Patent Application: US-201715585005-A

Abstract:
a serum free cell culture media , wherein the media is adapted to be conditioned by culturing a first set of eukaryotic cells in the media , wherein the first set of eukaryotic cells use an expression vector to excrete levels of desired complex proteins into the media ; wherein said desired complex proteins include human growth hormone , growth hormone - like growth factors , insulin - like growth factors , insulin , modified insulins , cytokines , mitogenic proteases and mixtures thereof ; and wherein the media is adapted to grow a set of eukaryotic cells .

Description:
the most preferred embodiment of the present invention is cell culture media produced by use of modified cho cells to secrete growth factors in the cell media to improve the potential growth characteristics of the conditioned cell culture media . preferably , the first embodiment of the present invention uses neucho cell which are modified cho dg44 cells that include an expression vector to secrete human growth hormone ( hgh ) into the cell culture media that they are used to condition . the neucho cell line , deposited under the provisions of the budapest treaty with the cell bank australia located at 214 hawkesbury rd , westmead , nsw , 2145 , australia as of 4 feb . 2013 and assigned accession no . cba20130024 , as is particularly suitable for use in pharmaceutical manufacture as described within the present application . the advantage of using neucho cells is that hgh is excreted into the cell culture media where it can be utilised by other microorganisms which are typically difficult to grow or lack suitable cell viability for in vitro growth . one embodiment of the present invention describes the use of conditioned media from neucho cell cultures to improve the efficiency of transfection in mammalian cells . neucho cells secrete human - growth hormone ( hgh ). transfection efficiency is defined here as the number of cells surviving transfection , dna integration and selection before the individual cells are allowed to expand to form a stable pool . transfection efficiency is improved with the addition of conditioned media from neucho cell cultures . the use of neucho conditioned media maximizes the number of high producing clones that can be isolated from a stable transfected cell population . the method results in a population of cells with greater genetic heterogeneity which significantly increases the likelihood of identifying high expressing clones more quickly and with more certainty than conventional methods . this embodiment simplifies cell line development by enabling rapid identification , selection , isolation and collection of high - value clones . it improves cell line productivity , shortens timelines and reduces cost . in a further embodiment of the present invention the neucho cells may be utilised as feeder cell layer for improving efficiency of single cell cloning . single cell cloning methods are generally inefficient but may be greatly improved by the use of conditioned cell culture media as described within the present invention or embodiment . the expansion from a single cell to a culture can be improved through the use of neucho cells and / conditioned media from neucho cells . in another embodiment of the present invention relates to the use of neucho as feeder cells to facilitate the growth and expansion of high clones . the use of neucho feeder cells increases the survival rate and number of clones that can be isolated in a single cloning procedure . this invention relates to methods of transient gene expression for the production of recombinant bio - pharmaceuticals and other desirable proteins , polypeptides and peptides using mammalian cell cultures . in particular , the methods of the invention involve the use of specially bioengineered cell culture media which maintains very high viable cell densities during and after transient gene expression . cells cultured in the mentioned media have the ability to maintain high growth in cheap , reproducible , fully - defined protein - free medium . the number of recombinant proteins used for therapeutic applications in recent years has increased dramatically , a market expected to reach approximately $ 70 billion by 2010 ( walsh 2006 ). recombinant antibodies currently represent over 20 % of biopharmaceuticals in clinical trials as highlighted by the us food and drug administration ( pavlou and betsey 2005 ). however , the production of recombinant proteins is itself expensive and time consuming and the biotechnology industry is already experiencing a shortage of manufacturing capacity ( garber 2001 ; dyck , lacroix et al . 2003 ). thus , factors such as scale up , total annual manufacturing capacity , post translational modifications , choice of expression system for the biosynthesis of therapeutic proteins and speed of process set up need to be evaluated in order to make both upstream and downstream production of therapeutic proteins a cost - effective process ( verma , boleti et al . 1998 ; werner , noe et al . 1998 ; fischer , drossard et al . 1999 ; bulleid , john et al . 