Invented by Chetan Goudar, Sean COLE, Nicole Sabo, Henry Lin, Jonathan LULL, Tharmala Tharmalingam, Amgen Inc

The market for methods of harvesting mammalian cell cultures has witnessed significant growth in recent years. Mammalian cell cultures are widely used in various fields such as biopharmaceutical production, regenerative medicine, and research and development. These cultures provide a valuable tool for studying cell behavior, disease mechanisms, and drug discovery. The process of harvesting mammalian cell cultures involves separating the cells from the growth medium and preparing them for further analysis or use. There are several methods available for this purpose, each with its own advantages and limitations. The market for these methods is driven by the increasing demand for mammalian cell cultures and the need for efficient and reliable harvesting techniques. One of the most commonly used methods for harvesting mammalian cell cultures is trypsinization. Trypsin is an enzyme that breaks down proteins, including those that hold the cells together. By treating the cell culture with trypsin, the cells can be detached from the culture vessel and collected for further processing. This method is relatively simple and cost-effective, making it a popular choice in many laboratories and biopharmaceutical companies. Another widely used method is mechanical dissociation, which involves physically disrupting the cell culture to release the cells. This can be achieved through techniques such as scraping, pipetting, or using a cell scraper. Mechanical dissociation is particularly useful for fragile or adherent cell lines that may be sensitive to enzymatic treatments. However, it can be time-consuming and may result in lower cell viability compared to other methods. Filtration is another method commonly employed for harvesting mammalian cell cultures. This technique involves passing the cell culture through a filter to separate the cells from the growth medium. Filtration is often used when large volumes of cell culture need to be processed quickly. It is a gentle method that preserves cell viability, but it may not be suitable for all cell types and can be limited by the size of the filter pores. In recent years, automated cell harvesting systems have gained popularity in the market. These systems utilize advanced technologies such as robotics and image analysis to streamline the cell harvesting process. They offer higher throughput, improved reproducibility, and reduced labor requirements compared to manual methods. Automated systems are particularly beneficial for large-scale production or high-throughput screening applications. The market for methods of harvesting mammalian cell cultures is driven by factors such as the increasing demand for biopharmaceuticals, advancements in cell-based therapies, and the growing need for reliable and efficient cell culture techniques. Additionally, the emergence of new technologies and the integration of automation in cell harvesting systems are expected to further propel market growth. However, challenges such as the high cost of advanced harvesting systems, the need for skilled personnel to operate these systems, and the variability in cell culture requirements across different applications may hinder market growth to some extent. In conclusion, the market for methods of harvesting mammalian cell cultures is witnessing significant growth due to the increasing demand for cell-based research and production. Various methods, including trypsinization, mechanical dissociation, filtration, and automated systems, are available to meet the diverse needs of researchers and biopharmaceutical companies. As technology continues to advance, the market is expected to expand further, offering more efficient and reliable solutions for harvesting mammalian cell cultures.

The Amgen Inc invention works as follows

The invention provides materials and methods for culturing cells of mammalian origin and harvesting recombinant proteins.

Background for Methods of harvesting mammalian cells cultures

As the demand for therapeutic recombinant protein continues to increase, efforts are being made on optimizing processes, especially methods and strategies that have a positive effect on cell viability.

The development of manufacturing processes to produce recombinant protein is a complex undertaking where many variables need to be balanced. This is especially true for upstream processes where each element of the cell-culture process can have an impact on later stages of production.

A typical cell-culture undergoes a phase of growth, which is a period where the cell density increases exponentially. After the growth phase, a transitional phase occurs when exponential cell proliferation slows and protein production increases. The stationary phase begins, which is a phase of production, when cell density usually levels off, and product titer typically increases. In batch harvesting systems, in which the culture of cells is maintained for a certain number of days, followed by the harvesting of the entire culture at once, most of the product can be produced during the final few days before harvest, when the culture has typically reached its maximum output. This may lead to a high-titer single harvest but at the cost of a long turnaround time and a lag in reaching peak production. The cell culture is prolonged in continuous harvest systems where the product containing the permeate is continuously collected throughout the production phase. However, this is at the cost of lower product titers, and larger volumes of waste fluid during the harvesting and purification stage.

Cell culture and harvest processes are ultimately an exercise in optimizing the process, trading variables like processing speed for product quality and titer. These challenges include maintaining cell viability and achieving a product titer that can be used, as well as balancing outputs from the upstream and downstream processes.

New process methods which provide incremental improvements to recombinant proteins production and recovery will be valuable. This is because large-scale cell culture processes are expensive and there is a growing demand for more biological products and at lower prices. Cell culture improvements that lead to higher product recovery and lower costs are needed. This invention meets these needs because it provides methods and materials that can extend cell culture duration, while increasing protein recoveries.

