WO1994006458A1 - Compositions mcsf a haute concentration - Google Patents

Compositions mcsf a haute concentration Download PDF

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Publication number
WO1994006458A1
WO1994006458A1 PCT/US1993/008897 US9308897W WO9406458A1 WO 1994006458 A1 WO1994006458 A1 WO 1994006458A1 US 9308897 W US9308897 W US 9308897W WO 9406458 A1 WO9406458 A1 WO 9406458A1
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WO
WIPO (PCT)
Prior art keywords
mcsf
concentration
concentrated
buffer
protein
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Application number
PCT/US1993/008897
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English (en)
Inventor
Jay A. Schrier
Richard P. Northey, Jr.
Original Assignee
Genetics Institute, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Genetics Institute, Inc. filed Critical Genetics Institute, Inc.
Priority to AU49310/93A priority Critical patent/AU4931093A/en
Publication of WO1994006458A1 publication Critical patent/WO1994006458A1/fr

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/193—Colony stimulating factors [CSF]

Definitions

  • Hematopoietins i.e., hematopoietic growth factors
  • Colony stimulating factors are a subset of these hematopoietic growth factors that are characterized by the ability to support the growth, in vitro, of colonies of hematopoietic cells arising from progenitor cells of bone marrow, fetal liver and other hematopoietic organs.
  • CSF-1 also known as macrophage colony stimulating factor (MCSF).
  • MCSF monocytic lineage type cells
  • Clark et al. U.S. Pat. No. 4,879,227
  • ADCC antibody-dependent cellular cytotoxicity
  • Mufson et al. Cellular Immunology.119:182-192 (1989)
  • Young etal. J. Immunology. 145:607-615 (1990)
  • lipoprotein cholesterol profiles in mammals Garnick, United States
  • Stability of proteins during concentration is often problematic, resulting in alterations due to such factors as pH, temperature, freezing/thawing and shear. As a result, during concentration, protein may be lost due to hydrolysis, aggregation and the formation of high molecular weight species.
  • stability of protein pharmaceuticals see Manning et al., Pharmaceutical Research. 6_:903-918 (1989).
  • the present invention comprises highly concentrated compositions of MCSF, preferably in a concentration sufficient for subcutaneous administration.
  • the present invention comprises bulk compositions of MCSF, preferably sufficiently concentrated to allow the preparation of MCSF in finished dosage forms for subcutaneous administration.
  • the present invention provides methods for concentrating pharmaceutical proteins, in particular, MCSF protein.
  • the present invention includes compositions of MCSF of a concentration sufficient for subcutaneous administration.
  • the compositions of the present invention contain MCSF in a concentration of at least about 20 mg/ml, and more preferably at least about 35 mg/ml.
  • the present invention further includes bulk compositions of MCSF in a concentration suitable for use in preparing finished dosage form compositions of MCSF suitable for subcutaneous administration.
  • the preformulated bulk drug composition may be concentrated to at least about 45 mg/ml MCSF, and more preferably up to at least about 120 mg/ml.
  • the present invention comprises finished dosage form compositions of MCSF suitable for subcutaneous administration.
  • the present invention further comprises methods for concentrating MCSF to concentrations sufficient for subcutaneous administration.
  • compositions of the present invention are suitable for therapeutic compositions for treating conditions such as leukopenia, and atherosclerosis, as well as other conditions related to high cholesterol levels.
  • Such compositions comprise a therapeutically effective amount of one or more additional members of the family of CSF-1-like polypeptides of the present invention in admixture with a pharmaceutically acceptable carrier.
  • This composition can be systemically administered either parenterally, intravenously or subcut- aneously.
  • the therapeutic composition for use in this invention is, of course, in the form of a pyrogen-free, parenterally acceptable aqueous solution.
