Patent Issued for Methods for Enhanced Protein Production

By a News Reporter-Staff News Editor at Life Science Weekly — According to news reporting originating from Alexandria, Virginia, by NewsRx journalists, a patent by the inventors Arunakumari, Alahari (Pennington, NJ); Dai, Xiao-Ping (Flemington, NJ); Garcia, Javier (Palmerton, NJ); Martel, Richard P. (Allentown, PA), filed on November 17, 2010, was published online on July 1, 2014 (see also Medarex, L.L.C.).
The assignee for this patent, patent number 8765415, is Medarex, L.L.C. (Princeton, NJ).
Reporters obtained the following quote from the background information supplied by the inventors: “Animal cell culture, particularly mammalian cell culture, is commonly used for the expression of recombinantly produced proteins for therapeutic, prophylactic and diagnostic purposes. Although mammalian cell culture methods are preferred over microbial expression systems (e.g., bacterial or yeast expression systems), because they are better suited to express high molecular weight proteins and proteins having complex steric structures, protein expression levels from mammalian cell culture-based systems are generally considerably lower than those from microbial expression systems. While numerous approaches have tried to increase protein expression from mammalian cell culture, these methods have been deficient in optimizing conditions for cell growth, maintaining high cell viability and producing large volumes of high quality protein, thereby limiting their practical application in the biopharmaceutical industry.
“As such, there is a need in the art to develop improved large-scale animal cell culture methods for producing and purifying proteins (e.g., antibodies and antibody fragments, peptides, enzymes, growth factors, hormones, interleukins, interferons, and vaccines) to achieve reliable and cost-efficient protein production at high yields.”
In addition to obtaining background information on this patent, NewsRx editors also obtained the inventors’ summary information for this patent: “In certain embodiments, the present invention provides methods for increasing production of a protein in a cell culture by growing cells that produce the protein in perfusion to a high cell density (e.g., at least to above about 40.times.10.sup.6 cells/mL, more preferably above about 60 to 80.times.10.sup.6 cells/mL, and most preferably around about 100.times.10.sup.6 cells/mL), also referred to herein as ‘super high density’, then switching the cell culture to fed-batch, such that the cells enter a high protein production phase. The present invention is based, at least in part, on the unexpected discovery that it is possible to grow cells in culture to a much higher density than previously thought possible. This, in turn, yields significantly more protein than is typically produced using known cell culture techniques. Specifically, the maximum cell density achieved using known cell cultures techniques is generally thought to be limited by the presence of particular levels of waste products (e.g., lactate, ammonium, etc.), since it is known that such waste products can be toxic to cell growth when they accumulate to critical concentrations (see, for example, Schumpp, B. et al., J. Cell Sci., 7(Pt 4):639-647 (December 1990)). As such, known cell culture techniques often limit the maximum density to which cells are initially grown, so that the cells are not adversely affected by particular concentrations of waste products.
“For example, the cell culture method described in WO 2009/023562 relies on lactate levels to signal the maximum cell density that can theoretically be achieved before initiating perfusion cell culture. As taught in WO 2009/023562, cells are grown to a cell density of about 1 million to 9 million cells/mL, which results in a lactate concentration of about 1 g/L to about 6 g/L. According to WO 2009/023562, this signals the optimum time point to begin perfusion. After perfusion, cells are then grown to roughly 5 to 40 million cells/mL and maintained in fed-batch cell culture to yield antibody titers between 8-10 g/L (between days 14-17).
“In contrast to such known cell culture methods, no particular level of waste products restricts the density of cell growth according to the methods of the present invention. Instead, it was discovered that when cells are initially grown only in a perfusion cell culture, the cells are able to grow to a much higher cell density (e.g., at least above 40.times.10.sup.6 cells/mL) than previously reported for known cell culture techniques, regardless of the presence of waste products. In a particular embodiment, the cells are grown to a density of at least above about 40.times.10.sup.6 cells/mL to 60.times.10.sup.6 cells/mL, preferably above about 70.times.10.sup.6 cells/mL to 90.times.10.sup.6 cells/mL, more preferably above about 100.times.10.sup.6 to 130.times.10.sup.6, and most preferably above about 140.times.10.sup.6 to 200.times.10.sup.6 cells/mL or more. This ‘super’ high density of cells, in turn, enables the production of significantly higher quantities of protein (e.g., 13.8 g/L at day 6) in a shorter period of time than has been previously reported.
