Antibody purification
阅读说明：本技术 抗体纯化 (Antibody purification ) 是由 T·伊斯克拉 A·M·萨克拉莫 于 2019-02-25 设计创作，主要内容包括：提供了用于纯化抗体的方法。所提供的纯化方法涉及使用羟基磷灰石树脂(HA)以使感兴趣的抗体与一种或多种杂质物质分离。(Methods for purifying antibodies are provided. The provided purification methods involve the use of hydroxyapatite resin (HA) to separate the antibody of interest from one or more impurity species.)
1. A method of purifying an antibody, the method comprising:
A) loading the antibody preparation in a loading buffer onto a Hydroxyapatite (HA) resin, wherein
The antibody formulation comprises: I) an intact antibody of interest and II) a trimmed form of the antibody of interest, wherein the trimmed form of the antibody of interest is a degradation product of the intact antibody of interest and has a mass that differs from the mass of the intact antibody of interest by less than 10%; and
B) eluting the intact antibody of interest from the HA resin with an elution buffer comprising ions, wherein the concentration of ions in the elution buffer increases during the elution.
2. A method of purifying a bispecific antibody, the method comprising:
A) loading the antibody preparation in a loading buffer onto a Hydroxyapatite (HA) resin, wherein:
the antibody formulation comprises: I) an intact bispecific antibody of interest; and II) at least one impurity species, wherein the impurity species is selected from: a) a trimmed form of a bispecific antibody of interest, wherein the trimmed form of the bispecific antibody of interest is a degradation product of the intact bispecific antibody of interest and has a mass that differs from the mass of the intact bispecific antibody of interest by less than 10%; b) a first parent antibody, wherein the first parent antibody is a monospecific antibody having the same antigen specificity as the first arm of the intact bispecific antibody; c) a second parent antibody, wherein the second parent antibody is a monospecific antibody having the same antigen specificity as the second arm of the intact bispecific antibody; and d) a high molecular mass material (HMMS); and
B) eluting the intact bispecific antibody of interest from the HA resin with an elution buffer comprising ions, wherein the concentration of ions in the elution buffer increases during elution.
3. A method of purifying a bispecific antibody, the method comprising:
A) loading the antibody preparation in a loading buffer onto a Hydroxyapatite (HA) resin, wherein:
I) the antibody formulation comprises: a) an intact bispecific antibody of interest and b) a trimmed form of the bispecific antibody of interest, wherein the trimmed form of the antibody of interest is a degradation product of the intact bispecific antibody of interest and has a mass that differs from the mass of the intact bispecific antibody of interest by less than 10%; and
II) the ratio of molecules of the pruned bispecific antibody to molecules of the intact bispecific antibody in the antibody preparation is between at least 1:50 and not more than 1: 5;
B) eluting the intact bispecific antibody from the HA resin with an elution buffer comprising ions, wherein the concentration of ions in the elution buffer increases during elution, and optionally,
C) collecting a purified fraction eluted from the HA resin, wherein the purified fraction comprises the intact bispecific antibody.
4. The method of claim 1, wherein the antibody is a heterodimeric bispecific antibody.
5. The method of claim 1 or 2, further comprising collecting a purified fraction eluted from the HA resin, wherein the purified fraction comprises the intact antibody of interest, and wherein the purified fraction comprises at least 95%, 96%, 97%, 98%, or 99% by mass of the intact antibody of interest.
6. The method of claim 3, wherein the purified fraction comprises the intact bispecific antibody and the trimmed bispecific antibody, further wherein the ratio of trimmed bispecific antibody molecules to intact bispecific antibody molecules in the purified fraction is no greater than 1: 100.
7. The method of claim l, wherein the antibody of interest is an anti-CD 3 antibody, and wherein the antibody comprises at least one of: i) comprises the amino acid sequence shown as SEQ ID NO: 1; ii) comprises the sequence as set forth in SEQ ID NO: 2; iii) comprises the sequence as set forth in SEQ ID NO: 1 and a VH region comprising an amino acid sequence as set forth in seq id NO: 3, VL region of an amino acid sequence set forth in seq id no; or iv) comprises the sequence set forth in SEQ ID NO: 2 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 4, or a light chain of the amino acid sequence shown in seq id no.
8. The method of any one of claims 2-6, wherein the bispecific antibody is: i) an anti-BCMA/anti-CD 3 bispecific antibody comprising an anti-BCMA arm and an anti-CD 3 arm, or ii) an anti-FLT 3/anti-CD 3 bispecific antibody comprising an anti-FLT 3 arm and an anti-CD 3 arm.
9. The method of claim 8, wherein the anti-CD 3 arm comprises at least one of: i) comprises the amino acid sequence shown as SEQ ID NO: 1; ii) comprises the sequence as set forth in SEQ ID NO: 2; iii) comprises the sequence as set forth in SEQ ID NO: 1 and a VH region comprising an amino acid sequence as set forth in SEQ ID NO: 3, VL region of an amino acid sequence set forth in seq id no; or iv) comprises the sequence set forth in SEQ ID NO: 2 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 4, or a light chain of the amino acid sequence shown in seq id no.
10. The method of claim 8 or 9, wherein the anti-BCMA arm comprises at least one of: i) comprises the amino acid sequence shown as SEQ ID NO: 5; ii) comprises the sequence as set forth in SEQ ID NO: 6; iii) comprises the sequence as set forth in SEQ ID NO: 5 and a VH region comprising the amino acid sequence shown in SEQ ID NO: 7, VL region of an amino acid sequence set forth in seq id no; or iv) comprises the sequence set forth in SEQ ID NO: 6 and the amino acid sequence shown in SEQ ID NO: 8, or a light chain of the amino acid sequence shown in seq id No. 8.
11. The method of claim 8 or 9, wherein the anti-FLT 3 arm comprises at least one of: i) comprises the amino acid sequence shown as SEQ ID NO: 9, VH region of the amino acid sequence shown in seq id no; ii) comprises the sequence as set forth in SEQ ID NO: 10; iii) comprises the sequence as set forth in SEQ ID NO: 9 and a VH region comprising the amino acid sequence shown in SEQ ID NO: 11, VL region of the amino acid sequence set forth in seq id no; or iv) comprises the sequence set forth in SEQ ID NO: 10 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 12, or a light chain of the amino acid sequence shown in seq id no.
12. The method of any one of claims 1-11 wherein the antibody preparation is loaded onto the HA resin such that the density on the resin is from 5g/L to 20 g/L.
13. The method of any one of claims 1-12 wherein at least 1 gram of antibody preparation is loaded onto the HA resin.
14. The method of any one of claims 1-13, wherein the antibody preparation comprises at least 50% but less than 95% by mass of intact antibody of interest.
15. The method of any one of claims 1-14, wherein the mass of the trimmed antibody differs from the mass of the intact antibody by less than 1%.
16. The method of any one of claims 1-15, wherein the mass of the trimmed antibody is about 5 to 100 daltons greater than the mass of the intact antibody.
17. The method of any one of claims 1-16, wherein said trimmed antibody has a cleaved peptide bond in the polypeptide chain of said antibody, and wherein said cleaved peptide bond is in the heavy chain of said antibody.
18. The method of any one of claims 1-17, wherein the trimmed antibody comprises the same number of amino acids and the same amino acid sequence as the intact antibody.
19. The method of any one of claims 1-17, wherein the trimmed antibody comprises a different number of amino acids than the intact antibody.
20. The method of any one of claims 1-19, wherein the antibody of interest comprises VH and VL domains that specifically bind CD3, and wherein the trimmed antibody comprises a cleaved peptide bond in the VH domain that specifically binds CD 3.
21. The method of any one of claims 1-20, wherein the HA resin is a ceramic hydroxyapatite (cHA) resin.
22. The method of any one of claims 1-21, wherein after loading the antibody preparation onto the HA resin but before eluting the intact bispecific antibody, the resin is washed with a wash buffer comprising phosphate ions at a concentration of 10-50 mM.
