CN117607450A - Method for determining protein FcRn binding, screening drugs, determining sample stability and predicting PK/PD and half-life in vivo - Google Patents
Method for determining protein FcRn binding, screening drugs, determining sample stability and predicting PK/PD and half-life in vivo Download PDFInfo
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6845—Methods of identifying protein-protein interactions in protein mixtures
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
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Abstract
Methods for determining protein FcRn binding, screening drugs, determining sample stability and predicting PK/PD and half-life in vivo are disclosed. Wherein the method of determining protein FcRn binding comprises: (a) immobilizing FcRn expressing cells on a solid support; (b) incubating the solid support with a blocking agent; (c) Labeling the protein with a first molecule and incubating the labeled protein with the solid support at an acidic pH; (d) Incubating a second molecule with the solid support, the second molecule specifically binding to the first molecule; and (e) detecting binding of the first molecule to the second molecule.
Description
Technical Field
The present application relates to the field of protein binding assays, and in particular to methods for determining protein FcRn binding, screening protein drugs, determining protein sample stability, and predicting PK/PD and/or half-life in vivo of Fc-containing macromolecular drugs.
Background
Antibodies are components that link specific antigen recognition to different effector mechanisms of the immune system. Antibodies have two functional domains, a Fab (antigen binding fragment) region and an Fc region. The Fab region is responsible for antigen recognition, while the Fc region couples antibodies to immune effector cells. The process of antibody class switching causes a change in the expression of the heavy chain constant region, and thus the Fc region, of B cells, resulting in an Fc region with different effector functions. The different localization of B cell blast and plasma cells expressing these different heavy chains brings about different localization and effector cell recognition for the five antibody classes (IgA, igD, igE, igG and IgM).
Of these five antibody classes, igG is the most widely distributed class in serum and non-mucosal tissues. IgG antibodies play an important role in protective immunity against a variety of pathogens and toxins. As a demonstration of its important role in protective immunity, igG has long been considered the only class of antibodies that actively migrate from the parent to the offspring, conferring short-term passive immunity, and this particular IgG transport is accomplished through neonatal Fc receptors (FcRn). FcRn transfers IgG from the mother to the fetus through the placenta and proximal small intestine.
At present, the mainstream of products in the biopharmaceutical and biotechnology industries is to use mammalian engineering cell lines for expression and production of monoclonal antibodies or bispecific antibodies. The Fc-terminus of antibodies has a strong correlation with neonatal receptor affinity and drug metabolism, i.e., effective plasma concentration. At present, the affinity between the Fc end of the antibody and the neonatal receptor is mainly measured by means of protein levels such as ELISA and SPR, and a detection means closer to a physiological state is lacking, and the detection of the neonatal receptor affinity at the protein level sometimes fails to directly reflect the change of some macromolecules in the space conformation due to the limitation of the detection means, so that a high-sensitivity detection means closer to the physiological state is lacking.
The Fc region of IgG-based molecules plays an important role in determining their in vivo, and pharmacokinetic profiles are achieved by its pH-dependent binding to neonatal Fc receptor (FcRn). Because of this specific pH, fcRn also transfers maternal IgG from mother to fetus through placenta by maintaining IgG homeostasis, serum IgG levels in human adults upon interaction with IgG-Fc. The Fc-FcRn interaction is also critical to maintaining circulation of IgG-based therapeutic molecules. At present, a method for releasing and characterizing the combination of monoclonal antibodies for treating neonatal Fc receptor based on cell homogeneity detection, which is close to physiological state, is lacking, can be used as an analysis tool between evaluation batches, and can be used for testing the consistency and the stability of different batches of the same molecule.
Chinese patent application number CN202010822793.0 discloses a method for preparing cell lines stably expressing hFcRn and its application in drug screening. Although this application uses hFcRn-expressing cells, it is not used to directly detect antibody binding to FcRn, but rather to detect antibodies that are transported across membranes and to screen compounds that have an effect on hFcRn-mediated endocytosis, exocrine recycling, and transport across membranes. The application still does not address the problem of providing a detection means that more accurately and sensitively detects biological macromolecules, particularly antibodies, and is closer to physiological conditions.
