FIELD OF THE INVENTION

The invention provides method and materials for culturing mammalian cells and harvesting recombinant proteins.

BACKGROUND OF THE INVENTION

As the demand for greater quantities of therapeutic recombinant proteins continues to grow, much effort is being placed on process optimization, particularly methods and strategies for growing, feeding, and maintaining production cell cultures which have a positive impact on cell viability and protein recovery.

Developing manufacturing processes for production of recombinant proteins is a complex endeavor where many variables must be balanced. This is particularly true for upstream processes, where every element of the cell culture process can have a large impact on the later stages of production, particularly harvest and downstream processing.

A typical cell culture undergoes a growth phase, this is a period of exponential growth where cell density is increased. The growth phase is followed by a transition phase when exponential cell growth is slowing and protein production starts to increase. This marks the start of the stationary phase, a production phase, where cell density typically levels off and product titer increases. In batch harvest systems, where the cell culture is maintained for a set number of days followed by harvesting the entire culture all at once, the majority of the product may be produced in the last few days prior to harvest when the cell culture typically has reached its greatest output. While this may result in a single high titer harvest, it is at the expense of a non-productive turnaround time to initiate the next run and the lag time to once again achieve peak production. In a continuous harvest systems, where product containing permeate is collected from the cell culture on a continuous basis throughout the production phase, the cell culture duration is extended, but at the expense of lower product titers and higher volumes of waste cell culture fluid to be dealt with during the harvest and purification stages.

Cell culture and harvest process development is ultimately is an exercise in process optimization, trading variables such as processing speed for product titer and product quality. The challenges include, for example, maintaining cell viability, achieving a workable product titer, and balancing the output from the upstream process with what the harvest and downstream processes can handle.

New process methods that provide even incremental improvements in recombinant protein production and recovery are valuable, given the expense of large scale cell culture processes and the growing demand for greater quantities of and lower costs for biological products. Improvements to cell culture processes that can lead to greater product recovery, thereby reducing the costs associated with manufacturing protein therapeutics are needed. The invention fulfills these needs by providing such methods and materials for extending cell culture duration while increasing protein recovery.

SUMMARY OF THE INVENTION

The invention provides a method for an extended periodic harvest comprising establishing a cell culture by inoculating a bioreactor with mammalian cells expressing a recombinant protein, maintaining the cell culture by perfusing fresh cell culture medium into the bioreactor, passing the cell culture through a filter and collecting a permeate, wherein a null permeate is initially collected until a first predetermined parameter is reached, at which time a harvest permeate is collected for a predetermined time, this is followed by alternately collecting a null permeate until a second predetermined parameter is reached, then collecting a harvest permeate for a predetermined time, wherein the alternating collection of null permeate and harvest permeate continues until the cell culture is terminated.

In one embodiment the predetermined parameter is selected from time, viable cell density, packed cell volume or titer.

In one embodiment the first predetermined parameter is at least 12 hours to 25 days following the establishment of the cell culture. In one embodiment the first predetermined parameter is at least 24 to 72 hours following the establishment of the cell culture. In one embodiment the first predetermined parameter is at least 4 days following the establishment of the cell culture. In one embodiment the first predetermined parameter is at least 5 or more days following the establishment of the cell culture. In one embodiment the first predetermined parameter is at least 25 days following the establishment of the cell culture. In one embodiment the first predetermined parameter is at least 5 to 25 days following the establishment of the cell culture. In one embodiment the first predetermined parameter is at least 10 to 12 days following the establishment of the cell culture.

In one embodiment the second predetermined parameter is at least 12 to 72 hours following the collection of the harvest permeate. In one embodiment the second predetermined parameter is at least 24 to 72 hours following the collection of the harvest permeate. In one embodiment the second predetermined parameter is at least 24 to 48 hours following the collection of the harvest permeate.

In one embodiment in the predetermined time is at least 12 to 72 hours. In one embodiment the predetermined time is at least 24 to 72 hours. In one embodiment the predetermined time is at least 24 to 48 hours.

