Modulating product quality of asymmetric multispecific antibodies through the use of temperature
AssigneeAmgen Inc.
InventorsLi ZHANG, Hedieh BARKHORDARIAN, Ewelina ZASADZINSKA, Jonathan DIEP
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Abstract
The present invention relates to the field of biopharmaceutical manufacturing. In particular, the invention relates to using temperature as a lever to modulate product quality during upstream operations.
Claims62
A method for modulating the product quality of a recombinant asymmetric multispecific antibody expressed by a cell during cell culture comprising the steps:
- a) establishing at least two cell cultures each inoculated with the same cell line expressing the asymmetric multispecific antibody;
- b) culturing at least one cell culture at a first temperature regime that consists of a single temperature and at least one cell culture at a second temperature regime;
- c) comparing at least one product-related impurity in the cell culture at each temperature regime;
- d) selecting the temperature regime that reduces the expression of at least one product-related impurity comprising an unpaired or mis-paired long heavy chain;
- e) culturing the cell line at the selected temperature regime;
- and
- f) harvesting the recombinant asymmetric multispecific antibody.
A method for modulating the product quality of a recombinant asymmetric multispecific antibody comprising a mutein expressed by a cell during cell culture comprising the steps:
- a) establishing at least two cell cultures each inoculated with the same cell line expressing the asymmetric multispecific antibody;
- b) culturing at least one cell culture at a first temperature regime that consists of a single temperature and at least one cell culture at a second temperature regime;
- c) comparing at least one product-related impurity comprising an unpaired or mis-paired long heavy chain produced by a cell culture at each temperature regime;
- and
- d) selecting the temperature regime that reduces the expression of at least one product-related impurity comprising an unpaired or mis-paired long heavy chain.
A method for modulating the product quality of a conjugate of an IL-21 mutein linked to the C-terminus of one of the two antibody heavy chains of an anti-PD-1 antibody expressed by a cell during cell culture comprising the steps:
- a) establishing a cell culture inoculated with a cell line expressing the antibody;
- b) culturing the cells at 36+1° C. for the duration of the culture;
- and
- c) harvesting the antibody;
- wherein amount of at least one product-related impurity resulting from an imbalance in the ratio of long heavy chain to short heavy chain in the harvested cell culture is decreased compared to the amount of the same product-related impurity in the harvest from a cell culture exposed to a temperature of 32° C. to 34° C. at some point during the culture.
A method for selecting a cell line expressing an asymmetric multispecific antibody comprising a mutein comprising the steps:
- a) establishing at least one clonally derived cell line expressing the antibody;
- b) establishing at least two cell cultures from one or more of the clonally derived cell lines, wherein at least one cell culture from each cell line is cultured at a first temperature regime and at least one cell culture from each cell line is cultured at a second temperature regime;
- c) comparing the expression of at least one product-related impurity comprising an unpaired or mis-paired long heavy chain produced by the cells cultured at each temperature regime;
- and
- d) selecting a cell line that resulted in reduced expression of at least one product-related impurity comprising an unpaired or mis-paired long heavy chain.
A method for modulating production of at least one recombinant asymmetric multispecific antibody product-related impurity comprising an antibody heavy chain having an attached IL-21 mutein during cell culture through cell culture temperature comprising the steps:
- a) selecting the temperature regime that reduces the expression of at least one product-related impurity comprising an unpaired or mis-paired long heavy chain comprising a long heavy chain;
- b) culturing the cell line at the selected temperature regime;
- and
- c) harvesting the recombinant asymmetric multispecific antibody.
A method for producing an isolated, purified, recombinant asymmetric multispecific antibody, the method comprising the steps:
- a) isolating at least one single stably transformed cell expressing the asymmetric multispecific antibody and establishing a clonally derived culture;
- b) establishing at least two cell cultures from one or more of the clonally derived cultures;
- c) culturing at least one cell culture from each clonally derived culture at a first temperature regime comprising a single temperature and culturing at least one cell culture from each clonally derived culture at a second temperature regime;
- d) comparing the amount of at least one product-related impurity comprising an unpaired or mis-paired long heavy chain produced by the cells cultured at each temperature regime;
- e) selecting the cell culture that modulated production of the product related impurity;
- f) establishing a cell line expressing the asymmetric multispecific antibody from the selected cell culture;
- g) inoculating a bioreactor with the cell line expressing the asymmetric multispecific antibody;
- h) culturing the cells to express the asymmetric multispecific antibody at the temperature regime that modulated the production of the product-related impurity;
- i) harvesting the recombinant asymmetric multispecific antibody from the cell culture;
- j) processing the recombinant multispecific antibody through one or more chromatography unit operations;
- and
- k) obtaining an isolated, purified, recombinant asymmetric multispecific antibody.
A method for controlling the growth of cells in a nanofluidic chamber of a nanofluidic chip comprising the steps of:
- (a) isolating a single cell in to a nanofluidic chamber of a nanofluidic chip, wherein said cell comprises an expression construct capable of expressing a recombinant protein;
- (b) culturing the cell at a first temperature;
- (c) at a predetermined point, culturing the cells at a second temperature;
- and
- (d) exporting the cells out of the nanofluidic chamber and into a culture vessel.
A method for minimizing cell cross contamination during export of cells grown in a nanofluidic chamber of a nanofluidic chip comprising the steps of:
- (a) isolating a single cell in to a nanofluidic chamber of a nanofluidic chip, wherein said cell comprises an expression construct capable of expressing a recombinant protein;
- (b) culturing the cell at a first temperature;
- (c) at a predetermined point, culturing the cells at a second temperature;
- and
- (d) exporting the cells out of the nanofluidic chamber and into a culture vessel;
- wherein the number of cells per chamber at export is lower compared to a single cell cultured under similar conditions a constant temperature for the duration of the culture.
A method to improve clone selection for cells grown in a nanofluidic chamber of a nanofluidic chip comprising the steps of:
- (a) isolating a single cell in to a nanofluidic chamber of a nanofluidic chip, wherein said cell comprises an expression construct capable of expressing a recombinant protein;
- (b) culturing the cell at a first temperature;
- (c) lowering the temperature of the culture to a second temperature no earlier than day 3 of the culture;
- and
- (d) exporting the cells out of the nanofluidic chamber at least one to four days after the temperature shift.
Dependencies are inferred from the claim text.