Antibodies have a long half-life in vivo compared to other proteins or small molecules, an important reason being the ability to bind to a specific Fc receptor FcRn. The structure of FcRn receptor is composed of an alpha chain and beta microglobulin. FcRn is widely expressed in vivo, including epithelium of many tissues, endothelial cells, and various immune cells (e.g., monocytes macrophages), among others. FcRn binding to IgG is pH dependent: binding was only around weakly acidic pH6.0, whereas no binding was found at neutral pH. When free antibodies in blood are non-specifically endocytosed by cells into the endosome (endosome), igG binds to FcRn through the Fc region of the antibody due to the weak intra-endosome pH (5-6). The combined antibody is transported to the surface of a cell membrane along with the FcRn, and the pH of extracellular fluid is near neutral (about 7.4), so that the IgG loses binding with the FcRn and reenters the blood circulation, thereby prolonging the half-life of the IgG in vivo. While other proteins that do not bind FcRn cannot be recovered by FcRn in the intracellular space and eventually enter lysosomal degradation, resulting in a shorter half-life.
There remains a need in the art for a method that can more accurately and sensitively detect biological macromolecules, particularly antibodies, in closer physiological conditions, screen protein drugs and determine protein sample stability, and help predict PK/PD and/or half-life in vivo of Fc-containing macromolecular drugs.
Disclosure of Invention
To solve the above technical problem, one aspect of the present application provides a method for determining protein FcRn binding, the method comprising:
(a) Immobilizing FcRn expressing cells on a solid support;
(b) Incubating the solid support with a blocking agent;
(c) Labeling the protein with a first molecule and incubating the labeled protein with the solid support at an acidic pH;
(d) Incubating a second molecule with the solid support, the second molecule specifically binding to the first molecule; and
(e) Detecting binding of the first molecule to the second molecule.
Another aspect of the present application provides a method of screening for a protein drug, the method comprising:
(1) FcRn binding of a plurality of protein candidates is determined by a method comprising the steps of:
(a) Immobilizing FcRn expressing cells on a solid support;
(b) Incubating the solid support with a blocking agent;
(c) Labeling the protein with a first molecule and incubating the labeled protein with the solid support at an acidic pH;
(d) Incubating a second molecule with the solid support, the second molecule specifically binding to the first molecule; and
(e) Detecting binding of the first molecule to the second molecule; and
(2) Screening the protein drug for FcRn binding of the plurality of protein candidates.
Another aspect of the present application provides a method of determining the stability of a protein sample, the method comprising:
(1) FcRn binding of a plurality of protein samples is determined by a method comprising the steps of:
(a) Immobilizing FcRn expressing cells on a solid support;
(b) Incubating the solid support with a blocking agent;
(c) Labeling the protein with a first molecule and incubating the labeled protein with the solid support at an acidic pH;
(d) Incubating a second molecule with the solid support, the second molecule specifically binding to the first molecule; and
(e) Detecting binding of the first molecule to the second molecule; and
(2) Comparing FcRn binding of the plurality of protein samples, thereby determining stability of the plurality of protein samples.
Another aspect of the present application provides a method of predicting in vivo PK/PD and/or half-life of an Fc-containing macromolecular drug, said method comprising:
(1) Determining FcRn binding of said Fc-containing macromolecular drug by a method comprising the steps of:
(a) Immobilizing FcRn expressing cells on a solid support;
(b) Incubating the solid support with a blocking agent;
(c) Labeling the protein with a first molecule and incubating the labeled protein with the solid support at an acidic pH;
(d) Incubating a second molecule with the solid support, the second molecule specifically binding to the first molecule; and
(e) Detecting binding of the first molecule to the second molecule; and
(2) Predicting the in vivo PK/PD and/or half-life of said Fc-containing macromolecular drug.