In one embodiment the null permeate is initially collected for at least 24 hours to 25 days, at which time a harvest permeate is collected for 12 to 72 hours, followed by alternately collecting a null permeate for at least 24 hours to 25 days, then collecting a harvest permeate for 12 to 72 hours.

In one embodiment when the null permeate is collected, the filter is a hollow fiber filter having a pore size or molecular weight cut off (MWCO) that retains the recombinant protein in the bioreactor. In a related embodiment the molecular weight cutoff is 300 kDa or less. In a related embodiment the hollow fiber filter is an ultrafilter.

In one embodiment when the harvest permeate is collected, the filter is a hollow fiber filter having a pore size or molecular weight cut off (MWCO) that does not retain the recombinant protein in the bioreactor. In a related embodiment the molecular weight cutoff is at least 500 kDa. In a related embodiment the hollow fiber filter is a microfilter.

In one embodiment the filter is a single unit filter system. In a related embodiment rein the single unit filter system comprises at least one hollow fiber filter component having a pore size or molecular weight cut off (MWCO) that retains the recombinant protein in the bioreactor and at least one hollow fiber filter component having a pore size or molecular weight cut off (MWCO) that does not retain the recombinant protein in the bioreactor. In a related embodiment the molecular weight cutoff of at least one hollow fiber filter component that retains the recombinant protein in the bioreactor is 300 kDa or less. In a related embodiment the molecular weight cutoff of at least one hollow fiber filter component that does not retain the recombinant protein in the bioreactor is at least 500 kDa. In a related embodiment at least one hollow fiber filter component that retains the recombinant protein in the bioreactor is an ultrafilter and at least one hollow fiber filter component that does not retain the recombinant protein in the bioreactor is a microfilter. In a related embodiment the single unit filter system is contained within a housing. In a related embodiment the single unit filter system further comprises a spacer between at least two of the hollow fiber filter components.

In one embodiment when the null permeate is collected it is drawn from at least one hollow fiber filter component having a pore size or molecular weight cut off (MWCO) that retains the recombinant protein in the bioreactor.

In one embodiment when the harvest permeate is collected, it is drawn from at least one hollow fiber filter component having a pore size or molecular weight cut off (MWCO) that does not retain the recombinant protein in the bioreactor.

In one embodiment when the permeate is collected from a filter that is a hollow fiber filter having a pore size or molecular weight cut off that does not retain the recombinant protein in the bioreactor, the fresh cell culture medium is formulated with or supplemented to achieve at least 5 g/L of a non-ionic block copolymer. In a related embodiment the non-ionic block copolymer is a polyoxypropylene-polyoxyethylene block copolymer. In a related embodiment the non-ionic block copolymer is poloxamer 188.

In one embodiment the above method further comprising taking samples during the cell culture processes, evaluating the samples to quantitatively and/or qualitatively monitor characteristics of the recombinant protein and/or the cell culture process. In a related embodiment the samples are quantitatively and/or qualitatively monitored using process analytical techniques.

In one embodiment the perfusing is continuous perfusion. In one embodiment the rate of perfusion is constant. In one embodiment the perfusing is performed at a rate of less than or equal to 1.0 working volume per day. In one embodiment the perfusing is accomplished by a peristaltic pump, a double diaphragm pump, a low shear pump or alternating tangential flow. In a related embodiment the perfusing is accomplished by alternating tangential flow.

In one embodiment the method above further comprises subjecting the cell culture to a temperature shift wherein the cells are cultured a) at first temperature for a first period of time and b) at second temperature for a second period of time. In a related embodiment the temperature shift occurs at the transition between the growth phase and production phase. In a related embodiment the temperature shift occurs during the production phase. In a related embodiment the temperature shift is in response to a predetermined parameter. In a related embodiment the temperature shift is in response to a predetermined parameter wherein achieving the predetermined parameter is determined using a capacitance based biomass probe.

In one embodiment the cell culture is established by inoculating the bioreactor with at least 0.1×106viable cells/mL. In a related embodiment the inoculum was grown by means of a perfusion process using alternating tangential flow filtration.

