Systems, materials, and methods for reversed-phase high-performance liquid chromatography (RP-HPLC) to monitor the formation of multispecific molecules

Abstract

Provided herein is a high-pressure liquid chromatography (HPLC) method for detecting multispecific molecule formation after the multispecific molecule manufacturing or development process. The HPLC method provides a rapid, quantitative, real-time analysis of multispecific molecule formation.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of U.S. Provisional Application Nos. 63 / 010,907, 63 / 010,912, 63 / 010,920, and 63 / 010,926, filed April 16, 2020. The entire contents of the foregoing applications are incorporated herein by reference in their entirety.

[0002] FIELD OF THE INVENTION The present invention relates to a high-pressure liquid chromatography (HPLC) method for detecting multispecific molecule formation after the multispecific molecule manufacturing or development process. The HPLC method provides a rapid, quantitative, real-time analysis of multispecific molecule formation. [Background technology]

[0003] During the manufacturing or process research and development (R&D) of multispecific molecules (e.g., multispecific antibodies), the possibility of incomplete formation of multispecific molecules (e.g., multispecific antibodies) remains regardless of the manufacturing process. Non-reduced capillary sodium dodecyl sulfate (NR cSDS) has previously been employed to monitor the formation of incomplete multispecific antibodies. However, this technique is not practical for providing the rapid, quantitative, real-time results required to study multispecific formation in a process manufacturing environment or during multispecific molecule (e.g., multispecific antibody) development.

[0004] Therefore, there is a need for a fast, quantitative, real-time process to monitor the formation of multispecific molecules (e.g., multispecific antibodies). Summary of the Invention

[0005] Provided herein are methods for detecting the formation of multispecific molecules. The methods include: (a) obtaining a multispecific molecule sample; (b) obtaining a reversed-phase high performance liquid chromatography (RP-HPLC) column; (c) contacting the multispecific molecule sample in a polar aqueous mobile phase A with the RP-HPLC column, where the polar aqueous mobile phase comprises an ion-pairing agent; (d) contacting the RP-HPLC column with an organic non-polar mobile phase B, where the organic non-polar phase comprises an ion-pairing agent; (e) eluting the multispecific molecule sample; and (f) monitoring the amount of multispecific molecule formation in the eluted multispecific molecule sample. In certain embodiments, the multispecific molecule is a multispecific antibody.

[0006] Also provided herein is a method for detecting the formation of a multispecific antibody while performing a manufacturing process to produce the multispecific antibody, the method comprising: (a) performing a manufacturing process to produce the multispecific antibody, the manufacturing process resulting in a multispecific antibody sample containing a quantity of the multispecific antibody, (b) setting up a reversed-phase high-performance liquid chromatography (RP-HPLC) column, (c) injecting the multispecific antibody sample into a polar aqueous mobile phase A in the RP-HPLC column, the polar aqueous mobile phase comprising an ion-pairing agent, (d) applying an organic non-polar mobile phase B to the RP-HPLC column to form a gradient elution, the organic non-polar mobile phase comprising an ion-pairing agent, (e) eluting the multispecific molecule sample, and (f) monitoring the amount of the multispecific antibody in the eluted sample.

[0007] Also provided herein is a method for detecting bispecific antibody formation while performing controlled FAB arm exchange (cFAE) to generate a bispecific antibody. The method includes: (a) performing controlled FAB arm exchange (cFAE) to generate a cFAE sample, the cFAE sample containing a certain amount of bispecific antibody; (b) setting up a reversed-phase high-performance liquid chromatography (RP-HPLC) column; (c) injecting the cFAE sample into a polar aqueous mobile phase A in the RP-HPLC column, the polar aqueous mobile phase containing an ion-pairing agent; (d) applying an organic non-polar mobile phase B to the RP-HPLC column to form a gradient elution, the organic non-polar mobile phase containing an ion-pairing agent; (e) eluting the cFAE sample; and (f) monitoring the amount of bispecific antibody formation in the eluted sample.

[0008] Also provided herein is a method for designing a stable multispecific antibody, comprising: (a) performing a manufacturing process for producing a multispecific antibody, wherein the manufacturing process results in a multispecific antibody sample containing a quantity of the multispecific antibody; (b) configuring a reversed-phase high-performance liquid chromatography (RP-HPLC) column; (c) injecting the multispecific antibody sample into a polar aqueous mobile phase A in the RP-HPLC column, wherein the polar aqueous mobile phase comprises an ion-pairing agent; (d) applying an organic non-polar mobile phase B to the RP-HPLC column to form a gradient elution, wherein the organic non-polar mobile phase comprises an ion-pairing agent; (e) eluting the multispecific antibody sample; and (f) monitoring the amount of multispecific antibody formation in the eluted sample, wherein an increase in the amount of multispecific antibody formation compared to a control indicates the design of a stable multispecific antibody.

[0009] Also provided herein is a method for obtaining a stability profile of a multispecific antibody during a manufacturing process for producing the multispecific antibody, the method comprising: (a) performing a manufacturing process for producing the multispecific antibody, wherein amounts of multispecific antibody samples are obtained at different time points during the manufacturing process; (b) setting up a reversed-phase high-performance liquid chromatography (RP-HPLC) column; (c) injecting the multispecific antibody sample from each time point into a polar aqueous mobile phase A in the RP-HPLC column, wherein the polar aqueous mobile phase contains an ion pairing agent; (d) applying an organic nonpolar mobile phase B to the RP-HPLC column to form a gradient elution, wherein the organic nonpolar mobile phase contains an ion pairing agent; (e) eluting the multispecific antibody sample; and (f) monitoring the amount of multispecific antibody formation in the eluted sample, wherein the amount of multispecific antibody formation at each time point provides a stability profile of the multispecific antibody.

[0010] Also provided herein is a method for obtaining a degradation fragment species profile of a multispecific antibody during a manufacturing process for producing the multispecific antibody. The method includes: (a) performing a manufacturing process for producing a multispecific antibody, wherein amounts of multispecific antibody samples are obtained at different time points during the manufacturing process; (b) setting up a reversed-phase high-performance liquid chromatography (RP-HPLC) column; (c) injecting the multispecific antibody sample from each time point into a polar aqueous mobile phase A in the RP-HPLC column, wherein the polar aqueous mobile phase contains an ion pairing agent; (d) applying an organic nonpolar mobile phase B to the RP-HPLC column to form a gradient elution, wherein the organic nonpolar mobile phase contains an ion pairing agent; (e) eluting the multispecific antibody sample; and (f) monitoring the amount of degradation fragment species in the eluted sample, wherein the amount of degradation fragment species at each time point provides a degradation fragment species profile of the multispecific antibody manufacturing process.

[0011] Also provided herein is a system comprising a reversed-phase high-performance liquid chromatography (RP-HPLC) component for detecting the formation of multispecific molecules. The system comprises: (a) a multispecific molecule sample in a polar aqueous mobile phase A, the polar aqueous mobile phase comprising an ion-pairing agent; (b) an RP-HPLC column; and (c) an organic non-polar mobile phase B, the organic non-polar mobile phase B comprising an ion-pairing agent. The RP-HPLC column is configured to contact the multispecific molecule sample in the polar aqueous mobile phase A, and the RP-HPLC is further configured to contact the multispecific molecule sample with the organic non-polar mobile phase B to elute the multispecific molecule sample. The amount of multispecific molecule formation can be determined in the eluted multispecific molecule sample.

[0012] Also provided is a means for reversed-phase high performance liquid chromatography (RP-HPLC) detection of multispecific molecule formation, comprising: (a) a multispecific molecule sample in a polar aqueous mobile phase A, the polar aqueous mobile phase comprising an ion-pairing agent; (b) an RP-HPLC column; and (c) an organic non-polar mobile phase B, the organic non-polar mobile phase comprising an ion-pairing agent. The RP-HPLC column is configured to contact the multispecific molecule sample in the polar aqueous mobile phase A, and the RP-HPLC column is further configured to contact the RP-HPLC column with the organic non-polar mobile phase B to elute the multispecific molecule sample. The amount of multispecific molecule formation can be determined in the eluted multispecific molecule sample.

[0013] In certain embodiments, the polar aqueous mobile phase A is a solution containing about 1% to about 5% isopropanol. The solution may contain, for example, about 2% isopropanol.

[0014] In certain embodiments, the ion pairing agent in the polar aqueous mobile phase A is selected from the group consisting of trifluoroacetic acid (TFA), difluoroacetic acid (DFA), and formic acid (FA). In certain embodiments, the ion pairing agent is TFA. TFA can be present in the polar aqueous mobile phase A at about 0.1% to about 2%. In certain embodiments, TFA is present in the polar aqueous mobile phase A at about 0.1%.

[0015] In certain embodiments, the organic non-polar mobile phase is a solution containing about 60% to about 80% isopropanol and about 15% to about 25% acetonitrile. The solution can contain, for example, about 70% isopropanol. The solution can contain, for example, about 20% acetonitrile.

[0016] In certain embodiments, the ion pairing agent in organic non-polar mobile phase B is selected from the group consisting of trifluoroacetic acid (TFA), difluoroacetic acid (DFA), and formic acid (FA). In certain embodiments, the ion pairing agent is TFA. TFA may be present in organic non-polar mobile phase B at, for example, about 0.1% to about 2%. In certain embodiments, TFA is present in organic non-polar mobile phase B at about 0.1%.

[0017] In certain embodiments, the gradient elution comprises a solution comprising about 75% polar mobile phase A solution and about 25% organic nonpolar mobile phase B solution at the time the multispecific (e.g., bispecific) antibody sample is injected onto the RP-HPLC column. The gradient elution may comprise, for example, a solution comprising about 66% polar mobile phase A solution and about 34% organic nonpolar mobile phase B solution at about 16 minutes after the multispecific antibody sample is injected onto the RP-HPLC column. The gradient elution may comprise, for example, a solution comprising about 5% polar mobile phase A solution and about 95% organic nonpolar mobile phase B solution at about 20 minutes after the multispecific antibody sample is injected onto the RP-HPLC column. The gradient elution may comprise, for example, a solution comprising about 75% polar mobile phase A solution and about 25% organic nonpolar mobile phase B solution at about 21 minutes after the multispecific antibody sample is injected onto the RP-HPLC column.

[0018] In certain embodiments, the manufacturing process for producing multispecific antibodies is selected from the group consisting of the knobs-in-holes process, the strand-exchange engineered domain process, the chemically linked bispecific antibody (BsAb) process, the immunoglobulin domain crossover process, and the controlled Fab arm exchange (cFAE) and dual variable domain process.