23. The method of any one of claims 1-22, wherein the ion in the elution buffer is phosphate.
24. The method of any one of claims 1-23, wherein the concentration of phosphate ions increases from about 40mM to 100mM during the elution.
25. The method of any one of claims 1-24, wherein the pH of at least one of the loading buffer, wash buffer, and elution buffer is about pH7.0 and 8.0 or between about pH7.0 and 8.0.
26. The method of any one of claims 1-25, wherein the antibody preparation comprises a protein that is preloaded onto and eluted from at least one of: i) protein a resin and ii) ion exchange resin.
27. The method of any one of claims 1-26, wherein the antibody is isolated and/or purified for use as a medicament or for use in the preparation of a medicament.
Drawings
Figure 1 depicts a schematic of a process for making a bispecific antibody that can be purified according to the methods provided herein.
Figure 2 depicts a schematic diagram illustrating i) an intact bispecific antibody (left panel) and ii) a trimmed version of the bispecific antibody (right panel), wherein the trimmed bispecific antibody is an impurity that may be present in an antibody preparation with the intact bispecific antibody.
Figure 3 depicts a chromatogram showing the isolation of an anti-BCMA/anti-CD 3 bispecific antibody ("POI") of interest from a variety of different impurities by elution from HA resin.
Figure 4 depicts a graph showing the relative amounts of different proteins (including the antibody of interest and various impurities) in different fractions eluted from the HA resin according to the HA chromatographic run as depicted in the chromatogram of figure 3.
Figure 5 depicts a chromatogram showing the isolation of an anti-BCMA/anti-CD 3 bispecific antibody ("POI") of interest from a variety of different impurities by elution from HA resin.
Figure 6 depicts a graph showing the relative amounts of different proteins (including the antibody of interest and various impurities) in different fractions eluted from an HA resin according to an HA chromatographic run as depicted in the chromatogram of figure 5, in which an anti-BCMA/anti-CD 3 bispecific antibody of interest was separated from a plurality of different impurities by elution from the HA resin.
Figure 7 depicts a graph showing the relative amounts of different proteins (including the antibody of interest and various impurities) in different fractions eluted from the HA resin according to HA chromatographic runs, where the anti-FLT 3/anti-CD 3 bispecific antibody of interest was separated from a variety of different impurities by elution from the HA resin.
Detailed Description
Provided herein are methods for purifying an antibody of interest from one or more impurities. The methods provided herein relate to the separation of antibodies of interest from impurities using hydroxyapatite resins. In some embodiments, the antibody of interest is a bispecific antibody. In some embodiments, the impurity is an antibody that is related to (i.e., has a similar or identical amino acid sequence to) the antibody of interest, but is altered in one or more ways compared to the antibody of interest, and is of a different quality than the antibody of interest. Optionally, the mass of antibody impurity species is very similar to the mass of the antibody of interest. For example, in some embodiments, the mass of antibody impurity species differs from the mass of the antibody of interest by less than 5%, 2%, 1%, 0.5%, 0.2%, 0.1%, 0.05%, 0.02%, or 0.01%. Optionally, the methods provided herein can be used for large scale purification of an antibody of interest from one or more impurities.
Definition of
Unless defined otherwise, all technical terms, symbols, and other scientific terms or terminology used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In some instances, terms having commonly understood meanings are defined herein for clarity and/or for ease of reference, and the inclusion of such definitions herein is not necessarily to be construed as representing a substantial difference from what is commonly understood in the art.
Unless otherwise indicated, the following terms are to be understood to have the following meanings:
an "antibody" is an immunoglobulin molecule capable of specifically binding a target (e.g., a carbohydrate, polynucleotide, lipid, polypeptide, etc.) through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, the term includes not only intact polyclonal or monoclonal antibodies, but also fragments thereof (e.g., Fab ', F (ab')2The term "antibody" includes monospecific, bispecific and multispecific antibodies including antibodies of any class, such as IgG, IgA or IgM (or subclasses thereof), and which need not be of any particular class depending on the antibody amino acid sequence of the constant region of its heavy chain, the immunoglobulins can be assigned to different classes there are five major classes of immunoglobulins, IgA, IgD, IgE, IgG and IgM, and several of which can be further sub-classified (isotype), e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and different IgA2, the constant regions of the heavy chains corresponding to immunoglobulins of different classes are respectively designated α, gamma and mu.
As used herein, the terms "heavy chain," "light chain," "variable region" or "variable domain," "framework region," "constant domain," and similar terms have their ordinary meaning in the immunological arts and refer to domains in naturally occurring immunoglobulins and corresponding domains of recombinant binding proteins (e.g., humanized antibodies, bispecific antibodies, single chain antibodies, chimeric antibodies, etc.). The basic building block of naturally occurring immunoglobulins is a tetramer of two light and two heavy chains, usually represented by a glycoprotein of about 150,000 Da. The amino-terminal (N-terminal) portion of each chain comprises a variable region of about 100-110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal (C-terminal) portion of each chain defines a constant region. Each light chain comprises a light chain variable domain (VL) and a light chain constant domain (CL). Each heavy chain comprises a heavy chain variable region (VH) and a heavy chain constant region having CH1, hinge, CH2 and CH3 domains. The variable regions of an IgG molecule comprise regions of high denaturation (called Complementarity Determining Regions (CDRs) comprising residues that are in contact with the antigen and non-CDR segments (called Framework Regions (FRs) which generally maintain structure and determine the positioning of the CDR loops (although certain framework residues may also be in contact with the antigen.) each VH and VL comprises three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following structure n-FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4-c. the immunoglobulin molecule can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) and class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass.
"bispecific" or "dual specificity" is a hybrid antibody having two different antigen-binding sites. The two antigen binding sites of a bispecific antibody bind two different epitopes, which may be located on the same or different protein targets.
By "intact" antibody is meant a recombinant antibody that comprises all of the peptide bonds and amino acids expected of a recombinant antibody (i.e., expected based on the nucleic acid sequence encoding the polypeptide of the antibody). Conversely, a "trimmed" antibody refers to a form of the corresponding "intact" antibody that lacks at least one peptide bond as compared to the corresponding "intact" antibody. Reference herein to "an antibody of interest" generally refers to an intact antibody of interest, unless the context clearly dictates otherwise.
Reference herein to "about" a value or parameter includes embodiments for the value or parameter itself as well as embodiments for values or parameters that may be 10% lower or 10% higher than the stated value for the parameter. For example, reference to "about 5 mg" includes 5mg and any value between 4.5mg and 5.5 mg.
Method of producing a composite material
The methods provided herein can be used to purify an antibody of interest from one or more impurities. In the methods provided herein, an antibody preparation comprising an antibody of interest and one or more impurity molecules (also referred to herein as a "starting sample") is loaded onto a Hydroxyapatite (HA) resin that binds the antibody of interest and optionally one or more impurity molecules. The HA resin is then washed to remove any loosely bound impurities. (in some embodiments, all impurity molecules may flow through but not bind to the HA resin.) next, the antibody of interest is eluted from the HA resin using a phosphate elution buffer, which is typically introduced onto the resin by an increasing phosphate ion concentration gradient. Elution of the antibody of interest from the HA resin yields a purified sample comprising the antibody of interest and fewer (or no) impurities than were present with the antibody of interest in the starting sample. Any impurity molecules that bind to the HA resin may also elute at a point during the increasing phosphate ion concentration gradient during elution of the antibody of interest from the HA resin. However, the impurity molecules elute from the HA resin under conditions that are significantly different from the elution conditions of the antibody of interest, so that the antibody of interest can be efficiently separated from the impurity molecules during the elution process. Additional details regarding the above-described method steps and related materials and steps are provided below.
Hydroxyapatite resin
Various hydroxyapatite resins are commercially available, and any useful form of material may be used with the methods provided herein. Optionally, the hydroxyapatite is in crystalline form. Optionally, the hydroxyapatite is agglomerated to form particles and sintered at high temperature into a stable porous ceramic mass.
