FI103342B - A method for isolating and purifying G-CSF - Google Patents

Description

, 103342

Method for Isolating and Purifying G-CSF This application is divided by FI application 900178.

5

Field of the Invention

The invention relates to a method for isolating and purifying a granulocyte colony growth stimulating factor (G-CSF) from a G-CSF producing microorganism.

G-CSF can be used to treat or prevent infections in animals. In particular, G-CSF may be human G-CSF (hG-CSF) or bovine G-CSF (bG-CSF). G-CSF may be derived from a natural source or may be derived from genetically modified prokaryotic or euka-15 host cells containing a recombinant plasmid or viral DNA vectors containing the human or bovine G-CSF gene or genetically modified human or bovine bovine G-CSF genes or synthetic human or bovine G-CSF genes.

Background of the Invention

Animal infections lead to billions of dollars in losses every year in the meat and dairy industry. Although nowadays antibiotics are used in animal infections. With some success, huge losses happen 25. Examples of such infections are mastitis in cows and stroke in cattle transport.

Cattle A. Ulcerative infection

The most expensive problem in the dairy industry today is the • 30 udder infection. Ulcerative inflammation is defined as an inflammation of the mammary gland. It can occur in any mammal, such as cows, sheep and goats, but mastitis in bovine animals is of major economic importance. Bovine mastitis is an infection of ruminant uta-35 in ruminants, such as cows, caused primarily by gram-2 103342 positive and gram-negative bacteria, and especially by cows in intensive milk production units. Bacterial infection leads to inflammation of the mammary gland (namely the nipples and the udder). The disease is particularly problematic and economically significant because the pathogen is easily transferred from one animal to another during the milking process. Some of the major pathogenic microorganisms causing mastitis in bovine animals are Staphylococcus aureus, Streptococcus agalactiae.

10 Streptococcus uberis. Streptococcus dvsgalactiae.

Escherichia coli. Aerobacter aeroaenes. Klebsiella pneumoniae and Pseudomonas aeruginosa. See also Bovine Mastitis. edited by Glenys Bloomfield, V&O Publications 1987, which is incorporated herein by reference. These microorganisms invade the udder through the nipple canal and cause inflammation of the milk-producing tissue, resulting in scar tissue formation, which, once formed, can lead to a reduction in the permanent milk production in the cow. Inflammation can also change the composition, volume, appearance and quality of the milk.

There are different forms or types of bovine mastitis, varying in severity and symptoms, including: (1) Urticaria: Invasion of the udder cavity by micro-or-25 ganism, which is distributed within the gland and causes inflammation; (2) Non-clinical or subclinical mastitis; A form of mastitis with no glandular swelling or detectable milk abnormality, although there are changes in the milk that can be detected with specialty teas. This type of mastitis is by far the most common and causes the largest total loss in most herds. It is often referred to as "hidden" mastitis; 3 103342 (3) Clinical mastitis; A form of urticaria with abnormal conditions of the udder and secretion. Mild clinical mastitis includes changes in the milk such as flakes, clots and watery or unusual appearance. Urethral warming and sensitivity are low or non-existent, but there may be signs of swelling. Severe clinical mastitis includes a rapid onset of swelling of the infected quarter, which is hot, hard and sensitive. Mai-10 to is abnormal and milk yields are falling. Sometimes, in addition to the local effects of the udder, the cow himself becomes ill. There are signs of fever, rapid pulse, depression, weakness and loss of appetite. The combination of these conditions is often referred to as acute mastitis. because the effect applies not only to the udder but to the whole animal; and (4) chronic mastitis; A form of mastitis caused by persistent mastitis, which occurs most of the time as a non-clinical form but may occasionally develop into an active clinical form.

After these "bursts", the non-clinical form returns temporarily. (See generally Current Concepts of Bovine Mastitis, published by The National Mastitis Council, Inc., 2nd Edition, 1978, page 5).

25 Urticaria is a major contributor to the dairy industry. Urticaria affects livestock productivity in many ways, both directly and indirectly, including: (1) loss of milk production; (2) higher rejection rates of infected cows; (3) depreciation of milk; (4) antibiotic post-treatment milk rejection; (5) veterinary costs (antibiotics and veterinary visits); and (6) Deaths. (Bovine Mastitis. Glenys Bloomfield, supra page 33). The estimated annual cost in the United States is $ 1 billion for milk 35 alone. Treatment and rejection costs increase this estimate to a total cost of about $ 1.5 to $ 2 billion a year in the United States, representing the impact of mastitis on only 5% of the world's dairy fauna. (Current Concepts of Bovine Mastitis.

5 straight. page 7). As the world figures are sketching, the drafts prepared by the USDA reveal global losses amounting to over $ 20 billion annually.

Effective compounds used to treat or prevent mastitis in bovine animals should give the following results: (1) most or all of the above pathogens should be sensitive to the compound when the pathogen is present in milk and other udder fluids; (2) the therapeutic effect should be relatively rapid; (3) Whereas, on the active or other ingredients of the composition, no significant irritation should be caused to the cow's udder or nipples; and (4) the active compound should not remain in the milk for much longer than necessary for therapeutic activity, to reduce the loss of milk that the milk must discard as long as the foreign compound is present. There are other requirements for the composition used to treat bovine mastitis, but the four criteria above are some of the most important.

25 Antibiotic medication has been a major part of the control strategy for UAE. Table 1 summarizes the various antibiotics used to treat mastitis: 5 103342

table 1

Antimicrobial agents used in the treatment of UTI 5 Group Compounds 1. Beta-lactam antibiotics

Penicillins Ampicillin

cloxacillin

Hethacillin 10 Nafsillin

Penicillin G (benzylpenicillin) Procaine penicillin

Cephalosporins Cefoperazone **

Keturoxime Kefalonium Cefapirin 20 Cefoxazole1

Cephrasetil1 2. Aminoquinocidal Antibiotics Framysetin

neomycin

Novobiosin 25 Streptomycin 3. Macrolide antibiotics Erythromycin 4. Tetraslinics Chlorotetracycline

oxytetracycline

5. Polvoeotide antibiotics Polymyxin B

First generation; ** Third Generation Bovine Mastitis, supra, page 70.

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Antibiotic treatment for mastitis is usually given by intravenous infusion of the mammary gland either to dairy cows when mastitis has been diagnosed or to cows that have become depleted (dry cow therapy). (Bovine Mastitis.

5 supra, page 69). In cases of severe clinical disease, antibiotics must be administered parenterally (intravenous infusions are ineffective due to clogging of the mammary glands) (Ibid.).

Previous hopes that antibiotics would allow complete control of the disease have not materialized. None of the above-mentioned antibiotics used so far has been completely satisfactory. In addition, replacement of antibiotic therapy with non-antibiotic chemotherapeutic drugs has been found to be highly desirable for the following reasons: (1) Antibiotics effective in human medicine should not be used in veterinary medicine in order to avoid the development of resistant bacterial strains found in human diseases; (2) Antibiotics should be reserved for diseases for which no chemotherapeutic drug compound exists because bacterial strains have been shown to develop resistance to the antibiotic after extensive use; and 25 (3) Staphylococcus aureus. one of the above pathogens has already developed resistance to most of the antibiotics used to treat mastitis in cattle.

One method of treating a non-antibiotic chemotherapeutic drug compound is described in U.S. Patent 4,610,993, which discloses a method of treating animals with an effective amount of at least one pyridine N-oxide disulfide compound for bovine mastitis. Another method of the same inventors is described in U.S. Patent 7,103,342,440,666, which discloses a method of treating animals with an effective amount of at least one metal salt of pyridine N-oxide for bovine mastitis.

Despite these many published methods, it remains very important to find methods that have a good cost / benefit ratio, employ non-antibiotic compounds, and are substantially capable of resolving the drawbacks of the antibiotics used to date and yet are effective in treating and in the prevention of mastitis.

B. Transport stroke

Another common disease affecting the livestock industry is transport stroke (bovine respiratory disease). Respiratory diseases are a major cause of disease loss in meat-15 livestock. In a one-year disease study of 407,000 year-old beef cattle, respiratory disease was found to be responsible for about 3/4 of clinical diagnoses and about 2/3 of autopsy diagnoses.

The term "transport stroke" is used to describe a combination of respiratory tract disease found in cattle 6 months or older after transport to either feed sites or pasture. Stress caused by weaning, castration, horn removal, fasting, crowding, exposure to infectious agents, dietary changes, extreme temperatures and other stress factors, combined with viral, bacterial, mycoplasma and / or chlamydial infections, contribute to the delivery of the disease. Mixing calves on different farms and / or farms greatly contributes to the susceptibility to infectious material. Mixing populations can be a more important predisposing factor to transport destruction than stress factors, although the disease can occur without mixing and stress factors, which usually exacerbate respiratory disease. Attempts to reduce the stress caused by weaning, castration, horn removal, etc., and to accustom the herd to new diets days or weeks prior to transportation, have sometimes succeeded (but may not be economical) in reducing the incidence of stroke. Vaccination against some infectious agents involved in transport stroke sometimes helps, but vaccines are available and are effective against only a few known agents involved in the disease-10 complex.