[0019] In certain embodiments, the multispecific antibody is a bispecific antibody. [Brief explanation of the drawings]

[0020] The foregoing summary, as well as the following detailed description of preferred embodiments of the present application, will be better understood when read in conjunction with the appended drawings. It should be understood, however, that the present application is not limited to the precise embodiments shown in the drawings. [Figure 1] 1 shows a schematic diagram of a reversed-phase high-performance liquid chromatography (RP-HPLC) single stack configuration. [Figure 2] A graph demonstrating a representative blank chromatogram is shown. [Figure 3] Graphs demonstrating representative chromatograms of anti-GPRC5D / anti-CD3 bispecific antibody (fully oxidized) reference material are shown. [Figure 4] Graphs demonstrating representative chromatograms of partially oxidized anti-GPRC5D / anti-CD3 bispecific antibodies are shown. [Figure 5] Graphs demonstrating representative chromatograms of anti-CD33 / anti-CD3 bispecific antibody (fully oxidized) reference material are shown. [Figure 6] Graphs demonstrating representative chromatograms of partially oxidized anti-CD33 / anti-CD3 bispecific antibodies are shown. [Figure 7] Graphs demonstrating representative chromatograms of anti-TMEFF2 / anti-CD3 bispecific antibody (fully oxidized) reference material are shown. [Figure 8] Graphs demonstrating representative chromatograms of partially oxidized anti-TMEFF2 / anti-CD3 bispecific antibodies are shown. [Figure 9] Graphs demonstrating representative chromatograms of anti-CD123 / anti-CD3 bispecific antibody (fully oxidized) reference material are shown. [Figure 10] Graphs demonstrating representative chromatograms of partially oxidized anti-CD123 / anti-CD3 bispecific antibodies are shown. [Figure 11] 1 shows a graph demonstrating a representative chromatogram of an anti-BCMA / anti-CD3 bispecific antibody (fully oxidized) reference material. [Figure 12] 1 shows a graph demonstrating a representative chromatogram of a partially oxidized anti-BCMA / anti-CD3 bispecific antibody. DETAILED DESCRIPTION OF THE INVENTION

[0021] Various publications, articles, and patents are cited or described in the Background and throughout this specification, and each of these references is incorporated herein by reference in its entirety. Any discussion of documents, operations, materials, devices, articles and the like which has been included in the specification is for the purpose of providing a context for the present invention. Such discussion is not an admission that any or all of these items constitute part of the prior art to any invention disclosed or claimed.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Unless otherwise defined, certain terms used herein have the meanings described herein.

[0023] It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0024] Unless otherwise specified, all numerical values, such as concentrations or concentration ranges, described herein should be understood in all instances to be modified by the term "about." Thus, numerical values ​​typically include ±10% of the stated value. For example, a concentration of 1 mg / mL includes 0.9 mg / mL to 1.1 mg / mL. Similarly, a concentration range of 1% to 10% (w / v) includes 0.9% (w / v) to 11% (w / v). As used herein, the use of numerical ranges explicitly includes all possible subranges, including integers and fractions of values ​​within that range, and all individual numerical values ​​within that range, unless the context clearly indicates otherwise.

[0025] Unless otherwise indicated, the term "at least" preceding a series of elements should be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0026] As used herein, the terms "comprises," "comprising," "includes," "including," "has," "having," "contains," or "containing," or any other variation thereof, are intended to include the stated integer or group of integers, but not to exclude other integers or groups of integers, and are understood to be non-exclusive or non-limiting. For example, a composition, mixture, process, method, article, or device comprising a list of elements is not necessarily limited to only those elements and may include other elements not expressly listed or not inherent in such composition, mixture, process, method, article, or device. Further, unless expressly stated to the contrary, "or" refers to an inclusive "or" and not an exclusive "or." For example, condition A or B is satisfied by one of the following: A is true (or exists) and B is false (or does not exist); A is false (or does not exist) and B is true (or exists); and both A and B are true (or exist).

[0027] As used herein, the connective term "and / or" between multiple listed elements is understood to encompass both individual and combined alternatives. For example, when two elements are connected by "and / or," the first alternative refers to the first element being applicable without the second element. The second alternative refers to the second element being applicable without the first element. The third alternative refers to the first and second elements being applicable together. Any one of these alternatives is understood to be within the meaning and therefore meets the requirements of the term "and / or" as used herein. The simultaneous applicability of one or more of the alternatives is also understood to be within the meaning and therefore meets the requirements of the term "and / or."

[0028] As used herein, the term "consists of" or variations such as "consist of" or "consisting of," as used throughout the specification and claims, indicates that any enumerated integer or group of integers is inclusive, but that no additional integer or group of integers is added to the specified method, structure, or composition.

[0029] As used herein, the term "consists essentially of" or variations such as "consist essentially of" or "consisting essentially of," as used throughout the specification and claims, refers to the inclusion of any recited integer or group of integers, optionally including any recited integer or group of integers that does not materially alter the basic or novel characteristics of the specified method, structure, or composition. See MPEP §2111.03.

[0030] It should also be understood that the terms "about," "approximately," "generally," "substantially," and the like, used herein when referring to dimensions or characteristics of components of the preferred invention, indicate that the described dimensions / characteristics are not precise boundaries or parameters, but do not exclude minor variations therefrom that are functionally the same or similar, as would be understood by one of ordinary skill in the art. At a minimum, such references involving numerical parameters will include variations that do not change the least significant digit using mathematical and industrial principles accepted in the art (e.g., rounding, measurement or other systematic errors, manufacturing tolerances, etc.).

[0031] As used herein, the term "polynucleotide," also referred to interchangeably as "nucleic acid molecule," "nucleotide," or "nucleic acid," refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. "Polynucleotide" includes, but is not limited to, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is a mixture of single- and double-stranded regions, and hybrid molecules containing DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. Additionally, "polynucleotide" refers to triple-stranded regions containing RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNA or RNA containing one or more modified bases and DNA or RNA with backbones modified for stability or other reasons. "Modified" bases include, for example, tritylated bases and unusual bases, such as inosine. Various modifications can be made to DNA and RNA. Thus, "polynucleotide" encompasses chemically, enzymatically, or metabolically modified forms of polynucleotides typically found in nature, as well as chemical forms characteristic of viral and cellular DNA and RNA. "Polynucleotide" also encompasses relatively short nucleic acid strands, often called oligonucleotides.

[0032] As used herein, the term "vector" refers to a replicon into which another nucleic acid segment may be operatively inserted so as to bring about the replication or expression of the segment.

[0033] As used herein, the term "host cell" refers to a cell that contains a nucleic acid molecule of the invention. A "host cell" can be any type of cell, for example, a primary cell, a cell in culture, or a cell from a cell line. In one embodiment, a "host cell" is a cell that has been transfected with a nucleic acid molecule of the invention. In another embodiment, a "host cell" is the progeny or potential progeny of such a transfected cell. The progeny of a cell may not be identical to the parent cell due, for example, to mutations or environmental influences that may occur in subsequent generations, or to integration of the nucleic acid molecule into the host cell genome.

[0034] As used herein, the term "expression" refers to the biosynthesis of a gene product. The term includes transcription of a gene into RNA. The term also includes translation of RNA into one or more polypeptides, and further includes all naturally occurring post-transcriptional and post-translational modifications. The expressed bispecific antibody may be present in the cytoplasm of a host cell, in an extracellular environment such as the growth medium of a cell culture, or may be anchored to the cell membrane.

[0035] As used herein, the terms "peptide," "polypeptide," or "protein" can refer to a molecule composed of amino acids and recognized as a protein by those skilled in the art. Conventional one-letter or three-letter codes for amino acid residues are used herein. The terms "peptide," "polypeptide," and "protein" can be used interchangeably herein to refer to polymers of amino acids of any length. The polymers can be linear or branched, can contain modified amino acids, and can be interrupted by non-amino acids. The term also encompasses amino acid polymers that are naturally modified or modified by intervention. Examples of intervention include, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. The definition also includes, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids), as well as other modifications known in the art.

[0036] In the broadest sense, as used herein, the terms "detecting," "detect," or variations thereof, include both qualitative and quantitative measurements of a target molecule (e.g., a multispecific antibody). Detecting includes identifying the mere presence of a target molecule in a sample, as well as determining whether the target molecule is present in a sample at detectable levels.

[0037] As used herein, the term "sample" refers to a small portion of a larger amount of material. Generally, testing according to the methods described herein is performed on a sample. Samples are typically obtained from multispecific molecule preparations (e.g., multispecific antibody preparations) obtained from a manufacturing process for producing multispecific molecules, e.g., as described below.

[0038] As used herein, the term "inject" or "injecting" may refer to the loading of a sample into an HPLC column in the aqueous phase. The sample may be equilibrated with an equilibration buffer prior to the injection and / or loading of the composition to be detected and / or purified.

[0039] As used herein, the terms "elute," "eluting," and / or "elution" can refer to the removal of a product (e.g., a multispecific antibody) from a chromatographic material. An elution buffer is a buffer used to elute the multispecific antibody from the chromatographic material.

[0040] Systems, materials, and methods for detecting multispecific molecule formation Provided herein are methods for detecting multispecific molecule formation. The methods include: (a) obtaining a multispecific molecule sample; (b) obtaining a reversed-phase high-performance liquid chromatography (RP-HPLC) column; (c) contacting the multispecific molecule sample in a polar aqueous mobile phase A with the RP-HPLC column, where the polar aqueous mobile phase comprises an ion-pairing agent; (d) contacting the RP-HPLC column with an organic non-polar mobile phase B, where the organic non-polar mobile phase comprises an ion-pairing agent; (e) eluting the multispecific molecule sample; and (f) determining the amount of multispecific molecule formation in the eluted multispecific molecule sample. In certain embodiments, the multispecific molecule is a multispecific antibody. In certain embodiments, the multispecific antibody is a bispecific antibody.

[0041] Provided herein is a method for detecting the formation of a multispecific antibody while performing a manufacturing process to produce the multispecific antibody, the method comprising: (a) performing a manufacturing process to produce the multispecific antibody, the manufacturing process resulting in a multispecific antibody sample containing a quantity of the multispecific antibody; (b) setting up a reversed-phase high-performance liquid chromatography (RP-HPLC) column; (c) injecting the multispecific antibody sample into a polar aqueous mobile phase A in the RP-HPLC column, the polar aqueous mobile phase comprising an ion-pairing agent; (d) applying an organic non-polar mobile phase B to the RP-HPLC column to form a gradient elution, the organic non-polar mobile phase comprising an ion-pairing agent; (e) eluting the multispecific molecule sample; and (f) monitoring the amount of the multispecific antibody in the eluted sample.

[0042] Also provided herein is a method for detecting bispecific antibody formation during controlled FAB arm exchange (cFAE) to generate a bispecific antibody. The method includes: (a) performing controlled FAB arm exchange (cFAE) to generate a cFAE sample, the cFAE sample containing a certain amount of bispecific antibody; (b) setting up a reversed-phase high-performance liquid chromatography (RP-HPLC) column; (c) injecting the cFAE sample into a polar aqueous mobile phase A in the RP-HPLC column, the polar aqueous mobile phase containing an ion-pairing agent; (d) applying an organic non-polar mobile phase B to the RP-HPLC column to form a gradient elution, the organic non-polar mobile phase containing an ion-pairing agent; (e) eluting the cFAE sample; and (f) monitoring the amount of bispecific antibody formation in the eluted sample.

[0043] In certain embodiments, the amount of formed multispecific antibodies can be determined by preparing and testing a reference sample. The amount of partially formed multispecific antibodies can be determined by comparison to a reference sample (fully formed). The reference sample can include a sample having 80-100% fully formed multispecific antibodies. By comparing a process manufacturing sample to a reference sample, the amount of both fully formed multispecific antibodies and their partially formed antibody fragments can be determined, providing a measure of the efficiency and effectiveness of the manufacturing process for producing multispecific antibodies.