In some embodiments, the HA resin provided herein is a ceramic hydroxyapatite (cHA) resin. "ceramic hydroxyapatite"/"cHA" means a compound of the formula Ca10(PO4)6(OH)2Which has been sintered at high temperatures to form a spherical macroporous ceramic form. Unless otherwise stated, all references to "a", "an", and "the" are intended to mean that the elements are not in any way limitingAs used herein, "ceramic hydroxyapatite"/"cHA" includes, but is not limited to, type I and type II ceramic hydroxyapatite, and also includes any suitable particle size. Typical cHA particle sizes that can be used with the methods provided herein include, for example, particle sizes between 1 to 100 μm or 1 to 1000 μm in diameter, e.g., 20 μm, 40 μm, or 80 μm. Exemplary cHA resins that can be used with the methods provided herein include CHTTMType I and type II resins (Bio-Rad). Any reference herein to "HA resin" or the like includes cHA resin.
Typically, in the methods provided herein, the HA resin is provided in one or more chromatography columns. Column properties (e.g., column diameter, length, and packing density) may be selected according to various factors, including the requirements of a particular purification project (i.e., the amount of protein intended to be purified) and factors related to the HA resin to be used in the column (e.g., its pore size, particle size, compressibility, loading capacity, and dynamic binding capacity). Further, the methods provided herein are generally related to the description of HA resins in chromatography columns; however, other suitable relevant configurations of the resin are not excluded. Further, reference herein to an "HA column" or the like refers to a chromatography column packed with HA resin.
Equilibration of HA columns prior to protein Loading
In some embodiments, prior to loading a sample comprising an antibody of interest onto an HA column, the methods provided herein can include the step of pre-equilibrating the column with one or more equilibration buffers. Equilibration buffer may be introduced onto the column, for example, to ensure that the HA resin is clean at the start of the process (i.e., to ensure that the resin is free of impurities that have bound to the resin) and/or to ensure that the solution surrounding the HA resin is compatible with the sample to be loaded onto the resin.
In some embodiments, the equilibration buffer is a phosphate buffer comprising, for example, sodium phosphate, wherein the concentration of phosphate ions in the buffer is about 100 to 500 mM. Such equilibration buffers may also be referred to herein as "high phosphate equilibration buffers" or similar terms. For example, in one embodiment, a high phosphate equilibration buffer may comprise about 250 to 450mM phosphate ions; in other embodiments, it may comprise about 200, 250, 300, 350, 400 or 450mM phosphate ion. The equilibration buffer contains a relatively high concentration of phosphate ions in the buffer to elute any contaminants/impurities already present on the HA resin (i.e., contaminants/impurities present prior to loading the sample containing the protein of interest onto the resin; such impurities may be present, for example, if the HA resin HAs been previously used in a purification process, and the resin HAs not been completely washed after previous use). The high phosphate equilibration buffer may have a pH of about 6.0 to 9.0. For example, in one embodiment, the high phosphate equilibration buffer may have a pH of about 7.0 to 8.0; in other embodiments, it may have a pH of about 7.0, 7.5, or 8.0. In one embodiment, the high phosphate equilibration buffer comprises about 400mM phosphate ions and has a pH of about 7.5. In some embodiments, the high phosphate equilibration buffer may also be referred to herein as "
In some embodiments, the equilibration buffer is a phosphate buffer comprising, for example, sodium phosphate, wherein the concentration of phosphate ions in the buffer is about 1 to 20 mM. Such equilibration buffers may also be referred to herein as "low phosphate equilibration buffers" or similar terms. For example, in one embodiment, the low phosphate equilibration buffer may comprise about 1 to 10mM phosphate ions; in other embodiments, it may comprise about 1, 2, 3, 4, 5, or 10mM phosphate ion. The equilibration buffer contains a relatively low concentration of phosphate ions in the buffer to create conditions around the HA resin that favor the binding of the protein of interest to the resin. Optionally, the low phosphate equilibration buffer may further comprise HEPES at a concentration of about 1 to 50 mM. For example, in one embodiment, the low phosphate equilibration buffer may comprise about 2 to 30mM HEPES; in other embodiments, it may comprise about 5, 10, 15, 20, or 25mM HEPES. The low phosphate equilibration buffer may have a pH of about 6.0 to 9.0. For example, in one embodiment, the low phosphate equilibration buffer may have a pH of about 7.0 to 8.0; in other embodiments, it may have a pH of about 7.0, 7.5, or 8.0. In one embodiment, the low phosphate equilibration buffer comprises about 2mM phosphate ions, 20mM HEPES, and has a pH of about 7.5. In some embodiments, the low phosphate equilibration buffer may also be referred to herein as "
Loading of HA columns with samples containing antibodies of interest
Once the HA column is ready to be loaded with protein, a sample containing the antibody of interest and impurities is loaded onto the HA column. The buffer in which the sample is loaded onto the HA column may be referred to herein as the "loading buffer". In some embodiments, when a sample comprising an antibody of interest is initially obtained for use with a method as provided herein, the sample is already in a suitable loading buffer for loading the sample onto an HA column. However, in other embodiments, prior to loading the sample onto the HA column, the sample may be treated (e.g., diluted, concentrated, or buffer exchanged) to change the buffer conditions of the sample so that the sample is in a suitable buffer for loading onto the column. For example, using the methods provided herein, the loading buffer cannot have a high concentration of phosphate ions, which would prevent the antibody of interest from binding to the HA resin. Thus, if the initial sample containing the antibody of interest contains a high concentration of phosphate ions, the sample needs to be subjected to (for example) dilution or buffer exchange until the concentration of phosphate ions in the sample is reduced to a suitably low concentration that allows the antibody of interest to bind to the HA resin.
In some embodiments, the loading buffer comprises no more than about 10mM phosphate ions. For example, in some embodiments, the loading buffer comprises less than about 10mM, 5mM, 4mM, 3mM, 2mM, or 1mM phosphate ions. In some embodiments, the loading buffer comprises 0mM phosphate ions. Optionally, the loading buffer may comprise various other salts or buffer components (e.g., Tris, glycine). The loading buffer may have a pH of about 6.0 to 9.0. For example, in one embodiment, the loading buffer may have a pH of about 7.0 to 8.0; in other embodiments, it may have a pH of about 7.0, 7.5, or 8.0.
In some embodiments, a sample comprising an antibody of interest can be loaded onto an HA resin such that the density on the resin is at least 1g/L, 2g/L, 3g/L, 4g/L, 5g/L, 6g/L, 7g/L, 8g/L, 9g/L, 10g/L, 12g/L, 15g/L, 20g/L, 25g/L, or 30 g/L. In some embodiments, a sample comprising an antibody of interest can be loaded onto an HA resin such that the density on the resin is at least 1g/L, 2g/L, 3g/L, 4g/L, 5g/L, 6g/L, 7g/L, 8g/L, 9g/L, 10g/L, 12g/L, 15g/L, 20g/L, or 25g/L to no greater than 2g/L, 3g/L, 4g/L, 5g/L, 6g/L, 7g/L, 8g/L, 9g/L, 10g/L, 12g/L, 15g/L, 20g/L, 25g/L, or 30g/L, wherein the second value is greater than the first value.
Washing HA column
After loading a sample comprising the antibody of interest and impurities onto the HA column, but prior to eluting the antibody of interest, the methods provided herein can optionally include another step of washing the loaded column with one or more wash buffers, e.g., to remove non-specifically immobilized impurities or to otherwise prepare or equilibrate the column for the elution step. The nature of any wash buffer can be determined by one of ordinary skill in the art. In one embodiment, the wash buffer is a phosphate buffer comprising, for example, sodium phosphate, and the concentration of phosphate ions in the buffer is about 5 to 50 mM. For example, in one embodiment, the wash buffer may comprise about 10 to 40mM phosphate ions; in other embodiments, it may comprise about 10, 20, 30, 40 or 50mM phosphate ion. Optionally, the wash buffer may further comprise HEPES at a concentration of about 1 to 50 mM. For example, in one embodiment, the wash buffer may comprise about 2 to 30mM HEPES; in other embodiments, it may comprise about 5, 10, 15, 20, or 25mM HEPES. The wash buffer may have a pH of about 6.0 to 9.0. For example, in one embodiment, the wash buffer may have a pH of about 7.0 to 8.0; in other embodiments, it may have a pH of about 7.0, 7.5, or 8.0. In one embodiment, the wash buffer comprises about 40mM phosphate ions, 20mM HEPES, and has a pH of about 7.5.