It is generally known that bacterial pneumonia (usually Pasteurella) is the root cause of most transport stroke deaths. Pasteurella haemo-lytica. especially Type IA, is the most common bacterium isolated from respiratory tract disease in North America. Attempts to experimentally produce bacterial pneumonia in cattle have generally failed without severe stress and respiratory distress. It is generally believed that during times of stress, viruses, mycoplasma and / or 20 Chlamydia are most often the cause of the inhalation of the airway, which exposes the injury to severe bacterial infection and disease.

The onset of a typical clinical respiratory disease usually begins within hours or days of the arrival of the litter at the feed site. Freshly transported cattle weighing between 300 and 500 pounds typically have a morbidity of 20-80% and a mortality of 1-10% or more. By analyzing bovine serum for a fourfold rise in antibody (serocon-30 version) and exposing the airway and its secretions to microbiological isolates, a myriad of etiological agents can be identified. Many animals, diseased and apparently healthy, can be shown to have undergone infection by one or more substances (respiratory tract infection).

road sickness is probably rarely due to a single infectious agent). Although the bovine respiratory disease complex is clinically identifiable upon arrival at the feed site, infections caused by clinical disease 5 may possibly commence in commercial drives, where cattle from different farms are first pooled. See also Bovine Respiratory Disease. Loan, R.W., Texas A&M

University Press, 1984, which is incorporated herein by reference. Pigs 10 Respiratory diseases, especially pneumonia, cost the pig industry hundreds of millions of dollars each year. It is a problem during the rest of the number one rearing, which takes up half of the pig's life. Some common pathogens associated with pneumonia are Pasteurella multocida.

15 Mycoplasma hvopneumonia. Haemophilus (Actinobacillus) pleuropneumonia. Streptococcus suis. Salmonella choler-suis. Bordetella bronchiseptica. pseudorabies virus and swine flu virus.

Haemophilus Actinobacillus pleuropneumoniae is the major cause of swine pneumonia. The disease has spread worldwide and is one of the most economically important pig diseases. In 1985, swine respiratory disease was estimated to have cost producers $ 208 million. Pleuropneumonia is the most feared human-25 gastrointestinal disease because vaccines fail to prevent infections or the occurrence of continuously infected carrier animals.

The economic impact of an acute outbreak of pleuropneumonia in an unprotected herd is ≥ 30 years, with mortality ranging from 0.4% to 24% and morbidity between 8.5% and 40%. The treatment of acute disease has relied on the use of antibiotics. Routine use of antibiotics is rapidly being restricted due to public concern and government emphasis.

10 103342

Detailed histomorphological and bacteriological studies of swine pleuropneumonia reveal that (i) a large number of neutrophils are localized at the site of infection at the onset of the disease; ii) there is a relationship between the ability of pigs to calm the infection into the lungs, thereby preventing bacteremia, and survival; and iii) experimental reduction of circulating neutrophils leads to more severe disease.

Equines 10 The first two weeks of life (neonatal period) are the time when the foals develop severe disease or die due to infection (blood poisoning). Neonatal blood poisoning represents a significant loss for the horse industry. In foals affected by blood poisoning, the neutrophil count is usually very low (neutropenia) and this is one of the causes of 60-75% mortality in neonatal foals.

Problems less severe than Gram-negative blood poisoning, such as bone and joint injuries, inflammation of the bowels, inflammation of the 20 polar veins, respiratory disease and poor development, also result in significant losses in the equine industry. Immunoglobulin levels did not correlate well with protection against infection in 20% of foals affected by blood poisoning, with twice-favored IgG levels (400 mg / ml). Even with effective treatment of a critically ill blood poison, the survival rate is only 25-40%. Neutrophil activity has been determined to be significantly reduced in foals that are not fed enough colostrum. Newborn foal. This, together with neutropenia, which often develops with blood poisoning, are important causes of high mortality and possibly the onset of infection.

Granulosa colony growth stimulating factor

Granulocyte colony stimulating factor (G-35 CSF) is one of several glycoprotein growth factors known as "103342" known as colony stimulating factors (CSFs) because they maintain the growth of haemopoietic stem cells. G-CSF stimulates the growth of bone marrow-specific stem cells and their differentiation into granulocytes. It is distinguished from the other CSFs by its ability to both stimulate neutrophilic granulocyte colony formation on semi-solid agar and to induce terminal differentiation of rodent myelomonocytic leukemia cells in vitro. Granulocyte colony stimulating factor is a potent stimulator of neutrophil growth and maturation in vivo [Cohen et al., Proc. Natl. Acad. Sci. (1987) 84: 2484-2488]. G-CSF is also capable of inducing functional activity or "priming" of mature neutrophils in vitro [Weisbart, R.H., Gasson, C.G., and D.W. Golde, Annals of 15 Internal Medicine (1989) 110: 297-303]. G-CSF has been shown to activate human granulocytes and to enhance superoxide release stimulated by a chemotactic peptide, N-formylmethionyl-leucyl-phenylalanine [S. Kitagawa et al., Biochem. Biophys. Res. Commun. (1987) 20 144: 1143-1146 and C.F. Nathan, Blood (1989) 74: 301-306] and activating IgA-mediated phagocytosis of human neutrophils [Weisbart, R.H. et al., Nature (1988) 332: 647-649].

Neutrophils are a critical component of the host defense mechanism against bacterial and fungal infections. G-CSF is capable of inducing elevation in total circulating neutrophils and improving neutrophil function.

Cloning and expression of recombinant human G-CSF cDNA has been described and verified that recombinant G-CSF possesses most, if not all, of the biological properties of a natural molecule [Souza, L. et al. Science 232: 61-65 (1986)]. Sequence analyzes of cDNA and genomic DNA clones have allowed deduction of the amino acid sequence and reveal that the protein has a length of 204 amino acids of 12 103342 and has a signal sequence of 30 amino acids. The mature protein is 174 amino acids in length and has no potential N-linked glycosylation sites but several possible sites for O-linked mediated glycosylation.

The cloning of human G-CSF and expression of cDNA encoding it have been described by two groups [Nagata, S. et al. . Nature 319: 415-418 (1986), Souza, L.M. et al., Science 232: 61-65 (1986)]. The first publication, 10 depicting a G-CSF cDNA clone, suggested that the mature protein was 177 amino acids long. The authors announced that they had also identified a cDNA clone for G-CSF, which encoded a protein lacking a three amino acid sequence. This shorter G-CSF cDNA form-15 expresses the expected G-CSF activity. Another publication describes a cDNA sequence identical to this short form and makes no mention of any other modification. Because these authors confirmed that short cDNA expresses G-CSF with the expected biological activity profile, it is likely that this is an important form of G-CSF and that the longer form is either a minor variation upon mRNA generation or artificially derived by cloning. result.

Matsumoto et al., Infection and Immunity, Vol. 55, Vol. 25, No. 11 (1987) 2715, reported the protective effect of human G-CSF against microbial infection in a neutropenic mouse.

The following patents relate to G-CSF: WO-A-8 703 689, Kirin / Amgen, describes hybridomas that * produce monoclonal antibodies specific for human G-CSF, and their use in purifying G-CSF, WO-A-8702060, Biogen, discloses human G-CSF-like polypeptides and methods for their production, U.S. Patent 4,810,643 to Amgen, discloses human G-CSF-like 13,103,342 polypeptides, encoding sequences and methods thereof. and WO-A-8 604 605 and WO-A-8 604 506 to Chugai Seiyaku Kabushiki Kaisha disclose a gene encoding human G-CSF and infection inhibitors containing human G-CSF.

The invention relates to a process for isolating and purifying G-CSF from a G-CSF producing microorganism.

Other features and features of the invention will become apparent upon consideration of the following description and claims.

Description of the Invention

The invention thus relates to a process for isolating and purifying G-CSF from a G-SCF producing microorganism.

The process according to the invention is characterized in that 1) the microorganism is disrupted and the insoluble G-CSF-containing material is separated from the soluble protein material, 2) G-CSF is dissolved and oxidized in the presence of a denaturing solvent and an oxidizing agent, 3 a) the denaturing solvent is separated from G-CSF using an anion exchange resin containing quaternary ammonium groups such as Dowex® anion exchange resin, 4) G-CSF is subjected to anion exchange chromatography followed by cation exchange chromatography, and 5) recovered.

