CN115066258A - Purification of bispecific antibodies - Google Patents

Abstract

A method of purifying a bispecific antibody of interest having only one kappa light chain constant region from a liquid comprising the bispecific antibody of interest and at least one by-product having two kappa Light Chain (LC) constant regions, the method comprising the step of separating the bispecific antibody of interest from the at least one by-product by kappa electric affinity chromatography.

Description

Purification of bispecific antibodies

Technical Field

The present invention relates to a method for purifying bispecific antibodies (bsabs), in particular bsabs containing only one kappa Light Chain (LC) constant region, using kappa-electric affinity chromatography.

Background

The therapeutic potential of bispecific antibodies (bsabs) has long been recognized. Moreover, they have also found use in detection and diagnosis. Many different forms of bispecific antibodies have been constructed, roughly divided into two classes: IgG-like bispecific antibodies and Fc-free bispecific antibodies. Each with advantages and limitations. For example, Fc-free bispecific antibodies are more permeable to solid tumors, and IgG-like bispecific antibodies have the advantage of having a longer circulating half-life and supporting secondary immune function.

In bispecific antibody production, the random assembly of four different polypeptide chains, i.e., Heavy Chains (HC) and LCs from two parent mabs, may yield up to 10 different products, only one of which has the desired function. Two techniques, namely the knob-and-hole technique ("KiH") and CrossMab, were developed for promoting HC heterodimerization and promoting homologous HC-LC pairing, respectively. Nevertheless, bispecific antibody production is often accompanied by considerable amounts of by-products due to inconsistent expression of each chain leading to residual HC homodimerization and incomplete assembly. In particular, although the use of the KiH technique significantly improves heterodimerization, homodimerization cannot be completely eliminated. Homodimerization between the "knob" variants generally does not occur, but homodimerization between the "hole" variants still occurs. The "mortar-hole" homodimer can be up to 5% of the total mass. In addition, good heterodimerization yield requires that the expression of both HCs be approximately equal. In the case of KiH, the amount of homodimers increased significantly if the "mortar" chain was overexpressed.

WuXiBody ^TM (hereinafter also referred to as "WuXiBody") is a novel bispecific antibody platform developed by the pharmacogenomics, in which the CH1 and CL regions of one of the Fab arms are replaced by the T Cell Receptor (TCR) constant region [1]. Such modifications ensure homologous LC-HC pairing. In the WuXiBody design, the KiH technique was used to promote HC heterodimerization [2]]. Examples of the present inventionOne, as shown in fig. 1A, the chain in which CH1 was replaced had a "knob" mutation ("knob" mutation) and the chain containing CH1 had a "hole" mutation ("hole" mutation). However, although this strategy greatly improved heterodimer formation, a small amount of hole-hole homodimer (hole-hole homodimer) was formed. Steric hindrance exists between the two knob structures, so knob-knob homodimers (knob-knob homomodimers) are rare. At this time, the target bsAb contained only one LC constant region, while the potential mortar-mortar homodimer contained two LC constant regions (fig. 1B).

Of all impurity by-products, homodimers were the most difficult to remove because of their physicochemical properties close to the target bsAb. Protein a chromatography, which is commonly used to capture monoclonal antibodies and Fc fusion proteins, cannot distinguish between homodimers and target bsabs. However, it has been reported that protein a chromatography separates half-antibodies containing one Fc domain from intact antibody portions containing an intact Fc region when eluted with a linear pH gradient [3 ].

