1. Field of the Invention
The present invention relates generally to the fields of production of natively glycosylated mammalian biological molecules. Specifically, the present invention relates to a system and process for producing natively glycosylated mammalian biological molecules produced by using electromagnetic fields. More specifically, the present invention relates to a process for producing natively glycosylated mammalian biological molecules by electromagnetically stimulating mammalian cells.
The preferred embodiment utilizes introducing mammalian cells and a carrier medium into a cylindrical chamber and rotating the cylindrical chamber about its axis at a rotational speed sufficient to prevent the cells from substantially contacting the cylindrical walls of the cylindrical chamber and continuing the rotation until the supernatant liquid containing the cells has a significantly increased amount of a mixture of natively glycosylated mammalian biological molecules, and then separating the natively glycosylated mammalian biological molecules into individual molecular entities in significant quantities to be used for therapeutic purposes.
Subjecting the original cell mixture to an electromagnetic field, preferably a time varying electromagnetic field may enhance the process.
2. Description of the Prior Art
In order to more fully understand this invention, a brief discussion of definitions and terms is useful including the following:
Mouse granulocyte colony stimulating factor (G-CSF) was first recognized and purified in Australia in 1983, and groups from Japan and the U.S.A. cloned the human form in 1986. The natural human glycoprotein exists in two forms of 174 and 177 amino acids. The more abundant and more active 174 amino acid form has been used in the development of pharmaceutical products by recombinant DNA technology.
The recombinant human G-CSF synthesized in an E. coli expression system is called filgrastim. The structure of filgrastim differs slightly from the natural glycoprotein. Most published studies have used filgrastim and it was the first form of G-CSF to be approved for marketing in Australia.
Another form of recombinant human G-CSF called lenograstim is synthesized in Chinese hamster ovary (CHO) cells. As this is a mammalian cell expression system, lenograstim is indistinguishable from the 174 amino acid natural human G-CSF. No clinical or therapeutic consequences of the differences between filgrastim and lenograstim have yet been identified, but there are no formal comparative studies. G-CSF should not be confused with granulocyte macrophage colony stimulating factor (GM-CSF), which is a distinctly different hematopoietic growth factor also under clinical development.
G-CSF (filgrastim) is indicated for the prevention of febrile neutropenia in patients receiving myelosuppressive chemotherapy for non-myeloid malignancies. It reduces the duration and severity of post-chemotherapy neutropenia.
G-CSF (lenograstim) is also approved for use to reduce the incidence of infection associated with established cytotoxic chemotherapy.
There are extensive publications on techniques to increase natively glycosylated mammalian biological molecules in humans and laboratory animals and the therapeutic effect derived there from. However, like the problem associated with obtaining commercial quantities of reasonably priced G-CSF, the obtaining of reasonably priced quantities of GM-CSF, cytokines, interleukins, and other desired natively glycosylated mammalian biological molecules has not been accomplished.
The present invention overcomes the problems of prior processes and systems and provides an economical system of producing commercial quantities of natively glycosylated mammalian biological molecules.
The present invention relates to a process for producing natively glycosylated mammalian biological molecules, such as mammalian cells, human cells, within a culture medium. The cells are preferably exposed to an electromagnetic field, which, in the preferred embodiment, is a time-varying electromagnetic field.
The cells are preferably grown in a bioreactor in a manner so that they maintain their three dimensional geometry. In a preferred embodiment, the presence of time varying electromagnetic field potentiates the rapid growth of cells.
The system and process are utilized in combination with tissue culture processes to produce growth of natively glycosylated mammalian biological molecules. In this environment, growth-promoting genes are up regulated and growth inhibitory genes are down regulated. The effect is shown to persist over a period of time after termination of the process. It is an object of the present invention to provide a process for producing natively glycosylated mammalian biological molecules.
Another object of this invention is to provide a composition comprising a mixture of natively glycosylated mammalian biological molecules produced by electromagnetically stimulating living mammalian cells.