Also described are pharmaceutical compositions containing an effective amount of the polypeptide product of the invention together with suitable diluents, adjuvants and / or carriers useful in the treatment of animals.

G-CSF obtained according to the invention, preferably G-CSF derived from the gene of the animal to be treated, can be used for the treatment and prevention of infections in animals such as bovine animals. In particular, G-CSF may be used for the treatment or prevention of mastitis or transport stroke in bovine animals, for the treatment or prevention of neonatal foal poisoning, and for the treatment or prevention of swine infections such as pleuropneumonia. Thus, G-CSF is effective in treating or preventing various infections in animals, particularly mammals, bovine animals, poultry, pigs, horses, dogs and cats.

G-CSF can also be used as a prophylactic treatment to increase host defense in animals at risk of bacterial, yeast or fungal infection. For example, G-CSF can be used as a prophylactic treatment in normal human patients at risk for pneumonia 15 (e.g., hospital pneumonia), surgical wound infections, urinary tract infections and intestinal infections. The term "normal" as used herein refers to an animal with normal immune function and normal white blood cell count and cell differentiation. A description of the various hospital infections for which prophylactic treatment can be used is given in Nelson et al., J-Critical Illness, pages 12-24 (April 1988), which is incorporated herein by reference. Prophylactically, G-CSF is typically administered 0-3 days prior to the event (e.g., surgery, vein cannulation, 25 catheter insertion, throat tube insertion) at risk of infection. G-CSF is administered subcutaneously or intravenously daily at a dose ranging from 1 to 20 µg / kg, more preferably from 3 to 15 µg / kg. One skilled in the art will know how to adjust the dose.

The G-CSF of the invention may also be used to treat a patient shortly after, preferably within eight hours, most preferably within four hours, of a patient sustaining an injury (e.g., a human or animal bite, a firearm 35 or knife wound or other puncture wound) that requires medical 103342 occlusion or surgery. G-CSF is administered prior to the identification of infection (eg by microbiological or histological examination of the patient's tissues or body fluids) or clinical manifestations and symptoms (eg purulent inflammation, chills, fever).

Cattle are treated prophylactically before transportation or other events that may weaken cattle to enhance and prim their ability to fight infections. Administration of G-CSF can be performed when cattle 10 is treated, namely, vaccinated, labeled, etc. Treatment with G-CSF can also be performed during the dry cow's grain and / or just before the cow gives birth to intrauterine infections and mastitis. to reduce the likelihood of early breastfeeding. See Kehrli et al., Am. J. Vet. Res., 50, No. 2 (1989) 207, which describes the function of bovine neutrophils during parturition. Conventionally, no antibiotic treatment is given just before delivery because the residues that appear in cow's milk render it unusable.

G-CSF refers to one of the hematopoietic growth factors known as granulocyte colony stimulating factors. Biological activities of G-CSF include: stimulating the differentiation of small stem cells into different blood cell lines, stimulating the growth of these blood cell lines, and stimulating the final differentiation of mature blood cells from these lines. Preferred sources of the G-CSF polypeptide used to treat or prevent UTI are human and. · 30 bovine animals, and may be the product of host cells of natural origin or recombinant DNA containing the DNA sequence encoding G-CSF.

The DNA encoding the G-CSF gene is a genomic DNA sequence, a cDNA sequence, or a prepared (or synthetic) 1 «103342 DNA sequence expressed in a prokaryotic or eukaryotic host cell that contains some or all of the naturally occurring G-CSF. conformations of the primary structure and one or more of its biological properties. A biologically active plasmid or viral DNA vector containing a DNA sequence encoding G-CSF can be used to transform or transfect a prokaryotic or eukaryotic host cell to produce cell lines expressing the 10 G-CSF polypeptide, glycosylated or glycosylated. .

Various forms of human G-CSF useful in a method of treating or preventing mastitis, and their preparation and purification, are described in detail in U.S. Patent 4,810,643, which is incorporated herein by reference. U.S. Patent 4,810,643 describes novel gene segments, biologically active recombinant plasmids and viral DNA vectors, and prokaryotic and eukaryotic host cells containing the G-CSF gene or the G-CSF gene produced by recombinant DNA technology. conversion. The host cells express biologically active G-CSF or a G-CSF variant produced by recombinant DNA technology.

, FI Patent Application 900178 describes the isolation and characterization of the bovine G-CSF gene and specifically describes novel gene segments, biologically active recombinant plasmids and viral DNA vectors, and prokaryotic and eukaryotic host cells containing the bovine G-CSF gene or a 30 g G-CSF gene variant produced by recombinant DNA technology. Host cells transformed or transfected with recombinant plasmids or viral DNA vectors express biologically active bovine G-CSF or a bovine G-CSF variant produced by recombinant DNA techniques.

Purification of G-CSF G-CSF is produced in an insoluble form in E. coli. A purification process was developed to ensure obtaining the monomeric oxidized product. To achieve this, the protein is solubilized in the presence of a denaturing agent and allowed to oxidize in the presence of this denaturing agent. The oxidized protein is then removed from the denaturing agent and purified to homogeneity. The following describes a method for purifying human G-CSF, indicating differences in purification of bovine G-CSF.

Cell lysis 350 g of frozen cell paste is combined with 2-3 liters of 1 mM dithiothreitol (DTT) and carefully dispersed. The cell suspension is then passed through a Gaulin homogenizer for an appropriate number of cycles to achieve greater than 95% cell lysis. The broth is cooled to 5 ± 3 ° C prior to homogenization and to less than 18 ° C between cycles passed through a homogenizer. The homogenization-20 broth is centrifuged to recover a pellet containing more than 95% of the G-CSF originally present in the cell paste. (DTT is not present in the bovine G-CSF lysis mixture).

Extraction. The homogenized pellet is then dispersed in about 3 to 25 liters of 1% deoxycholate (DOC), 5 mM DTT, 5 mM EDTA, 50 mM Tris, pH 9. The suspension is stirred at 15 to 20 for 30 to 40 minutes and then centrifuged. (This DOC extraction step is not used for bovine G-CSF). The pellet is then resuspended in about 3 liters of cold water and centrifuged again. G-CSF is recovered from the pellet in a total yield of more than 80%. As an alternative to deoxycholate, another bile salt or non-ionic detergent is used.

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Solubilization and oxidation

The final pellet obtained from the above extracts is suspended in about 2.5 liters of 2% Sarkosyl, 40 mM Tris (pH 8.0) and allowed to dissolve at 15-20 ° C for 30 ml-5 noodles. Add copper sulphate to give a final concentration of 40 μΜ and allow the mixture to stir for at least 12 hours at 15 to 20 ° C. About 70% of G-CSF is oxidized to the correct monomeric form during this process.

Removal of Sarcosyl The resulting soluble protein mixture is centrifuged to remove insoluble cell debris and then diluted four times with 13.3 mM Tris pH 7.7. Then about 2000 g Dowex equilibrated in 20 mM Tris pH 7.7 are added. Dowex is a 15 ion exchange resin. See Dowex: Ion Exchange. The Dow Chemical Company, Midland, MI (1988), which is incorporated herein by reference. Preferably, Dowex 1X4 mesh type-1 having a mesh size 20 to 50 is a strong basic anion-exchange resin, chloride form. The resin is a 4% cross-20 dot styrene-divinylbenzene polymer matrix containing quaternary functional ammonium groups which serve as ion exchange sites. "Type 1" means that the ammonium group consists of a nitrogen atom which is bonded. a polymeric benzyl group attached to the matrix; and three methyl groups. The mesh size is measured on a wet resin using a U.S. standard sieve. The ammonium group is positively charged; this functional group replaces its attached chloride ion with other negatively charged ions by a reversible reaction. Dowex 2 resin is a type II resin in which one of the methyl groups is replaced by an ethanol group.

19, 103342

Type I

5 1 ΓfH3 Ί ° <- Γ V CHi — Ν —CHa ί UJ CHa ίο

Type II

15 _ __, CHa ~ 1 +> ί \ ι Cl

5 - ('VCHa-N-CHa-CHaOH

"\ = / U

20 '-H *

Type I and Type II resins differ mainly in their affinity for the hydroxide ion relative to the other anions and their chemical stability. Type II resins are more efficiently converted to the hydroxide form than type I resins, but type I resins are inherently more chemically stable, especially in the hydroxide form.

The quaternary ammonium anion exchange resins are highly ionized and can be used throughout the pH range. They can also be used in salt decomposition reactions where the neutral salt is converted to the corresponding 35 bases.

20 1 0 3 3 4 2

Other ion-exchange resins having strong anion-exchange properties in which the charge is inaccessible to the proteins of interest are alternatives to the use of Dowex in the present invention.