[0044] Also provided is a method of utilizing this assay in a process development kinetic study for developing a multispecific antibody, the method comprising: (a) conducting a research and development process for producing a multispecific antibody, the research and development process resulting in a multispecific antibody sample containing a quantity of the multispecific antibody; (b) setting up a reversed-phase high-performance liquid chromatography (RP-HPLC) column; (c) injecting the multispecific antibody sample into a polar aqueous mobile phase A in the RP-HPLC column, the polar aqueous mobile phase comprising an ion-pairing agent; (d) applying an organic non-polar mobile phase B to the RP-HPLC column to form a gradient elution, the organic non-polar mobile phase comprising an ion-pairing agent; (e) eluting the multispecific antibody sample; and (f) monitoring the amount of multispecific antibody formation in the eluted sample, wherein an increased amount of multispecific antibody formation compared to a reference sample indicates successful multispecific antibody design.

[0045] Also provided herein is a method for obtaining a stability profile of a multispecific antibody during a manufacturing process for producing the multispecific antibody, the method comprising: (a) performing a manufacturing process for producing the multispecific antibody, wherein amounts of multispecific antibody samples are obtained at different time points during the manufacturing process; (b) setting up a reversed-phase high-performance liquid chromatography (RP-HPLC) column; (c) injecting the multispecific antibody sample from each time point into a polar aqueous mobile phase A in the RP-HPLC column, wherein the polar aqueous mobile phase contains an ion pairing agent; (d) applying an organic nonpolar mobile phase B to the RP-HPLC column to form a gradient elution, wherein the organic nonpolar mobile phase contains an ion pairing agent; (e) eluting the multispecific antibody sample; and (f) monitoring the amount of multispecific antibody formation in the eluted sample, wherein the amount of multispecific antibody formation at each time point provides a stability profile of the multispecific antibody.

[0046] By comparing a process production sample with a reference sample, the amount of both fully formed multispecific antibody and its partially formed antibody fragments can be determined, thereby providing a measure of the efficiency and effectiveness of the production process for producing multispecific antibodies. An increase in the amount of fully formed multispecific antibody compared to the reference sample can indicate that the design of the multispecific antibody is favorable for producing multispecific antibodies that are free of unformed and / or partially formed multispecific antibody fragments.

[0047] Also provided herein is a system comprising a reversed-phase high-performance liquid chromatography (RP-HPLC) component for detecting the formation of multispecific molecules. The system comprises: (a) a multispecific molecule sample in a polar aqueous mobile phase A, the polar aqueous mobile phase comprising an ion-pairing agent; (b) an RP-HPLC column; and (c) an organic non-polar mobile phase B, the organic non-polar mobile phase B comprising an ion-pairing agent. The RP-HPLC column is configured to contact the multispecific molecule sample in the polar aqueous mobile phase A, and the RP-HPLC is further configured to contact the multispecific molecule sample with the organic non-polar mobile phase B to elute the multispecific molecule sample. The amount of multispecific molecule formation can be determined in the eluted multispecific molecule sample.

[0048] Also provided is a means for reversed-phase high performance liquid chromatography (RP-HPLC) detection of multispecific molecule formation, comprising: (a) a multispecific molecule sample in a polar aqueous mobile phase A, the polar aqueous mobile phase comprising an ion-pairing agent; (b) an RP-HPLC column; and (c) an organic non-polar mobile phase B, the organic non-polar mobile phase comprising an ion-pairing agent. The RP-HPLC column is configured to contact the multispecific molecule sample in the polar aqueous mobile phase A, and the RP-HPLC column is further configured to contact the RP-HPLC column with the organic non-polar mobile phase B to elute the multispecific molecule sample. The amount of multispecific molecule formation can be determined in the eluted multispecific molecule sample.

[0049] As disclosed herein, reversed-phase high-pressure liquid chromatography (RP-HPLC) is a chromatographic technique that uses a hydrophobic stationary phase (e.g., a stationary phase containing alkyl chains covalently attached to the stationary phase particles). The use of a hydrophobic stationary phase is the opposite of normal-phase chromatography because the polarity of the mobile phase and stationary phase is reversed. RP-HPLC employs a polar aqueous mobile phase. As a result, hydrophobic molecules in the polar mobile phase tend to adsorb to the hydrophobic stationary phase, while hydrophilic molecules in the mobile phase pass through the column and are eluted first. Hydrophobic molecules can be eluted from the column by decreasing the polarity of the mobile phase using an organic (nonpolar) solvent that reduces hydrophobic interactions. The more hydrophobic a molecule is, the more strongly it binds to the stationary phase, and the higher the concentration of organic solvent required to elute the molecule.

[0050] In certain embodiments, the polar aqueous mobile phase is a solution containing about 1% to about 5% isopropanol. The solution may contain, for example, about 1%, about 2%, about 3%, about 4%, about 5%, or any value therebetween. In a preferred embodiment, the polar aqueous mobile phase contains about 2% isopropanol.

[0051] In certain embodiments, the polar aqueous mobile phase is a solution containing at least one component selected from the group consisting of acetonitrile, propanol, and tetrahydrofuran (THF). The solution may contain, for example, about 1%, about 2%, about 3%, about 4%, about 5%, or any value therebetween of acetonitrile, propanol, and / or tetrahydrofuran (THF). In a preferred embodiment, the polar aqueous mobile phase contains about 2% acetonitrile, propanol, and / or tetrahydrofuran (THF).

[0052] In certain embodiments, the ion pairing agent in mobile phase A is selected from the group consisting of trifluoroacetic acid (TFA), difluoroacetic acid (DFA), and formic acid (FA).

[0053] In certain embodiments, the ion pairing agent is TFA. TFA may be present, for example, in the polar aqueous mobile phase at about 0.1% to about 2%. TFA may be present, for example, in the polar aqueous mobile phase at about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2.0%, or any value therebetween. In a preferred embodiment, TFA is present in the polar aqueous mobile phase at about 0.1%.

[0054] In certain embodiments, the ion pairing agent is DFA. DFA may be present, for example, in a polar aqueous mobile phase at about 0.1% to about 2%. DFA may be present, for example, in a polar aqueous mobile phase at about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2.0%, or any value therebetween. In a preferred embodiment, DFA is present in a polar aqueous mobile phase at about 0.1%.

[0055] In certain embodiments, the ion pairing agent is FA. FA may be present, for example, in a polar aqueous mobile phase at about 0.5% to about 5%. FA may be present, for example, in a polar aqueous mobile phase at about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2.0%, about 2.1%, about 2.2%, about 2.3%, about 2.4%, about 2.5%, about 2.6%, about 2.7%, or It may be present at about 2.8%, about 2.9%, about 3.0%, about 3.1%, about 3.2%, about 3.3%, about 3.4%, about 3.5%, about 3.6%, about 3.7%, about 3.8%, about 3.9%, about 4.0%, about 4.1%, about 4.2%, about 4.3%, about 4.4%, about 4.5%, about 4.6%, about 4.7%, about 4.8%, about 4.9%, about 5.0%, or any value therebetween.

[0056] In certain embodiments, the organic nonpolar mobile phase is a solution containing about 60% to about 100% isopropanol and about 0% to about 25% acetonitrile. The organic nonpolar mobile phase solution may contain, for example, about 60%, about 65%, about 70%, about 75%, about 80%, about 90%, about 100% isopropanol, or any value therebetween. In a preferred embodiment, the organic nonpolar mobile phase solution contains about 70% isopropanol. The organic nonpolar mobile phase solution may contain, for example, about 0%, about 5%, about 10%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, or any value therebetween. In a preferred embodiment, the organic nonpolar mobile phase contains about 20% acetonitrile.

[0057] In certain embodiments, the organic non-polar mobile phase is a solution containing at least one component selected from the group consisting of acetonitrile, isopropanol, and propanol (e.g., normal propanol (n-propanol)). The solution can contain, for example, about 0%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100% acetonitrile, isopropanol, and / or propanol, or any value therebetween. In certain embodiments, the organic non-polar mobile phase comprises about 70% isopropanol, about 20% acetonitrile, and about 10% water; about 70% n-propanol, about 20% acetonitrile, and about 10% water; about 70% acetonitrile, about 20% isopropanol, and about 10% water; about 80% isopropanol and about 20% acetonitrile; about 80% acetonitrile and about 20% isopropanol; about 75% isopropanol and about 25% acetonitrile; about 25% isopropanol and about 75% acetonitrile; or about 50% isopropanol and about 50% acetonitrile.

[0058] In certain embodiments, the ion pairing agent in mobile phase B is selected from the group consisting of trifluoroacetic acid (TFA), difluoroacetic acid (DFA), and formic acid (FA).

[0059] In certain embodiments, the ion pairing agent is TFA. TFA may be present, for example, in the polar aqueous mobile phase at about 0.1% to about 2%. TFA may be present, for example, in the polar aqueous mobile phase at about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2.0%, or any value therebetween. In a preferred embodiment, TFA is present in the polar aqueous mobile phase at about 0.1%.

[0060] In certain embodiments, the ion pairing agent is DFA. DFA may be present, for example, in a polar aqueous mobile phase at about 0.1% to about 2%. DFA may be present, for example, in a polar aqueous mobile phase at about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2.0%, or any value therebetween. In a preferred embodiment, DFA is present in a polar aqueous mobile phase at about 0.1%.

[0061] In certain embodiments, the ion pairing agent is FA. FA may be present, for example, in a polar aqueous mobile phase at about 1.0% to about 5%. FA may be present, for example, in a polar aqueous mobile phase at about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2.0%, about 2.1%, about 2.2%, about 2.3%, about 2.4%, about 2.5%, about 2.6%, about 2.7%, about 2.8%, about 2.9%, or about 3. It may be present at 0%, about 3.1%, about 3.2%, about 3.3%, about 3.4%, about 3.5%, about 3.6%, about 3.7%, about 3.8%, about 3.9%, about 4.0%, about 4.1%, about 4.2%, about 4.3%, about 4.4%, about 4.5%, about 4.6%, about 4.7%, about 4.8%, about 4.9%, about 5.0%, or any value therebetween.

[0062] In certain embodiments, the gradient elution comprises a solution comprising about 75% polar mobile phase A solution and about 25% organic nonpolar mobile phase B solution at the time the multispecific antibody sample is injected onto the RP-HPLC column. The gradient elution may, for example, comprise a solution comprising about 66% polar mobile phase A solution and about 34% organic nonpolar mobile phase B solution at about 16 minutes after the multispecific antibody sample is injected onto the RP-HPLC column. The gradient elution may, for example, comprise a solution comprising about 5% polar mobile phase A solution and about 95% organic nonpolar mobile phase B solution at about 20 minutes after the multispecific antibody sample is injected onto the RP-HPLC column. The gradient elution may, for example, comprise a solution comprising about 75% polar mobile phase A solution and about 25% organic nonpolar mobile phase B solution at about 21 minutes after the multispecific antibody sample is injected onto the RP-HPLC column.

[0063] antibody Provided herein are methods for detecting multispecific antibody formation while performing a manufacturing process to generate a multispecific antibody, methods for designing stable multispecific antibodies, and methods for obtaining a stability profile during a manufacturing process to generate a multispecific antibody or a degradation fragment species profile during a manufacturing process. In certain embodiments, the multispecific antibody is a bispecific antibody.

