For use in dairy cows

Somatotropin, also known as growth hormone, is a protein produced in the pituitary of vertebrate species that promotes postnatal growth.1 It shares similar tertiary structure to related polypeptide hormones prolactin and placental lactogen.1 The amino acid sequence of somatotropin is similar for nonprimates but has diverged more during vertebrate evolution of primates. The amino acid sequence for primate somatotropin differs by approximately 35% from that of nonprimates such as the bovine.1

bST is made up of 190-191 amino acids (Figure 7.1). Cows produce two or four natural variants of bST that differ from one another by one or two amino acids (e.g., ala-phe-pro or phe-pro at the amino terminus and either leucine or valine at position 126 or 127 in the molecule).3 The leucine or valine difference is due to a single nucleotide polymorphism (SNP) and the terminal variants result from random post-translational cleavage between alanine and phenylalanine.45 In the bovine, bST not only supports growth but also directs the partition of nutrients from the body to the mammary gland to support lactation.6 Other species such as primates and rabbits are much more dependent on prolactin, rather than somatotropin, to sustain lactation. Rodents require both somatotropin and prolactin for maintenance of lactation.1

The discovery of somatotropins began more than 80 years ago when extracts of pituitary glands were shown to promote growth, increase muscle mass, and reduce fat content when injected into rats.7-9 As a consequence, the pituitary extract was named somatotropin from the Greek words soma (body tissue) and tropin (growth). Subsequent studies showed that bovine pituitary extracts could also stimulate lactation. In France, it was reported that milk yield increased when lactating laboratory animals and goats were injected with pituitary extracts.1011 Russian scientists treated more than 500 lactating dairy cows with subcutaneous injections of a crude extract from ox anterior pituitaries and observed a substantial increase in milk yield.12 During World War II, food shortages prompted British scientists to examine the possibility of using bST to increase the milk supply.13 They established that bST was the galactopoietic factor in crude bovine pituitary extracts and evaluated several dimensions of the milk response in dairy cows.14 Unfortunately, the amount of bST

40

Recombinant (L-Met-1, L-Leu126) Bovine Somatotropin

Legend

A

Ala

Alanine

R

Arg

Arginine

N

Asn

Asparagine

D

Asp

Aspartic acid

C

Cys

Cysteine

Q

Gln

Glutamine

E

Glu

Glutamic acid

G

Gly

Glycine

H

His

Histidine

I

Ile

Isoleucine

L

Leu

Leucine

K

Lys

Lysine

M

Met

Methionine

F

Phe

Phenylalanine

P

Pro

Proline

S

Ser

Serine

T

Thr

Threonine

W

Trp

Tryptophan

Y

Tyr

Tyrosine

V

Val

Valine

O"

FIGURE 7.1 Bovine somatotropin (bST). 6

that could be extracted from the pituitary gland of cattle was limited and would not provide enough to meaningfully increase the nation's milk supply. Research on the galactopoietic effects of bST continued over the next 20 years.1516

Machlin, who worked at Monsanto Company (St. Louis, MO), reported a 40% increase in milk yield of dairy cows following injection of bST over a 10- to 12-week treatment period.16 However, extraction of sufficient quantities of bST from bovine pituitaries to support commercial use was still not feasible. Since it was not possible to synthesize large proteins like bST at that time, Machlin made smaller pep-tide fragments derived from pituitary bST and injected them into dairy cows to see whether they had any galactopoietic activity. He was encouraged by preliminary reports of a few investigators that large, proteolytic-derived fragments of bST had anabolic effects in animal models. Since the absence of intact bST in these preparations could not be ruled out due to limitations in analytical methods available at the time, the accuracy of these preliminary reports has been questioned.17 Machlin found no evidence that bST fragments could increase milk yield, and the project was subsequently abandoned. Later research showed that the intact somatotropin molecule was required for binding to the somatotropin receptor on tissues to exert its hormonal effects.17,18 Scientists would have to wait until the advent of biotechnology before large-scale production of somatotropin was possible.

With the advent of recombinant DNA technology, it became possible to clone bacteria with the genes that code for the production of therapeutically important proteins. Large quantities of bacteria could be produced during fermentation, and gram to kilogram quantities of the protein expression product of the cloned gene could be harvested from the bacteria. Genentech Inc. pioneered the cloning of human genes into E. coli bacteria for the production of protein therapeutics such as human insulin and somatotropin.19 This group also cloned bacteria with a bovine gene for bST.20 The bST molecule developed by Genentech had the same amino acid sequence as one of the natural bST variants, with the exception of a methionine residue added to the amino terminus of the molecule by E. coli. Tryptic peptide mapping of bacterial-derived bST and pituitary bST are almost identical, with the exception of methio-nyl- instead of alanyl-containing fragments generated at the amino terminus of the bST protein.21 Similar findings have been reported for tryptic peptide mapping of pituitary and bacterial-derived methionyl human somatotropin.22