This mixture is allowed to stir for at least one hour at 15-20 ° C to remove Sarkosyl (N-lauroyl sarcosine sodium salt) (Sigma) from the protein solution and then Dowex is filtered off. More than 80% of the properly oxidized G-CSF form can be recovered. (Bovine G-CSF 10 is treated at 2-8 ° C during this step).

Anion Exchange Chromatography (This step is not used for bovine G-CSF)

The resulting protein solution is added to DEAE cellulose (Whatman DE-52 or equivalent) on a column equilibrated in 20 mM Tris pH 7.7. The column is washed with 20 mM Tris pH 7.7 until the absorbance at 280 nm is approximately zero. G-CSF is then eluted from the column with 20 mM Tris, 40 mM NaCl, pH 7.7.

Cation exchange chromatography The pH of the eluate from the DEAE column is adjusted to pH 5.4 with 50% acetic acid and diluted at least twice with 5 mM sodium acetate, pH 5.4. The solution is applied to a CM-Sepharose high-speed column equilibrated in 20 mM sodium acetate, pH 5.4. After addition, the column is washed with 20 mM sodium acetate, pH 5.4, until the absorbance at 280 nm is approximately zero. G-CSF is then eluted with 20 mM sodium acetate, 37.5 mM NaCl, pH 5.4. (Bovine G-CSF is eluted with 30 150 mM NaCl in the same buffer).

Brief description of the figures

Figure 1 shows a restriction map of bovine G-CSF;

Figure 2a depicts the mature protein coding region of bovine G-CSF; Figure 2b is the amino acid sequence of a mature protein; 2i 103342

Figure 3 is a genomic sequence of human G-CSF;

Figure 4 is the DNA sequence of the first bovine G-CSF synthetic gene (bG-CSF dna);

Figure 5 depicts the oligos used in construct-5 subunits of the second bovine G-CSF synthetic gene (bG-CSF dna4);

Figure 6 shows two subunits of the bG-CSF synthetic gene bG-CSF dna4 in bovine G-CSF;

Figure 7 shows the amino acids encoded by dna4 in the bovine bG-CSF synthetic gene;

Figures 8-12 are graphical representations of the results obtained in Example 4 for the treatment of Pasteurella hemolvtica-infected cows with G-CSF; Figures 13-17 show the results obtained in Example 5 concerning the use of G-CSF in the regulation of bovine airway disease;

Figures 18-20 illustrate the results obtained in Example 6 investigating the effect of G-CSF on respiratory tract disease;

Figures 21 to 23 are graphical representations of the results obtained in Example 7 for infecting cows with a strain of Klebsiella pneumonia: and

Figures 24-26 are graphical representations of the results obtained in Example 8 in which cows with coliform mastitis were treated with G-CSF.

Figures 27 to 29 are graphical representations of the results obtained in Examples 9-11.

Example 1 a) Screening of a bovine G-CSF genomic library cDNA clone encoding human G-CSF as described in U.S. Patent No. 4,810,643 was used to screen for a genomic clone containing the bovine G-CSF gene. -nin. The bovine genomic booklet-35 to in the phage (Charon 28), prepared by Woychick et al., Nucleic Acids 22 1 0 3 3 4 2

Research 10 (22): 7197-7210 (1982) and obtained from J. Bloom and F. Rothman, were plated in E. coli strain K802 and screened using a nick translated probe containing human G. 5 cDNA fragment of CSF isolated from HgiAI site to StuI site. A total of about 1.2 x 10 6 phages were plated on 12 15 cm Petri dishes, plaques were collected and hybridized to the probe using the methods described in Maniatis et al., Molecular Cloning, A Laboratory Manual 10 (1982). A total of 7 positive clones were found. The three clones which gave the strongest signals in autoradiography after the second screening were grown in 1 liter cultures and phage DNA was prepared as described in Maniatis et al., Molecular Cloning. A Laboratory 15 Manual (1982). This DNA was mapped by restriction enzyme digestion and Southern blotting using a HAIAI-StuI radioactive probe. The mapping results showed that a BamHI fragment of about 3000 bases contained the entire G-CSF region. DNA from clone 2 was digested with BamHI-ent-20 enzymes, releasing an approximately 3000 bp bovine G-CSF containing fragment, which was subsequently subcloned into pUC9 and further mapped by restriction endonuclease digestion and Southern blotting.

A restriction endonuclease map of genomic DNA containing the bovine G-CSF gene (about 3.0 Kb) is shown in Figure 1. The entire sequence of the coding region for bovine mature G-CSF was determined by subcloning the fragments into M13 and sequencing them by the dideox method Sanger et al., Proc. Natl. Acad. Sci. 30 USA. 74: 5463 (1977). The sequences were confirmed or expanded using the primers within the same clones. The sequence of the coding region was deduced by direct comparison with the human genomic G-CSF sequence (Figure 3) and is shown in Figure 2a. The mRNA formation cut-off sites and protein amino acid processing were expected to occur at the same sites as human G-CSF. The DNA sequence encodes a mature protein of the same length as human G-CSF (174 amino acids), and the proteins are 82% homologous.

5 b) Construction of bovine synthetic 6-CSF genes and expression of bovine G-CSF

The preparation of produced genes encoding bovine G-CSF and containing codons favored by E. coli and Expression of bovine G-CSF are described.

The synthetic genes were designed to allow expression of bovine granulocyte colony stimulating factor (bG-CSF) in E. coli (bG-CSF dna (Fig. 4) and bG-CSF dna4 (Figs. 5-7)). bG-CSF is 174 amino acids long and 82% homologous to human G-CSF (174 amino acids). The bovine protein contains five more charged residues (arg and Glu) than hG-CSF and is therefore believed to be more hydrophilic.

The bG-CSF dna (Fig. 4) and bG-CSF dna4 (Figs. 5-7) 20 genes were designed to have the highest possible tendency for codons favored by E. coli. In addition to the coding region, the bG-CSF dna gene included an initiating ATG, a ribosome binding site, two Lope termination codons, and a 5 'Xba I and 3' Bam HI restriction site. The bG-CSF dna4 gene was also designed to have the smallest possible secondary interactions and enough single restriction sites to insert subunits and handle the gene. Individual BamHI and PstI sites were included in positions identical to those found in the hG-CSF gene disclosed in U.S. Patent No. 4,810,643. This allows unique human / bovine hybrid genes and their protein products.

The gene was designed as two subunits (subunit-35 unit I XbaI-HindIII and subunit II HindIII-EcoRI) for expression / expression vectors for cloning (Figure 6). Subunit I contains a short 'leader' sequence having an XbaI cloning head and a ribosome binding site (RBS). Subunit II contains a redundant stop codon pair and an EcoRI cloning head.

Briefly, the methods used were generally as described in the jointly owned disclosure with Alton et al., PCT Publication No. WO83 / 04353, which is incorporated herein by reference. The genes in the mouth were constructed by first assembling the partial oligonucleotides (Figure 5) into multiple duplexes, which in turn were assembled into two distinct regions (Figure 6). These regions were designed to be easily amplified and so that when removed from the amplification system, they can be sequentially assembled or through several fragment ligation steps into a suitable expression vector.

The construction of regions I and II is illustrated in Figures 5 and 6. When constructing region I as shown in Figures 5 and 6, 16 oligonucleotides were assembled into 8 duplexes. 8 duplexes 20 were then ligated to form region I. From Figure 6, it can also be seen that region I contains an upstream end of Xbal enzyme of various lengths and a downstream HindIII enzyme having useful lengths of amplitude, useful for amplification and amplification. expression vectors and region II for ligation.

Region II was constructed as shown in Figures 5 and 6. For this construct, 16 oligonucleotides were assembled into 8 duplexes. The 8 duplexes were then ligated to form region II as shown in Figure 6. As also shown in Figure 6, region II contains an upstream HindIII enzyme having different lengths of strand length and downstream an EcoRI enzyme having different lengths of strand length, useful for amplification. and expression vectors and region I for ligation.

25 103342

Although any suitable vector can be used to express this DNA, the expression plasmid pCFM1156 can be easily constructed from plasmid pCFM836, the construction of which is described in published European Patent Application Publication No. 136,490 and U.S. Patent 4,710,473, both of which are incorporated herein by reference. pCFM836 is first cleaved with NdeI and then made flat with Poli so that both existing NdeI sites are destroyed. Next, the vector kat-10 is banded with Clal and SacII to remove the existing poly-linker region before ligation with the replacement polylinker region. This replacement polylinker region may be constructed according to the method described by Alton et al., Direct. The expression control is effected in pCFM1156 by the lambda PL promoter, which may itself be under the control of the CI857 repressor gene (one in E. coli strain K12 Htrp or FM5). FM5 is described in Burnette et al., BIO / TECH NOLOGY Volume 6, page 699 (1988).