[0064] As used herein, the term "antibody" is used broadly to include immunoglobulins, or antibody molecules, including monoclonal or polyclonal, human, humanized, composite, and chimeric antibodies, as well as antibody fragments. Generally, antibodies are proteins or peptide chains that exhibit binding specificity to a specific antigen. The structure of antibodies is known. Immunoglobulins can be assigned to five major classes (i.e., IgA, IgD, IgE, IgG, and IgM) depending on the amino acid sequence of the heavy chain constant domain. IgA and IgG are further subdivided into isotypes IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. Thus, antibodies of the present invention can be of any of the five major classes or corresponding subclasses. Preferably, antibodies of the present invention are IgG1, IgG2, IgG3, or IgG4. Antibody light chains of vertebrate species can be assigned to one of two clearly distinct types, kappa and lambda, based on the amino acid sequence of their constant domains. Thus, antibodies of the present invention can contain either a kappa or lambda light chain constant domain. According to certain embodiments, antibodies of the present invention comprise heavy and / or light chain constant regions derived from rat or human antibodies. In addition to the heavy and light constant domains, the antibodies contain an antigen-binding region consisting of a light chain variable region and a heavy chain variable region, each of which contains three domains (i.e., complementarity-determining regions 1 to 3; CDR1, CDR2, and CDR3). The light chain variable region domains are alternatively referred to as LCDR1, LCDR2, and LCDR3, and the heavy chain variable region domains are alternatively referred to as HCDR1, HCDR2, and HCDR3.

[0065] As used herein, the term "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated multispecific antibody that specifically binds to two or more antigens is substantially free of multispecific antibodies that do not bind to the two or more antigens). Moreover, an isolated antibody is substantially free of other cellular material and / or chemicals.

[0066] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible minor naturally occurring mutations. Monoclonal antibodies of the invention can be produced by hybridoma methods, phage display techniques, single lymphocyte gene cloning techniques, or recombinant DNA methods. For example, monoclonal antibodies can be produced by hybridomas comprising B cells obtained from a transgenic non-human animal, e.g., a transgenic mouse or rat, having a genome comprising human heavy chain transgenes and light chain transgenes.

[0067] As used herein, the term "antigen-binding fragment" refers to an antibody fragment, such as, for example, a diabody, Fab, Fab', F(ab'), Fv fragment, disulfide-stabilized Fv fragment (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabody (dsdiabody), single-chain antibody molecule (scFv), single-domain antibody (sdab) scFv dimer (bivalent diabody), multispecific antibody formed from a portion of an antibody comprising one or more CDRs, camelized single-domain antibody, nanobody, domain antibody, bivalent domain antibody, or any other antibody fragment that binds to an antigen but does not comprise the complete antibody structure. An antigen-binding fragment can bind to the same antigen to which the parent antibody or parent antibody fragment binds. According to certain embodiments, the antigen-binding fragment comprises a light chain variable region, a light chain constant region, and an Fd fragment of a heavy chain. According to other particular embodiments, the antigen-binding fragment comprises Fab and F(ab').

[0068] As used herein, the term "single-chain antibody" refers to a single-chain antibody conventionally known in the art, which comprises a heavy-chain variable region and a light-chain variable region connected by a short peptide of about 15 to about 20 amino acids. As used herein, the term "single-domain antibody" refers to a single-domain antibody conventionally known in the art, which comprises a heavy-chain variable region and a heavy-chain constant region, or which comprises only a heavy-chain variable region.

[0069] As used herein, the term "human antibody" refers to an antibody produced by a human or an antibody having an amino acid sequence corresponding to an antibody produced by a human, made using any technique known in the art. This definition of a human antibody includes intact or full-length antibodies, fragments thereof, and / or antibodies comprising at least one human heavy and / or light chain polypeptide.

[0070] As used herein, the term "humanized antibody" refers to a non-human antibody that has been modified to increase sequence homology to human antibodies such that the antigen-binding properties of the antibody are retained but the antigenicity of the antigen in the human body is reduced.

[0071] As used herein, the term "chimeric antibody" refers to an antibody in which the amino acid sequences of the immunoglobulin molecules are derived from two or more species. The variable regions of both the light and heavy chains often correspond to the variable regions of antibodies derived from one species of mammal (e.g., mouse, rat, rabbit, etc.) having the desired specificity, affinity, and capacity, while the constant regions correspond to the sequences of antibodies derived from another species of mammal (e.g., human) to avoid eliciting an immune response in that species.

[0072] As used herein, the term "multispecific antibody" refers to an antibody comprising a plurality of immunoglobulin variable domain sequences, wherein a first immunoglobulin variable domain sequence of the plurality has binding specificity for a first epitope and a second immunoglobulin variable domain sequence of the plurality has binding specificity for a second epitope. In one embodiment, the first and second epitopes are on the same antigen, e.g., the same protein (or subunit of a multimeric protein). In one embodiment, the first and second epitopes overlap or substantially overlap. In certain embodiments, the first and second epitopes do not overlap or substantially do not overlap. In one embodiment, the first and second epitopes are on different antigens, e.g., different proteins (or different subunits of a multimeric protein). In one embodiment, the multispecific antibody comprises a third, fourth, or fifth immunoglobulin variable domain. In one embodiment, the multispecific antibody is a bispecific antibody molecule, a trispecific antibody molecule, or a tetraspecific antibody molecule.

[0073] As used herein, the term "bispecific antibody" refers to a multispecific antibody that binds to no more than two epitopes or no more than two antigens. A bispecific antibody is characterized by a first immunoglobulin variable domain sequence that has binding specificity for a first epitope and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope. In one embodiment, the first and second epitopes are on the same antigen, e.g., the same protein (or subunit of a multimeric protein). In one embodiment, the first and second epitopes overlap or substantially overlap. In one embodiment, the first and second epitopes are on different antigens, e.g., different proteins (or different subunits of a multimeric protein). In certain embodiments, a bispecific antibody comprises heavy and light chain variable domain sequences that have binding specificity for a first epitope and heavy and light chain variable domain sequences that have binding specificity for a second epitope. In certain embodiments, a bispecific antibody comprises a half antibody or fragment thereof that has binding specificity for a first epitope and a half antibody or fragment thereof that has binding specificity for a second epitope, hi certain embodiments, a bispecific antibody comprises an scFv or fragment thereof that has binding specificity for a first epitope and an scFv or fragment thereof that has binding specificity for a second epitope.

[0074] Provided herein are methods for detecting the formation of a multispecific antibody after performing a manufacturing process for producing the multispecific antibody. Also provided are methods for obtaining a stability profile of the multispecific antibody and a degradation fragment species profile during the manufacturing process for producing the multispecific antibody. In certain embodiments, the manufacturing process for producing the multispecific antibody is selected from the group consisting of a controlled arm exchange (cFAE) process, a knobs-in-holes process, a heavy chain heterodimerization process, and an in vitro disulfide bond isomerization process.

[0075] Full-length bispecific antibodies of the present invention can be generated, for example, using Fab arm exchange (or half molecule exchange) between two monospecific bivalent antibodies by introducing substitutions in the heavy chain CH3 interface in each half molecule to favor heterodimerization of two antibody half molecules with distinct specificities, either in vitro in a cell-free environment or by co-expression. The Fab arm exchange reaction is the result of disulfide bond isomerization and dissociation-association of the CH3 domains. Heavy chain disulfide bonds in the hinge region of the monospecific parent antibody are reduced. The resulting free cysteine ​​in one of the parent monospecific antibodies forms an intra-heavy chain disulfide bond with a cysteine ​​residue in the second parent monospecific antibody molecule, while the CH3 domain of the parent antibody is released and reformed by dissociation-association. The CH3 domain of the Fab arm can be engineered to favor heterodimerization over homodimerization. The resulting product is a bispecific antibody with two Fab arms or half molecules, each binding to a different epitope, ie, an epitope on the first antigen and an epitope on the second antigen.

[0076] As used herein, "homodimerization" refers to the interaction of two heavy chains with identical CH3 amino acid sequences. As used herein, "homodimer" refers to an antibody having two heavy chains with identical CH3 amino acid sequences.

[0077] As used herein, "heterodimerization" refers to the interaction of two heavy chains with non-identical CH3 amino acid sequences. As used herein, "heterodimer" refers to an antibody having two heavy chains with non-identical CH3 amino acid sequences.

[0078] A "knob-in-hole" strategy (see, e.g., WO 2006 / 028936) can be used to generate full-length bispecific antibodies. Briefly, selected amino acids that form the interface of the CH3 domain in human IgG can be mutated at positions that affect CH3 domain interactions to promote heterodimer formation. Amino acids with small side chains (holes) are introduced into the heavy chain of an antibody that specifically binds to a first antigen, and amino acids with large side chains (knobs) are introduced into the heavy chain of an antibody that specifically binds to a second antigen. After co-expression of the two antibodies, heterodimers form as a result of the preferential interaction of the heavy chain with the "hole" and the heavy chain with the "knob." Exemplary pairs of CH3 substitutions that form knobs and holes are T366Y / F405A, T366W / F405W, F405W / Y407A, T394W / Y407T, T394S / Y407A, T366W / T394S, F405W / T394S, and T366W / T366S_L368A_Y407V (expressed as altered position in the first CH3 domain of the first heavy chain / altered position in the second CH3 domain of the second heavy chain).

[0079] Other strategies, such as promoting heavy chain heterodimer formation using electrostatic interactions by substituting positively charged residues on one CH3 surface and negatively charged residues on the second CH3 surface, can be used as described in U.S. Patent Application Publication No. 2010 / 0015133, U.S. Patent Application Publication No. 2009 / 0182127, U.S. Patent Application Publication No. 2010 / 028637, or U.S. Patent Application Publication No. 2011 / 0123532. In another strategy, heterodimer formation can be achieved using the following substitutions: L351Y_F405A Y407V / T394W, T366I_K392M_T394W / F405A_Y407V, T366L_K392M_T394W / F405A_Y407V, L351Y_Y407A / T366A_K409F, L351Y_Y407A / T366V K409F Y407A / T366A_K409F, or T350V_L351Y_F405A This can be facilitated by Y407V / T350V_T366L_K392L_T394W (expressed as modified position in the first CH3 domain of the first heavy chain / modified position in the second CH3 domain of the second heavy chain).

[0080] In addition to the above methods, bispecific antibodies of the present invention can be generated in vitro in a cell-free environment by introducing asymmetric mutations into the CH3 regions of two monospecific homodimeric antibodies and forming a bispecific heterodimeric antibody from the two parent monospecific homodimeric antibodies under reducing conditions that cause disulfide bond isomerization, according to the method described in WO 2011 / 131746. In this method, a first monospecific bivalent antibody (e.g., an anti-CD33 antibody) and a second monospecific bivalent antibody (e.g., an anti-CD3 antibody) are engineered to have specific substitutions in the CH3 domains that promote heterodimer stability. These antibodies are incubated together under reducing conditions sufficient to cause cysteines in the hinge regions to isomerize disulfide bonds, thereby generating a bispecific antibody by Fab arm exchange. The incubation conditions can optimally be returned to non-reducing conditions. Exemplary reducing agents that can be used include 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris(2-carboxyethyl)phosphine (TCEP), L-cysteine, and beta-mercaptoethanol, preferably a reducing agent selected from the group consisting of 2-mercaptoethylamine, dithiothreitol, and tris(2-carboxyethyl)phosphine. For example, incubation at a temperature of at least 20°C, in the presence of at least 25 mM 2-MEA or at least 0.5 mM dithiothreitol, at a pH of 5 to 8, e.g., pH 7.0 or 7.4, for at least 90 minutes can be used.

[0081] Embodiment The present invention provides the following non-limiting embodiments.