KappaSelect is an affinity medium designed for purification of Fab (kappa) fragments [4 ]. It specifically binds to the kappa (. kappa.) LC constant region. It is commonly used to separate antibodies containing kappa-type LC constant regions from antibodies without kappa-type LC constant regions. For example, some bsabs take the form of Fab x single chain variable fragments (scFv), where one of the Fab arms is replaced by an scFv [5 ]. scFv is constructed by linking HC to the variable region of LC via a linker peptide, and does not contain the constant region of LC. During recombinant production of Fab x scFv type bsAb, scFv-Fc chains may be overexpressed and become a major impurity. At this point, kappa electrolyte provides a direct solution to the purification problem, allowing for rapid removal of scFv-Fc and the corresponding homodimers. As shown in fig. 2, the Fab x scFv bispecific antibody of interest binds to kappaselelect via its Fab arm, while the scFv-Fc chain and its homodimer, which lacks the LC constant region, remain in the flow-through fluid (fig. 2). While kappa-LC constant regions can be used to separate antibodies containing kappa-type LC constant regions from antibodies without kappa-type LC constant regions, it is not clear whether the medium can easily separate antibodies containing one kappa-type LC constant region from antibodies containing two kappa-type LC constant regions. Although it is presumed that an antibody containing one kappa-type LC constant region binds to kappa-type LC constant region less strongly than an antibody containing two kappa-type LC constant regions, it is not clear whether the difference in affinity is large enough to separate them.

Impurity by-products may exhibit undesirable activity if they remain in the final purified product. For example, monospecific by-products in bsAb production may reduce the efficacy of the final bispecific formulation if not isolated. Therefore, there is a need for new and/or improved protocols for the purification of bispecific antibodies of interest from culture harvest products, which allow the final purified product to meet the production requirements of the biotechnology industry for diagnostic and therapeutic products.

Disclosure of Invention

The present invention is based on the following findings: antibodies with two kappa-type LC constant regions bind more tightly to the kappa-electric mediator than antibodies with only one kappa-type LC constant region. Thus, these two substances can be easily separated with kappa-type LC constant region binding affinity medium kappa-select resin.

In general, the present invention provides a method of purifying a bispecific antibody of interest having only one kappa light chain constant region from a liquid comprising the bispecific antibody of interest and at least one by-product having two kappa Light Chain (LC) constant regions, the method comprising the step of separating the bispecific antibody of interest from the at least one by-product by kappa electric affinity chromatography; the separating step comprises:

(a) loading a liquid comprising the bispecific antibody of interest and the at least one byproduct onto a KappaSelect affinity media packed column;

(b) leaching the medium; and

(c) eluting the bispecific antibody of interest under conditions wherein the at least one byproduct remains bound to the column.

In some embodiments, the bispecific antibody of interest can be an IgG-like bispecific antibody or bispecific F (ab) ₂ And (3) fragment. In particular, in some embodiments, the bispecific antibody is an IgG-like bispecific antibody based on a knob-hole (also known as "KiH") model. More specifically, in some embodiments, one of the Fab arms of the bispecific antibody of interest comprises a kappa-type LC constant region and the CH1 and CL regions of the other Fab arm are TCR-constantRegion substitutions, and the strand in which CH1 was substituted had a "knob" mutation and the strand containing CH1 had a "hole" mutation.

In some embodiments, the side product can be a homodimeric side product in bispecific antibody production. In particular, in some embodiments, the byproduct may be a mortar-mortar homodimer byproduct in the production of a KiH bispecific antibody.

In other embodiments, the bispecific antibody of interest can be a kappa lambda antibody (kappa lambda-body), and the at least one byproduct includes a mono lambda homodimer and a mono kappa homodimer.

In some embodiments, the eluting step (c) comprises a linear pH gradient elution. For example, a linear pH gradient may be from pH 5.5 to pH 3.0.

In other embodiments, the eluting step (c) may be a step pH gradient elution. For example, a step pH gradient elution may include a step at pH 3.0, pH3.2, or pH 3.5. Preferably, the staged pH gradient elution may comprise a pH 3.5 stage.

In some embodiments, the washing step (b) may be performed by sequentially washing the column with buffers of different pH values. For example, the washing step (b) may be performed by washing the column sequentially with buffers of pH7.4 and pH 5.5, respectively. In one of the examples, the column was washed 3 times with buffers of pH7.4, pH 5.5 and pH 5.5, respectively.

Drawings

FIGS. 1A-1B: based on WuXiBody ^TM Schematic representation of asymmetric IgG-like bispecific antibodies of the platform. FIG. 1A: a heterodimer of interest; FIG. 1B: a latent mortar-mortar homodimer.