The Genentech recombinant DNA technology for production of bST was licensed to Monsanto in 1981. Monsanto subsequently filed an Investigational New Animal Drug Application (INAD) with the U.S. Food and Drug Administration's (FDA's) Center for Veterinary Medicine (CVM) to undertake clinical trials in dairy cows with bST. FDA scientists reviewed and approved protocols for studies to investigate the safety and effectiveness of bST to increase milk production in dairy cows. The USAN (United States Adopted Name) designation for bST manufactured by Monsanto is "sometribove." The chemical formula of sometribove is C978H1537N265O286S9 with a molecular weight of 21,872.29 Daltons. In the early days of scale-up work, each gram of bST cost several thousand dollars to produce. Remarkable advances in manufacturing during the next 10 years made it possible to bring down production costs to levels that would make commercial production of kilogram quantities feasible. A state of the art bST manufacturing facility was constructed at Kundl, Austria that became the largest recombinant protein pharmaceutical manufacturing facility in the world. Production has since been expanded to include a new manufacturing facility in Augusta, Georgia.

bST has to be administered to the dairy cow by injection since it would be digested like other dietary proteins if added to the feed. Daily injection of bST was possible but was considered to be too labor-intensive for commercial use in larger dairy herds. A prolonged-release delivery system was needed that would prevent rapid degradation of bST by tissue proteases and permit controlled release of the product into the circulation to support sustained milk production. A prolonged-release delivery system was developed by Monsanto that consists of the zinc salt of bST (sometribove) suspended in food-grade vegetable oil [sesame oil (ALMS) aluminum monostearate]. This afforded gradual, non-zero-order release of bST when injected into subcutaneous tissues and sustained blood levels of bST during the 14-day injection cycle. The currently approved commercial formulation of bST, known as sometribove zinc suspension (POSILACĀ®, Monsanto, LLC) consists of 1.4 ml of the food-grade oil formulation containing 500 mg of the zinc salt of sometribove. The formulation is injected subcutaneously under the skin of dairy cows every two weeks during lactation using a short (5/8-in.) needle which minimizes the potential for intramuscular injection. These injection sites are removed with the hide at slaughter and do not end up in muscle used for food.

Many studies have been conducted with dairy cattle administered biotechnology-derived bST to evaluate its effects on milk production as well as on animal health.23-27 These studies were conducted under normal dairy practices in many locations in the United States and other countries; some were conducted over multiple lactations (years). These studies have consistently shown an increase in milk production without meaningful effects on cow health, including reproduction. bST increases milk production by acting as a "homeorhetic controller that shifts the partitioning of nutrients so that more are used for milk synthesis. Thus, effects are primarily, perhaps exclusively, on directing the use of absorbed nutrients. This involves coordinating the metabolism of various organs and tissues."23

Other companies (Eli Lilly, Upjohn, American Cyanamid) also developed biotechnology-derived bST proteins. Depending on their respective manufacturing processes, from zero to nine additional amino acids were present on the N-terminus of the bST molecule (Table 7.1). However, when the same purification techniques are used, biotechnology-derived and pituitary-derived bST have similar potencies in biological test systems.21,29

These companies also conducted many safety and efficacy studies with dairy cows which demonstrated that biotechnology-derived bST, whether injected daily or in oil-based, controlled-release formulations, consistently increased milk production and was well tolerated by dairy cattle.14,30-33 Given the fact that not one, but four companies carried out extensive safety and efficacy studies on biotechnology-derived bST, it was the most thoroughly studied animal drug that has ever been approved for use in the United States.34 It was estimated that by 1992, more than 1000 research studies had been carried out with bST involving more than 20,000 dairy cows.23 At the time, this amount of published work, supported by the efforts of universities, government agencies, and private industry, was considered to be unprecedented for a

TABLE 7.1

bst varieties Developed by Different companies;

Amino Acid Additions at the Amino Terminus product Name of Ala (191) bsT pituitary variant

Met-Asp-Gln

Met-Phe-Pro-Leu-Asp-Asp-Asp-Asp-Lys

None

Somagrebove Somidobove Sometribove Somavubove new technology, and greater than for most dairy technologies in use.23 Following its approval for commercial use by FDA in 1993, millions of dairy cows have received bST. The safety and efficacy of bST demonstrated in the many precommercial trials continues to be evident today.

At this time, the only form of biotechnology-derived bST approved for use in dairy cows in the United States is Monsanto's sometribove marketed under the trade name POSILACĀ®. Upjohn and American Cyanamid companies no longer exist due to mergers with and/or acquisitions by other companies. Monsanto Company has licensed Eli Lilly to market POSILAC in countries outside of the United States.

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