“The present invention further provides yet another unexpected advantage over known cell culture techniques in that it optimizes the production of large amounts of high quality protein. For example, in one embodiment, protein harvested according to the methods of the present invention is obtained only from the fed-batch cell culture. This increases the freshness, stability and quality of the protein, in contrast to protein obtained from both perfusion and fed-batch cultures subjected to longer periods of culture, which is thus more susceptible to degradation and generally of poorer quality. Notwithstanding, the present invention also contemplates harvesting protein from both the perfusion and fed-batch cultures (e.g., when the cells are cultured in a single bioreactor).
“In certain embodiments, the present invention employs recombinant cells which express a protein of interest. For example, cells are transfected with at least one nucleic acid that encodes a protein of interest.
“The methods of the present invention can be used to enhance production of any protein of interest, including, without limitation, enzymes, receptors, fusion proteins, cytokines, regulatory factors, hormones, diagnostic proteins, therapeutic proteins, and antigen-binding agents. In a particular embodiment, the protein is an antibody (or antigen binding fragment thereof). Similarly, a wide variety of cells can be cultured according to the methods of the present invention. In a particular embodiment, the cells are animal cells. In a preferred embodiment, the cells are mammalian cells, such as CHO, NSO, BHK and Per C6 cells.
“The conditions of the cell culture (e.g., pH, temperature, media, particular vessel for growing the cultures, etc.) can be determined and adjusted by one of ordinary skill in the art depending on a number of factors, such as the particular cell line and protein to be produced. In one embodiment, the perfusion cell culture and fed-batch mode cell culture are performed in bioreactors. This can include a single bioreactor or multiple bioreactors (e.g., such that the perfusion cell culture and fed-batch mode cell culture are performed in separate bioreactors). For example, the perfusion cell culture can be performed in one bioreactor and then the cells can be transferred into multiple fed-batch bioreactors.
“Additionally, the cell culture conditions can be optimized to maximize protein production. For example, cell culture parameters, such as pH, DO, temperature, feed strategies, feed composition, etc. can be optimized.
“In other embodiments, the present invention provides methods for clarifying a protein from a cell culture. This is achieved by adjusting the pH of the cell culture to below neutral pH (i.e., below a pH of 7) and settling the cell culture, such that the cell culture separates to form a supernatant layer and a cell-bed layer, wherein the protein is present in (and can be isolated from) the supernatant layer. While the methods can be applied to cell cultures of the invention, they can be applied to clarify a protein from any cell culture, including high density cell cultures.
“In one embodiment, the method involves adjusting the pH of the cell culture to a low pH (i.e., below a neutral pH of 7 such as pH of 4-7). In a particular embodiment, the pH is adjusted to a pH between about 4.5 and 6.5. In other embodiment, the method involves settling the cell culture for at least about 30 minutes, so as to enhance clarification of the cell culture in advance of collecting the protein. However, the cell culture can be settled for longer periods of time at the discretion of the skilled artisan. In a particular embodiment, the cell culture is settled for between about 30 and 120 minutes.
“Other known purification techniques can optionally be employed in conjunction with the clarification method described above. For example, in one embodiment, the cell culture can be centrifuged before settling the cell culture. In another embodiment, the cell culture can be centrifuged after settling the cell culture. In yet another embodiment, the cell culture can be centrifuged before and after settling the cell culture.
“Another optional step that can be taken to enhance the clarification method is washing the cell-bed layer that forms as a result of the settling process with a low pH buffer. In a particular embodiment, the pH of the buffer is the same as the pH of the cell culture, i.e., when the cell culture is adjusted to a low pH (i.e., below a neutral pH of 7).
“Additionally, filtration techniques can be used to further clarify the cell culture. For example, the cell culture can be filtered before the cell culture is settled. In another embodiment, the cell culture can be filtered after the cell culture is settled. In yet a further embodiment, the cell culture can be filtered before and after the cell culture is settled. Filtration can be performed using any number of techniques known in the art, including, but not limited to depth filtration and polishing filtration.
“Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.”
For more information, see this patent: Arunakumari, Alahari; Dai, Xiao-Ping; Garcia, Javier; Martel, Richard P.. Methods for Enhanced Protein Production. U.S. Patent Number 8765415, filed November 17, 2010, and published online on July 1, 2014. Patent URL: http://patft.uspto.gov/netacgi/nph-Parser?Sect1=PTO1&Sect2=HITOFF&d=PALL&p=1&u=\%2Fnetahtml\%2FPTO\%2Fsrchnum.htm&r=1&f=G&l=50&s1=8765415.PN.&OS=PN/8765415RS=PN/8765415
Keywords for this news article include: Antibodies, Peptides, Immunology, Amino Acids, Blood Proteins, Medarex L.L.C., Immunoglobulins.
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