Drawings
The present application is described in more detail below with reference to the attached drawing figures, wherein:
FIG. 1 is a schematic illustration of a detection process employing a graphite electrode high adsorption 96-well plate in a method according to one embodiment of the present application;
FIG. 2 is a schematic representation of detection results using different pH conditions in a method according to one embodiment of the present application.
Detailed Description
The present application relates to a method for determining protein FcRn binding. The method comprises the following steps: (a) immobilizing FcRn expressing cells on a solid support. In one embodiment of the present application, the protein includes a protein comprising an antibody Fc region. In one embodiment of the present application, the protein comprises an Fc fusion protein. In one embodiment of the present application, the protein comprises an antibody. In a preferred embodiment of the present application, the protein comprises a monoclonal antibody. In a preferred embodiment of the present application, the protein comprises IgG. In this application, the solid support may be any solid material known in the art capable of immobilizing cells. In one embodiment of the present application, the solid support comprises a multi-well plate. In one embodiment of the present application, the solid support comprises a high-adsorptivity porous plate. In a preferred embodiment of the present application, the solid support comprises a graphite electrode highly adsorptive porous plate. In one embodiment of the present application, immobilization includes adsorption, coupling, covalent attachment, van der Waals forces, electrostatic interactions, or adhesion. In one embodiment of the present application, immobilizing includes adsorption. In the present application, the FcRn expressing cell may be any cell known in the art that can express FcRn on its cell surface. In one embodiment of the present application, fcRn is mammalian FcRn. In one embodiment of the present application, fcRn is human FcRn. In one embodiment of the present application, fcRn includes Uniprot: the amino acid sequence of P55899 or the amino acid sequence homologous thereto. In one embodiment of the present application, the FcRn expressing cells comprise eukaryotic cells. In one embodiment of the present application, the FcRn expressing cells comprise mammalian cells. In one embodiment of the present application, the FcRn expressing cells comprise human cells. In a preferred embodiment of the present application, the FcRn expressing cells comprise human embryonic kidney cells. In one embodiment of the present application, the expression of FcRn in FcRn expressing cells is endogenous. In one embodiment of the present application, the expression of FcRn in the FcRn expressing cells is exogenous. In one embodiment of the present application, the time for fixing FcRn expressing cells on a solid support is about 1 minute to 5 hours. In one embodiment of the present application, the time for fixing FcRn expressing cells on a solid support is about 10 minutes to 2 hours. In one embodiment of the present application, the time for fixing FcRn expressing cells on a solid support is about 30 minutes to 1.5 hours. In one embodiment of the present application, the time to immobilize FcRn expressing cells on a solid support is about 1 hour. In one embodiment of the present application, the temperature at which FcRn expressing cells are immobilized on a solid support is about 10 to 40 ℃. In one embodiment of the present application, the temperature at which FcRn expressing cells are immobilized on a solid support is about 20 to 30 ℃. In one embodiment of the present application, the temperature at which FcRn expressing cells are immobilized on a solid support is about 25 ℃.
The method of determining protein FcRn binding of the present application further comprises: (b) incubating the solid support with a blocking agent. In this application, the blocking agent may be any agent known in the art that prevents the immobilization of the unrelated protein to the surface of the solid support. In one embodiment of the present application, the blocking agent comprises a protein solution. In one embodiment of the present application, the blocking agent comprises milk or a dilution thereof, such as animal milk, e.g. cow milk. In one embodiment of the present application, the blocking agent comprises a solution comprising Bovine Serum Albumin (BSA). In one embodiment of the present application, the blocking agent comprises a buffer comprising BSA. In one embodiment of the present application, the buffer comprises a PBS solution. In one embodiment of the present application, the concentration of BSA in the capping reagent is about 1-5%. In one embodiment of the present application, the concentration of BSA in the blocking agent is about 2%. In one embodiment of the present application, the solid support is incubated with the blocking agent for a period of time ranging from about 1 minute to 5 hours. In one embodiment of the present application, the solid support is incubated with the blocking agent for a period of about 10 minutes to 2 hours. In one embodiment of the present application, the solid support is incubated with the blocking agent for a period of about 30 minutes to 1.5 hours. In one embodiment of the present application, the solid support is incubated with the blocking agent for a period of about 1 hour. In one embodiment of the present application, the temperature at which the solid support is incubated with the blocking agent is about 10 to 40 ℃. In one embodiment of the present application, the temperature at which the solid support is incubated with the blocking agent is about 20 to 30 ℃. In one embodiment of the present application, the temperature at which the solid support is incubated with the blocking agent is about 25 ℃.