  • the preparation of such a parenterally acceptable protein solution having due regard to pH, isotonicity, and the like, is within the skill of the art.
  • the preferred pharmaceutical composition comprises MCSF.
  • MCSF produces colonies that contain primarily macrophages.
  • Long form MCSF (LCSF-1) has been specifically described by Wong et al., Science. 235:1504-1508 (1987). Its production by recombinant DNA techniques is described in WO87/06954. See also EP 0,261,592;
  • a truncated version of CSF-1 is described in WO86/04607.
  • Kaswasaki et al., Science. 230:291-296 (1985) describe the cDNA sequence encoding that truncated protein. Variations on that truncated version having deletions or substitutions of hydrophobic amino acids characteristic of a transmembrane region are described in EP 0,249,477.
  • Methods for purifying CSF-1 proteins from natural sources are described in GB 2,016,477; and WO86/04587. See also, S. K. Das et al., Blood. 5.8:630 (1981); publications by E. R. Stanley; and references cited in WO86/04587.
  • An MCSF protein for purposes of this invention, ⁇ icludes the natural proteins, recombinant versions thereof, and derivatives and analogues thereof, which may contain amino acid deletions, substitutions and/or insertions, but retain the characteristic biological activity of MCSF; which is the ability to proliferate the growth of cells predominantly of the monocyte/macrophage lineage in the standard bone marrow assay of Wong, et al.,
  • compositions of this invention may be administered systemically, e.g. intravenously.
  • the therapeutic preparations for use in this invention should be in the form of non-pyrogenic, sterile, parenterally acceptable aqueous solutions.
  • a therapeutically effective dose of MCSF i.e. an amount sufficient to increase the
  • HDL cholesterol/LDL cholesterol ratio is in the range of 1-200 micrograms ⁇ g)/kg/day.
  • the presently preferred dose is in the range of about 5-150 ⁇ g/kg/day.
  • the methods of the present invention may involve a series of administrations of the pharmaceutical compositions. Such a series may take place over a period of about 7-14 days. One or more series may be administered. Additionally, it is contemplated that the methods of the present invention may be used in chronic treatment for maintaining an acceptable lipoprotein cholesterol profile in a mammal.
  • PVDF 0.22 ⁇ m filter The diluted protein concentration was 0.125 mg/ml by RP-HPLC and stored at 2-8 °C until use.
  • the filtrate fluxes ranged between 0.35 and 0.95 mL/min- cm -psi.
  • New filters were used with no pre-wetting. As filtration progressed, observed fluxes dropped slightly across all the filters tested. The reduced flux might indicate protein interactions with the filter materials. The bubble points were monitored.
  • the MCSF concentration in this solution was 4.4 mg/mL.
  • the solution was divided into two portions. One portion was sterile filtered. The other portion was subdivided again into two portions and concentrated by UF to 20 mg/mL.
  • the 20.0 mg/ml solutions were sterile filtered and set up on a stability protocol, using standard assays.
  • This material was split into four 2 L fractions and each sterile filtered with four commonly used sterile filters.
  • This sterile filtered material was pooled and concentrated by ultrafiltration (UF) to about 9x (to 1.08 mg/ml).
  • This concentrate was split again and sterile filtered, pooled again and concentrated to approximately 20 mg/ml.
  • a final sterile filtration was done on the second concentrate. Samples were saved following each step in the protocol for assay by SDS-PAGE, RPHPLC and size exclusion chromatography (SEC). The final concentrate was tested by differential scanning calorimetry (DSC) for retention of MCSF tertiary structure.
  • the filtration experiments were all conducted at room temperature and samples stored at 2-8 °C.
  • MCSF preformulated bulk in 10 mM sodium citrate buffer at pH 6.0 was subjected to a similar test protocol using the Amicon ® and Millipore ® filters.
  • MCSF was concentrated using a 43mm diameter YM10 membrane (40mm EFD) to 30 mg/ml while stirring under 30 psi TMP.