Region I was initially cloned into M13 from the XbaI site

HindIII and sequenced by the dideoxy method described by Sanger et al., Proc. Natl. Acad. Sci. U.S. 74: 5463 (1977). Region II was cloned into M13 from HindIII to EcoRI and also sequenced by the dideoxymene method. Region I was cleaved from M13 at Xbal site

The HindIII site and region II were cleaved from M13 HindIII site to EcoRI site. The two fragments were then ligated with pCFM1156 cleaved from XbaI to EcoRI and transformed into E. coli strain FM5 (or other suitable E. coli host cells known to those skilled in the art) to form pCFM1156bG-. CSF.

This plasmid contains the lambda PL promoter / operator region and has a heat sensitive replication. When e.

35 coli strain FM5, containing pCFM1156bG-CSF, blots at 28 ° C, plasmid copy number remains at 10-20 copies / cell, and transcription from the lambda PL promoter is regulated by a thermosensitive repressor . Growth at 42 ° C results in increased copy number and release of the lambda PL promoter from repression. Recombinant bovine G-CSF begins to accumulate at elevated temperatures as a result of promoter activation and plasmid amplification. The Lambda PL promoter is located just upstream of the bovine G-CSF ribosome binding site and the start methionine codon. The trans-10 transcriptional terminator, t-oop, is located just downstream of the two translational termination codons near the 31 end of the gene. Strains containing plasmid pCFM1156bG-CSF express f-metbG-CSF such that up to 5% of the total cellular protein is present.

To increase expression of bovine G-CSF, the synthetic gene was cloned from pCFM1156bG-CSF to pCFM536 from XbaI to Koni to form pCFM536bG-CSF1. This construct was transformed into FM5 cells and bovine G-CSF was expressed such that up to 10% of the total protein was present. Using the oligonucleotide TTA ATA ATG ATC CCA TTA GGT CCT GCA CGT TCT, targeted mutagenesis (see Method 3) was performed to convert the proline, leucine and glycine codons at positions 3, 4 and 5 from CCG, CTC and GGC. from CCA to TTA to GGT. This new construct, pCFM536bG-CSF2, was transformed into FM5 cells and with this construct bovine G-CSF was expressed such that up to 30% of the total protein was present.

c) Purification of bovine G-CSF produced by E. coli 1. Cell lysis and Sarkosyl lysis and oxidation

About 100 grams of cellular paste were weighed out and resuspended in 2.5 liters of water. The cells were dispersed in a suitable mixer until completely dispersed. The suspension was run through a Gaulin Homo 103342 generator four times at 8000 psig while maintaining the temperature below 18 ° C. The homogenate was centrifuged in a Beckman J2-21 centrifuge using a JA-10 rotor at 10,000 rpm at 4 ° C for 30 minutes. The supernatant was poured out and discarded. The pellet was resuspended in 2.5 liters of cold water and centrifuged in a J2-21 centrifuge using a JA-10 rotor at 10,000 rpm at 4 ° C for 30 minutes. The supernatant was discarded and discarded. The pellets were resuspended in 380 ml of 10 water and added with 20 ml of 1 M Tris pH 8, 100 ml of 10% Sarkosyl and 0.5 ml of 1% copper sulfate pentahydrate. The mixture was stirred overnight at room temperature. The material was centrifuged in a J2-21 centrifuge using a JA-10 rotor for 30 minutes at 10,000 rpm at 15 ° C. The supernatant was discarded and saved.

The pellets were rejected.

2. Removal of Sarcosyl by Dowex Approximately 500 ml of cold water and 1 liter of cold 20 mM Tris solution, pH 8, plus 400 grams of Dowex (equilibrated in 20 mM Tris solution, pH 8) were added to the supernatant. The mixture was stirred at 4 ° C for 90 minutes. The broth was filtered through a column and the effluent was collected. The resin was washed with 400 ml of a 20 mM Tris pH 7.7 solution and this wash was added to the flow-through liquid to give 2.4 liters.

3. Fast-flow CM-Sepharose chromatography

The pH of the material was adjusted to 5.4 using 50% acetic acid and then centrifuged in a J2-21 centrifuge using a JA-10 rotor at 10,000 rpm at 4 ° C for 25 minutes. The supernatant was discarded and saved. The pellet was rejected. The supernatant was directly added to an 80 ml CM-Sepharose ion exchange column equilibrated in 20 mM sodium acetate, pH 5.4. Pyl-35 resin was washed with 100 mM NaCl in starter buffer and then eluted with 28 103342 in 150 mM NaCl in starter buffer. Approximately 850 mL of eluate containing bovine G-CSF was collected.

4. Diafiltration The pH of the CM-Sepharose column material 150mM NaCl eluate was adjusted to 3.5 with 0.1N HCl and then diafiltered using a tangential flow ultrafiltration device (Pellicon) equipped with 10,000 MW membranes. (0.35 mM HCl-water, pH 3.5). The final volume was adjusted to obtain a concentration of bovine G-CSF of 2 mg / ml.

Example 2

Construction of Bovine G-CSF Analogs This example relates to the use of recombinant DNA methods for the generation of bovine G-CSF analogs in which 15 at cysteine at 17 were replaced by serine.

The site-directed mutagenesis methods described by Souza et al., PCT WO85 / 00817, published Feb. 28, 1985, incorporated herein by reference, were performed using the oligonucleotide CTGCTGAAATCCCTCGAACAG.

Purification of 20 G-CSF Bovine Analogues Produced by E. coli 1. Cell lysis and urea lysis and oxidation

About 100 grams of cellular paste were weighed out and resuspended in 2.5 liters of water. The cells were dispersed in a suitable mixer until they were completely dispersed. The suspension was passed through a Gaulin homo generator four times at 8000 psig, keeping the temperature below 18 ° C. The homogenate was centrifuged in a J2-21 centrifuge using a JA-10 rotor at 10,000 rpm for 30 minutes at 4 ° C for 30 minutes. The supernatant was discarded and discarded. The pellets were resuspended in 2.5 liters of cold water and centrifuged in a J2-21 centrifuge using a JA-10 rotor at 10,000 rpm at 4 ° C for 30 minutes. The supernatant was discarded and discarded. The pellets were resuspended in 120 ml of 29 1 0 3 3 4 2 water and 40 ml of 1 M Tris pH 8.5 and 640 ml of 10 M urea were added. The mixture was stirred for 2-3 hours at room temperature and then diluted 1:10 with 20 mM Tris, pH 8.5. The mixture was then stirred overnight at room temperature. The material was centrifuged in a Beckman J6-B centrifuge in a JS 4.2 rotor at 4200 rpm for 4 minutes at 4 ° C. The supernatant was discarded and saved. The pellets were rejected. The supernatant was adjusted to pH 5.4 with 50% acetic acid and the material was centrifuged in a J6B centrifuge using a JS 4.2 rotor at 4200 rpm for 4 minutes at 4 ° C. The supernatant was discarded and saved. The mixture was then directly applied onto a 100 mL CM-Sepharose ion exchange column equilibrated in 20 mM sodium acetate, pH 5.4. The column was washed with 100 mM NaCl in the initial buffer and then eluted with 150 mM NaCl in the initial buffer. Approximately 775 ml of bovine G-CSF containing eluate was collected.

2. Diafiltration The pH of the CM-Sepharose column material, 150 mM NaCl, was adjusted to 3.5 with 0.1 N HCl and then diafiltered using a tangential flow ultrafiltration device (Pellicon) equipped with 10,000 MW membranes. , 0.35 mM HCl-water, pH 3.5. The final volume was adjusted so that the concentration of bovine G-CSF &gt; 25 was 2 mg / ml.

Example 3

Treatment of Pasteurella hemolytica infection A G-CSF study was performed on cattle exposed to Pasteurella hemolvtica. Calves weighing approximately 60 kg 30 were divided into two groups of 11 animals. Both groups were given 5 ml of bacteria (3 x 10 7 / ml) intranasally on day 1. Four hours prior to challenge, the calves were treated with dilute acetic acid (pH about 5.5) by gavage. In the group receiving 35 subcutaneously administered human G-CSF (10 μg / kg), 30 103342 were pretreated 4 days before infection and treated daily for the remaining 9 days of the study.