2000 ; morton and potter 2000 ). the high throughput screening required in the drug discovery process has intensified the need for a rapid technique to produce milligram amounts of recombinant protein . in order to achieve this , transient gene expression technology has attracted much interest over the traditional stable expression technology . the speed of transient gene expression represents its major economic advantage over standard stable cell line development ( durocher , perret et al . 2002 ; meissner , pick et al . 2001 ; girard , derouazi et al . 2002 ; kunaparaju , liao et al . 2005 ). however many transfection procedures result in massive cell death of the transfected cell line from a very early stage ( within hours ), which leads to a concomitant reduction in recombinant protein production . in order to counteract this problem , many cell lines are transfected in the presence of serum containing media . the use of such media however also poses other drawbacks . front a regulatory perspective , there are concerns regarding the use of animal derived materials and the inherent possibility of introducing adventitious agents to the culture ( sunstrom , sugiyono et al . 2000 ). the use of serum is also associated with high costs , batch to batch variability , and product purification difficulties associated with the use of such media ( zang , trautmann et al . 1995 ). alternatively , cho cells can be cultured in the presence of media containing growth factors which confer protection to the cells during the transfection procedure , thereby allowing the cells to maintain high viable cell densities consequently leading to concomitant increase in the expression of recombinant proteins . the embodiments of the present results in increasing recombinant proteins , polypeptides and peptides production by utilizing defined neucho media capable of maintaining high viable cell densities during gene expression in mammalian cells . host cells are transfected using neucho media which contains human - growth hormone ( hgh ) so that mammalian cells transfected in the presence of neucho media have very high viable cell densities post transfection consequently leading to increased protein production . the embodiment may also a method for producing high levels of desired recombinant protein , polypeptide or peptide comprising the step of : culturing a mammalian host cell in neucho media wherein said media : ( i ) neucho media may be used in culturing or transfecting any of those commonly used cell lines used in the art of expressing recombinant proteins , polypeptides and peptides . for example , the host cell may be a chinese hamster ovary ( cho ) cell line such as cho - k1 , cho - dg44 dhfr - and cho - s . these include both adherent and suspension cell lines . fig1 is a graph depicting viable cell density plotted against time of various cell cultures . expression of human growth hormone increases transfection efficiency by increasing survival rate of cultures following transfection . the graph represents the number of viable cells 48 hrs following transfection with plasmids encoding different recombinant proteins . dg44 cells transfected with plasmid encoding human growth hormone gene , ( dg44 - hgh ), has the highest viable cell density after control ( no dna ) post transfection implying that hgh confers protection to dg44 cells from harsh conditions during transfection thereby leading to a higher number of cells surviving transfection . viable cell density is plotted on the y - axis in cells / ml , and the number of days in culture is plotted on the x - axis . six line graphs are shown in the figure , namely line graph a ( negative control , dg44 cells — freestyle reagent only , no dna , b ( dg44 pnas - hgh or neucho ), c ( dg44 rmab ), d ( dg44 - epo ), e ( dg44 - imab ) and f ( dg44 ifn ). fig2 is a set of two graphs depicting an increasing hit rate of finding a high producer clone cell line wherein various proteins are plotted against protein concentration . neucho conditioned media or neucho cells as feeder layer increases efficiency of single cell cloning . in an embodiment , neucho cells were seeded in single wells of microtitre plates prior to single cell cloning of a stable transfected pool to form a layer in the cell culture media . secretion of human growth hormone from neucho cells results in an increased survival rate of single cells following limiting dilution cloning . various additional modifications and variations are possible within the scope of the foregoing specification and accompanying drawings without departing from the scope of the invention . although the invention has been described with reference to specific examples , it will be appreciated by those skilled in the art that the invention may be embodied in many other forms , in keeping with the broad principles and the spirit of the invention described herein . the present invention and the described preferred embodiments specifically include at least one feature that is industrial applicable . pavlou , a . k . and m . j . belsey ( 2005 ). “ the therapeutic antibodies market to 2008 .” eur pharm biopharm 59 ( 3 ): 389 - 96 . garber , k . 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