[0082] Embodiment 1 is a method for detecting the formation of a multispecific molecule, comprising: a. obtaining a multispecific molecule sample; b. Obtaining a reversed-phase high-performance liquid chromatography (RP-HPLC) column; c. contacting the multispecific molecule sample in a polar aqueous mobile phase A with an RP-HPLC column, wherein the polar aqueous mobile phase comprises an ion pairing agent; d. contacting an organic non-polar mobile phase B with an RP-HPLC column, wherein the organic non-polar phase comprises an ion pairing agent; e. Eluting the multispecific molecule sample; f. monitoring the amount of multispecific molecule formation in the eluted multispecific molecule sample.

[0083] Embodiment 2 is the method of embodiment 1, wherein the polar aqueous mobile phase A is a solution containing about 1% to about 5% isopropanol.

[0084] Embodiment 3 is the method of embodiment 2, wherein the solution comprises about 2% isopropanol.

[0085] Embodiment 4 is the method according to any one of embodiments 1 to 3, wherein the ion pairing agent in the polar aqueous mobile phase A is selected from the group consisting of trifluoroacetic acid (TFA), difluoroacetic acid (DFA), and formic acid (FA).

[0086] Embodiment 5 is the method of embodiment 4, wherein TFA is present in the polar aqueous mobile phase A at about 0.1% to about 2%.

[0087] Embodiment 6 is the method of embodiment 5, wherein TFA is present in the polar aqueous mobile phase A at about 0.1%.

[0088] Embodiment 7 is the method of embodiment 1, wherein the organic non-polar mobile phase B is a solution comprising about 60% to about 80% isopropanol and about 15% to about 25% acetonitrile.

[0089] Embodiment 8 is the method of embodiment 7, wherein the solution comprises about 70% isopropanol.

[0090] Embodiment 9 is the method of embodiment 7 or 8, wherein the solution comprises about 20% acetonitrile.

[0091] Embodiment 10 is the method of any one of embodiments 7 to 9, wherein the ion pairing agent in the organic non-polar mobile phase B is selected from the group consisting of trifluoroacetic acid (TFA), difluoroacetic acid (DFA), and formic acid (FA).

[0092] Embodiment 11 is the method of embodiment 10, wherein TFA is present in the organic non-polar aqueous mobile phase B at about 0.1% to about 2%.

[0093] Embodiment 12 is the method of embodiment 11, wherein TFA is present in the organic non-polar aqueous mobile phase B at about 0.1%.

[0094] Embodiment 13 is the method according to any one of embodiments 1 to 12, wherein the multispecific molecule is a multispecific antibody, preferably the multispecific antibody is a bispecific antibody.

[0095] Embodiment 14 is a method of detecting the formation of a multispecific antibody while performing a manufacturing process for producing the multispecific antibody, comprising: a. performing a manufacturing process for producing a multispecific antibody, the manufacturing process resulting in a multispecific antibody sample comprising a quantity of the multispecific antibody; b. setting up a reversed-phase high-performance liquid chromatography (RP-HPLC) column; c. Injecting the multispecific antibody sample into a polar aqueous mobile phase A in an RP-HPLC column, the polar aqueous mobile phase comprising an ion pairing agent; d. applying an organic non-polar mobile phase B to the RP-HPLC column to form a gradient elution, wherein the organic non-polar mobile phase comprises an ion pairing agent; e. Eluting the multispecific antibody sample; f. monitoring the amount of multispecific antibody formation in the eluted sample.

[0096] Embodiment 15 is the method of embodiment 14, wherein the polar aqueous mobile phase A is a solution containing about 1% to about 5% isopropanol.

[0097] Embodiment 16 is the method of embodiment 15, wherein the solution comprises about 2% isopropanol.

[0098] Embodiment 17 is the method of any one of embodiments 14 to 16, wherein the ion pairing agent in the polar aqueous mobile phase A is selected from the group consisting of trifluoroacetic acid (TFA), difluoroacetic acid (DFA), and formic acid (FA).

[0099] Embodiment 18 is the method of embodiment 17, wherein TFA is present in the polar aqueous mobile phase A at about 0.1% to about 2%.

[0100] Embodiment 19 is the method of embodiment 18, wherein TFA is present in the polar aqueous mobile phase A at about 0.1%.

[0101] Embodiment 20 is the method of embodiment 14, wherein the organic non-polar mobile phase is a solution comprising about 60% to about 80% isopropanol and about 15% to about 25% acetonitrile.

[0102] Embodiment 21 is the method of embodiment 20, wherein the solution comprises about 70% isopropanol.

[0103] Embodiment 22 is the method of embodiment 20 or 21, wherein the solution comprises about 20% acetonitrile.

[0104] Embodiment 23 is the method of any one of embodiments 20 to 22, wherein the ion pairing agent in the organic non-polar mobile phase B is selected from the group consisting of trifluoroacetic acid (TFA), difluoroacetic acid (DFA), and formic acid (FA).

[0105] Embodiment 24 is the method of embodiment 23, wherein TFA is present in the non-polar aqueous mobile phase at about 0.1% to about 2%.

[0106] Embodiment 25 is the method of embodiment 24, wherein TFA is present at about 0.1% in the non-polar aqueous mobile phase.

[0107] Embodiment 26 is the method of any one of embodiments 14 to 25, wherein the gradient elution comprises a solution comprising about 75% polar mobile phase A solution and about 25% organic non-polar mobile phase B solution when the multispecific antibody sample is injected onto the RP-HPLC column.

[0108] Embodiment 27 is the method of embodiment 26, wherein the gradient elution comprises a solution comprising about 66% polar mobile phase A solution and about 34% organic non-polar mobile phase B solution at about 16 minutes after the multispecific antibody sample is injected onto the RP-HPLC column.

[0109] Embodiment 28 is the method of embodiment 26, wherein the gradient elution comprises a solution comprising about 5% polar mobile phase A solution and about 95% organic non-polar mobile phase B solution at about 20 minutes after the multispecific antibody sample is injected onto the RP-HPLC column.

[0110] Embodiment 29 is the method of embodiment 26, wherein the gradient elution comprises a solution comprising about 75% polar mobile phase A solution and about 25% organic non-polar mobile phase B solution at about 21 minutes after the multispecific antibody sample is injected onto the RP-HPLC column.

[0111] Embodiment 30 is the method of any one of embodiments 14 to 29, wherein the manufacturing process for producing the multispecific antibody is selected from the group consisting of: knobs-in-holes process, strand-exchange engineered domain process, chemically linked bispecific antibody (BsAb) process, immunoglobulin domain crossover process, and controlled Fab arm exchange (cFAE) and dual variable domain process.

[0112] Embodiment 31 is the method of any one of embodiments 14 to 30, wherein the multispecific antibody is a bispecific antibody.

[0113] Embodiment 32 is a method for detecting the formation of a bispecific antibody while performing controlled FAB arm exchange (cFAE) to generate the bispecific antibody, comprising: a. performing controlled FAB arm exchange (cFAE) to generate a cFAE sample, the cFAE sample comprising a quantity of a bispecific antibody; b. setting up a reversed-phase high-performance liquid chromatography (RP-HPLC) column; c. Injecting the cFAE sample into a polar aqueous mobile phase A in an RP-HPLC column, the polar aqueous mobile phase including an ion pairing agent; d. applying an organic non-polar mobile phase B to the RP-HPLC column to form a gradient elution, wherein the organic non-polar mobile phase comprises an ion pairing agent; e. Eluting the cFAE sample; f. monitoring the amount of multispecific antibody formation in the eluted sample.

[0114] Embodiment 33 is the method of embodiment 32, wherein the polar aqueous mobile phase A is a solution comprising about 1% to about 5% isopropanol.

[0115] Embodiment 34 is the method of embodiment 33, wherein the solution comprises about 2% isopropanol.

[0116] Embodiment 35 is the method of any one of embodiments 32 to 34, wherein the ion pairing agent in the polar aqueous mobile phase A is selected from the group consisting of trifluoroacetic acid (TFA), difluoroacetic acid (DFA), and formic acid (FA).

[0117] Embodiment 36 is the method of embodiment 35, wherein TFA is present in the polar aqueous mobile phase A at about 0.1% to about 2%.

[0118] Embodiment 37 is the method of embodiment 36, wherein TFA is present in the polar aqueous mobile phase A at about 0.1%.

[0119] Embodiment 38 is the method of embodiment 32, wherein the organic non-polar mobile phase is a solution comprising about 60% to about 80% isopropanol and about 15% to about 25% acetonitrile.

[0120] Embodiment 39 is the method of embodiment 38, wherein the solution comprises about 70% isopropanol.

[0121] Embodiment 40 is the method of embodiment 38 or 39, wherein the solution comprises about 20% acetonitrile.

[0122] Embodiment 41 is the method of any one of embodiments 38 to 40, wherein the ion pairing agent in the organic non-polar mobile phase B is selected from the group consisting of trifluoroacetic acid (TFA), difluoroacetic acid (DFA), and formic acid (FA).

[0123] Embodiment 42 is the method of embodiment 41, wherein TFA is present in the non-polar aqueous mobile phase at about 0.1% to about 2%.

[0124] Embodiment 43 is the method of embodiment 42, wherein TFA is present at about 0.1% in the non-polar aqueous mobile phase.

[0125] Embodiment 44 is a system comprising a reversed-phase high performance liquid chromatography (RP-HPLC) component for detecting the formation of a multispecific molecule, comprising: a. a sample of polyspecific molecules in a polar aqueous mobile phase A, the polar aqueous mobile phase comprising an ion pairing agent; b. an RP-HPLC column; c. an organic non-polar mobile phase B, wherein the organic non-polar mobile phase comprises an ion pairing agent; an RP-HPLC column configured to contact the multispecific molecule sample in a polar aqueous mobile phase A; The RP-HPLC is further configured to elute the multispecific molecule sample in contact with an organic non-polar mobile phase B, and the amount of multispecific molecule formation can be determined in the eluted multispecific molecule sample.

[0126] Embodiment 45 is the system of embodiment 44, wherein the polar aqueous mobile phase A is a solution comprising about 1% to about 5% isopropanol.

[0127] Embodiment 46 is the system of embodiment 45, wherein the solution comprises about 2% isopropanol.

[0128] Embodiment 47 is the system of any one of embodiments 44 to 46, wherein the ion pairing agent in the polar aqueous mobile phase A is selected from the group consisting of trifluoroacetic acid (TFA), difluoroacetic acid (DFA), and formic acid (FA).

[0129] Embodiment 48 is the system of embodiment 47, wherein TFA is present in the polar aqueous mobile phase A at about 0.1% to about 2%.

[0130] Embodiment 49 is the system of embodiment 48, wherein TFA is present in the polar aqueous mobile phase A at about 0.1%.

[0131] Embodiment 50 is the system of embodiment 44, wherein the organic non-polar mobile phase B is a solution comprising about 60% to about 80% isopropanol and about 15% to about 25% acetonitrile.

[0132] Embodiment 51 is the system of embodiment 50, wherein the solution comprises about 70% isopropanol.

[0133] Embodiment 52 is the system of embodiment 50 or 51, wherein the solution comprises about 20% acetonitrile.

[0134] Embodiment 53 is a system according to any one of embodiments 50 to 52, wherein the ion pairing agent in the organic non-polar mobile phase B is selected from the group consisting of trifluoroacetic acid (TFA), difluoroacetic acid (DFA), and formic acid (FA).

[0135] Embodiment 54 is the system of embodiment 53, wherein TFA is present in the organic non-polar aqueous mobile phase B at about 0.1% to about 2%.

[0136] Embodiment 55 is the system of embodiment 54, wherein TFA is present in the organic non-polar aqueous mobile phase B at about 0.1%.

