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US 2016/0108111 A1Published application
Methods of shifting an isoelectric profile of a protein product and uses thereof
AssigneeAlexion Pharmaceuticals, Inc.
InventorsHunter F. Malanson, Pratik Jaluria, Rachael Alford
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Abstract
Provided herein are methods shifting the isoelectric profile of a recombinant protein product and the use of such methods in the manufacture of recombinant protein products. Also provided are recombinant protein products produced by such methods.
Claims55
A method of shifting the isoelectric profile of a recombinant protein product, the method comprising:
- (a) culturing mammalian cells comprising a nucleic acid encoding a recombinant protein in a production bioreactor under conditions sufficient to produce the product for a first period of time that consists of a growth phase and/or a stationary phase of the mammalian cells;
- (b) assaying the product isoelectric profile at the end of the first period of time;
- and
- (c) incubating the product under conditions sufficient to shift the isoelectric profile of the product toward a more acidic profile for a second period of time that consists of at least 6 hours of a decline phase of the mammalian cells, or
- incubating the product under conditions sufficient to shift the isoelectric profile of the product toward a more basic profile for a second period of time of a decline phase of the mammalian cells;
- and,
- (d) assaying the product to determine the isoelectric profile and repeating step (c) if the isoelectric profile requires further shifting.
4 more elements
A method of shifting the isoelectric profile of a recombinant protein product, the method comprising:
- (a) culturing mammalian cells comprising a nucleic acid encoding a recombinant protein in a production bioreactor under conditions sufficient to produce the product for a first period of time that comprises a growth phase of the mammalian cells and terminates at a critical time point;
- (b) incubating the product under conditions sufficient to shift the isoelectric profile of the product toward a more acidic profile for a second period of time that consists of at least 6 hours of a decline phase of the mammalian cells;
- and
- (c) assaying the product to determine the isoelectric profile and repeating step (b) if the isoelectric profile requires further shifting toward a more acidic profile.
1 more element
A method of shifting the isoelectric profile of a recombinant protein product, the method comprising:
- (a) culturing mammalian cells comprising a nucleic acid encoding a recombinant protein in a production bioreactor under conditions sufficient to produce the product for a first period of time that that comprises a growth phase of the mammalian cells and terminates at a critical time point;
- (b) incubating the product under conditions sufficient to shift the isoelectric profile of the product toward a more basic profile for a second period of time of a decline phase of the mammalian cells;
- and
- (c) assaying the product to determine the isoelectric profile and repeating step (b) if the isoelectric profile requires further shifting toward a more basic profile.
1 more element
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25-27. (canceled)
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31-32. (canceled)
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37-39. (canceled)
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46-49. (canceled)
A method of shifting the isoelectric profile of a recombinant protein product having seven protein subpopulations with an isoelectric point between about 5.45 and about 6.55, the method comprising:
- (a) fed batch culturing mammalian cells comprising a nucleic acid encoding a recombinant protein in a production bioreactor under conditions sufficient to produce the product for a first period of time that consists of a growth phase or a growth phase and a stationary phase of the mammalian cells;
- (b) incubating the product under conditions sufficient to shift the isoelectric profile of the product toward a more acidic profile for a second period of time that consists of at least 6 hours of a decline phase of the mammalian cells, wherein the more acidic profile comprises an increase in the quantity of the fourth and fifth most acidic protein subpopulations of the seven protein subpopulations, and a decrease in the quantity of the first and second most basic protein subpopulations of the seven protein subpopulations;
- and optionally
- (c) assaying the product to determine the isoelectric profile and repeating step (b) if the isoelectric profile requires further shifting toward the more acidic profile.
1 more element
A method of shifting the isoelectric profile of a recombinant protein product having seven protein subpopulations with an isoelectric point between about 5.45 and about 6.55, the method comprising:
- (a) fed batch culturing mammalian cells comprising a nucleic acid encoding a recombinant protein in a production bioreactor under conditions sufficient to produce the product for a first period of time that comprises a growth phase of the mammalian cells and terminates at a critical time point;
- (b) incubating the product under conditions sufficient to shift the isoelectric profile of the product toward a more acidic profile for a second period of time that consists of at least 6 hours of a decline phase of the mammalian cells, wherein the more acidic profile comprises an increase in the quantity of the fourth and fifth most acidic protein subpopulations of the seven protein subpopulations, and a decrease in the quantity of the first and second most basic protein subpopulations of the seven protein subpopulations;
- and optionally
- (c) assaying the product to determine the isoelectric profile and repeating step (b) if the isoelectric profile requires further shifting toward the more acidic profile.
1 more element
(canceled)
A recombinant protein product produced by a method comprising:
- (a) culturing mammalian cells comprising a nucleic acid encoding a recombinant protein in a production bioreactor under conditions sufficient to produce the product for a first period of time that consists of a growth phase and/or a stationary phase of the mammalian cells;
- (b) assaying the product at the end of the first period of time to the isoelectric profile;
- and
- (c) incubating the product under conditions sufficient to shift the isoelectric profile of the product toward a more acidic profile for a second period of time that consists of at least 6 hours of a decline phase of the mammalian cells, or
- incubating the product under conditions sufficient to shift the isoelectric profile of the product toward a more basic profile for a second period of time of a decline phase of the mammalian cells;
- and,
- (d) assaying the product to determine the isoelectric profile and repeating step (c) if the isoelectric profile requires further shifting.
4 more elements
55-56. (canceled)
Dependencies are inferred from the claim text.