All livestock except one of the 5 animals in the control group showed clinical (subjective scoring) and histological signs of pneumonia. Although G-CSF treated cattle were not protected against infection, their clinical status at the end of the study was superior to that of the control group based not only on their clinical pips but also on their lung pathological scores (%). In addition, 3 cows from the control group died while none of the treated groups died. Figure 8 shows white blood cell count (WBC) as a function of time. Figure 9 shows the neutrophil count as a function of time. Ku-15 Figure 10 depicts the number of band cells as a function of time. The amount of neutrophils includes the total number of Seg's (the most mature neutrophils) and Band's (slightly less mature than the Seg's). Figure 11 shows the morbidity as a function of time and Figure 12 shows the percentage of lung effect (% -20 th lungs affected) as a function of time.

Example 4 Use of G-CSF as a preventive / therapeutic agent for controlling bovine respiratory disease The purpose of this study was to demonstrate the efficacy of human G-25 CSF in controlling bovine respiratory disease (BRD) caused by Pasteurella hemolytica. The groups were as follows (10 calves / group):

Group 1: infected, non-medicated Group 2: infected, medicated (started 5 days-30 before exposure)

Group 3: Infected, Medication (started 12 hours prior to challenge)

1-2 day-old Holstein bull calves were obtained. The calves were housed in separate calf-35 calves and their diet started with a 1 gallon of milk at a daily dose of 10,103,342 divided into two doses per day. At 7 days, free calf grain was provided and at 7 days water was provided freely.

Five days prior to challenge (day -5), all calves in Group 2 received 10 mg / kg / calf G-CSF administered SQ, SID. 12 hours prior to challenge (day -1/2), Group 3 calves received 10 µg / kg / calf G-CSF administered SQ, SID. Group 1 calves received solution control after al-10 hand exposure (day 0). The calves received medication until day 9 (the day before the end of the study).

The drug was administered as a 0.27 mg / ml solution and injected with 10 µg / kg, SQ, SID. Solution control was administered by injection of 2 ml, SQ, SID.

The treatment levels were as follows: Group 1: Solution Control, Group 2: G-CSF 10 Mg / kg, SQ, SID from day -5, Group 3: 10 µg / kg, SQ, SID.

Calves were challenged at 25 days (day 0). Four hours prior to challenge, the calves were treated with 20 dilute acetic acid (pH about 5.5) to give a human horn. Exposure to Pasteurella hemolvtica occurred by injection of 5 ml of overnight broth culture with 1 x 108 exposure organisms. Calves were observed for a total of 10 days.

On day 10, the calves were sacrificed and autopsy was performed. The lungs were scored for pneumonia and cultured for bacteriology. Figure 14 (see Figure 13) shows the relative morbidity of the three groups. The morbidity was based on body temperature, food intake and mood. Figures 15, 16, and 17 depict WBC (white blood cell count), PMN (neutrophils), and the number of band cells as a function of time, respectively.

32 1 0 3 3 4 2

Example 5 Effect of G-CSF on Frequency and Duration of Calf Respiratory Disease and Immunological and Clinical Pathology Parameters The purpose of this study was to determine the effect of administration of human G-CSF on the frequency and duration of respiratory disease of calves. Parameters checked include morbidity, mortality, complete blood counts and antigen response and antigen recall. The study was performed on a commercial calf cattle.

Sixty (60) Holstein oxen calves, approximately 21 days old, were acquired and placed in their calf duct over a period of three days (day -3 to day 15 -1). When the calves were placed in the barn, they were assigned to one of two groups. The calves were placed in separate calf boxes with 30 pens per row, two rows and a 4-foot wide corridor between rows. Calves were placed alternately. Every day you place -20 an even number of calves. The calves were examined on the day they were placed in the barn. Day 1 of the study was when the calf was actually placed in its barn. In other words, there was 1 day for 3 days. I'm stating the actual start date. All of the following 25 days came down to the actual start date.

Starting on day 1, Group 1 calves received a daily injection of 10 µg / kg G-CSF. Group 2 calves received daily injections of solution control. Injections were continued for 21 consecutive days.

30 Starting day 1, the animal health technician monitored the calves daily for signs of respiratory disease. Physical signs observed were body temperature, appetite, respiratory rate, and mood. Monitoring was continued for 30 consecutive 35 days (Day 1 to Day 30). All calves showing signs of respiratory disease (see criteria for diagnosis below) were placed in therapy according to the treatment schedule below. All treatments were recorded. All calves that died during study AI-5 were autopsied.

On days 1 (before the start of injections), 7, 14, 21 and 28, all calves were bled for complete blood counts (CBC). In addition, blood was drawn for CBC on days 1 and 4 of respiratory disease. The 10 experiments performed included packed cell volume decrease (PCV), total red and white cell counts, and differentiated cell counts.

All calves who were ill were treated with therapy. Duration and type of therapy were recorded in 15 records.

Comparisons were made between groups 1 and 2 regarding the effects of G-CSF on complete blood counts, respiratory disease morbidity as determined by daily examinations, and mortality.

34 103342

RESPIRATORY DISEASE MONITORING CRITERIA

NORMAL ABNORMAL

BODY TEMPERATURE <103.5> 103.5 5 appetite 1 2, 3 and 4 1 = drink whole amount of milk 2 = drink 3/4 of milk 3 = drink 1/2 of milk 10 4 = drink <1/2 of milk INHALATION RATE 1 2 and 3 1 = normal 2 = slightly elevated 15 3 = extremely elevated SENSE 1 2, 3 and 4 1 = normal 2 = slightly depressed 20 3 = moderately depressed 4 = severely depressed Treatment schedule Treatment number 1: 5 mg oxytetracycline / nail,. IM. Duration of therapy was based on calf response. Therapy was continued for 2 days after the parameters had normalized with the shortest treatment being 4 days and the longest 8 days. If calves did not respond to treatment number 1, treatment number 2 was initiated.

30 Treatment number 2: 25 mg sulfadimethoxin / pound, • PO, on the first day, 12.5 mg / pound on the second and following days. The maximum treatment time was 5 days.

Figure 18 shows treatment days for Group 1 calves (who received G-CSF) who became ill on the first day of treatment (T1), on the second day of treatment (T2), ... on the first day of the second treatment (T1B). , ...

another treatment on day 5 (T5B). The treated group consistently had a smaller number of size-5 female treatments at each treatment point. There is little tendency between treated and controls for larger differences as the number of treatments increased. For example, starting at T4, the difference between treated and control calves increases by about 40% compared to 15% in prior treatments.

Figure 19 depicts the total number of treatments per day for controls compared to treated calves. This again shows a decrease in the number of treatments the treated calves need compared to controls. 15 Healing sometimes ranges from almost 50% to 0%. The total recovery percentage for treated calves is 36%.

Figure 20 depicts the number of calves per day with temperatures above 103.5 ° F. The results are very consistent with the total treatment days. There is a consistent improvement in the temperature of the treated calves compared to the controls. The percentage improvement is approximately the same as the total treatment days (39.9%).

. G-CSF, when administered to young fattened calves before and during infection with BRD, significantly reduces the severity and length of respiratory disease compared to controls treated with a pseudo-drug. Calves treated with G-CSF had less respiratory disease (27%) compared to controls and also became healthier faster (47% improvement).

Example 6

Klebsiella pneumonia The subject of this study was to determine whether the proliferation of circulating neutrophil group cells would allow the dairy cow to respond more appropriately to bacterial exposure to the mammary gland.

G-CSF PROTOCOL: 1) Human G-CSF administration started two days after arrival and initial screening (evening milking) and continued for a total of 15 days during evening milking.

2) Dose / route: 10 μg / kg / day administered subcutaneously.

3) The control exposure group received 1.0 µg / kg G-CSF (daily) by subcutaneous administration for 8 days starting on day 12.

UTITUDE EXPOSURE MODEL: 1) Organism: Klebsiella pneumoniae was administered on the morning of the sixth day after the start of G-CSF administration.

2) Dose: 1.0 ml (about 200,000 CFU) was aseptically administered via the viral duct to the left hindquarter (D). The remaining quarters were used as controls.

3) No milk samples were taken for 3 hours after exposure to allow the colonization of the bacteria to a quarter.

4) Antibiotic therapy was not used during the experiment, except for fluid therapy if necessary.

DOG GROUPS: • · 25 Tx exposure (n = 3) cow ear tag numbers: 736, 924, 964

Tx control (n = 3) cow ear tag numbers: 248, 293, 790

Control Exposure (n = 3) Cow ear tag numbers: 30 124, 771, 4179 'Representative results are shown in Figures 21-23.

24 hours after exposure, an independent physician performed a palpation test on all 36 udders. The doctor extracted the infected quarters from a total of 36 quarters; 37 103342 these were exposed quarters of three untreated cows.

Example 7

Method for the prevention of coliform mastitis This example relates to a method using G-CSF for the prevention of coliform mastitis in dairy cows. To test the efficacy of human G-CSF in preventing coliform mastitis in 10 dairy dairy cows, the cows were subjected to experimental challenge with pathogenic E. coli.