The invention provides a technique for extended periodic harvest, which comprises establishing a culture of mammalian cell expressing a recombinant proteins, maintaining it by supplying fresh medium to the bioreactor and passing the culture through a filtre, collecting the permeate. Initially, a null-permeate is collected, until a certain parameter is met, after which a harvest-permeate is collected, for a set period, followed by collecting alternately a null-

In one embodiment, the predetermined parameter can be selected from among time, viable cell densities, packed cell volumes or titer.

In one embodiment, the first parameter predetermined is 12 hours or more to 25 days after the establishment of the cellular culture. In one embodiment, the first predetermined parameters is between 24 and 72 hours after the establishment of a cell culture. In one embodiment, the first predetermined parametr is at least four days after the establishment of cell culture. In one embodiment, the first predetermined parameters is 5 days or more after the establishment of cell culture. In one embodiment, the first predetermined parametr is at least 25days after the establishment of cell culture. In one embodiment, the first predetermined parametr is between 5 and 25 days after the establishment of cell culture. In one embodiment, the first predetermined parameters is at least 10-12 days after the establishment of cell culture.

In one embodiment, the second parameter predetermined is between 12 and 72 hours after the harvest permeate has been collected. In one embodiment, the second predetermined para is at least 12 to 72 hours after the collection of harvest permeate. In one embodiment, the second predetermined parameters is between 24 and 48 hours after the harvest permeate has been collected.

In one embodiment, the predetermined period is between 12 and 72 hours. In one embodiment, the predetermined period is between 24 and 72 hours. In one embodiment, the predetermined time ranges from 24 to 72 hours.

In one embodiment, the harvest permeate will be collected from 12 to 72 hrs., after which a null-permeate will be collected from 12 to 24 hrs.

In one embodiment, when the null-permeate is collected the filter is hollow fiber filter with a molecular weight or pore size cutoff (MWCO) which retains the recombinant proteins in the bioreactor. In one embodiment, the molecular mass cutoff is less than 300 kDa. The hollow fiber filter in a related embodiment is an ultrafilter.

In one embodiment, when harvest permeate (or harvested liquid) is collected, the hollow fiber filter has a molecular weight or pore size cutoff (MWCO), which does not retain recombinant proteins in the bioreactor. In one embodiment, the molecular mass cutoff is 500 kDa or greater. The hollow fiber filter in a related embodiment is a microfilter.

In one embodiment, the filter is a unit filter system. In a similar embodiment, the single-unit filter system contains at least one filter component with a molecular mass cutoff (MWCO), which retains the recombinant proteins in the bioreactor. At least one other filter component has a molecular mass cutoff (MWCO), which does not retain recombinant proteins in the bioreactor. In a similar embodiment, the molecular mass cutoff for at least one hollow fibre filter component that retains recombinant proteins in the bioreactor must be 300 kDa. In a similar embodiment, the molecular mass cutoff for at least one hollow fibre filter component that does NOT retain the recombinant proteins in the bioreactor must be at least 500kDa. In a similar embodiment, at least one of the hollow fiber filters that retains the protein in the reactor is an ultrafilter. At least one other hollow fiber filter that does not retain protein is a microfilter. In one embodiment, the single unit filters system is enclosed within a housing. In one embodiment, the single unit filters system includes a spacer that is positioned between two or more of the hollow fibre filter components.

In one embodiment, the null filtrate is collected from at least one hollow fibre filter component that has a pore-size or MWCO (molecular weight cutoff) that retains recombinant proteins in the bioreactor.

In one embodiment, the harvest permeate collected is taken from at least one hollow fibre filter component with a pore-size or molecular weight-cut off (MWCO), which does not retain recombinant proteins in the bioreactor.

In one embodiment, when the permeate collected from a hollow-fiber filter has a molecular weight or pore size cutoff that does not retain recombinant proteins in the bioreactor is formulated or supplemented with at least 5g/L non-ionic copolymer. In a related embodiment the non-ionic block copolymer is a polyoxypropylene-polyoxyethylene block copolymer. In a similar embodiment, the non-ionic copolymer used is poloxamer.

In one embodiment, the method comprises taking samples while the cell cultures are being performed and evaluating them to monitor quantitatively or qualitatively characteristics of the recombinant proteins and/or cell culture processes. In one embodiment, the samples are monitored quantitatively or qualitatively using process analytical techniques.

In one embodiment, the perfusing is continuous. The rate of perfusion in one embodiment is constant. In one embodiment, the rate of perfusion is less than or equal 1.0 working volume every day. In one embodiment, the perfusing can be accomplished using a peristaltic or double diaphragm pumps, low shear pumps, or alternate tangential flow. In another embodiment, the perfusing can be accomplished using alternating tangential flows.

In one embodiment, the method includes subjecting the cell cultures to a temperature change. The cells are then cultured at a first temperature for the first period and at a second temperature for the second period. In one embodiment, the temperature change occurs during the transition from the growth phase to the production phase. In a similar embodiment, the temperature shift takes place during the production phase. In one embodiment, the temperature change is based on a predetermined factor. In a similar embodiment, the temperature shift occurs in response to an established parameter. The predetermined parameter can be determined by a capacitance-based biomass probe.

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