[0137] Embodiment 56 is the system of any one of embodiments 44 to 55, wherein the multispecific molecule is a multispecific antibody, preferably wherein the multispecific antibody is a bispecific antibody.

[0138] Embodiment 57 is a means for reversed-phase high performance liquid chromatography (RP-HPLC) detection of multispecific molecule formation, comprising: a. a sample of polyspecific molecules in a polar aqueous mobile phase A, the polar aqueous mobile phase comprising an ion pairing agent; b. an RP-HPLC column; c. an organic non-polar mobile phase B, wherein the organic non-polar mobile phase comprises an ion pairing agent; an RP-HPLC column configured to contact the multispecific molecule sample in a polar aqueous mobile phase A; The RP-HPLC column is further configured to contact an organic non-polar mobile phase B to elute the multispecific molecule sample, whereby the amount of multispecific molecule formation can be determined in the eluted multispecific molecule sample.

[0139] Embodiment 58 is a method for reversed-phase high performance liquid chromatography (RP-HPLC) detection of multispecific molecule formation according to embodiment 57, wherein the polar aqueous mobile phase A is a solution comprising about 1% to about 5% isopropanol.

[0140] Embodiment 59 is a means for reversed-phase high performance liquid chromatography (RP-HPLC) detection of multispecific molecule formation according to embodiment 58, wherein the solution comprises about 2% isopropanol.

[0141] Embodiment 60 is a method for reversed-phase high performance liquid chromatography (RP-HPLC) detection of the formation of a multispecific molecule according to any one of embodiments 57 to 59, wherein the ion pairing agent in the polar aqueous mobile phase A is selected from the group consisting of trifluoroacetic acid (TFA), difluoroacetic acid (DFA), and formic acid (FA).

[0142] Embodiment 61 is a method for reversed-phase high performance liquid chromatography (RP-HPLC) detection of polyspecific molecule formation according to embodiment 60, wherein TFA is present in a polar aqueous mobile phase A at about 0.1% to about 2%.

[0143] Embodiment 62 is a means for reversed-phase high performance liquid chromatography (RP-HPLC) detection of multispecific molecule formation according to embodiment 61, wherein TFA is present at about 0.1% in a polar aqueous mobile phase A.

[0144] Embodiment 63 is a method for reversed-phase high performance liquid chromatography (RP-HPLC) detection of polyspecific molecule formation as described in embodiment 57, wherein the organic non-polar mobile phase B is a solution comprising about 60% to about 80% isopropanol and about 15% to about 25% acetonitrile.

[0145] Embodiment 64 is a means for reversed-phase high performance liquid chromatography (RP-HPLC) detection of multispecific molecule formation according to embodiment 63, wherein the solution comprises about 70% isopropanol.

[0146] Embodiment 65 is a means for reversed-phase high performance liquid chromatography (RP-HPLC) detection of multispecific molecule formation according to embodiment 63 or 64, wherein the solution comprises about 20% acetonitrile.

[0147] Embodiment 66 is a method for reversed-phase high performance liquid chromatography (RP-HPLC) detection of the formation of a multispecific molecule according to any one of embodiments 63 to 65, wherein the ion pairing agent in the organic non-polar aqueous mobile phase B is selected from the group consisting of trifluoroacetic acid (TFA), difluoroacetic acid (DFA), and formic acid (FA).

[0148] Embodiment 67 is a method for reversed-phase high performance liquid chromatography (RP-HPLC) detection of polyspecific molecule formation according to embodiment 66, wherein TFA is present in an organic non-polar aqueous mobile phase B at about 0.1% to about 2%.

[0149] Embodiment 68 is a means for reversed-phase high performance liquid chromatography (RP-HPLC) detection of multispecific molecule formation according to embodiment 67, wherein TFA is present at about 0.1% in an organic non-polar aqueous mobile phase B.

[0150] Embodiment 69 is a means for reversed-phase high performance liquid chromatography (RP-HPLC) detection of the formation of a multispecific molecule according to any one of embodiments 57 to 68, wherein the multispecific molecule is a multispecific antibody, preferably the multispecific antibody is a bispecific antibody.

[0151] Embodiment 70 is a method for designing a stable multispecific antibody, comprising the steps of: a. performing a manufacturing process for producing a multispecific antibody, the manufacturing process resulting in a multispecific antibody sample comprising a quantity of the multispecific antibody; b. setting up a reversed-phase high-performance liquid chromatography (RP-HPLC) column; c. Injecting the multispecific antibody sample into a polar aqueous mobile phase A in an RP-HPLC column, the polar aqueous mobile phase comprising an ion pairing agent; d. applying an organic non-polar mobile phase B to the RP-HPLC column to form a gradient elution, wherein the organic non-polar mobile phase comprises an ion pairing agent; e. Eluting the multispecific antibody sample; f. monitoring the amount of multispecific antibody formation in the eluted sample; An increased amount of multispecific antibody formation compared to a control indicates the design of a stable multispecific antibody.

[0152] Embodiment 71 is the method of embodiment 70, wherein the polar aqueous mobile phase A is a solution comprising about 1% to about 5% isopropanol.

[0153] Embodiment 72 is the method of embodiment 71, wherein the solution comprises about 2% isopropanol.

[0154] Embodiment 73 is the method of any one of embodiments 70 to 72, wherein the ion pairing agent in the polar aqueous mobile phase A is selected from the group consisting of trifluoroacetic acid (TFA), difluoroacetic acid (DFA), and formic acid (FA).

[0155] Embodiment 74 is the method of embodiment 73, wherein TFA is present in the polar aqueous mobile phase A at about 0.1% to about 2%.

[0156] Embodiment 75 is the method of embodiment 74, wherein TFA is present in the polar aqueous mobile phase A at about 0.1%.

[0157] Embodiment 76 is the method of embodiment 70, wherein the organic non-polar mobile phase is a solution comprising about 60% to about 80% isopropanol and about 15% to about 25% acetonitrile.

[0158] Embodiment 77 is the method of embodiment 76, wherein the solution comprises about 70% isopropanol.

[0159] Embodiment 78 is the method of embodiment 76 or 77, wherein the solution comprises about 20% acetonitrile.

[0160] Embodiment 79 is the method of any one of embodiments 76 to 78, wherein the ion pairing agent in the organic non-polar mobile phase B is selected from the group consisting of trifluoroacetic acid (TFA), difluoroacetic acid (DFA), and formic acid (FA).

[0161] Embodiment 80 is the method of embodiment 79, wherein TFA is present in the non-polar aqueous mobile phase at about 0.1% to about 2%.

[0162] Embodiment 81 is the method of embodiment 80, wherein TFA is present at about 0.1% in the non-polar aqueous mobile phase.

[0163] Embodiment 82 is the method of any one of embodiments 70 to 81, wherein the manufacturing process for producing the multispecific antibody is selected from the group consisting of: knobs-in-holes process, strand-exchange engineered domain process, chemically linked bispecific antibody (BsAb) process, immunoglobulin domain crossover process, and controlled Fab arm exchange (cFAE) and dual variable domain process.

[0164] Embodiment 83 is the method of any one of embodiments 70 to 82, wherein the multispecific antibody is a bispecific antibody.

[0165] Embodiment 84 is a method for obtaining a stability profile of a multispecific antibody during a manufacturing process for producing the multispecific antibody, comprising: a. performing a manufacturing process for producing a multispecific antibody, wherein amounts of multispecific antibody samples are obtained at different times during the manufacturing process; b. setting up a reversed-phase high-performance liquid chromatography (RP-HPLC) column; c. Injecting the multispecific antibody sample from each time point into a polar aqueous mobile phase A in an RP-HPLC column, the polar aqueous mobile phase including an ion pairing agent; d. applying an organic non-polar mobile phase B to the RP-HPLC column to form a gradient elution, wherein the organic non-polar mobile phase comprises an ion pairing agent; e. Eluting the multispecific antibody sample; f. monitoring the amount of multispecific antibody formation in the eluted sample; The amount of multispecific antibody formation at each time point provides a stability profile of the multispecific antibody.

[0166] Embodiment 85 is the method of embodiment 84, wherein the polar aqueous mobile phase A is a solution comprising about 1% to about 5% isopropanol.

[0167] Embodiment 86 is the method of embodiment 85, wherein the solution comprises about 2% isopropanol.

[0168] Embodiment 87 is the method of any one of embodiments 84 to 86, wherein the ion pairing agent in the polar aqueous mobile phase A is selected from the group consisting of trifluoroacetic acid (TFA), difluoroacetic acid (DFA), and formic acid (FA).

[0169] Embodiment 88 is the method of embodiment 87, wherein TFA is present in the polar aqueous mobile phase A at about 0.1% to about 2%.

[0170] Embodiment 89 is the method of embodiment 88, wherein TFA is present in the polar aqueous mobile phase A at about 0.1%.

[0171] Embodiment 90 is the method of embodiment 84, wherein the organic non-polar mobile phase is a solution comprising about 60% to about 80% isopropanol and about 15% to about 25% acetonitrile.

[0172] Embodiment 91 is the method of embodiment 90, wherein the solution comprises about 70% isopropanol.

[0173] Embodiment 92 is the method of embodiment 90 or 91, wherein the solution comprises about 20% acetonitrile.

[0174] Embodiment 93 is the method of any one of embodiments 90 to 92, wherein the ion pairing agent in the organic non-polar mobile phase B is selected from the group consisting of trifluoroacetic acid (TFA), difluoroacetic acid (DFA), and formic acid (FA).

[0175] Embodiment 94 is the method of embodiment 93, wherein TFA is present in the non-polar aqueous mobile phase at about 0.1% to about 2%.

[0176] Embodiment 95 is the method of embodiment 94, wherein TFA is present at about 0.1% in the non-polar aqueous mobile phase.

[0177] Embodiment 96 is the method according to any one of embodiments 84 to 95, wherein the manufacturing process for producing the multispecific antibody is selected from the group consisting of: a knobs-in-holes process, a strand-exchange engineered domain process, a chemically linked bispecific antibody (BsAb) process, an immunoglobulin domain crossover process, and a controlled Fab arm exchange (cFAE) and dual variable domain process.

[0178] Embodiment 97 is the method of any one of embodiments 84 to 96, wherein the multispecific antibody is a bispecific antibody.

[0179] Embodiment 98 is a method for obtaining a degradation fragment species profile of a multispecific antibody during a manufacturing process for producing the multispecific antibody, comprising the steps of: a. performing a manufacturing process for producing a multispecific antibody, wherein amounts of multispecific antibody samples are obtained at different times during the manufacturing process; b. setting up a reversed-phase high-performance liquid chromatography (RP-HPLC) column; c. Injecting the multispecific antibody sample from each time point into a polar aqueous mobile phase A in an RP-HPLC column, the polar aqueous mobile phase including an ion pairing agent; d. applying an organic non-polar mobile phase B to the RP-HPLC column to form a gradient elution, wherein the organic non-polar mobile phase comprises an ion pairing agent; e. Eluting the multispecific antibody sample; f. monitoring the amount of degradation fragment species in the eluted sample; The method wherein the amount of degradation fragment species at each time point provides a degradation fragment species profile of the multispecific antibody production process.

[0180] Embodiment 99 is the method of embodiment 98, wherein the polar aqueous mobile phase A is a solution comprising about 1% to about 5% isopropanol.

[0181] Embodiment 100 is the method of embodiment 99, wherein the solution comprises about 2% isopropanol.