The method of determining protein FcRn binding of the present application further comprises: (c) The protein is labeled with a first molecule and the labeled protein is incubated with the solid support at an acidic pH. In one embodiment of the present application, the first molecule comprises biotin. In one embodiment of the present application, the acidic pH is about 5.0 to 7.0. In one embodiment of the present application, the acidic pH is about 5.5 to 6.5. In a preferred embodiment of the present application, the acidic pH is about 6.0. In one embodiment of the present application, step (c) comprises diluting the labeled protein with a buffer of acidic pH. In one embodiment of the present application, the dilution buffer comprises a buffer. In one embodiment of the present application, the buffer comprises PBS. In one embodiment of the present application, the dilution buffer comprises BSA. In one embodiment of the present application, the concentration of BSA in the dilution buffer is about 0.1-1%. In one embodiment of the present application, the concentration of BSA in the blocking agent is about 0.5%. In one embodiment of the present application, dilution includes gradient dilution. In one embodiment of the present application, gradient dilution includes serial gradient dilution. In one embodiment of the present application, serial gradient dilution includes 1.2-4 fold serial gradient dilution. In one embodiment of the present application, serial gradient dilution includes 1.5-2 fold serial gradient dilution. In one embodiment of the present application, serial gradient dilution includes about 2-fold serial gradient dilution. In one embodiment of the present application, serial gradient dilution includes about 1.5 times serial gradient dilution. In one embodiment of the present application, serial gradient dilution includes a combination of about 1.5-fold serial gradient dilution in part and about 2-fold serial gradient dilution in part. In one embodiment of the present application, serial gradient dilution includes serial gradient dilution where the concentration of the labeled protein ranges from about 200 to 0.01 μg/mL. In one embodiment of the present application, serial gradient dilution includes serial gradient dilution where the concentration of the labeled protein ranges from about 100 to 0.1 μg/mL. In one embodiment of the present application, serial gradient dilution includes serial gradient dilution where the concentration of the labeled protein ranges from about 80-0.1234 μg/mL. In one embodiment of the present application, the labeled protein is incubated with the solid support for a period of about 1 minute to 5 hours. In one embodiment of the present application, the labeled protein is incubated with the solid support for a period of about 10 minutes to 2 hours. In one embodiment of the present application, the labeled protein is incubated with the solid support for a period of about 30 minutes to 1.5 hours. In one embodiment of the present application, the time for which the labeled protein is incubated with the solid support is about 1 hour. In one embodiment of the present application, the temperature at which the labeled protein is incubated with the solid support is about 10 to 40 ℃. In one embodiment of the present application, the temperature at which the labeled protein is incubated with the solid support is about 20 to 30 ℃. In one embodiment of the present application, the temperature at which the labeled protein is incubated with the solid support is about 25 ℃.
The method of determining protein FcRn binding of the present application further comprises: (d) Incubating a second molecule with the solid support, the second molecule specifically binding to the first molecule. In one embodiment of the present application, the second molecule comprises avidin or streptavidin. In one embodiment of the present application, the second molecule comprises streptavidin. In one embodiment of the present application, the second molecule is incubated with the solid support for a period of time ranging from about 1 minute to 5 hours. In one embodiment of the present application, the second molecule is incubated with the solid support for a period of about 10 minutes to 2 hours. In one embodiment of the present application, the second molecule is incubated with the solid support for a period of time ranging from about 30 minutes to 1.5 hours. In one embodiment of the present application, the second molecule is incubated with the solid support for a period of about 1 hour. In one embodiment of the present application, the temperature at which the second molecule is incubated with the solid support is about 10 to 40 ℃. In one embodiment of the present application, the temperature at which the second molecule is incubated with the solid support is about 20 to 30 ℃. In one embodiment of the present application, the temperature at which the second molecule is incubated with the solid support is about 25 ℃.