Elution of antibodies of interest from HA columns
The methods provided herein include the step of eluting bound antibody of interest from the HA resin. The bound antibody of interest is eluted by one or more elution buffers. Typically, the elution buffer comprises one or more salts or ions, and the concentration of the salt or ion increases during elution.
In some embodiments, the elution buffers provided herein comprise phosphate ions. Optionally, the concentration of phosphate ions in the elution buffer is increased from an initial concentration of about 20mM to about 200mM during elution. For example, in one embodiment, the concentration of phosphate ions in the elution buffer is increased from an initial concentration of about 40mM to about 80mM or from an initial concentration of about 40mM to about 100mM during elution. The particular method and rate of increasing the concentration of phosphate ions in the elution buffer may be determined to be appropriate for the antibody of interest, and taking into account the type of impurity molecules that also bind to the HA resin. For example, the concentration of phosphate ions in the elution buffer may increase in a gradual/shallow linear gradient. The use of a shallow gradient may allow for efficient separation of one or more molecules that elute from the HA resin under similar but different conditions. Alternatively, in some embodiments, the concentration of phosphate ions in the elution buffer may be increased in a steep gradient, or it may be increased stepwise. Optionally, the elution buffer may further comprise HEPES at a concentration of about 1 to 50 mM. For example, in one embodiment, the elution buffer may comprise about 2 to 30 mh epes; in other embodiments, it may comprise about 5, 10, 15, 20, or 25mM HEPES. The elution buffer may have a pH of about 6.0 to 9.0. For example, in one embodiment, the elution buffer may have a pH of about 7.0 to 8.0; in other embodiments, it may have a pH of about 7.0, 7.5, or 8.0. In one embodiment, the elution buffer comprises about 40-80mM phosphate ions (increasing on the gradient), 20mM HEPES, and has a pH of about 7.5.
Elution conditions include, but are not limited to, the properties of the elution buffer (e.g., buffer composition, pH, concentration, ionic strength, and the like) suitable for use with the HA resin; any necessary step or gradient change in the properties of the elution buffer can be determined; the number of column volumes of elution buffer to be used; flow rate and the like to optimize elution of the antibody of interest from the HA column, and separation of the antibody of interest from impurity molecules.
After elution, one or more peak fractions containing the antibody of interest are optionally collected separately or separately and optionally combined, optionally pH adjusted, optionally filtered and then optionally stored prior to additional processing as needed. The peak fraction collected can be identified by any suitable method, for example using uv light at a280 and collection is initiated when the uv signal is above a desired amount and/or when at a desired point in the elution conditions.
The material that elutes from the HA resin and contains the antibody of interest may optionally be referred to herein as a "purified fraction" or similar term. The purified fraction may comprise material from a single fraction eluted from the HA resin, or it may be a combination of multiple fractions eluted from the HA resin that have been pooled together. Typically, purified fractions are prepared so that an equilibrium is made between collecting a large amount of the antibody of interest and a small amount of impurity molecules. Such competing targets must often be balanced, for example, because there may be at least partial overlap between conditions when the antibody of interest is eluted from the HA resin, and when one impurity molecular species is eluted from the HA resin.
Any buffer used in the methods provided herein (e.g., equilibration, loading, washing, or elution buffers) may also comprise additional or alternative suitable components, such as acetate, succinate, MES, ACES, MOPSO, PIPES, BES, TAPSO, AMPSO, TRICINE, EPPS, Bicine, DIPSO, HEPPSO, imidazole, Tris, Bis-Tris, TAPS, arginine, glycine, acetonitrile, ethanol, methanol, 1% sodium lauryl sulfate (SDS), or other surfactants, and the like.
In some embodiments, any buffer provided herein can have a pH of about 6.0 to 9.0. In other embodiments, any buffer provided herein can have a pH of about 5.0 to 9.0, 5.5 to 9.0, 6.5 to 9.0, 7.0 to 9.0, 7.5 to 9.0, 7.0 to 8.0, or 6.5 to 8.5.
In buffers provided herein that are described as comprising "phosphate ions," the phosphate ions can be generated in the buffer from any suitable phosphate (e.g., sodium or potassium phosphate). In addition, solutions provided herein that are described as being prepared with "sodium phosphate" can be prepared with any suitable sodium phosphate salt (e.g., sodium dihydrogen phosphate or disodium hydrogen phosphate).
After the antibody of interest HAs been eluted from the HA column, the HA column is optionally washed to remove impurities and degrade other components of the column resin and is ready for subsequent storage for use. In one embodiment, the column is first regenerated using a buffer (e.g., a buffer comprising sodium phosphate at a concentration of about 0.4M and a pH of about 7.5); an optional disinfection step is then performed using a cleaning solution (e.g., about 1M NaOH and about 0.5M potassium phosphate), and then a storage solution (e.g., about 0.1M NaOH) is used to prepare for storage.
Antibodies
The methods provided herein can be used to purify an antibody of interest from one or more impurities. For example, the purified antibody may be used as a medicament or in the manufacture of a medicament.
In some embodiments, an antibody purified according to the methods provided herein is any type of antibody provided herein. For example, an antibody purified according to the methods provided herein can be a full-length antibody or an antibody fragment (e.g., scFv or Fab), and it can be monospecific or bispecific. Typically, the antibody of interest purified according to the methods provided herein is a recombinant antibody.
IgG antibodies
In some embodiments, the antibody purified according to the methods provided herein is an immunoglobulin g (igg) antibody. As known in the art, IgG antibodies comprise two heavy chains and two light chains, and have a common "Y" shape. In a standard IgG molecule, the two heavy chains have the same amino acid sequence and the two light chains have the same amino acid sequence. IgG antibodies can be described as having two "arms" (i.e., "first arm" and "second arm"), wherein each arm comprises one heavy chain and one light chain linked together by a disulfide bond. In a standard IgG molecule, the first arm of the antibody is identical to the second arm of the antibody (due to each arm comprising a heavy chain and a light chain having the same amino acid sequence as the heavy chain and the light chain, respectively, in the other arm). The N-terminal region of the heavy chain comprises the heavy chain variable region (VH) and the N-terminal region of the light chain comprises the light chain variable region (VL). The VH and VL regions comprise portions of the antibody that specifically bind antigen. Thus, each arm of an IgG antibody can specifically bind to an antigen. In a standard IgG molecule, both the first and second arms of an IgG antibody bind to the same antigen (due to the fact that both arms comprise heavy and light chains with identical respective amino acid sequences). Based on having the same 2 arms, a standard IgG antibody can be referred to as "homodimerization". The IgG antibody purified according to the methods provided herein can be of subclass IgG1, IgG2, IgG3, or
Bispecific IgG antibodies
In some embodiments, the antibody purified according to the methods provided herein is a bispecific IgG antibody. In bispecific IgG antibodies, each of the two arms of the antibody specifically binds a different antigen. In addition, the amino acid sequence of the heavy chain in the first arm of a bispecific IgG antibody differs from the amino acid sequence of the heavy chain in the second arm of the same bispecific IgG antibody, and similarly, the amino acid sequence of the light chain in the first arm of a bispecific IgG antibody typically differs from the amino acid sequence of the light chain in the second arm of the same bispecific IgG antibody. Thus, based on having different 2 arms, bispecific IgG antibodies can be referred to as "heterodimeric". The first arm of the bispecific IgG antibody can be described as specific for a "first antigen", and the second arm of the bispecific IgG antibody can be described as specific for a "second antigen". In some embodiments, the bispecific antibody has an IgG1, IgG2, IgG3, or IgG4 isotype. In some embodiments, the bispecific antibody comprises an immunologically inert Fc region.