FIG. 2 is a schematic diagram: schematic representation of Fab X scFv type bsAb purification using KappaSelect affinity medium.

FIG. 3: WuXiBody ^TM Protein a affinity chromatogram of the bispecific antibody harvest. Illustration is shown: non-reducing SDS-PAGE analysis of the relevant fractions, including lane 1: isolated mortar-mortar homodimers as reference, lane 2: loading, lane 3: and (4) eluting the product.

FIG. 4: one of the examples herein elutes WuXiBody with a linear pH gradient ^TM Kappa-select chromatogram of bispecific antibody harvest.The column was eluted at 20CV from pH 5.5 to pH 3.0.

FIGS. 5A-5B: one of the examples herein elutes WuXiBody with a step pH gradient ^TM Kappa-select chromatogram of bispecific antibody harvest. FIG. 5A: superposition of three chromatograms of different elution pH values; FIG. 5B: non-reducing SDS-PAGE analysis of relevant fractions for three experiments, including lane 1: isolated mortar-mortar homodimers as reference, lane 2: loading, lane 3: eluate 1(E1), lane 4: eluate 2(E2), lane 5: protein a eluate (for comparison), lane 6: strong eluate (strip).

FIG. 6: analytical HIC patterns for protein a eluate and kappa electrolyte eluate of the examples herein were superimposed.

FIGS. 7A-7B: AEX chromatograms of protein a eluate (fig. 7A) and of KappaSelect eluate (fig. 7B). Illustration is shown: non-reducing SDS-PAGE analysis of the relevant fractions, including lane 1: isolated mortar-mortar homodimers as reference, lane 2: sample loading, lanes 3-5: flow through fractions 1-3 (F1-F3).

FIGS. 8A-8B: purification of kappa lambda antibodies. FIG. 8A: existing purification strategies; FIG. 8B: an alternative strategy to kappaselelect is used according to the present invention.

Detailed Description

As used herein, "a" and "the" are to be understood as including both the singular and the plural, unless expressly stated otherwise.

Herein, "or" should be understood as being inclusive and interchangeable with "and/or" synonymously, unless expressly stated otherwise.

KappaSelect is an affinity medium designed for the purification of Fab (kappa) fragments. It binds specifically to the kappa-type LC constant region. It is commonly used to separate antibodies containing kappa-type LC constant regions from antibodies without kappa-type LC constant regions. However, it is not clear whether such a medium can easily separate a species containing one kappa-type LC constant region from a species containing two kappa-type LC constant regions. Although it is presumed that an antibody containing one kappa-type LC constant region binds to kappa-type LC constant region less strongly than an antibody containing two kappa-type LC constant regions, it is not clear whether the difference in affinity is large enough to separate them.

Recently, we have an opportunity to investigate this problem when purifying asymmetric IgG-like bsabs based on the WuXiBody platform. We have thereby had the opportunity to achieve the feasibility of separating the target bsAb from the impure mortar-hole homodimer with KappaSelect. In practice, we found that mortar-mortar homodimers (containing two kappa-type LC constant regions) bound more tightly to the kappa-electric medium than bsAb containing only one kappa-type LC constant region and eluted at a lower pH. Thus, under common elution conditions, only bsAb was eluted, whereas homodimers appeared in the strong eluate. Thus, kappa-select provides a convenient means to separate antibodies containing one kappa-type LC constant region from antibodies containing two kappa-type LC constant regions. Not only does this enrich our knowledge of kappa area but it has significant utility value. In addition to WuXiBody-based bsAb, KappaSelect can also be used to purify bsAb-based antibodies in other forms, such as kappa lambda antibodies. Furthermore, in the case of kappa lambda antibody purification, the efficiency can be greatly improved with the new information investigated in the present invention.

Accordingly, disclosed herein is a method of purifying a bispecific antibody of interest having only one kappa light chain constant region from a liquid comprising the bispecific antibody of interest and at least one by-product having two kappa Light Chain (LC) constant regions, the method comprising the step of separating the bispecific antibody of interest from the at least one by-product by kappa electric affinity chromatography.