In one embodiment prior to entering the bioreactor, the cell culture medium is treated using nanofiltration, high temperature short time (HTST), or UV in combination with filtration.

In one embodiment the bioreactor is a production bioreactor. In a related embodiment the bioreactor has a capacity of at least 500 L. In a related embodiment the bioreactor has a capacity of at least 500 L to 2000 L. In a related embodiment the bioreactor has a capacity of at least 1000 L to 2000 L.

In one embodiment the cell culture medium is a serum-free cell culture medium. In one embodiment the cell culture medium is a serum-free chemically defined cell culture medium. In one embodiment the cell culture medium is a perfusion cell culture medium.

In one embodiment the mammalian cells are Chinese Hamster Ovary (CHO) cells. In one embodiment the recombinant protein is selected from the group consisting of a human antibody, a humanized antibody, a chimeric antibody, a recombinant fusion protein, or a cytokine.

In one embodiment the recombinant protein is purified from the harvest permeate by one or more of flocculation, precipitation, centrifugation, depth filtration, affinity chromatography, size exclusion chromatography, ion exchange chromatography, mixed mode anion exchange chromatography, hydrophobic interaction chromatography or hydroxyapatite chromatography. In one embodiment the method above further comprises taking samples during the purification process, evaluating the samples to quantitatively and/or qualitatively monitor characteristics of the recombinant protein and the purification process.

In one embodiment the recombinant protein is formulated into a pharmaceutically acceptable formulation. In one embodiment is provided a recombinant protein produced by the above method.

In invention also provides a method for harvesting a recombinant protein comprising establishing a cell culture by inoculating a bioreactor with mammalian cells expressing a recombinant protein, maintaining the cell culture by perfusing the cell culture with fresh cell culture medium formulated or supplemented to achieve a concentration of at least 5 g/L of a non-ionic block copolymer and passing the cell culture through a hollow fiber filter having a pore size or molecular weight cut off (MWCO) that does not retain the recombinant protein in the bioreactor and collecting a permeate containing the recombinant protein.

In one embodiment the molecular weight cutoff is at least 500 kDa. In one embodiment the hollow fiber filter is a microfilter.

The invention also provides a method for harvesting a recombinant protein comprising establishing a cell culture by inoculating a bioreactor with mammalian cells expressing a recombinant protein, maintaining the cell culture by perfusing the cell culture with fresh cell culture media formulated or supplemented to achieve a concentration of at least 1 g/L of a non-ionic block copolymer and passing the cell culture through a hollow fiber filter having a pore size or molecular weight cut off (MWCO) that retains the recombinant protein in the bioreactor, and collecting a permeate; once a predetermined parameter is reached perfusing the cell culture with fresh cell culture medium formulated or supplemented to achieve a concentration of at least 5 g/L of a non-ionic block copolymer and passing the cell culture through a hollow fiber filter having a pore size or molecular weight cut off (MWCO) that does not retain the recombinant protein in the bioreactor and collecting a permeate containing the recombinant protein.

In one embodiment the molecular weight cutoff of the hollow fiber filter having a pore size or molecular weight cut off that retains the recombinant protein in the bioreactor is 300 kDa or less. In one embodiment the hollow fiber filter having a pore size or molecular weight cut off that retains the recombinant protein in the bioreactor is an ultrafilter.

In one embodiment the molecular weight cutoff of the hollow fiber filter having a pore size or molecular weight cut off (MWCO) that does not retain the recombinant protein in the bioreactor is at least 500 kDa. In one embodiment the hollow fiber filter having a pore size or molecular weight cut off (MWCO) that does not retain the recombinant protein in the bioreactor is a microfilter.

In one embodiment the a hollow fiber filter having a pore size or molecular weight cut off that retains the recombinant protein in the bioreactor and the hollow fiber filter having a pore size or molecular weight cut off that does not retain the recombinant protein in the bioreactor are components of a single unit filter system.