Bispecific IgG antibody-preparation method
Methods for making bispecific antibodies are known in the art (see, e.g., Suresh et al, methods in Enzymology 121:210,1986). Traditionally, recombinantly produced bispecific antibodies have been based on the co-expression of two immunoglobulin heavy-light chain pairs, where the two heavy chains have different specificities (Millstein and Cuello, Nature 305,537-539, 1983).
Recently, methods have been developed to prepare bispecific heterodimeric antibodies taking the following general steps:
1) the first homodimeric antibody (also referred to herein as the "first parent antibody") and the second homodimeric antibody (also referred to herein as the "second parent antibody") were expressed separately and purified. The first homodimeric antibody is specific for a first target antigen of the bispecific antibody prepared, and the second homodimeric antibody is specific for a second target antigen of the bispecific antibody prepared. Thus, for example, if the goal is to make bispecific antibodies specific for BCMA and CD3, monoclonal anti-BCMA antibody ("primary parent antibody") and monoclonal anti-CD 3 antibody ("secondary parent antibody") are expressed separately and purified.
2) Next, the purified first homodimer/parent antibody and the purified second homodimer/parent antibody are mixed and incubated together under conditions that promote antibody arm exchange such that a heterodimeric bispecific antibody is formed comprising a first arm from the first parent antibody and a second arm from the second parent antibody. Such conditions typically involve a series of reducing conditions followed by oxidizing conditions. The reducing conditions promote cleavage of disulfide bonds linking the two heavy chains of the homodimeric antibody together and thereby allow antibody arm exchange between the first and second parent antibodies. Subsequent oxidation conditions then form new disulfide bridges that stabilize the newly formed bispecific antibody. Such a general method for generating bispecific antibodies is outlined in figure 1. In fig. 1, a first parent antibody ("parent antibody a"; grey) and a second parent antibody ("parent antibody B"; black) are depicted, each being a monospecific homodimer, and comprising a first arm and a second arm. Typically, the first parent antibody and the second parent antibody are specific for different antigens. The first and second parent antibodies are then mixed together and exposed to reduction and oxidation steps, which result in the formation of a bispecific antibody of interest comprising the respective specificities of the first arm from parent antibody a and the second and both arms from parent antibody B.
Optionally, the amino acid sequence of the antibody heavy chain may be modified in one or more ways to facilitate formation of a bispecific antibody. For example, the heavy chain of one arm of the bispecific antibody may comprise an amino acid modification in the first dumpling region such that the substituted/substituted amino acids in this first dumpling region have an opposite charge to the corresponding amino acids in the dumpling region of the other arm of the bispecific antibody formed. This is described, for example, in international patent application No. PCT/US2011/036419(WO 2011/143545). In another approach, formation of a desired heteromultimeric or heterodimeric protein (e.g., a bispecific antibody) is enhanced by altering or engineering the interface between the first and second immunoglobulin-like Fc regions (e.g., the dumpling chain region and/or the CH3 region). In this method, the bispecific antibody can comprise a CH3 region, wherein the CH3 region comprises a first CH3 polypeptide and a second CH3 polypeptide that interact together to form a CH3 interface, wherein one or more amino acids within the CH3 interface destabilizes and are not electrostatically detrimental to homodimer formation. This process is also described in international patent application No. PCT/US2011/036419(WO 2011/143545).
The above and other methods for making bispecific antibodies are further described in (for example): international patent application Nos. PCT/IB2011/054899(WO2012/059882), PCT/US2011/036419(WO2011/143545) and Giese et al, Biotechnology Progress, "Bispecific Antibody Process Development: assemblyand Purification of Knob and Hole Bispecific Antibodies ", 17Jan 2018 and the references cited therein, each of which is incorporated herein by reference for all purposes. The methods provided herein for purifying antibodies can be used to purify bispecific antibodies prepared by any suitable method.
Bispecific IgG antibody-specificity
In some embodiments, an antibody that can be purified according to the methods provided herein is a full length human bispecific IgG antibody, wherein a first antibody variable domain of a first arm of the bispecific antibody is capable of binding a first antigen and a second antibody variable domain of a second arm of the bispecific antibody is capable of binding a second antigen. The first antigen and the second antigen may have any of the characteristics of the antigens as described herein. In some embodiments, the first antigen is present on a first cell type and the second antigen is present on a second cell type.
In some embodiments, an antibody that can be purified according to the methods provided herein is a full-length human bispecific IgG antibody, wherein a first antibody variable domain of the antibody is capable of recruiting the activity of human immune effector cells by specifically binding to an effector antigen located on the human immune effector cells, and wherein a second antibody variable domain of the antibody is capable of specifically binding to a target antigen.
The human immune effector cells that can be bound by the antibodies provided herein can be any of a variety of immune effector cells known in the art. For example, the immune effector cell may be a member of the human lymphocyte lineage, including, but not limited to, T cells (e.g., cytotoxic T cells), B cells, and Natural Killer (NK) cells. Immune effector cells may also be, for example, members of the human myeloid lineage, including, but not limited to, monocytes, neutrophils, and dendritic cells. Such immune effector cells may have a cytotoxic or apoptotic effect on target cells or have other desired effects upon activation by binding of effector antigens. The effector antigen is an antigen (e.g., a protein or polypeptide) expressed on human immune effector cells. Examples of effector antigens that can be bound by the antibodies provided herein include, but are not limited to, human CD3 (or CD3 (cluster of differentiation) complex), CD16, NKG2D, NKp46, CD2, CD28, CD25, CD64, and CD 89.
The target antigen is expressed on a target cell in a diseased condition (e.g., an inflammatory disease, a proliferative disease (e.g., cancer), an immune disease, a neurological disease, a neurodegenerative disease, an autoimmune disease, an infectious disease (e.g., a viral infection or a parasitic infection), an allergic reaction, a graft-versus-host disease, or a host-versus-graft disease). The target antigen is not an effector antigen. Examples of target antigens include, but are not limited to, BCMA, EpCAM (epithelial cell adhesion molecule), CCR5 (chemokine receptor type 5), CD19, HER (human epidermal growth factor receptor) -2/neu, HER-3, HER-4, EGFR (epidermal growth factor receptor), FLT3 (Fms-like tyrosine kinase 3), PSMA, CEA, MUC-1 (mucin), MUC2, MUC3, MUC4, MUC5AC, MUC5B, MUC7, CIhCG, Lewis-Y, CD20, CD33, CD30, ganglioside GD3, 9-O-acetyl-GD 3, GM2, Globoh, glycosyl GM 8, Poly SA, GD2, carbonic anhydrase IX (MN/CA IX), CD44v6, Shh (sonic Hedge) 1, plasma-1, plasma cell antigen, (MCAP-membrane bound) antigen, fucoidan-derived protein precursor IgE, TNF alpha-TNF 27, TNF alpha-TNF 8, TNF- α -glycoprotein antigen, TNF- α -glycoprotein precursor 8, and fragment 27, PSCA (prostate stem cell antigen), Ly-6; desmocollin 4, E-cadherin neo-epitopes, fetal acetylcholine receptors, CD25, CA19-9 marker, CA-125 marker and type II MIS (Muellian inhibitory substance) receptors, sTn (sialyl Tn antigen; TAG-72), FAP (fibroblast activation antigen), endosialin (endosialin), EGFRvIII, LG, SAS and CD 63.
In some embodiments, an antibody purified according to the methods provided herein can be any antibody as described in U.S. application No. 15/085,644 (publication No. US20160297885) filed 3/30/2016 or U.S. application No. 15/993,874 (publication No. US20180346601) filed 5/31/2018, the entire contents of which are incorporated herein by reference for all purposes.
In some embodiments, an antibody purified according to the methods provided herein can be a bispecific IgG antibody, wherein one arm of the antibody specifically binds cluster of differentiation 3(CD 3). Information about CD3 is provided by, for example, UniProtKB ID # P07766.