It will be appreciated that bispecific antibodies may be IgG-like or Fc-free and may be of any form comprising LC constant regions of the kappa type, including, for example, Triomabs/quadroma, DVD-Ig (double variable Ig), CrossMAb, Two-in-one IgG, Fab × scFv bispecific antibodies, especially WuXiBody bispecific antibodies (TCR constant region replacement of the CH1 and CL region of one of the Fab arms) and kappa λ antibodies and their F (ab) ₂ And (3) fragment. Alternatively, bispecific antibodies may have a KiH design to facilitate heterodimerization.

It will be appreciated that by-products include those commonly found in bispecific antibody production, including homodimers, 3/4 antibodies (antibodies lacking one LC), half antibodies, HC dimers, free HC, and free LC.

In one embodiment, the bispecific antibody of interest has only one kappa-type LC constant region, e.g., Fab x scFv bispecific antibody, WuXiBody, and kappa λ antibody, while the by-product comprises two kappa-type LC constant regions, e.g., an undesired homodimer.

Typically, the bispecific antibody of interest and the by-product are contained in a liquid to be loaded onto the chromatography column (i.e., a "loading liquid" herein). For example, the liquid can be a cell culture harvest from recombinant production of bispecific antibodies. In one embodiment, the cell culture harvest is pretreated by filtration (e.g., depth filtration) to obtain the liquid or "loading solution".

The purification method of the present invention is based mainly on the separation of the target bispecific antibody into the eluate with kappa-select medium, optionally separating the side products into a strong eluate, whereby the target diabody can be collected from the corresponding eluted fraction. Optionally, the method further comprises one or more upstream or downstream purification processes, such as filtration and other chromatography.

In the method of the present invention, the separating step may comprise:

(a) loading a liquid comprising the bispecific antibody of interest and the at least one byproduct onto a KappaSelect affinity media packed column;

(b) leaching the medium; and

(c) eluting the bispecific antibody of interest under conditions in which the at least one byproduct remains bound to the column.

In some embodiments, the eluting step (c) is a linear pH gradient elution. For example, a linear pH gradient may be from pH 5.5 to pH 3.0 over a CV factor or period of time. One of ordinary skill in the art will be able to determine an appropriate CV multiple or length of time. One of ordinary skill in the art will be able to determine an appropriate buffer. In some embodiments, the eluting step (c) is a step pH gradient elution. For example, a step pH gradient elution may comprise a step of pH 3.0, pH3.2 or pH 3.5, preferably pH 3.5.

In some embodiments, the washing step (b) may be performed sequentially with buffers of different pH values (e.g., pH7.4 and pH 5.5). In some examples, the elution volume is 5CV per time. One of ordinary skill in the art will be able to determine an appropriate buffer.

Although the present invention is based on the discovery in the purification of WuXiBody bispecific antibodies, it is clear from the above that the basic concept of the present invention can be extended to other forms of antibodies. It has been demonstrated that the method of the present invention provides a more efficient method for purifying a bispecific antibody of interest. In one embodiment, the bispecific antibody of interest is a kappa lambda antibody, and the method of the invention using kappa shell is more effective than conventional protocols. The bispecific in the form of κ λ antibodies contains the same HC to avoid HC pairing problems, the bispecific being from two different LCs: one kappa type (. kappa.) and one lambda type (. lamda.) [6 ]. Thus, the construction of kappa λ antibodies requires three chains: HC. Kappa type LC and lambda type LC. In addition to the desired kappa lambda antibody, when these three chains are co-expressed, mono-kappa and mono-lambda homodimers are also produced. According to the prior art, kappa lambda antibodies can be purified by sequential kappa-as-select affinity chromatography and LambdaFabSelect affinity chromatography, wherein LambdaFabSelect is an affinity medium that binds to the lambda-type LC constant region. Thus, bispecific κ λ antibody bound to both media, mono λ homodimer and mono κ homodimer were removed with flow through kappa electric column and LambdaFabSelect column, respectively, and κ λ antibody eluted from the column as the final purified product [6] (fig. 8A). However, with the kappa lambda related knowledge obtained in this study, kappa lambda antibodies could be purified by another method that requires only kappa lambda (fig. 8B). As shown in fig. 8B, single λ homodimers were removed with flow through, κ λ antibodies were collected by elution, and single κ homodimers were only present in the strong eluate by kappa electric chromatography alone. This new process is more efficient in terms of operability and economy than the conventional process, since it requires only one step of affinity chromatography rather than two steps.