12 Holstein dairy cows in the middle of milk production were randomly assigned to one of two groups. In the pilot study, only cows whose milk cultures were negative for coliforms and negative for CMT (California Mastitis Test) were used. The two groups were: Group 1 (infected, non-medicated controls) and group 2 (infected, treated with 3 μg G-CSF / kg).

In the basic procedure, the cows were treated with G-CSF (Group 2) on days 1 to 17, then challenged on day 10, and finally monitored for mortality and morbidity on days 11 to 21. Group 1 received saline injections on days 1-17.

• «38 103342

The specific timetable was as follows:

DAY G-CSF CBC1 CMI2 SCC3 MILK-PLASEBO BODY

CULTIVATION TEMPERATURE

5 0 / / / / / 1 / / 2 / / 3 / / 4 / / 10 5 / / Il 6 / / 7 / / 8 / /

9 / / III

15 10 / // // // /

exposure

11 / / III

12 / / III

13 // Il 20 14 / / / / / / 15 / / / / / 16 / / Il 17 / / Il 18 / / 25 19 II / 20 / / 21 111 / / “• Complete Blood Bills, California Mastitis Test, 3Soma -30 cell counts obtained from HMS 'All cows were observed twice daily (morning and afternoon) and all abnormal clinical findings (namely, appetite, signs of inflammation, walking, mood, etc.) were recorded. The uta-35 thighs of cows were examined once daily (in the morning) throughout the 39 1 0 3 3 4 2 study period, recording abnormal findings. All injections of experimental and control configurations were administered between 8 and 10 am in the morning. The cows were milked twice a day and all milk was rejected. No other medications were given during the study.

The challenge included intravenous infusion of a virulent culture of E. coli into one quarter of a mammary gland. Exposure was titrated before the start of the study to produce a median morbidity.

10 Cows were fed until day 31 (14 days after the last dose of G-CSF) and then sold for slaughter. All cows that died during the study were necropsied and sold.

Comparisons between control and treatment groups were made regarding the efficacy of G-CSF in preventing cows from infecting coliform mastitis due to intracellular infusion of E. coli. The results are shown graphically in Figures 24-26. Milk culture points (1 = 1-100 E. coli colonies; 2 = 100-500 E. coli colonies; 3 = more than 500 E. coli colonies) ( 24), clinical points that are milk points (0 = normal milk, 1 = watery / lumpy, 2 = more watery / lumpy, 3 = very watery / lumpy), and · udder points (0 = normal, 1 = slightly swollen / hard, 2 25 = moderately swollen / hard, 3 = severely swollen / hard) (Figure 25) and California Mastitis Test1 in (CMT) score (negative and blur = 0) (Figure 26) indicates that treatment G -CSF under the experimental conditions described above was highly effective in reducing the severity of the disease and accelerating the healing of milk producing dairy cows. Thus, an effective treatment rate for treating mastitis was shown to be 3 μg / kg.

40 1 0 3 3 4 2

Example 8

Hemogram changes in milk-producing dairy cows administered granulocyte colony stimulating factor prior to induction of coliform (E. coli) breast inflammation The original purpose of this study was to determine whether treatment with G-CSF would accelerate the healing of experimental mastitis. Inoculations given in this experiment did not produce significant clinical signs and all cows cleared the infection within 24 hours after inoculation. Therefore, the therapeutic strength of G-CSF could not be evaluated. The final topic of this study was to evaluate the haematological changes induced by human G-CSF at a dose of 3 μg / kg in bovine peripheral blood.

Eight healthy Holstein cows, which were in the second half of their milk production, were infected by inoculation of an E. coli isolate of the mammary gland. An E. coli iso-20 plate from mastitis, grown in tryptocase-soy broth for 14-16 hours, was used. Four cows were given G-CSF, the remaining four formed an infected control group.

,, Initially, an inoculum containing 803.7 CFU was administered internally to the right 25 quarter of the thyroid gland. This failed to induce clinical mastitis. The second and third inoculations containing 44,000 CFUs and 842,000 CFUs, respectively, were administered 2 days later and every 6 hours. The second and • 30 third inoculations used the left hindquarters. Time zero was taken at the start of the second inoculation.

Four of these 8 cows were randomly selected and injected subcutaneously with human G-CSF approximately 6 hours after inoculation. A single injection of 35 was given to each of these cows after evening milking for 4 to 10 days. The dose used was 3 Mg / kg each time. The remaining 4 cows exposed to E. coli received no treatment.

Baseline hematology was obtained for three consecutive 5 days prior to treatment and days 0-12 after E. coli inoculation. Blood samples were collected from tail vein into EDTA containing tubes.

The hematogram included assays for total white blood cells (WBC), differentiated cells, total red blood cells (RBC), packed cell volume (PCV), and plasma proteins (PlPr). All specimens were treated within 3 hours of collection and separated from Wright-Giemsa-stained specimens. Red and white cell counts were performed using a Coulter-las-15 discipline model F.

None of the cows showed systemic signs of disease during the experiment. About 6 hours after inoculation, an infected quarter of some cows swelled, became red and solid. The swelling of the quarters to 20 of these cows was only mild at 12 hours.

Hemograms of the 4 cows administered G-CSF were characterized by moderate leukocytosis within 24 hours of the first injection. Observed in the jaw. cytosis consisted mainly of mature, non-toxic 25 neutrophils. On the second day of treatment, band neutrophils were detected in peripheral blood. The largest change to the left was 2.5 x 103 / μ1 band cells observed in cow number 768. The time at which the maximum change to the left occurred was proportional to the time at which segmented neutrophils reached their highest value. The dose interval was short enough to cause an additional effect of the drug on neutrophil counts that did not return to baseline levels between injections. When the injections were stopped, the cell count decreased at the same rate as the elevated-35 nuts.

42 103342

At about day 7, the bills returned to a level about 2-3 times the baseline value and remained at that level for the rest of the processing time.

Maximum white blood cell counts and segmented-5 neutrophil counts were observed at approximately day 5. The following table shows these cell counts for treated and untreated groups at day 0 and day 5. The magnitude of change in cell counts between days 0 and 5 is given only to the treated group.

10 UNREGISTERED QUANTITIES (x 103 / μ1) COW NUMBER Day 0 Day 5

Seg WBC Seg WBC

15,326 3.8 11.6 2.5 8.8 801 2.9 8.3 2.4 7.4 574 4.4 11.1 1.9 6.8 825 3.5 10.2 2.8 8.3

20 G-CSF PROCESSED

QUANTITIES (X 103 / μ1) COW NUMBER Day 0 Day 5% increase

Seg WBC Seg WBC Seg (%) WBC (%) 768 2.9 11.7 8.9 21.1 300 180 25 767 4.7 10.4 21.5 28.44 500 270 773 4.2 11 22, 6 32.3 500 290 324 4.6 10.3 21.8 32.6 500 320 The untreated group had significant changes in cell numbers between days 0 and 5. Treated: The group had 3-5 fold increases in neutrophil counts and 2-3 fold increases in WBC counts.

Some cows (326, 801, 574, 825, 767, 768) showed a slight decrease in their WBC levels 6 hours after the inoculation.

43 1 0 3 3 4 2 In the treated group, cow number 324 had high normal WBC and PIPr and low normal PCV before and during the experiment. These laboratory abnormalities, combined with mild mucous membrane nasal discharge, suggested the existence of a 5 chronic disease process. Interestingly, this cow had the highest increase in neutrophil count (500%) with the smallest maximum left change (<400 / μ1).

There were no changes in the other 10 white blood cell counts of treated and untreated cows during the experiment. In addition, no changes in red blood cell or plasma protein parameters were observed in either group.

It was concluded that: 1. Human G-CSF stimulates granulopoiesis in bovine neutrophils.

2. Human G-CSF administered at a daily dose of 3 mg / kg causes a 3-5 fold increase in segmented neutrophils after 4 days with only a slight leftward change.

3. Poor experimental udder inflammation in one quarter does not worsen G-CSF-induced bone marrow stimulation.

. The following examples were designed to determine whether G-CSF therapy could prevent mortality in animal models of infectious disease.

HAMSTER MODEL OF PSEUDOMONAS AERUGINOSA-PNEUMONIAN

Bacteria The P. aeruginosa strain PA02 used in these experiments was isolated from the blood of a human patient with a stomach infection.

The PAO2 strain was cultured for an exponential phase in trypticase-soy broth (TSB) (Difco) and then small amounts of this culture were stored frozen at 70 ° C in 40% glycerol until used. 1.5 ml volumes of 35 PAO2 strains were thawed, added to 50 ml TSB, and incubated in shaker flasks at 37 ° C for 44 hours. Logarithmic growth phase organisms were harvested by centrifugation at 2000 x g, washed twice with sterile 0.9% saline, and resuspended to the desired concentration as determined spectrophotometrically. All bacterial inocula were amplified by quantitative cultures using a spiral plating apparatus (Spiral Systems, Cincinnati).