  • a 1.0 ml aliquot of MCSF, 1.11 mg/mL in buffer (10 mM citrate, 500 mM glycine, 1 % sucrose, 0.005% polysorbate 80, pH 6.0) was sampled at 5, 10, 20 and 30 mg/ml for analysis.
  • the membrane was washed with buffer to remove any residual MCSF, and was quantitated by RPHPLC. All samples were run through the assays mentioned above to check the protein integrity.
  • the process was scaled up to 1 gram of MCSF, requiring the tangential flow unit.
  • This experiment was done concurrently with a sterile filtration study.
  • the MCSF solution collected from each of the four sterile filter runs, was pooled into one container.
  • Eight liters of MCSF solution at 0.125 mg/ml concentration circulated at 375 ml/min with no pressure on the downstream side of the retentate line for 10 min.
  • the TMP on the upstream inlet was maintained at 20 psi and 10 psi on the downstream retentate or an average TMP of 15 psi initially.
  • the flux was about 4.5 x 10 "3 ml/min-cm -psi over the course of the run.
  • the filtration was stopped after the volume had been reduced 10X by draining the system and measuring the volume and comparing this to the filtrate volume.
  • the membrane was rinsed with 50 ml of buffer and assayed.
  • the MCSF solution from sterile filtration study was again pooled for concentration.
  • the same procedure was used for flushing and sanitizing the UF system as outlined above, the initial flow conditions were the same except the TMP was increased to 25 psi.
  • the concentration was stopped after the limiting holdup volume of about 40ml was reached.
  • the system was again rinsed with 50 ml buffer and flushed with 200ml of RODI water twice. About 3 % of the MCSF filtered was hung-up on the filter, with 0.4% irreversibly adsorbed and 0.8% in the filtrate.
  • the final concentration reached was 19.5 mg/ml, 98.8% recovery after dilution with buffer rinse to 11.0 mg/ml in 90 ml.
  • the Amicon ® YM 1043mm membranes produced a flux of 2.5 x 10 3 ml/min-cm 2 -psi dropping to 1.0 x 10" 3 at the very end of the run. the concentration reached was 34 mg/ml, which in turn was diluted with 5ml of buffer used to rinse the membrane of residual protein. The final concentration reached was 20 mg/ml in about 15 ml for each batch. This material was maintained, along with the preconcentrate control, for stability monitoring.
  • the MCSF was concentrated and diluted with its filtrate three times using the same membrane that had been sanitized with caustic between each run. Beginning with 700ml of the preconcentrate solution, the UF was run until 650 ml had been filtered. A feed rate of 400 ml min was used throughout. Initial flux values averaged around 4.8 x ⁇ O 3 ml/min-cm 2 -psi dropping to as low as 2 x 10 "3 at the end of the run. the concentration of MCSF at this point was 44 mg/ml.
  • Dilution to 20 mg/ml with the buffer rinse of 60 ml was performed to recover 103% of the protein in the first run, 99% in the second, and 105% in the third, the average recovery for all the runs was 102%.
  • the first run was carried out at 15 psi with the second and third at 25 psi TMP. This material was then used in sterile filtration studies.
  • M-CSF (4.18 mg/ml) in buffer (10 mM citrate, 500mM glycine, 1 % sucrose,
  • the concentrate, 30 ml was then concentrated in a stirred cell to 120 mg/ml.
  • the flux dropped to 0.0003 ml/min-cm 2 -psi, 16% of the initial flux observed in this stirred cell experiment.
  • This super-concentrate was then assayed at dilution factors of 10, 100, and 1000 to see if there is an effect on dilution of dissociating aggregates formed on concentration.
  • the membrane was rinsed with three aliquots of 5 ml of buffer. These washes were assayed along with the filtrates and concentrates.
  • Samples were loaded on the gels by adding 20 ⁇ l of sample and 7 ⁇ l of sample buffer. These represent non-reducing conditions.
  • the MCSF appears to match the standard and control samples at all concentrations. No evidence of structure changes or aggregation was seen.
  • control MCSF sample (Example 2) contained 1.4% c-terminus and the final concentrated (20 mg.ml) and filtered sample contained 0.5 % c-terminus. This sample was actually concentrated in two steps, diafiltered into surfactant free buffer and sterile filtered four times.