The method of determining protein FcRn binding of the present application further comprises: (e) Detecting binding of the first molecule to the second molecule. In one embodiment of the present application, the second molecule is labeled with a detectable label. In one embodiment of the present application, step (e) comprises determining the signal intensity of the detectable label. In one embodiment of the present application, the detectable label comprises a luminescent label, a fluorescent label, or a radioactive label. In one embodiment of the present application, the second molecule is labeled with a catalyst. In one embodiment of the present application, the second molecule is labeled with an enzyme. In one embodiment of the present application, the enzyme comprises horseradish peroxidase. In one embodiment of the present application, step (e) further comprises adding a substrate for the enzyme and detecting the substrate reaction. In one embodiment of the present application, the substrate reaction comprises a chromogenic reaction. In one embodiment of the present application, the substrate comprises tetramethylbenzidine. In one embodiment of the present application, the incubation time of step (e) is about 1 minute to 1 hour. In one embodiment of the present application, the incubation time of step (e) is about 5 minutes to 20 minutes. In one embodiment of the present application, the incubation time of step (e) is about 10 minutes. In one embodiment of the present application, the incubation temperature of step (e) is about 10 to 40 ℃. In one embodiment of the present application, the incubation temperature of step (e) is about 20 to 30 ℃. In one embodiment of the present application, the incubation temperature of step (e) is about 25 ℃. In one embodiment of the present application, the incubation temperature of step (e) is room temperature. In one embodiment of the present application, step (e) further comprises adding a stop solution. In one embodiment of the present application, the stop solution comprises a sulfuric acid solution. In one embodiment of the present application, the stop solution comprises a 2M sulfuric acid solution.
In one embodiment of the present application, in the method of determining protein FcRn binding of the present application, one or more or all of steps (a), (b), (c) and (d) is further followed by a washing step. In one embodiment of the present application, in the method of determining protein FcRn binding of the present application, each of steps (a), (b), (c) and (d) is further followed by a washing step. In one embodiment of the present application, the washing step comprises washing with a wash solution of acidic pH. In one embodiment of the present application, the acidic pH of the wash liquor is in the range of about 5.0 to 7.0. In one embodiment of the present application, the acidic pH of the wash liquor is about 5.5 to 6.5. In a preferred embodiment of the present application, the acidic pH of the wash liquor is about 6.0. In one embodiment of the present application, the wash solution includes a buffer. In one embodiment of the present application, the wash solution comprises PBS.
The present application also relates to a method of screening for a drug. The method comprises the following steps: (1) FcRn binding of a plurality of protein candidates is determined by a method for determining protein FcRn binding. In one embodiment of the present application, the method of determining protein FcRn binding comprises the method described in any one of the preceding embodiments of the present application.