[0182] Embodiment 101 is the method of any one of embodiments 98 to 100, wherein the ion pairing agent in the polar aqueous mobile phase A is selected from the group consisting of trifluoroacetic acid (TFA), difluoroacetic acid (DFA), and formic acid (FA).

[0183] Embodiment 102 is the method of embodiment 101, wherein TFA is present in the polar aqueous mobile phase A at about 0.1% to about 2%.

[0184] Embodiment 103 is the method of embodiment 102, wherein TFA is present in the polar aqueous mobile phase A at about 0.1%.

[0185] Embodiment 104 is the method of embodiment 98, wherein the organic non-polar mobile phase is a solution comprising about 60% to about 80% isopropanol and about 15% to about 25% acetonitrile.

[0186] Embodiment 105 is the method of embodiment 104, wherein the solution comprises about 70% isopropanol.

[0187] Embodiment 106 is the method of embodiment 104 or 105, wherein the solution comprises about 20% acetonitrile.

[0188] Embodiment 107 is the method of any one of embodiments 104 to 106, wherein the ion pairing agent in the organic non-polar mobile phase B is selected from the group consisting of trifluoroacetic acid (TFA), difluoroacetic acid (DFA), and formic acid (FA).

[0189] Embodiment 108 is the method of embodiment 107, wherein TFA is present in the non-polar aqueous mobile phase at about 0.1% to about 2%.

[0190] Embodiment 109 is the method of embodiment 108, wherein TFA is present at about 0.1% in the non-polar aqueous mobile phase.

[0191] Embodiment 110 is the method of any one of embodiments 98 to 109, wherein the manufacturing process for producing the multispecific antibody is selected from the group consisting of: knobs-in-holes process, strand-exchange engineered domain process, chemically linked bispecific antibody (BsAb) process, immunoglobulin domain crossover process, and controlled Fab arm exchange (cFAE) and dual variable domain process.

[0192] Embodiment 111 is the method of any one of embodiments 98 to 110, wherein the multispecific antibody is a bispecific antibody. [Example]

[0193] Example 1: Reversed-phase high performance liquid chromatography to determine bispecific antibody formation after controlled FAB arm exchange (FAE). Reversed-phase high-performance liquid chromatography (RP-HPLC) separates molecules based on a hydrophobic absorption / desorption process across a hydrophobic solid support. Using increasingly polar mobile phases, hydrophobic binding interactions between proteins and the solid support led to desorption of analytes from the column. In this particular method, where antibody formation was established through reoxidation of the parent mAb, RP-HPLC was employed to quantify intact, fully oxidized bispecific DuoBody® (anti-GPRC5D / anti-CD3, anti-CD33 / anti-CD3, anti-TMEFF2 / anti-CD3, anti-7959 / anti-CD3, and anti-BCMA / anti-CD3) from partially reoxidized fragments. Test samples were sampled after controlled FAB arm exchange (FAE) and injected onto the column in a highly polar aqueous mobile phase containing an ion-pairing agent (trifluoroacetic acid) used to improve peak shape and molecule retention. An increasing amount of an organic nonpolar mobile phase, also containing trifluoroacetic acid, was then applied to the column in a gradient elution, allowing molecules to elute based on their hydrophobic properties. The column eluate was continuously monitored at 280 nm and the relative amount of each IgG was determined by comparing the peak area counts with the subfragmented IgG species.

[0194] Example 2: Preparation of reagents / solutions for bispecific antibodies NOTE A: Different total volumes of mobile phase A and mobile phase B can be prepared, but the ratio of each component must be maintained.

[0195] Note B: Trifluoroacetic acid (TFA) has a low boiling point and high volatility. It is recommended to store TFA at +2–8 °C to ensure accurate dispensing by pipetting.

[0196] [Table 1]

[0197] To prepare mobile phase A, approximately 800 mL of deionized water was added to a 1-liter beaker in a fume hood. In a 50-mL graduated cylinder, 20 mL of isopropanol was added. 20 mL of isopropanol was added to the 1-liter beaker, and the deionized water and isopropanol were mixed with a stir bar for 3 minutes. 1 mL of refrigerated TFA was carefully pipetted and added to the 1-liter beaker containing the isopropanol and deionized water. The TFA was delivered below the surface of the solution, and the mixture was stirred for an additional 3 minutes. (Note: Due to the high volatility of TFA, delivery of TFA below the surface of the solution over the assay retention time was important.) The solution from the 1-liter beaker was transferred to a 1-liter graduated cylinder and filled to the 1-liter mark with deionized water. The solution was then transferred to the mobile phase A bottle.

[0198] [Table 2]

[0199] To prepare mobile phase B, in a fume hood, approximately 99 mL of deionized water was added to a 1-liter beaker, followed by 200 mL of acetonitrile. The solution was mixed together with a stir bar for 3 minutes. 1 mL of refrigerated TFA was carefully pipetted and added to the 1-liter beaker containing acetonitrile and deionized water. The TFA was delivered below the surface of the solution, and the mixture was stirred for an additional 3 minutes. (Note: Due to the high volatility of TFA, delivery of TFA below the surface of the solution over the assay retention time was important.) The solution was transferred from the 1-liter beaker to a 1-liter graduated cylinder and filled to the 1-liter mark with isopropanol. The solution was then transferred to the mobile phase B bottle.

[0200] Example 3: Sample and standard preparations Bispecific antibodies are made by reducing the parent homodimeric bispecific antibody and reoxidizing these reactants via cFAE to generate the heterodimeric bispecific antibody. To prepare post-cFAE bispecific antibody process samples for testing, the process samples are 280From the cFAE UF / DF concentration determined by, it is diluted according to Table 3 to a final test concentration of 2 mg / mL (diluted with mobile phase A).

[0201] [Table 3]

[0202] To prepare the reference material sample at 2 mg / mL, the bispecific antibody at stock concentration was diluted in mobile phase A as sample diluent according to Table 4.

[0203] [Table 4]

[0204] Example 4: HPLC instrument settings The instrument parameters are provided in Tables 5 and 6. The column module thermostat was controlled at 80°C to heat both the right and left compartments. The connecting thermostat capillary was plumbed to the heating element on the left (3 μL). To avoid overpressurizing the LC system and damaging the column, the system was started to flow once the thermostat temperature of 80°C was reached.

[0205] [Table 5]

[0206] [Table 6]

[0207] [Table 7]

[0208] [Table 8] NOTE: The array can be extended for multiple samples.

[0209] Example 5: Integration Results NOTE: Manual integration was used and integrated with the processing method.

[0210] Peak integration of reference material. Integration of the main component: The reference material main component consisted of two peaks, including the main component peak and the main component shoulder peak (Figures 3, 5, 7, 9, and 11). These two peaks were integrated with each other between the valley of the last pre-peak and the main peak, and ended between the valley of the shoulder main peak and the first post-peak (Figures 3, 5, 7, 9, and 11). The pre- and post-peaks relative to the main peak and the main shoulder peak were composed of unoxidized fragments and / or residual parent homodimeric bispecific antibody contained in the reference material (control).

[0211] The integration of the pre-peak in the reference material (RM) was integrated as one peak. The integration of the post-peak in the reference material (RM) was integrated as one peak.

[0212] cFAE test sample peak integration Integration of the main component of the post-cFAE test article: The post-cFAE test bispecific antibody consisted of two peaks, including a main component peak and a main component shoulder peak (Figures 4, 6, 8, 10, and 12). These two peaks were integrated with each other between the valley of the last pre-peak and the main peak, and ended between the valley of the shoulder main peak and the first post-peak (Figures 4, 6, 8, 10, and 12).

[0213] Non-baseline resolved peak clusters preceding the principal component were integrated together as a single pre-peak, and individual baseline resolved peaks were integrated separately. All post-peaks following the shoulder peak of the principal component were integrated as a single peak. The pre- and post-peaks represent unoxidized bispecific species (Figures 4, 6, 8, 10, and 12).

[0214] Peak identification of the reference material: The two peaks corresponding to the bispecific antibody reference material were labeled as the main component and the shoulder peak of the main component. The cluster peaks preceding the bispecific antibody were integrated with each other (Figures 3-12) and labeled as the pre-peak. The post-peak relative to the main component and the shoulder peak of the main component were integrated with each other (Figures 3-12) and labeled as the post-peak.

[0215] Those skilled in the art will appreciate that changes could be made to the embodiments described above without departing from the broad inventive concept. It is understood, therefore, that the invention is not limited to the particular embodiments disclosed, but is intended to cover modifications within the spirit and scope of the invention as defined by this description. The present invention includes the following embodiments. [1] A method for detecting the formation of a multispecific molecule, comprising: a. obtaining a multispecific molecule sample; b. Obtaining a reversed-phase high-performance liquid chromatography (RP-HPLC) column; c. contacting the multispecific molecule sample in a polar aqueous mobile phase A with the RP-HPLC column, wherein the polar aqueous mobile phase comprises an ion pairing agent; d. contacting an organic non-polar mobile phase B with the RP-HPLC column, wherein the organic non-polar phase comprises an ion pairing agent; e. Eluting the multispecific molecule sample; f. monitoring the amount of multispecific molecule formation in said eluted multispecific molecule sample. [2] The method according to [1] above, wherein the polar aqueous mobile phase A is a solution containing about 1% to about 5% isopropanol. [3] The method of claim 2, wherein the solution comprises about 2% isopropanol. [4] The method according to any one of [1] to [3] above, wherein the ion pairing agent in the polar aqueous mobile phase A is selected from the group consisting of trifluoroacetic acid (TFA), difluoroacetic acid (DFA), and formic acid (FA). [5] The method according to [4] above, wherein the TFA is present in the polar aqueous mobile phase A at about 0.1% to about 2%. [6] The method according to [5], wherein the TFA is present in the polar aqueous mobile phase A at about 0.1%. [7] The method according to [1] above, wherein the organic non-polar mobile phase B is a solution containing about 60% to about 80% isopropanol and about 15% to about 25% acetonitrile. [8] The method according to [7], wherein the solution contains about 70% isopropanol. [9] The method according to [7], wherein the solution contains about 20% acetonitrile.

[10] The method according to any one of [7] to [9], wherein the ion pairing agent in the organic non-polar mobile phase B is selected from the group consisting of trifluoroacetic acid (TFA), difluoroacetic acid (DFA), and formic acid (FA).

[11] The method according to

[10] , wherein the TFA is present in the organic non-polar aqueous mobile phase B at about 0.1% to about 2%.

[12] The method according to

[11] , wherein the TFA is present in the organic non-polar aqueous mobile phase B at about 0.1%.

[13] A method for detecting the formation of a multispecific antibody while performing a manufacturing process for producing the multispecific antibody, comprising: a. performing a manufacturing process for producing a multispecific antibody, said manufacturing process resulting in a multispecific antibody sample comprising a quantity of the multispecific antibody; b. setting up a reversed-phase high-performance liquid chromatography (RP-HPLC) column; c. injecting the multispecific antibody sample into a polar aqueous mobile phase A in the RP-HPLC column, wherein the polar aqueous mobile phase comprises an ion pairing agent; d. applying an organic non-polar mobile phase B to the RP-HPLC column to form a gradient elution, wherein the organic non-polar mobile phase comprises an ion pairing agent; e. Eluting the multispecific antibody sample; f. monitoring the amount of multispecific antibody formation in the eluted sample.

[14] The method according to

[13] above, wherein the polar aqueous mobile phase A is a solution containing about 1% to about 5% isopropanol.

