Unprotected "Control" Animals

In spite of the gains that have been made, especially the changeover to embryonated eggs (from vaccine production on primary cell culture requiring the death of the animal donor) some animals still die painfully to guarantee the purity of biological products. The ones that suffer most are not those that are injected with the vaccine to be sure it contains no bacterial or viral contaminants (they might fall ill, of course, if there were a contaminant and they were infected - but that presumably is rare). The real martyrs are the mice and guinea pigs who do not receive the protective vaccine against diseases such as cholera, anthrax, typhoid and tetanus, but, along with a matched group of vaccinated animals, are inoculated with the disease-causing bacteria. Thus Bayvet Corporation, Shawnee Mission, Kansas, reporting to the USDA under the Animal Welfare Act, described pain or distress inflicted on 9,448 guinea pigs, 4,850 hamsters and 1,239 rabbits -"without pain-relieving drugs. Their explanation:

"These animals were utilized to test the efficacy of biological products, using virulent challenge material, by the procedures and methods outlined in the Federal Register, Title 9, CFR, 113-117. Using drugs in these animals would invalidate the test and render it useless." (USDA/APHIS,1976, Bayvet Corp.).

In a "successful" test, proving the efficacy of a vaccine - such as the one for tetanus toxoid described on p. 189 - the immunized animals survive and the unprotected controls die, painfully.


Methods of Polio Vaccine Manufacture: Monkey Tissue Versus Human Cell Culture

The reduction in available rhesus monkeys as a result of the ban on exports from India as well as from, other countries, calls for a reappraisal of the extravagant consumption of primates in research, and in the manufacture and testing of polio vaccine - this being the largest use of rhesus in the U.S. Lederle Laboratories of Pearl River, New York, is the sole producer in this country of the vaccine. They make the Sabin attenuated virus under the trade name "Orimune," growing it on primary monkey kidney tissue. Pfizer, unfortunately, has discontinued production of the polio vaccine which was grown on human cell culture, and which at least spared the monkey kidney tissue donors. Wellcome Laboratories in England still produces the vaccine on human cell culture, and A.M. Whitaker, describing the advantages of starting with human cells, has pointed out that cell cultures prepared from monkeys are often infected by one of the many diseases to which the simians are prone (60 monkey viruses have been identified), so that about 55% have to be discarded during vaccine manufacture. (Whitaker, A.,1977, p.25).


Polio Vaccine: Production, and Tests for Contamination and Neurovirulence

After the cell culture has been prepared and tested, the seed virus is placed on it, is allowed to multiply, is attenuated by dilution and the vaccine fluid is "harvested." (Antibiotics in small quantities have also been added). Table 3 outlines the steps of manufacture of attenuated oral polio vaccine in Great Britain, a process similar to that used in the U.S. (UK DHSS,1977).

The testing of the cell cultures, whether of monkey or human origin, and later of the vaccine, is carried out both in test tubes ("in vitro"), and in animals ("in vivo"). Rabbits, guinea pigs, suckling and adult mice are used to detect extraneous, contaminating viruses and, finally, neurovirulence is tested in monkeys. LeCornu and Rowan comment:

"It should be noted that the same tests for extraneous agents are required whether the vaccine is produced on monkey kidney cell cultures or human diploid cell strains. Extensive testing is understandable in the case of monkey cell cultures since monkeys harbor many unwanted pathogens, but the human cells are derived from a seed stock which has been thoroughly tested. Repeated testing is therefore wasteful of time, money and animals."

 Virus seed (Vaccine virus)

 Show seed to be free from extraneous agents, pass monkey kidney test.
 Standard method (monkey cells)  Alternative method (human cells)
 Grow virus:  Grow virus:
 75% as monkey kidney cell cultures (primary) from quarantined monkeys.  90% as human diploid cell cultures (strains) from cell bank.
 25% as controls.  10% as controls.
 Test for haemadsorbing viruses by addition of red blood cells.  Test for haemadsorbing viruses.
 Test supernatant fluids for extraneous agents on monkey and human cell cultures.  Test supernatant fluids for extraneous agents on monkey, human and rabbit cell cultures.
 Test for simian herpes B virus in rabbit kidney cell cultures.  
 Harvest virus.  Harvest virus.