The method for screening drugs of the present application further comprises: (2) Screening the drug for FcRn binding of the plurality of protein candidates. In one embodiment of the present application, step (2) comprises comparing FcRn binding of the plurality of protein candidates. In one embodiment of the present application, step (2) comprises screening protein candidates whose FcRn binding is greater than the average binding of the plurality of protein candidates. In one embodiment of the present application, step (2) comprises comparing the signal intensities of the detectable labels of the plurality of protein candidates. In one embodiment of the present application, step (2) comprises screening for protein candidates whose detectable label has a signal intensity greater than the signal intensity of the average detectable label of the plurality of protein candidates. In one embodiment of the present application, step (2) comprises screening for protein candidates whose detectable label has a signal intensity that is at least 10% greater than the signal intensity of the average detectable label of the plurality of protein candidates. In one embodiment of the present application, step (2) comprises screening for protein candidates whose detectable label has a signal intensity that is at least 20% greater than the signal intensity of the average detectable label of the plurality of protein candidates. Step (2) comprises screening for protein candidates whose detectable label has a signal intensity that is at least 30% greater than the signal intensity of the average detectable label of the plurality of protein candidates. Step (2) comprises screening for protein candidates whose detectable label has a signal intensity that is at least 40% greater than the signal intensity of the average detectable label of the plurality of protein candidates. In one embodiment of the present application, step (2) comprises screening for protein candidates whose detectable label has a signal intensity that is at least 50% greater than the signal intensity of the average detectable label of the plurality of protein candidates. In one embodiment of the present application, step (2) comprises comparing the binding affinities of FcRn of the plurality of protein candidates. In one embodiment of the present application, step (2) comprises screening protein candidates whose FcRn binding affinity is greater than the average binding affinity of the plurality of protein candidates. In one embodiment of the present application, step (2) comprises screening for protein candidates whose FcRn binding affinity is at least 10% greater than the average binding affinity of the plurality of protein candidates. In one embodiment of the present application, step (2) comprises screening for protein candidates whose FcRn binding affinity is at least 20% greater than the average binding affinity of the plurality of protein candidates. In one embodiment of the present application, step (2) comprises screening for protein candidates whose FcRn binding affinity is at least 30% greater than the average binding affinity of the plurality of protein candidates. In one embodiment of the present application, step (2) comprises screening protein candidates whose FcRn binding affinity is at least 40% greater than the average binding affinity of the plurality of protein candidates. In one embodiment of the present application, step (2) comprises screening for protein candidates whose FcRn binding affinity is at least 50% greater than the average binding affinity of the plurality of protein candidates.
The application also relates to a method of determining the stability of a sample. The method comprises the following steps: (1) FcRn binding is determined for a plurality of protein samples by the method of determining protein FcRn binding. In one embodiment of the present application, the method of determining protein FcRn binding comprises the method described in any one of the preceding embodiments of the present application. In one embodiment of the present application, step (1) comprises determining the signal intensity of the detectable label of the plurality of protein samples. In one embodiment of the present application, step (1) comprises determining the binding affinity of FcRn of the plurality of protein samples.
The method for determining the stability of a sample further comprises the following steps: (2) Comparing FcRn binding of the plurality of protein samples, thereby determining stability of the plurality of protein samples. In one embodiment of the present application, step (2) comprises comparing the signal intensities of the detectable labels of the plurality of protein samples. In one embodiment of the present application, step (2) comprises determining the degree of dispersion of the signal intensity of the detectable label of the plurality of protein samples. In one embodiment of the present application, step (2) comprises determining whether the degree of dispersion of the signal intensity of the detectable label of the plurality of protein samples exceeds a threshold. In one embodiment of the present application, step (2) comprises comparing the binding affinities of FcRn of the plurality of protein samples. In one embodiment of the present application, step (2) comprises determining the degree of discretization of FcRn binding affinity of the plurality of protein samples. In one embodiment of the present application, step (2) comprises determining whether the degree of dispersion of FcRn binding affinity of the plurality of protein samples exceeds a threshold. In one embodiment of the present application, in step (2), the plurality of protein samples is determined to be not stable if the degree of dispersion exceeds a threshold value, and the plurality of protein samples is determined to be stable if the degree of dispersion does not exceed the threshold value.
The present application also relates to a method of predicting in vivo PK/PD and/or half-life of an Fc-containing macromolecular drug. The method comprises the following steps: (1) FcRn binding of Fc-containing macromolecular drugs is determined by the method of determining protein FcRn binding. In one embodiment of the present application, the method of determining FcRn binding of an Fc-containing macromolecular drug comprises the method described in any one of the preceding embodiments of the present application. In one embodiment of the present application, step (1) comprises determining the signal intensity of the detectable label of the Fc-containing macromolecular drug. In one embodiment of the present application, step (1) comprises determining the binding affinity of FcRn of the Fc-containing macromolecular drug.