Still another object of this invention is to provide a composition comprising a mixture of natively glycosylated mammalian biological molecules including proteins, peptides, polypeptides, glycoproteins, cytokines, post-translational proteins, post-translational peptides, and post-translational polypeptides.
It is still another object of this invention to produce a mixture of natively glycosylated mammalian biological molecules that can be separated into its individual component parts for later research or therapeutic use.
It is a further object of this invention to provide a method of producing natively glycosylated mammalian biological molecules utilizing an electromagnetic force to produce a mixture of natively glycosylated mammalian biological molecules present in a harvestable amount in a liquid, and thereafter separating one or more of the natively glycosylated mammalian biological molecules from the mixture.
It is still another object of this invention to provide a method for producing natively glycosylated mammalian biological molecules in which mammalian cells and a carrier medium are introduced into a chamber capable of sustaining cell growth, maintaining the mammalian cells and carrier medium in the chamber under cell growing conditions until natively glycosylated mammalian biological molecules are present in a harvestable amount in the carrier liquid, and separating one or more of the natively glycosylated mammalian biological molecules from the carrier medium. It is a more specific object of this invention to provide a process for producing natively glycosylated mammalian biological molecules in which the natively glycosylated mammalian biological molecules are a member selected from the group comprising proteins, peptides, polypeptides, glycoproteins, cytokines, post-translational proteins, post-translational peptides, and post-translational polypeptides, including specifically G-CSF, G-MCSF, and the interleukins, and where a time varying electromagnetic force is utilized to effect the production.
Other aspects, features and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention given for the purpose of disclosure.
In
In operation, the cylinder is opened and the cell culture and carrier liquid placed therein. The cells are obtained from readily available sources. The rotation of the cylinder is adjusted visually so that the cell culture substantially remains at or about the longitudinal axis of the cylinder. The electromagnetic generating device is activated and adjusted so that the square wave output generates the desired electromagnetic field in the cylinder, from 0.05 gauss to 6 gauss. The electromagnetic field can be determined by the number of windings of the coils and the current used by using the formula for Fourier curves (square waves).
This invention may be more fully described by the preferred embodiment as hereinafter described.
The preferred embodiment of this invention produces a mixture of natively glycosylated mammalian biological molecules produced by electromagnetically stimulating living mammalian cells. Preferably, the natively glycosylated mammalian biological molecules are produced in three-dimensional conditions and the electromagnetic stimulation is provided by applying a time varying electromagnetic force, and, more specifically, a square wave. It is preferred that the natively glycosylated mammalian biological molecules are a member selected from the group comprising proteins, peptides, polypeptides, glycoproteins, cytokines, post-translational proteins, post-translational peptides, and post-translational polypeptides, including specifically G-CSF, GM-CSF, Interleukin-6, and Interleukin-8.
The stated mixture is a mixture found in a supernatant liquid produced by mixing a cell culture and a carrier medium together and subjecting it to the electromagnetic force until the supernatant liquid has a harvestable amount of the natively glycosylated mammalian biological molecules. The supernatant liquid is then separated into its component parts for research or therapeutic use. The mammalian biological material being stimulated is preferably human cells, such as progenitor cells or neuronal cells.
The preferred method for producing the natively glycosylated mammalian biological molecules comprises: (a) introducing mammalian cells and a carrier medium into a cylindrical chamber; (b) rotating the cylindrical chamber about its axis at a rotational speed sufficient to prevent the cells from substantially contacting the cylindrical walls of the cylindrical chamber; (c) continuing the rotation until natively glycosylated mammalian biological molecules are present in a harvestable amount in the carrier liquid; and (d) separating one or more of the natively glycosylated mammalian biological molecules from the carrier medium. This invention also includes a method of therapeutically treating mammals comprising producing the natively glycosylated mammalian biological molecules as described herein and thereafter administering a therapeutical amount of the natively glycosylated mammalian biological molecules to a mammal to achieve a therapeutical affect.
In a preferred embodiment of the invention, normal human neuronal progenitor cells (NHNP) were pooled from three donors to diminish donor-to-donor variations in response. As controls, NHNP were grown in conventional tissue culture following standard cell culturing procedures in tissue culture flasks obtained through Clonetics Corporation, San Diego, Calif.