 Virus harvest, by whichever method grown, then undergoes following tests.
 Monovalent virus harvest
 -Test for bacteria, fungi, mycoplasma.
-Test for extraneous viruses on monkey cell culture (and human cell culture for virus grown on human diploid cell culture).
-Test for simian herpes B virus on rabbit kidney cell cultures.
-Test for extraneous viruses in animals (rabbits, adult & suckling mice & guinea pigs).
 Pool virus harvests
 -Identity test. Measure virus concentration.
-Virus characteristics
- in vitro virulence tests.
-Monkey neurovirulence test (35 monkeys).
-Test for bacteria, fungi & mycoplasma.
 Blend monovalent vaccines - monovalent, bivalent, or trivalent vaccines (i.e. 1, 2 or 3 types).
 -Test for identity.
-Measure virus concentration (i.e. potency test).
-Sterility test.
-Abnormal toxicity.

Table 3: Production of Live, Attenuated Oral Polio vaccine (UK DHSS,1977).

For vaccine grown on human cell cultures, they suggest testing large pooled batches rather than numerous small ones. (LeCornu, A.,1928,p.10). Dr. J.C. Petricciani of the Bureau of Biologics, Food and Drug Administration, also mentioned this as a way of reducing animal wastage, but added that if one of the larger batches failed the test the manufacturer would have more expense to replace it. (Petricciani, J.,1978).

Alternatives in Polio Vaccine Production and Testing

A number of other alternatives to animals in Polio vaccine production are in use or are being developed. Here are several of them and the tests they might replace:

1. Test for extraneous viruses in animals. Unfortunately, some of these contaminating viruses can still only be detected in the whole animal: for example, Hepatitis A or B and most arboviruses. For others, there are alternatives.

Alternatives: "in vitro" tests have been developed, however. Some viruses will cause added red blood cells to adhere firmly (haemadsorption) to the cell culture which the viruses are contaminating. Others may be detected by electron microscopy. The haemadsorption test is already included in the regulatory requirements (cf. Table 3); electron microscopy is used by Wellcome Research Laboratories and, even though it is of low sensitivity, may become part of the official requirements in the future. (Whitaker, A.,1977, p.29).

Primary cell cultures are always associated with wastage of animal life since they involve the killing of the animal. Although official regulations specify the use of primary monkey kidney cell cultures in testing vaccines for contaminating viruses, A.M. Whitaker of Wellcome states that monkey kidney cell lines (cells which have been sub-cultured, proliferate and go through numerous divisions) are equally sensitive to these contaminants. (lbid., p.28). The use of such lines would save many valuable primate lives.

2. Tumorigenicity Test. Cells from the culture are injected into immunodeficient mice to see whether tumors are induced. If they are, the culture has lost its normal character and contains transformed (cancer) cells.

Alternatives: There are several short-term tests for tumorigenicity which might be substituted; for example, the ability of tumorigenic cells to grow in semi-solid medium.

3. Neurovirulence Test. This is done to confirm that the vaccine virus has not reverted to virulence. LeCornu and Rowan (LeCornu,A.,1978,p.11) have this comment:

"The neurovirulence test ... compares the pathogenicity of successive polio vaccine batches to the original seed vaccine virus .... Basically, it involves the injection of the vaccine and reference virus intraspinally and intracerebrally into [two groups of] at least 25 and 10 susceptible monkeys respectively."