Animals 10 Virus-free female Syrian hamsters, weighing 80-100 grams, were obtained from Charles River, Canada (Quebec) and kept in quarantine for 5 days. Groups of 5 animals were raised in cages equipped with separate swamp-15 data lids. The animals were given free access to water and standard hashster food (Tecklad, Winfield, Iowa) until evening before infection. Then the animals were fasted overnight. Recombinant human granulocyte colony stimulating factor (G-CSF) or administration solution (5% dextrose in sterile water) was administered subcutaneously at a total volume of 0.1 ml. Animals were given G-CSF or bid twice daily (bid) at about 8 am and 3 pm. G-CSF injections were continued in surviving animals for 3 days after infection. The animals were infected between 12-2 in the afternoon.

For intracerebral inoculation, the hamsters were anesthetized by intraperitoneal administration of 75 mg / kg Nembutal (50 mg / ml; Abbott). Animals were placed at a 30-degree inclined plane, jaws opened, and 0.25 ml of bacterial-30 suspension administered into the trachea using a bent 22-gauge needle with a 2.5-inch ground tip. After bacterial inoculation, the animals were gently shaken for 15 seconds to apply the inoculum. The animals were allowed to recover from anesthesia at 37 ° C on a heat-soft-35 pathway. Only animals that recovered from anesthesia were used in the experiment. The animals were inspected regularly. Animals showing very high morbidity symptoms (unable to walk or stand up) were painlessly blindfolded by an in-house observer and killed. Example 9 Use of G-CSF as a Prophylactic / Therapeutic Agent for the Prevention of Bacterial Pneumonia in Hamsters The purpose of this experiment was to determine the efficacy of various G-CSF an-10 knots in preventing lethal respiratory infection by Pseudomonas aeruginosa. The groups were as follows (15 hamsters / group):

Group 1: infected, non-medicated (anti-diluent only) Group 2: infected, medicated with G-CSF 5 µg / kg bid

Group 3: infected, medication with G-CSF 15 Mg / kg bid

Group 4: Infected, G-CSF Medication 50 µg / kg bid 20 The animals were dosed one day prior to bacterial challenge. Thus, the animals received 3 doses prior to infection and the surviving animals continued to receive G-CSF bid. Approximately 5 x 10 7 colony forming units (CFU) were administered to each hamster. Ku-25 Figure 27 shows the relative mortality of each group over time. All three hamster groups receiving G-CSF had significantly less mortality than animals receiving the administration solution alone.

All groups had a significant treatment effect of 30 (p <0.001) as calculated by Log Rank Chi Square analysis. Hamsters pretreated with G-CSF at 5 μg / kg bid prior to infection had no significantly reduced mortality compared to control animals. However, animals pretreated with 15 µg / kg G-CSF bid were significantly different from the controls (p <0.001). Animals <103342 receiving 50 μg / kg bid G-CSF also had significantly reduced mortality (p <0.001). There was no significant difference between 15 mg / kg bid and 50 µg / kg bid.

Example 10 Effect of G-CSF Dosage Schedule on Mortality of Hamsters with Pseudomonas aeruginosa Pulmonary Infection The purpose of this experiment was to determine the amount of time required between G-CSF therapy initiation and infection exposure to achieve reduced mortality.

Group 1: Infected, no medication (solution only)

Group 2: infected, medication 15 μg / kg bid, starting about 30 hours before infection. The animals received 3 to 15 doses of G-CSF prior to challenge.

Group 3: Infected, medication 15 μg / kg bid, starting 5 hours prior to challenge.

The animals were administered approximately 5 x 10 7 CFU of PAO 2 intranasally. G-CSF dosing continued according to bid schedule 20 for 3.5 days in surviving animals. Figure 28 shows mortality over time in all animal groups. Both treated groups had significantly reduced mortality compared to non-treated animals.

All groups had a significant treatment effect (p <0.001) as calculated by Log Rank Chi Square analysis. Hamsters pretreated 5 hours prior to infection had significantly reduced mortality (p <0.001) compared to control animals. Hamsters that were age-matched 30 hours before infection were also significantly different (p <0.001) from control animals.

Rat model of Candida albicans ovelonephritis Organism The clinical isolate of Candida ai-35 bicans used in this experiment was maintained on Sabouraud's dextrose agar 47 103342 oblique surfaces. C. albicans strain was grown overnight in Sabouraud dextrose broth. The C. albicans strain was isolated by centrifugation at 2000 x g for 10 minutes at 4 ° C. The pellet was washed twice with phosphate buffered saline (PBS), resuspended in PBS, and adjusted to the desired concentration after counting using a hemocytometer. Quantitative cultures were used to verify all inoculations.

Animals 10 Female Sprague-Dawley rats weighing 150 to 200 grams were obtained from Harlan-Sprague-Dawley,

Inc. (Indianapolis, IN) and were quarantined for 5 days. Groups of 4 animals were raised in hanging wire cages and allowed free access to standard rat food and water. The animals were given G-CSF or administration solution (PBS) twice daily starting on the day before challenge. The surviving animals were treated with G-CSF or the administration solution for bid for 10 days.

For intravenous inoculation, rats were anesthetized with ether. A volume of 0.1 ml of C. albicans was injected into the tail vein.

Group 1: infected, no medication (· solution for administration only) Group 2: infected, mediated with G-CSG 50 μg / kg bid starting approximately 30 hours prior to infection.

Figure 29 shows mortality over time in each animal group. The animals were given 1.3 x 10 6 CFU of C. al-bicans strain. There were no deaths in the group that received G-CSF.

All groups had a significant treatment effect (p <0.01) as calculated by Log Rank Chi Square analysis.

Animals pretreated with G-CSF had a significantly reduced mortality rate (p <0.001) compared to 4β 103342 control animals. Rats treated with G-CSF after infection did not differ significantly from controls.

Example 11 5 G-CSF administered to growing pigs

Bovine G-CSF was administered to growing pigs (about 30 kg) as follows:

Dose = 5 mg / kg body weight / day Route of administration: subcutaneous injection of 10 n = 6 pigs

Neutrophil levels were as shown below: Day Neutrophils (Mean) 0 3108 2 29922 15 4 24595 6 42713

The study in 6 normal growing pigs showed a dramatic increase in neutrophil levels within 2 days and a 13-fold increase after 6 daily dosing for 20 days.

Example 12 G-CSF administered to normal newborn foals

Bovine G-CSF was administered to normal newborn foals starting at 1 day of age as follows: Dose = 10 and 20 μg / kg body weight / day Route of administration: Intramuscularly n = 5 foals Day 0 = Day before first G-CSF administration The neutrophil level was as shown below: 49 103342

neutrophil count

Foal Dose Day Day Day Day Day Day Day number (uq / kg) 0 1 2 3 4 5 1 20 4950 17473 28072 35524 30874 38263 52 10 6708 16195 27807 28560 26960 26535 3 10 5016 10472 6715 16112 19975 16464 4 10 6480 6225 4400 5610 10332 15372 5 10 9156 10406 11692 11304 13588 17300

In a study in which G-CSF was administered to 5 newborn 10 foals daily, neutrophil levels increased up to 3.5-fold within 24 hours and up to 7.5-fold within 5 days, depending on dose.

While the present invention has been described in terms of preferred embodiments, it should be understood that modifications and modifications may be discovered by those skilled in the art. It is therefore intended that the appended claims cover all such variations within the scope of the invention described in the claims.

Claims (6)

A method for isolating and purifying G-CSF from a G-CSF producing microorganism, wherein the 1. microorganism is degraded and insoluble G-CSF-containing material is separated from the soluble teiinimateriaalista, Process according to claim 1, characterized in that G-CSF is met-hG-CSF. 2. dissolving and oxidizing G-CSF in the presence of denaturing solvent and oxidizing agent, 3. separating the denaturing solvent from G-CSF using an anion exchange resin containing quaternary ammonium groups, such as Dowex® anion exchange resin, Process according to claim 1, characterized in that the denaturing solvent used in step 2) is a sarcosyl. Process according to Claim 1, characterized in that the oxidizing agent is CuSO 4. 4. G-CSF is subjected to anion exchange chromatography followed by cation exchange chromatography, and 5. purified G-CSF is recovered. A process according to claim 1, characterized in that the anion exchange resin is a Dowex® anion exchange resin. Process according to claim 1, characterized in that G-CSF is hG-CSF and after step 1) there is a step of extracting the G-CSF material with deoxycholate. 51 103342