In some embodiments, in a bispecific IgG antibody, one of its arms specifically binds CD3, the VH region of the heavy chain of the CD3 binding arm has an amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYYMTWVRQAPGKGLEWVAFIRNRARGYTSDHNPSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDRPSYYVLDYWGQGTTVTVSS (SEQ ID NO: 1) comprising the following amino acid sequence. In some embodiments, in a bispecific IgG antibody, one arm of which specifically binds CD3, the heavy chain of the CD3 binding arm has an amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYYMTWVRQAPGKGLEWVAFIRNRARGYTSDHNPSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDRPSYYVLDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCRVRCPRCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSRLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 2) comprising the following amino acid sequence. In some embodiments, in a bispecific IgG antibody with one arm that specifically binds CD3, the VH region of the heavy chain of the CD3 binding arm has a heavy chain comprising SEQ ID NO: 1, CDR1, CDR2, and CDR3 of the VH sequence shown in fig. 1.
In some embodiments, in a bispecific IgG antibody, one of its arms specifically binds CD3, the VL region of the light chain of CD 3-binding arm has an amino acid sequence of DIVMTQSPDSLAVSLGERATINCKSSQSLFNVRSRKNYLAWYQQKPGQPPKLLISWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSYDLFTFGSGTKLEIK (SEQ ID NO: 3) comprising the following amino acid sequence. In some embodiments, in a bispecific IgG antibody, one arm of which specifically binds CD3, the light chain of the CD 3-binding arm has an amino acid sequence of DIVMTQSPDSLAVSLGERATINCKSSQSLFNVRSRKNYLAWYQQKPGQPPKLLISWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSYDLFTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 4) comprising the following amino acid sequence. In some embodiments, in a bispecific IgG antibody with one arm that specifically binds CD3, the VL region of the light chain of CD 3-binding arm has a heavy chain comprising seq id NO: 3, CDR1, CDR2, and CDR3 of the VL sequence shown in fig. 3.
In some embodiments, in a bispecific IgG antibody with one arm that specifically binds CD3, the VH region of the heavy chain of CD 3-binding arm has a heavy chain comprising SEQ ID NO: 1, the VL region of the light chain of the CD 3-binding arm has an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 3, or a pharmaceutically acceptable salt thereof. In some embodiments, in a bispecific IgG antibody with one arm specifically binding to CD3, the heavy chain of the CD 3-binding arm has an amino acid sequence comprising SEQ ID NO: 2, the light chain of the CD 3-binding arm has an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 4, or a pharmaceutically acceptable salt thereof. In some embodiments, in a bispecific IgG antibody with one arm that specifically binds CD3, the VH region of the heavy chain of CD 3-binding arm has a heavy chain comprising SEQ ID NO: 1, the amino acid sequences of CDR1, CDR2 and CDR3 of the VH sequence shown in fig. 1, the VL region of the light chain of CD 3-binding arm has an amino acid sequence comprising SEQ ID NO: 3, CDR1, CDR2, and CDR3 of the VL sequence shown in fig. 3.
In some embodiments, an antibody purified according to the methods provided herein can be a bispecific IgG antibody, wherein one arm of the antibody specifically binds a B-cell mutant antigen (BCMA). Information about BCMA is provided by, for example, UniProtKB ID # Q02223.
In some embodiments, in a bispecific IgG antibody that specifically binds BCMA in one of its arms, the VH region of the heavy chain of the BCMA-binding arm has an amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYPMSWVRQAPGKGLEWVSAIGGSGGSLPYADIVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYWPMDIWGQGTLVTVSS (SEQ ID NO: 5) comprising the following amino acid sequence. In some embodiments, in a bispecific IgG antibody that specifically binds BCMA in one of its arms, the heavy chain of the BCMA binding arm has an amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYPMSWVRQAPGKGLEWVSAIGGSGGSLPYADIVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYWPMDIWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCEVECPECPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCEVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 6) comprising the following amino acid sequence. In some embodiments, in a bispecific IgG antibody with one arm that specifically binds BCMA, the VH region of the heavy chain of the BCMA-binding arm has a heavy chain comprising SEQ ID NO: 5, CDR1, CDR2, and CDR3 of the VH sequence shown in fig. 5.
In some embodiments, in a bispecific IgG antibody that specifically binds BCMA in one of its arms, the VL region of the light chain of the BCMA-binding arm has amino acid sequence EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLMYDASIRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYQSWPLTFGQGTKVEIK (SEQ ID NO: 7) comprising the following amino acid sequence. In some embodiments, in a bispecific IgG antibody that specifically binds BCMA in one of its arms, the light chain of the BCMA-binding arm has an amino acid sequence EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLMYDASIRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYQSWPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 8) comprising the following amino acid sequence. In some embodiments, in a bispecific IgG antibody that specifically binds BCMA in one of its arms, the VL region of the light chain of the BCMA-binding arm has a heavy chain comprising seq id NO: 7, CDR1, CDR2, and CDR3 of the VL sequence shown in fig. 7.
In some embodiments, in a bispecific IgG antibody with one arm that specifically binds BCMA, the VH region of the heavy chain of the BCMA-binding arm has a heavy chain comprising SEQ ID NO: 5, the VL region of the light chain of the BCMA-binding arm has an amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 7, or a pharmaceutically acceptable salt thereof. In some embodiments, in a bispecific IgG antibody that specifically binds BCMA in one of its arms, the heavy chain of the BCMA-binding arm has a heavy chain comprising SEQ ID NO: 6, and the light chain of the BCMA-binding arm has an amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 8, or a pharmaceutically acceptable salt thereof. In some embodiments, in a bispecific IgG antibody with one arm that specifically binds BCMA, the VH region of the heavy chain of the BCMA-binding arm has a heavy chain comprising SEQ ID NO: 5, the VL region of the light chain of the BCMA-binding arm has an amino acid sequence comprising the CDR1, CDR2 and CDR3 of the VH sequence shown in SEQ ID NO: 7, CDR1, CDR2, and CDR3 of the VL sequence shown in fig. 7.
In some embodiments, in a bispecific IgG antibody, one arm of which specifically binds BCMA, the VH region of the heavy chain of the BCMA-binding arm has the amino acid sequence evqllesgglvqpggslrlscaasgftfssmswvrwvrqapgkglewvsaiggsggslpyadsvkgrgrtvsrtnskntlyqmnslraedtyycarywwpmdqgtlvtvss (SEQ ID NO: 13) comprising the following amino acid sequence. In some embodiments, in a bispecific IgG antibody in which one arm specifically binds BCMA, the VL region of the light chain of the BCMA-binding arm has an amino acid sequence comprising the amino acid sequence of: EIVLTQSPGTLSLSPGERATLSCRASQSVSSTYLAWYQQKPGQ APRLLMYDASIRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYY CQQYQEWPLTFGQGTKVEIK (SEQ ID NO: 14). In some embodiments, any reference herein to an antibody comprises a polypeptide having an amino acid sequence comprising SEQ ID NO: 5, the antibody may alternatively comprise a VH region comprising the amino acid sequence of the amino acid sequence shown in SEQ ID NO: 13, or a VH region of the amino acid sequence shown in figure 13. In some embodiments, any reference herein to an antibody comprises a polypeptide having an amino acid sequence comprising SEQ ID NO: 7, the antibody may alternatively comprise a VL region comprising the amino acid sequence of the amino acid sequence set forth in SEQ ID NO: 14, VL region of the amino acid sequence set forth in seq id no. Similarly, also included herein are nucleic acids comprising SEQ ID NOs: 13 and SEQ ID NO: 14, and anti-BCMA heavy and light chains of VH and VL sequences.
In some embodiments, an antibody purified according to the methods provided herein can be a bispecific IgG antibody, wherein one arm of the antibody specifically binds fms-like tyrosine kinase 3(FLT 3). Information about FLT3 is provided by, for example, UniProtKB ID # P36888.