In 1955, a year after the first mass trial of the Salk vaccine (containing inactivated virus in contrast to the Sabin attenuated virus) a large number of people contracted polio because of a technical fault in the inactivation process. Before this incident, vaccine safety had been assayed by a tissue culture test which had a greater margin of safety than the intracerebral injection of monkeys which was also being used. However, after 1955 it was decided to abandon the tissue culture test for safety in favor of more extensive monkey tests. LeCornu and Rowan point out that "this appears to be an instance where too much time has been spent trying to improve an unsatisfactory animal test when more effort to standardize and improve the tissue culture test would have been more productive." (Ibid., p.6).

As it is, the safety tests on monkeys, performed by the manufacturing company, are then repeated by the control authorities (in the U.S., the F.D.A.'s Bureau of Biologics). American regulations require 30 rather than 10 monkeys to be tested intracerebrally. Since all monkeys have to be killed after a certain number of days to allow tissue examination for evidence of polio, 140 are sacrificed for each batch of vaccine in Great Britain and 220 in the U.S.

Reduction of animal wastage: LeCornu and Rowan state: "Ideally, duplicate testing should be unnecessary and the practice needs to be reviewed. It certainly offers one opportunity of reducing the number of monkeys needed." The problem is to develop a very stable seed virus, in which case "it should only be necessary to carry out neurovirulence tests on the seed, as is now done with rubella and measles live attenuated vaccine." (Ibid., p.11-12). At present, the Sabin seed strains are unpredictable and continual passaging does not automatically decrease neurovirulence.

Other in vitro tests for neurovirulence: Two of these, the "d" and "t marker" tests, cannot be called alternatives because they are currently used only to complement the monkey tests. The "d test" detects the reduced ability of avirulent virus to replicate in the presence of sodium bicarbonate. The "t marker test" depends on the increased temperature sensitivity of the attenuated virus. A third test is in the experimental stage and is being investigated at Britain's National Institute for Biological Standards and Control. It relies on the differences in the protein bands of virulent and avirulent strains of polio virus, which can be identified through the use of isoelectric focussing. (Magrath, D.,1978).


Synthetic Vaccines

Each lot of polio vaccine is subjected to about 29 separate tests in order to be certain that the product is pure, potent, avirulent and of the required dosage. But since living cells are involved, and since these have to be grown in a medium which sustains life and promotes growth, some of the testing is to guard against this life growing too exuberantly (escape from attenuation), or abnormally (mutagenicity), and the rest of the process is to prevent other life - bacteria, extraneous viruses - from getting into the act. How much simpler it would be, and happier for the animals now sacrificed along the way, if these living elements could be banished and a completely pure synthetic vaccine could be created by the chemist.

It has been done: a totally synthetic vaccine against a natural virus has been produced by Dr. Michael Sela, of the Weizmann Institute in Israel, and was reported at an international conference held at the National Institutes of Health in Bethesda, Maryland, in Feb. 1979. According to an article in The New York Times (Feb. 27, 1979), the new product was a vaccine containing a synthetic copy of part of a virus linked chemically to a synthetic carrier molecule and to a synthetic "adjuvant" molecule which has the property of amplifying the recipient's response to the vaccine. "Injected in laboratory animals, it immunized them against the natural virus it was designed to resist. The vaccine itself is of no practical usefulness because it is directed against a type of virus called a bacteriophage, which infects bacteria, but not humans or animals." (Schmick, M. 1979).

Of course this research is only the first step toward the development of other vaccines. Diseases like malaria, and other parasitic diseases against which there is presently no effective vaccine, as well as the major venereal diseases, will no doubt have priority in the development of these synthetic substances, although one against Hepatitis B is already in the early stages of synthesis (cf.p.249). Thus it seems likely that a vaccine against Polio will also be synthesized. Such a substance would be standardized to a high degree of uniformity and purity, reducing the present painful "29 steps" of manufacture and assay to a minimum.


NOTES

1. If the rabbits were in fact experiencing pain or fear.
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2. The U.S. Food and Drug Administration's "Good Laboratory Practice Regulations" (US FDA,1978) are intended to assure thehigh quality of testing for safety of products such as these.
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