In one embodiment the non-ionic block copolymer is a polyoxypropylene-polyoxyethylene block copolymer. In one embodiment the non-ionic block copolymer is poloxamer 188.

In one embodiment the method above further comprises taking samples during the cell culture processes, evaluating the samples to quantitatively and/or qualitatively monitor characteristics of the recombinant protein and/or the cell culture process. In one embodiment the samples to quantitatively and/or qualitatively monitored using process analytical techniques.

In one embodiment the perfusing is continuous perfusion. In one embodiment the rate of perfusion is constant. In one embodiment the perfusing is performed at a rate of less than or equal to 1.0 working volume per day. In one embodiment the perfusing is accomplished by a peristaltic pump, a double diaphragm pump, a low shear pump or alternating tangential flow. In one embodiment the perfusing is accomplished by alternating tangential flow.

In one embodiment the method above further comprises subjecting the cell culture to a temperature shift wherein the cells are cultured a) at first temperature for a first period of time and b) at second temperature for a second period of time. In a related embodiment n the temperature shift occurs at the transition between the growth phase and production phase. In a related embodiment the temperature shift occurs during the production phase. In a related embodiment the temperature shift is in response to a predetermined parameter. In a related embodiment the temperature shift is in response to a predetermined parameter wherein reaching the predetermined parameter is determined using a capacitance based biomass probe.

In one embodiment the cell culture is established by inoculating the bioreactor with at least 0.1×106viable cells/mL. In one embodiment the inoculum was grown by means of a perfusion process using alternating tangential flow filtration.

In one embodiment prior to entering the bioreactor, the cell culture medium is treated using nanofiltration, high temperature short time (HTST), or UV in combination with filtration.

In one embodiment the bioreactor is a production bioreactor. In one embodiment n the bioreactor has a capacity of at least 500 L. In a related embodiment the bioreactor has a capacity of at least 500 L to 2000 L. In a related embodiment the bioreactor has a capacity of at least 1000 L to 2000 L.

In one embodiment the cell culture medium is a serum-free cell culture medium. In one embodiment the cell culture medium is a serum-free chemically defined cell culture medium. In one embodiment the cell culture medium is a perfusion cell culture medium.

In one embodiment the mammalian cells are Chinese Hamster Ovary (CHO) cells.

In one embodiment the recombinant protein is selected from the group consisting of a human antibody, a humanized antibody, a chimeric antibody, a recombinant fusion protein, or a cytokine.

In one embodiment the recombinant protein is purified from the harvest permeate by one or more of flocculation, precipitation, centrifugation, depth filtration, affinity chromatography, size exclusion chromatography, ion exchange chromatography, mixed mode anion exchange chromatography, hydrophobic interaction chromatography or hydroxyapatite chromatography.

In one embodiment the method above further comprises taking samples during the purification process, evaluating the samples to quantitatively and/or qualitatively monitor characteristics of the recombinant protein and the production process.

In one embodiment the recombinant protein is formulated into a pharmaceutically acceptable formulation.

In one embodiment is provided a recombinant protein produced by the above method.

The invention also provides a single unit filter system comprising two or more hollow fiber filter components of different pore sizes or molecular weight cut offs (MWCO), wherein the hollow fiber filter components are secured to one another in series such that a sterile flow path is maintained between the individual hollow fibers and the hollow fiber filter components of different pore size or molecular weight cut offs are isolated from one another with respect to their hollow shell sides from which the permeate is withdrawn, such that permeate can be removed independently from each respective hollow fiber filter component.