In some embodiments, in a bispecific IgG antibody, one arm of which specifically binds FLT3, the VH region of the heavy chain of FLT 3-binding arm has an amino acid sequence of EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVSAISGGGRSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLSPSDVGWGYGFDIWGQGTLVTVSS (SEQ ID NO: 9) comprising the following amino acid sequence. In some embodiments, in a bispecific IgG antibody with one arm specifically binding FLT3, the heavy chain of the FLT 3-binding arm has an amino acid sequence comprising the amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVSAISGGGRSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLSPSDVGWGYGFDIWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCEVECPECPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCEVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 10). In some embodiments, in a bispecific IgG antibody with one arm specifically binding FLT3, the VH region of the heavy chain of FLT 3-binding arm has a heavy chain comprising SEQ ID NO: 9, CDR1, CDR2, and CDR3 of the VH sequence shown in fig. 9.
In some embodiments, in a bispecific IgG antibody, one arm of which specifically binds FLT3, the VL region of the light chain of FLT 3-binding arm has an amino acid sequence of EIVLTQSPATLSLSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYDTFTRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQYGSSPPTFGQGTRLEIK (SEQ ID NO: 11) comprising the following amino acid sequence. In some embodiments, in a bispecific IgG antibody, one of its arms specifically binds FLT3, the light chain of the FLT 3-binding arm has an amino acid sequence of EIVLTQSPATLSLSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYDTFTRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQYGSSPPTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 12) comprising the following amino acid sequence. In some embodiments, in a bispecific IgG antibody with one arm that specifically binds FLT3, the VL region of the light chain of FLT 3-binding arm has a heavy chain comprising seq id NO: 11, CDR1, CDR2, and CDR3 of the VL sequence shown in fig. 11.
In some embodiments, in a bispecific IgG antibody with one arm specifically binding FLT3, the VH region of the heavy chain of FLT 3-binding arm has a heavy chain comprising SEQ ID NO: 9, the VL region of the light chain of FLT 3-binding arm has an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 11, or a pharmaceutically acceptable salt thereof. In some embodiments, a bispecific IgG antibody that specifically binds FLT3 in one of its arms, the heavy chain of FLT 3-binding arm has an amino acid sequence comprising SEQ ID NO: 10, the light chain of the FLT 3-binding arm has an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, in a bispecific IgG antibody with one arm specifically binding FLT3, the VH region of the heavy chain of FLT 3-binding arm has a heavy chain comprising SEQ ID NO: 9, the VL region of the light chain of FLT 3-binding arm has an amino acid sequence comprising the CDR1, CDR2 and CDR3 of the VH sequence shown in SEQ ID NO: 11, CDR1, CDR2, and CDR3 of the VL sequence shown in fig. 11.
In some embodiments, provided herein are bispecific anti-BCMA/
Also provided herein are methods of purifying monospecific antibodies having affinity for any of the above antigens and/or comprising any of the amino acid sequences described above. For example, also provided herein are nucleic acid molecules comprising SEQ ID NOs: 1, monospecific, homodimeric anti-CD 3 antibody.
Impurities
The methods provided herein can be used to purify an antibody of interest from one or more impurities.
Impurities include, for example, trimmed forms of the antibody of interest, protein aggregates, and, in the case of bispecific antibodies of interest, parent monospecific antibodies involved in the formation of bispecific antibodies of interest. Such different impurities may also be referred to herein as different "impurity species," "impurity molecules," or similar terms.
Trimmed forms of the antibodies of interest
"trimmed form of an antibody of interest," "trimmed antibody," or similar terms refer to a recombinant antibody in which one or more polypeptide bonds in the antibody have been cleaved as compared to the corresponding intact antibody of interest. Conversely, an "intact" antibody refers to a recombinant antibody that comprises all of the peptide bonds and amino acids expected of the recombinant antibody (i.e., expected based on the nucleic acid sequence encoding the polypeptide of the antibody).
Thus, the trimmed antibody can be considered a degradation product associated with the antibody of interest. Cleavage of a peptide bond in an antibody can occur, for example, by enzymatic (e.g., protease-mediated) or non-enzymatic activity.
In some embodiments, when a peptide bond in an antibody polypeptide is cleaved, the cleaved portion of the polypeptide chain may no longer be covalently linked to the remainder of the antibody after cleavage; in this case, the cleaved portion of the polypeptide chain can be dissociated from the remainder of the antibody. This most commonly occurs when cleavage of a peptide bond located near the N or C terminus of an alpha polypeptide chain occurs, and it results in a trimmed antibody that has lost one or more amino acids compared to the corresponding intact antibody. Such a trimmed antibody has less mass than the corresponding intact antibody due to the loss of one or more amino acids in the antibody.
Alternatively, in some other embodiments, when a peptide bond in an antibody polypeptide is cleaved, the cleaved portion of the polypeptide chain may still remain covalently linked to the remainder of the antibody after cleavage (e.g., via an intra-or inter-chain disulfide bond). In this case, even if the peptide bond of the antibody is cleaved, the cleaved portion of the polypeptide chain will remain tethered to the remainder of the antibody by the remaining intact covalent bonds linking the cleaved portion of the polypeptide chain to the remainder of the antibody. In this case, the trimmed antibody will still have the same number of amino acids and amino acid sequence as compared to the intact antibody. In addition, in at least some embodiments, this type of trimmed antibody may have a slightly greater mass than the corresponding intact antibody. Such mass increase may be the result of one or more chemical reactions that occur, for example, upon cleavage of a peptide bond. In such reactions, one or more atoms (e.g., H, O) can react with an atom of an antibody polypeptide chain and covalently attach to the antibody chain, which results in an increase in the mass of the trimmed antibody compared to the corresponding intact antibody.
Since a "trimmed" antibody is produced from a corresponding "intact" antibody, the "trimmed" form of the antibody has the same amino acid sequence (in the case where no amino acids are lost in the antibody via cleavage of a peptide bond) or nearly the same amino acid sequence (in the case where one or more amino acid sequences are lost in the antibody via cleavage of a peptide bond) as the corresponding "intact" form of the antibody.
Typically, the trimmed form of the antibody has a mass similar to that of the corresponding intact antibody. As described above, in some embodiments, a trimmed antibody may have a mass that is less than a corresponding intact antibody (e.g., in cases where the trimming results in the antibody losing one or more amino acids). Alternatively, in some embodiments, the trimmed antibody may have a mass greater than the corresponding intact antibody (in cases where the trimming does not result in the antibody losing any amino acids, but instead results in the antibody gaining at least one atom via one or more reactions that occur as a result of cleavage of a peptide bond).
In some embodiments, the mass of the trimmed form of the antibody differs from the mass of the corresponding intact antibody of interest by no more than 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%. In other words, in some embodiments, the mass of the trimmed form of the antibody of interest differs by no more than 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.05%, 0.04%, 0.03%, 0.02%, or 0.01% from the mass of the corresponding intact antibody of interest.
As described above, in some embodiments, the trimmed form of the antibody of interest has a mass that is less than the corresponding intact antibody of interest. For example, in some embodiments, the mass of a trimmed form of an antibody is no more than 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01% less than the mass of the corresponding intact antibody of interest. In other words, if the mass of the trimmed form of the antibody is no more than 10% less than the mass of the corresponding intact antibody of interest, then for example if the mass of the intact antibody of interest is 100,000Da, the mass of the trimmed form of the antibody is no more than 10,000Da less than 100,000Da (10% of 100,000 is 10,000) — i.e. it has a mass between 90,000Da and 100,000 Da.
As also described above, in some embodiments, the trimmed form of the antibody of interest has a mass greater than the corresponding intact antibody of interest. For example, in some embodiments, the mass of the trimmed form of the antibody is no more than 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.05%, 0.04%, 0.03%, 0.02%, or 0.01% greater than the mass of the corresponding intact antibody of interest. In other words, if the mass of the trimmed form of the antibody is no more than 1% greater than the mass of the corresponding intact antibody of interest, then for example if the mass of the intact antibody of interest is 100,000Da, the mass of the trimmed form of the antibody is no more than 1,000Da greater than 100,000Da (1% of 100,000 is 1,000) -i.e. it has a mass between 100,000Da and 101,000 Da.