The method for determining the stability of a sample further comprises the following steps: (2) Predicting the in vivo PK/PD and/or half-life of said Fc-containing macromolecular drug. In one embodiment of the present application, step (2) comprises predicting in vivo PK/PD and/or half-life of the Fc-containing macromolecular drug based on signal intensity of the detectable label of said Fc-containing macromolecular drug. In this application, an enhancement of the binding affinity of a drug to FcRn is strongly associated with an extension of the in vivo half-life of the drug. In one embodiment of the present application, step (2) comprises predicting in vivo PK/PD and/or half-life of the Fc-containing macromolecular drug based on the binding affinity of FcRn of said Fc-containing macromolecular drug.
The following description of the embodiments of the present application will be made clearly and fully with reference to the accompanying drawings, in which some, but not all embodiments of the present application are shown. All other embodiments, based on the embodiments herein, which are within the scope of the protection of the present application, will be within the purview of one of ordinary skill in the art without the exercise of inventive faculty.
Examples
The technical scheme of the invention will be described in more detail below with reference to specific examples. The following examples are only examples, and do not constitute any limitation or restriction on the technical solution of the present invention. The specific materials, steps, conditions, values or numerical ranges specified in the following examples are illustrative only and are not intended to be exhaustive or limiting.
In addition to the specific methods, devices, materials used in the embodiments, any methods, devices, and materials of the prior art similar or equivalent to those described in the embodiments of the present invention may be used to practice the present invention according to the knowledge of one skilled in the art and the description of the present invention.
Table 1: list of experimental facilities
Table 2: list of experimental reagents
In order to more clearly illustrate the technical scheme of the invention, the invention will be further described with reference to the embodiment.
Example 1
The experimental steps are as follows:
1. 50 μL of 1E6/mL FcRn over-expression engineered cells (HEK 293F-FcRn-D3G1 cells, humanized FcRn sequence information: uniprot: P55899 (1-365), which contained B2M protein sequence information: uniprot: P61769 (1-119)) suspension (cell suspension without serum) was added to a graphite electrode high adsorption 96 well plate. The plates were sealed with a sealing plate and incubated at 25℃for 60min.
2. mu.L of blocking solution (PBS containing 2% BSA) was added to each well, the plates were sealed with a sealing plate, and the plates were incubated at 25℃for 60min.
3. The blocking solution was discarded.
4. 50. Mu.L of a gradient diluted biotinylated human IgG1 Fc to be tested was added and diluted with a diluent (PBS containing 0.5% BSA, pH6.0, 7.0 or 8.0) in the following manner:
5. the plates were sealed with a sealing plate and incubated at 25℃for 60min.
6. The test plates were washed with wash solutions (PBS, pH6.0, 7.0 or 8.0), 300. Mu.L per well was added and washed 3 times.
7. mu.L of a test solution containing 50ng/mL of an HRP-labeled streptavidin detection antibody (PBS containing 0.5% BSA) was added and incubated at 25℃for 60min.
8. The test plates were washed with wash solutions (PBS, pH6.0, 7.0 or 8.0), 300. Mu.L per well was added and washed 3 times.
9. 100. Mu.L of TMB substrate per well was added and incubated for 10 minutes at room temperature.
10. 100. Mu.L of stop solution (2M H) was added 2 SO 4 Solution) to each well, transfer 150 μl to a bottom permeable 96 well plate.
11. Absorbance was read with a microplate reader at OD450 to obtain a dose-dependent curve.
Experimental results:
the results obtained are shown in FIG. 2. Three sets of combinations of dilutions and washes of different pH were used in the figure. For Plot #1, a dilution and wash solution at pH 8.0 was used. For Plot #2, a dilution and wash solution at pH 7.0 was used. For Plot #3, it uses a diluent and wash solution at pH 6.0. It can be seen from the graph that pH has a great influence on the detection sensitivity, the detection sensitivity of Plot #2 is better than that of Plot #1, and the detection sensitivity of Plot #3 is far better than that of Plot # 2.