In one embodiment at least one hollow fiber filter component has a pore size or molecular weight cut off that retains the recombinant protein in the bioreactor and at least one hollow fiber filter component has a pore size filter that is a hollow fiber filter having a pore size or molecular weight cut off that does not retain the recombinant protein in the bioreactor. In a related embodiment at least one hollow fiber filter component has a molecular weight cutoff of 300 kDa or less and at least one hollow fiber filter component has a molecular weight cutoff of at least 500 kDa. In a related embodiment at least one hollow fiber filter component is an ultrafilter and at least one hollow fiber filter component is a microfilter. In a related embodiment the single unit filter system is contained within a housing. In one embodiment the single filter unit further comprising a spacer between at least two of the hollow fiber filter components

The method also provides a method for culturing cells expressing a recombinant protein comprising establishing a cell culture by inoculating a bioreactor with mammalian cells expressing a recombinant protein, maintaining the cell culture by perfusing fresh cell culture medium into the bioreactor, passing the cell culture through a single unit filter system and collecting a permeate, wherein the single unit filter system is attached to the bioreactor and the cell culture is drawn out of the bioreactor and into the single unit filter system by a single pumping system, wherein the cell culture passes through the lumen side of the hollow fibers of the single unit filter system and back into the bioreactor and a permeate is withdrawn from one or more of the hollow fiber filters components.

In one embodiment the method above further comprise taking samples during the cell culture processes, evaluating the samples to quantitatively and/or qualitatively monitor characteristics of the recombinant protein and/or the cell culture process. In a related process the samples are quantitatively and/or qualitatively monitored by process analytical techniques.

In one embodiment the perfusing is continuous perfusion. In one embodiment the rate of perfusion is constant. In one embodiment the perfusing is performed at a rate of less than or equal to 1.0 working volume per day.

In one embodiment the perfusing is accomplished by a peristaltic pump, a double diaphragm pump, a low shear pump or alternating tangential flow. In one embodiment the perfusing is accomplished by alternating tangential flow.

In one embodiment when the permeate is collected from a hollow fiber filter component that has a pore size or molecular weight cut off that does not retain the recombinant protein in the bioreactor, the fresh cell culture medium is formulated with or supplemented to achieve at least 5 g/L of a non-ionic block copolymer. In a related embodiment the non-ionic block copolymer is a polyoxypropylene-polyoxyethylene block copolymer. In a related embodiment the non-ionic block copolymer is poloxamer 188.

In one embodiment the method above further comprises subjecting the cell culture to a temperature shift wherein the cells are cultured a) at first temperature for a first period of time and b) at second temperature for a second period of time. In one embodiment the temperature shift occurs at the transition between the growth phase and production phase. In a related embodiment the temperature shift occurs during the production phase. In a related embodiment the temperature shift is in response to a predetermined parameter wherein reaching the predetermined parameter is determined using a capacitance based biomass probe.

In one embodiment the cell culture is established by inoculating the bioreactor with at least 0.1×106viable cells/mL. In a related embodiment the inoculum was grown by means of a perfusion process using alternating tangential flow filtration.

In one embodiment prior to entering the bioreactor, the cell culture medium is treated using nanofiltration, high temperature short time (HTST), or UV in combination with filtration.

In one embodiment the bioreactor is a production bioreactor. In a related embodiment the bioreactor has a capacity of at least 500 L. In a related embodiment the bioreactor has a capacity of at least 500 L to 2000 L. In a related embodiment the bioreactor has a capacity of at least 1000 L to 2000 L.

In one embodiment the cell culture medium is a serum-free cell culture medium. In one embodiment the cell culture medium is a serum-free chemically defined cell culture medium. In one embodiment the cell culture medium is a perfusion cell culture medium.

In one embodiment the mammalian cells are Chinese Hamster Ovary (CHO) cells.

In one embodiment the recombinant protein is selected from the group consisting of a human antibody, a humanized antibody, a chimeric antibody, a recombinant fusion protein, or a cytokine.

In one embodiment the recombinant protein is purified from the harvest permeate by one or more of flocculation, precipitation, centrifugation, depth filtration, affinity chromatography, size exclusion chromatography, ion exchange chromatography, mixed mode anion exchange chromatography, hydrophobic interaction chromatography or hydroxyapatite chromatography

In one embodiment the method above further comprising taking samples during the purification process, evaluating the samples to quantitatively and/or qualitatively monitor characteristics of the recombinant protein and the production process.

In one embodiment the recombinant protein is formulated into a pharmaceutically acceptable formulation. In one embodiment is provided a recombinant protein produced the method above.