High Molecular Mass Species (HMMS)/protein aggregates
In some embodiments, the impurities associated with the methods provided herein are referred to as "high molecular mass materials" (HMMS). HMMS refers to any high molecular mass contaminant or impurity, but is typically an association of at least two proteins that form an aggregate. For example, HMMS may comprise multiple molecules of an antibody of interest that have been aggregated together and/or aggregates of proteins from a host cell used to produce the antibody of interest. Aggregates can be produced by any process, including, for example, covalent or non-covalent attachment of molecules.
Parent antibody
In some embodiments, the impurity associated with the methods provided herein is a "parent antibody" or similar term. This type of impurity molecule is relevant to the methods provided herein, wherein the antibody of interest is a bispecific antibody produced from two different parent antibodies, e.g., as outlined in fig. 1. Such parent antibodies are monospecific, homodimers. Due to a variety of possible mechanisms, parent antibodies can be present in antibody preparations with bispecific antibodies of interest, for example: i) in some cases, some parent antibody molecules do not separate into first and second arms during the reduction step to separate the parent antibody into separate first and second arms (and thus, the antibody remains as a monospecific homodimer); or ii) in some cases, isolated first and second arms from the same type of parent antibody are linked together such that they form a monospecific homodimer (rather than participating in the formation of heterodimeric bispecific antibodies). As used herein, "parent antibody" refers to a homodimeric molecule produced by any of the above mechanisms. In addition, an antibody preparation as provided herein may comprise parent antibodies from one or both parent species as impurities.
Purification of antibodies of interest from impurities
Provided herein are methods for purifying an antibody of interest from one or more impurities.
In some embodiments, in the methods provided herein, the antibody of interest is in an antibody preparation (also referred to herein as a "starting sample") comprising the antibody of interest and one or more impurity molecular species. In this starting material for the methods as provided herein, the antibody of interest can comprise, for example, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% (by mass) of the protein in the antibody preparation. Then, in some embodiments of the methods provided herein, the purified fraction (also referred to herein as "purified sample") is collected as the eluate from the HA resin. The purified fraction comprises the antibody of interest and, in some embodiments, still comprises one or more impurity molecules. In some embodiments, in a purified fraction provided herein, an antibody of interest can comprise, for example, at least about 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% (by mass) of the protein in the purified fraction.
In some embodiments, in the methods provided herein, the starting sample is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% by mass of the antibody of interest, and the subsequent purified sample in the same method is at least about 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% by mass of the antibody of interest, wherein the second value is greater than the first value.
In some embodiments, in the methods provided herein, the starting sample comprises at least about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, or 25% by mass of a trimmed form of the antibody of interest. In some embodiments, in the methods provided herein, the purified sample comprises no more than about 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, or 20% by mass of a trimmed form of the antibody of interest. In some embodiments, in the methods provided herein, a starting sample comprises at least about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, or 25% by mass of a trimmed form of the antibody of interest, and subsequent purified samples in the same method comprise no more than about 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, or 20% by mass of a trimmed form of the antibody of interest, wherein the second value is less than the first value.
In some embodiments, in the methods provided herein, the starting sample comprises an intact antibody of interest and a trimmed form of the antibody of interest, wherein the ratio of trimmed antibody to intact antibody is at least about 1:100, 1:50, 1:25, 1:20, 1:10, 1:5, 1:4, or 1: 3. In some embodiments, in the methods provided herein, the purified sample comprises an intact antibody of interest and a trimmed form of the antibody of interest, wherein the ratio of trimmed antibody to intact antibody does not exceed about 1:100, 1:50, 1:25, 1:20, or 1: 10. In some embodiments, in the methods provided herein, the starting sample comprises an intact antibody of interest and a trimmed form of the antibody of interest, wherein the ratio of trimmed antibody to intact antibody in the starting sample is at least about 1:100, 1:50, 1:25, 1:20, 1:10, 1:5, 1:4, or 1:3 and wherein the ratio of trimmed antibody to intact antibody in a subsequent purified sample in the same method does not exceed about 1:200, 1:100, 1:50, 1:25, 1:20, or 1:10, wherein the second ratio is less than the first ratio. In some embodiments, in the methods provided herein, the starting sample comprises an intact antibody of interest and a trimmed form of the antibody of interest, wherein the ratio of trimmed antibody to intact antibody in the starting sample is between about one in group a (group a ratio: 1:100, 1:50, 1:25, 1:20, 1:10, 1:5, or 1:4) and one in group B (group B ratio: 1:50, 1:25, 1:20, 1:10, 1:5, 1:4, or 1:3) and wherein the ratio of trimmed antibody to intact antibody in subsequent purified samples in the same method is no more than about 1:200, 1:100, 1:50, 1:25, 1:20, or 1:10, wherein the ratio in purified samples is less than in the starting sample.
In some embodiments, a starting sample provided according to a method provided herein comprises at least 1, 5, 10, 15, 20, 25, 50, 100, 200, 500, 1000, 2000, 5000, or 10,000 grams of an intact antibody of interest. In some embodiments, a purified sample provided according to a method provided herein comprises at least 1, 5, 10, 15, 20, 25, 50, 100, 200, 500, 1000, 2000, 5000, or 10,000 grams of an intact antibody of interest. In some embodiments, a starting sample provided according to a method provided herein comprises at least 5, 10, 15, 20, 25, 50, 100, 200, 500, 1000, 2000, 5000, or 10,000 grams of an intact antibody of interest. Subsequent purified samples in the same method comprise at least 1, 5, 10, 15, 20, 25, 50, 100, 200, 500, 1000, 2000, or 5000 grams of the intact antibody of interest, wherein the first value is greater than the second value.
In addition, any of the above descriptions regarding a) amount or b) purity of an antibody of interest in a starting sample or a purified sample can be considered together with reference to the same sample. For example, as described above, the starting sample can comprise at least about 80% by mass of the antibody of interest; in addition, as also described above, the starting sample may comprise at least about 10 grams of the antibody of interest. Accordingly, also provided herein is a starting sample comprising by mass at least about 80% of an antibody of interest and at least 10 grams of the antibody of interest, and the like.
General techniques
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are well described in the literature, for example, Molecular Cloning, A Laboratory Manual, second edition (Sambrook et al, 1989) Cold Spring Harbor Press; oligonucleotide Synthesis (m.j. gate, ed., 1984); methods in Molecular Biology, human Press; cell Biology A laboratory Notebook (J.E. Cellis, ed.,1998) Academic Press; animal Cell Culture (r.i. freshney, ed., 1987); introduction to Cell and Tissue Culture (J.P.Mather and P.E.Roberts,1998) Plenum Press; cell and Tissue Culture Laboratory Procedures (A.Doyle, J.B.Griffiths, and D.G.Newell, eds.,1993-1998) J.Wiley and Sons; methods in enzymology (Academic Press, Inc.); handbook of Experimental Immunology (d.m.weirland c.c.blackwell, eds.); gene Transfer Vectors for Mammalian Cells (J.M.Millerand M.P.Calos, eds., 1987); current Protocols in Molecular Biology (F.M. Ausubel et al, eds., 1987); PCR The Polymerase Chain Reaction, (Mullis et al, eds., 1994); current Protocols in Immunology (j.e. coligan et al, eds., 1991); short Protocols in molecular Biology (Wiley and Sons, 1999); immunobiology (c.a. janeway and p.travers, 1997); antibodies (p.finch, 1997); antibodies a practical prophach (D.Catty., ed., IRL Press, 1988-; monoclonal antigens a practical proproach (P.shepherd and C.dean, eds., Oxford University Press, 2000); using Antibodies, oral manual (e.Harlow and D.Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M.Zantetti and J.D.Capra, eds., Harwood academic publishers,1995) and subsequent versions of The above references and corresponding websites, if applicable.
The following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims.
Examples
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