  • the melting temperature, enthalpy, and folding reversibility of the preformulate after ultrafiltration decreased slightly.
  • the MCSF tertiary structure is essentially unaffected by shearing on filtration and flow through the pumps and tubing.
  • the apparent decrease in thermostability is due to the thermodynamics of the solution in that the molar fraction of MCSF is increased, resulting in an increase in specific partial molar volume.
  • the effect of concentration on the preformulate thermostability is less than on the formulated MCSF, with the Tm dropping only 0.9°C from 85.8°C to 64.9C and enthalpy dropping 26.5 kcal mole from 85.6 to 58.1 kcal/mole (Table 3).
  • the reversibility of folding was better than the formulate at 65% at 2mg/ml MCSF decreasing to 50% at 20 mg/ml.
  • the samples of concentrated MCSF at 120 mg/ml were diluted 10, 100, and 1000 times and assayed. Aggregate was detected at very low levels of 0.66%, 0.29%. and 0.09% of the main peak area for the three dilutions respectively. This aggregation may be due to the HPLC mobile phase of other factors.
  • MCSF can be safely concentrated to at least about 35 mg/ml as a final product solution.
  • the ultrafiltration operation may be carried out with no apparent change in the MCSF molecule.
  • Preformulated bulk drug substance consisting of MCSF in lOmM sodium citrate buffer has also been concentrated to at least about 45 mg/ml with no detectable degradation using both the bench scale and production scale processes.
  • One batch of bulk MCSF was repeatedly concentrated and diluted three times with no negative effects. These batches appear stable at -80°C for up to 3 months according to the stability protocol. These concentrates can also be sterile filtered safely with no changes in the protein.
  • the solubility of MCSF was estimated in the 80-150 mg/ml range for both the formulated and preformulate solutions by the membrane zero flux extrapolation method. Studies of the actual solubility of MCSF were done in a stirred cell reaching at least about
  • thermostability of MCSF does appear to decrease very slightly at high concentrations. However, on dilution of the concentrate, the protein thermostability returns to its original state. The decrease is an expected physical change that occurs as the MCSF is concentrated in a solution and is not expected to affect the shelf stability of the concentrate significantly.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Immunology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne des procédés et des compositions de MCSF (facteur de stimulation de colonies de macrophages) qui sont très concentrées. Les compositions MCSF concentrées sont appropriées à la préparation de formes de dosage finies pour une administration sous-cutanée.
PCT/US1993/008897 1992-09-22 1993-09-21 Compositions mcsf a haute concentration WO1994006458A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU49310/93A AU4931093A (en) 1992-09-22 1993-09-21 Highly concentrated mcsf compositions

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US94927892A 1992-09-22 1992-09-22
US07/949,278 1992-09-22

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WO1994006458A1 true WO1994006458A1 (fr) 1994-03-31

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0331088A2 (fr) * 1988-02-29 1989-09-06 The Green Cross Corporation Médicament pour le traitement des maladies des tissus hématopoiétiques contenant, comme agent actif, le facteur de stimulation des colonies de monocytes et macrophages
WO1990006743A1 (fr) * 1988-12-21 1990-06-28 Genetics Institute, Inc. Procede de retablissement de la croissance des cheveux

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0331088A2 (fr) * 1988-02-29 1989-09-06 The Green Cross Corporation Médicament pour le traitement des maladies des tissus hématopoiétiques contenant, comme agent actif, le facteur de stimulation des colonies de monocytes et macrophages
WO1990006743A1 (fr) * 1988-12-21 1990-06-28 Genetics Institute, Inc. Procede de retablissement de la croissance des cheveux

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