In the present application, the detection sensitivity is calculated as follows: detection sensitivity=ec 50 Value/molecular weight. For Plot #3, its EC was determined 50 2.83. Mu.g/mL, calculated on the basis of a molecular weight of 34kDa, with a detection sensitivity of about 80nM.
As described above, the present invention can screen antibodies having a certain FcRn binding ability during the establishment and screening stage of monoclonal antibody-producing cell lines. In the process optimization stage, the invention can efficiently detect and release the cell culture condition and the purification condition with proper monoclonal antibody proportion. In the clinical trial declaration or commercial production declaration stage of raw material medicine and finished product, the biological activity detection scheme which can be used for monoclonal antibody product release and stability research is provided.
In summary, compared with the prior art, the invention has the following advantages:
1. the method has the pH dependency characteristic, is closer to an in-vivo state, provides FcRn binding capacity detection (figure 1) close to a cell level in a physiological state, has high sensitivity and convenient operation, and does not need a special large detection instrument;
2. the method can be used as an analysis tool for evaluating batches, and can be used for testing the consistency and the stability of different batches of the same molecule and different molecules;
3. the detection scheme using FcRn expressing cells immobilized on a solid support has very high detection sensitivity, e.g., can reach 80nM or better;
4. the operation is convenient, the detection does not need to rely on a flow cytometer, and the transfer convenience of the QC method is improved.
The foregoing is merely a specific application example of the present application, and the protection scope of the present application is not limited in any way. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary or obvious to describe all embodiments. All such technical solutions formed by equivalent transformation or equivalent substitution fall within the protection scope of the claims of the present application.
Claims (10)
1. A method of determining protein FcRn binding, the method comprising:
(a) Immobilizing FcRn expressing cells on a solid support;
(b) Incubating the solid support with a blocking agent;
(c) Labeling the protein with a first molecule and incubating the labeled protein with the solid support at an acidic pH;
(d) Incubating a second molecule with the solid support, the second molecule specifically binding to the first molecule; and
(e) Detecting binding of the first molecule to the second molecule.
2. The method of claim 1, wherein the protein comprises a protein comprising an Fc region of an antibody, e.g., an Fc fusion protein, an antibody, preferably a monoclonal antibody, igG.
3. The method of claim 1, wherein the solid support comprises a porous plate, preferably a high-adsorptivity porous plate, e.g. a graphite electrode high-adsorptivity porous plate.
4. The method of claim 1, wherein the FcRn expressing cells comprise eukaryotic cells, preferably human cells, e.g. human embryonic kidney cells.
5. The method of claim 1, wherein the first molecule comprises biotin; and the second molecule comprises avidin or streptavidin.
6. The method of claim 1, wherein the acidic pH is 5.0-7.0, preferably 5.5-6.5, more preferably 6.0.
7. The method of claim 1, wherein the second molecule is labeled with an enzyme, and step (e) further comprises adding a substrate for the enzyme and detecting a substrate reaction, preferably the substrate reaction comprises a chromogenic reaction.
8. A method of screening for a drug, the method comprising:
(1) Determining FcRn binding of a plurality of protein candidates by the method of any one of claims 1-7; and
(2) Screening the drug for FcRn binding of the plurality of protein candidates.
9. A method of determining the stability of a protein sample, the method comprising:
(1) Determining FcRn binding of a plurality of protein samples by the method of any one of claims 1-7; and
(2) Comparing FcRn binding of the plurality of protein samples, thereby determining stability of the plurality of protein samples.
10. A method of predicting in vivo PK/PD and/or half-life of an Fc-containing macromolecular drug, said method comprising:
(1) Determining FcRn binding of the Fc-containing macromolecular drug by the method of any one of claims 1-7; and
(2) Predicting the in vivo PK/PD and/or half-life of said Fc-containing macromolecular drug.
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