For two-dimensional culture, GTSF-2 medium with 10% FBS, Ciprofloxacin and Fungizone was used to culture the cells (Goodwin et al., 1993a). 1×PBS, Collagenase, DNase and trypsin were purchased from Clonetics San Diego, Calif., and used Coming T-75 flasks (Coming Inc., Corning, N.Y.) for initial cell culture to obtain the appropriate number of cells for each experiment. Briefly, cells to be cultured were enzymatically dissociated with the referenced reagents from T-flasks, washed once with PBS-CMF and assayed for viability by trypan dye exclusion (GIBCO, Grand Island, N.Y.). Cells were grown on 100-mm petri dishes (tissue culture treated to prevent adherence) or grown on the actual electrodes inside the petri dishes. Electrodes were made of platinum and stainless steel. Cell cultures were maintained in a humidified Forma CO2 incubator (Forma, Inc.) at 37° C. at a CO2 concentration of 6%.
For three-dimensional culture, NHNP cells were prepared as described above and an RWV was sequentially inoculated with 5 mg/ml Cytodex-3 type I collagen-coated microcarriers (Pharmacia) and freshly digested NHNP cells, yielding a cell density of 2.5.×105 cells/ml in a 55-ml vessel. Tissues were cultured for 17 to 21 days or until 3- to 5-mm diameter tissue masses formed.
A waveform (TVEMF) generator of original design and capability was developed and used to generate the waveform in a strength of 1-6 mA (AC) square wave, 10 Hz variable duty cycle, which was pulse-width modulated as described in the description of the drawings above. NHNP cells were subjected to these extremely low-level magnetic fields (ELF waves) (˜10-200 mGauss), which are far less than the field strength of the Earth.
Initially, a metal electrode was placed inside a petri dish and centered. NHNP were seeded at 2.5·105 cells in 0.7 ml of media and carefully dropped on the electrode in a concentrated bubble. Cells were incubated for 2 days. The second day after cell inoculation is considered day 0 of the experiment protocol. At day 0, each dish was given 15 ml of media and waveform was applied to the electrodes. Cells were fed with 15 ml of media at day 3 and with 13 ml every three days thereafter at day 6, 9, and 12. At days 14 and 17, the cells were fed again with 15 ml of media. At days 17 to 21, the cells were incubated for 10 minutes in a Collagenase/DNase cocktail, then trypsin was directly applied to the cocktail and the cells were further incubated for 3 more minutes. Before the complete media was added to deactivate the trypsin, the cocktail mix was pipetted up and down several times. The cells were washed twice with 1×PBS, reapplied with the media, and placed on ice. The cells were observed under a dissecting microscope, counted, and assessed for viability.
An identical protocol was followed in similar experiments with the exception that, instead of the electrode being placed within the petri dishes, in media, it was attached to the underside of the TVEMF treated dishes, so that the cells had no direct contact with the metal surface.
Three-dimensional neural cells and tissues were cultured by the method described above, except that the TVEMF RWV was modified to incorporate an electromagnetic coil. The coil was wrapped around the core of the vessel so it emitted the same electromagnetic field strength as in the two-dimensional configuration. All other conditions were identical to the two-dimensional experimental conditions.
The supernatant liquid was removed from the mixture and analyzed. (describe analysis).
The analysis provided the following results. (Insert Excel spreadsheets).
The results clearly show that this invention provides a new and unique mixture of natively glycosylated mammalian biological molecules that can be separated into therapeutic amounts of highly desirable natively glycosylated mammalian biological molecules including growth factors.
The invention described herein was made in part by an employee of the United States Government and may be manufactured and used by and for the Government of the United States for governmental purposes without the payment of any royalties thereon or therefor.
Number | Date | Country | |
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60583976 | Jun 2004 | US |
Number | Date | Country | |
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Parent | 11170520 | Jun 2005 | US |
Child | 12107924 | US |