[15] The method according to

[14] , wherein the solution contains about 2% isopropanol.

[16] The method according to any one of

[13] to

[15] , wherein the ion pairing agent in the polar aqueous mobile phase A is selected from the group consisting of trifluoroacetic acid (TFA), difluoroacetic acid (DFA), and formic acid (FA).

[17] The method according to

[16] , wherein the TFA is present in the polar aqueous mobile phase A at about 0.1% to about 2%.

[18] The method according to

[17] , wherein the TFA is present in the polar aqueous mobile phase A at about 0.1%.

[19] The method according to

[13] above, wherein the organic non-polar mobile phase B is a solution containing about 60% to about 80% isopropanol and about 15% to about 25% acetonitrile.

[20] The method according to

[19] , wherein the solution contains about 70% isopropanol.

[21] The method according to

[19] , wherein the solution contains about 20% acetonitrile.

[22] The method according to any one of

[19] to

[21] , wherein the ion pairing agent in the organic non-polar mobile phase B is selected from the group consisting of trifluoroacetic acid (TFA), difluoroacetic acid (DFA), and formic acid (FA).

[23] The method according to

[22] , wherein the TFA is present in the organic non-polar aqueous mobile phase B at about 0.1% to about 2%.

[24] The method according to

[23] , wherein the TFA is present in the organic non-polar aqueous mobile phase B at about 0.1%.

[25] The method according to any one of

[13] to

[15] ,

[17] to

[21] ,

[23] , or

[24] , wherein the manufacturing process for producing a multispecific antibody is selected from the group consisting of a knobs-in-holes process, a strand-exchange engineered domain process, a chemically linked bispecific antibody (BsAb) process, an immunoglobulin domain crossover process, and a controlled Fab arm exchange (cFAE) and dual variable domain process.

[26] The method according to any one of

[13] to

[15] ,

[17] to

[21] ,

[23] , or

[24] , wherein the multispecific antibody is a bispecific antibody.

[27] A method for detecting the formation of a bispecific antibody while performing controlled FAB arm exchange (cFAE) to generate a bispecific antibody, comprising: a. performing controlled FAB arm exchange (cFAE) to generate a cFAE sample, wherein the cFAE sample comprises an amount of a bispecific antibody; b. setting up a reversed-phase high-performance liquid chromatography (RP-HPLC) column; c. injecting the cFAE sample into a polar aqueous mobile phase A in the RP-HPLC column, wherein the polar aqueous mobile phase includes an ion pairing agent; d. applying an organic non-polar mobile phase B to the RP-HPLC column to form a gradient elution, wherein the organic non-polar mobile phase comprises an ion pairing agent; e. Eluting the cFAE sample; f. monitoring the amount of bispecific antibody formation in the eluted sample.

[28] A system comprising a reversed-phase high performance liquid chromatography (RP-HPLC) component for detecting the formation of a multispecific molecule, a. a sample of polyspecific molecules in a polar aqueous mobile phase A, wherein the polar aqueous mobile phase comprises an ion pairing agent; b. an RP-HPLC column; c. an organic non-polar mobile phase B, wherein the organic non-polar mobile phase B comprises an ion pairing agent; the RP-HPLC column is configured to contact the multispecific molecule sample in a polar aqueous mobile phase A; wherein the RP-HPLC is further configured to contact the organic non-polar mobile phase B to elute the multispecific molecule sample, and the amount of multispecific molecule formation can be determined in the eluted multispecific molecule sample.

[29] A means for reversed-phase high performance liquid chromatography (RP-HPLC) detection of polyspecific molecule formation, comprising: a. a sample of polyspecific molecules in a polar aqueous mobile phase A, wherein the polar aqueous mobile phase comprises an ion pairing agent; b. an RP-HPLC column; c. an organic non-polar mobile phase B, wherein the organic non-polar mobile phase B comprises an ion pairing agent; the RP-HPLC column is configured to contact the multispecific molecule sample in a polar aqueous mobile phase A; wherein the RP-HPLC column is further configured to contact the RP-HPLC column with the organic non-polar mobile phase B to elute the multispecific molecule sample, and the amount of multispecific molecule formation can be determined in the eluted multispecific molecule sample.

Claims

1. 1. A method for detecting the formation of a multispecific molecule, comprising: a. obtaining a multispecific molecule sample; b. Obtaining a reversed-phase high performance liquid chromatography (RP-HPLC) column; c. contacting the polyspecific molecule sample in a polar aqueous mobile phase A with the RP-HPLC column, wherein the polar aqueous mobile phase A is a solution comprising about 1% to about 5% isopropanol and an ion pairing agent; d. contacting an organic non-polar mobile phase B with the RP-HPLC column, wherein the organic non-polar mobile phase B is a solution containing an ion pairing agent; e. Eluting the multispecific molecule sample; f. monitoring the amount of multispecific molecule formation in said eluted multispecific molecule sample.

2. 2. The method of claim 1, wherein the polar aqueous mobile phase A is a solution containing about 2% isopropanol.

3. 3. The method of claim 1, wherein the ion pairing agent in the polar aqueous mobile phase A is selected from the group consisting of trifluoroacetic acid (TFA), difluoroacetic acid (DFA), and formic acid (FA).

4. 4. The method of claim 3, wherein the TFA is present in the polar aqueous mobile phase A at about 0.1% to about 2%.

5. 5. The method of claim 4, wherein the TFA is present in the polar aqueous mobile phase A at about 0.1%.

6. 6. The method of claim 1, wherein the organic non-polar mobile phase B is a solution comprising about 60% to about 80% isopropanol and about 15% to about 25% acetonitrile.

7. 7. The method of claim 6, wherein the organic non-polar mobile phase B is a solution containing about 70% isopropanol.

8. 8. The method of claim 6 or 7, wherein the organic non-polar mobile phase B is a solution containing about 20% acetonitrile.

9. 9. The method of claim 6, wherein the ion pairing agent in the organic non-polar mobile phase B is selected from the group consisting of trifluoroacetic acid (TFA), difluoroacetic acid (DFA), and formic acid (FA).

10. 10. The method of claim 9, wherein the TFA is present in the organic non-polar mobile phase B at about 0.1% to about 2%.

11. 11. The method of claim 10, wherein the TFA is present in the organic non-polar mobile phase B at about 0.1%.

12. 1. A method for detecting the formation of a multispecific antibody while performing a manufacturing process for producing the multispecific antibody, comprising: a. performing a manufacturing process for producing a multispecific antibody, said manufacturing process resulting in a multispecific antibody sample comprising a quantity of the multispecific antibody; b. Setting up a reversed-phase high performance liquid chromatography (RP-HPLC) column; c. injecting the polyspecific antibody sample into a polar aqueous mobile phase A in the RP-HPLC column, wherein the polar aqueous mobile phase A is a solution comprising about 1% to about 5% isopropanol and an ion pairing agent; d. applying an organic non-polar mobile phase B to the RP-HPLC column to form a gradient elution, wherein the organic non-polar mobile phase B is a solution containing an ion pairing agent; e. Eluting the multispecific antibody sample; f. monitoring the amount of multispecific antibody formation in the eluted sample.

13. 13. The method of claim 12, wherein the polar aqueous mobile phase A is a solution containing about 2% isopropanol.

14. 14. The method of claim 12 or 13, wherein the ion pairing agent in the polar aqueous mobile phase A is selected from the group consisting of trifluoroacetic acid (TFA), difluoroacetic acid (DFA), and formic acid (FA).

15. 15. The method of claim 14, wherein the TFA is present in the polar aqueous mobile phase A at about 0.1% to about 2%.

16. 16. The method of claim 15, wherein the TFA is present in the polar aqueous mobile phase A at about 0.1%.

17. 17. The method of any one of claims 12 to 16, wherein the organic non-polar mobile phase B is a solution comprising about 60% to about 80% isopropanol and about 15% to about 25% acetonitrile.

18. 18. The method of claim 17, wherein the organic non-polar mobile phase B is a solution containing about 70% isopropanol.

19. 19. The method of claim 17 or 18, wherein the organic non-polar mobile phase B is a solution containing about 20% acetonitrile.

20. 20. The method of any one of claims 17 to 19, wherein the ion pairing agent in the organic non-polar mobile phase B is selected from the group consisting of trifluoroacetic acid (TFA), difluoroacetic acid (DFA), and formic acid (FA).

21. 21. The method of claim 20, wherein the TFA is present in the organic non-polar mobile phase B at about 0.1% to about 2%.

22. 22. The method of claim 21 , wherein the TFA is present in the organic non-polar mobile phase B at about 0.1%.

23. 23. The method of any one of claims 12, 13, 15-19, 21, or 22, wherein the manufacturing process for producing multispecific antibodies is selected from the group consisting of: knobs-in-holes process, strand-exchange engineered domain process, chemically linked bispecific antibody (BsAb) process, immunoglobulin domain crossover process, and controlled Fab arm exchange (cFAE) and dual variable domain process.

24. 23. The method of any one of claims 12, 13, 15-19, 21, or 22, wherein the multispecific antibody is a bispecific antibody.

25. 1. A method for detecting the formation of a bispecific antibody while performing controlled FAB arm exchange (cFAE) to generate the bispecific antibody, comprising: a. performing controlled FAB arm exchange (cFAE) to generate a cFAE sample, wherein the cFAE sample comprises an amount of a bispecific antibody; b. Setting up a reversed-phase high performance liquid chromatography (RP-HPLC) column; c. injecting the cFAE sample into a polar aqueous mobile phase A in the RP-HPLC column; the injecting, wherein the polar aqueous mobile phase A is a solution containing about 1% to about 5% isopropanol and an ion pairing agent; d. applying an organic non-polar mobile phase B to the RP-HPLC column to form a gradient elution, wherein the organic non-polar mobile phase B is a solution containing an ion pairing agent; e. Eluting the cFAE sample; f. monitoring the amount of bispecific antibody formation in the eluted sample.

26. 1. A system comprising a reversed-phase high performance liquid chromatography (RP-HPLC) component for detecting the formation of a multispecific molecule, comprising: a. a polyspecific molecule sample in a polar aqueous mobile phase A, wherein the polar aqueous mobile phase A is a solution comprising about 1% to about 5% isopropanol and an ion pairing agent; b. an RP-HPLC column; c. An organic non-polar mobile phase B, wherein the organic non-polar mobile phase B is a solution containing an ion pairing agent; the RP-HPLC column is configured to contact the multispecific molecule sample in a polar aqueous mobile phase A; wherein the RP-HPLC is further configured to contact the organic non-polar mobile phase B to elute the multispecific molecule sample, and wherein the amount of multispecific molecule formation can be determined in the eluted multispecific molecule sample.

27. 1. A means for reversed-phase high performance liquid chromatography (RP-HPLC) detection of polyspecific molecule formation, comprising: a. a polyspecific molecule sample in a polar aqueous mobile phase A, wherein the polar aqueous mobile phase A is a solution comprising about 1% to about 5% isopropanol and an ion pairing agent; b. an RP-HPLC column; c. An organic non-polar mobile phase B, wherein the organic non-polar mobile phase B is a solution containing an ion pairing agent; the RP-HPLC column is configured to contact the multispecific molecule sample in a polar aqueous mobile phase A; wherein the RP-HPLC column is further configured to contact the RP-HPLC column with the organic non-polar mobile phase B to elute the multispecific molecule sample, and the amount of multispecific molecule formation can be determined in the eluted multispecific molecule sample.

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