Examples

The present invention will be explained in more detail below with reference to specific examples. It is to be understood that these examples are for illustration only, and are not to be construed as limiting the scope of the invention. The experiments are carried out as indicated or according to conventional methods, for example as taught in the technical manual molecular cloning: a laboratory manual, or according to the manufacturer's instructions.

Materials and methods

Material

Ammonium sulfate, ethanol, glycine, sodium acetate trihydrate, sodium chloride, sodium hydroxide, sodium dihydrogen phosphate monohydrate, disodium hydrogen phosphate dehydrate, and tris (hydroxymethyl) aminomethane were purchased from Merck (darmstadt, germany). Acetic acid and hydrochloric acid were purchased from JT Baker corporation (philips burgh, new jersey, usa). MabSelect prism A, KappaSelect and tricorn 5/200 chromatography columns (5 mm ID, 200mm length) were purchased from GE Healthcare, Uppsala, Sweden. POROS 50HQ and MAbPac HIC-20 (4.6X 100mm) columns were purchased from Thermo Fisher Scientific, Inc. (Walserm, Mass.). N-glycosidase (PNGase F) was purchased from New England Biolabs, Inc. (Epstein, Mass.). A30% acrylamide/bisacrylamide solution (37.5:1) and TEMED were purchased from Bio-Rad Laboratories, Inc. (Heracles, Calif., USA). Ammonium persulfate, Coomassie blue R-250, glycerol, Sodium Dodecyl Sulfate (SDS), iodoacetamide, and bromophenol blue were purchased from Sigma-Aldrich (St. Louis, Mo.).

The bispecific antibody of interest is based on WuXiBody ^TM An asymmetric IgG-like bispecific antibody for platform. Expressed from CHO-K1 cells, cultured in Hyclone ActiPro medium, Cell Boost 7a and 7b fed (medium and feed purchased from GE Healthcare). Cells were cultured for 14 days and then harvested. The culture harvest was clarified by depth filtration. The resulting filtrate contained the target bsAb heterodimer, potential mortar-mortar homodimer and mortar half antibody (hole half-antibody). The target bsAb heterodimer was a kuxibody designed by KiH ^TM IgG-like bispecific antibodies in which the CH1 region and CL region in the knob half were replaced by the C β domain and C α domain of the TCR, and the hole half without CH1/CL replacement was overexpressed in comparison to the formation of hole-hole homodimers (fig. 1A-1B).

Device for measuring the position of a moving object

Column chromatography was performed using an AKTA pure 150 system (GE Healthcare, Uppsala, Sweden) equipped with Unicorn software version 7.3. The pH and conductivity were measured using a SevenExcellence S470 pH/conductivity meter (Mettler-Toledo, Columbus, Ohio, USA). Protein concentration was measured using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Mass.) analytical grade Hydrophobic Interaction Chromatography (HIC) was performed using an Agilent 1260 liquid chromatograph (Agilent Technologies, Inc., Santa Clara, Calif., USA).

Method

Protein a chromatography

MabSelect prism A (a protein A affinity medium) was loaded into a 0.5cm diameter column with a bed height of 16.0 cm. The Column Volume (CV) was about 3 ml. Culture harvest clarified by depth filtration was loaded onto the column. The protein was loaded at 30mg per milliliter (ml) of medium and run in bind-elute mode. The flow rate of the system was 190cm/hr (residence time: 5 minutes (min)). After loading, the column was washed with 50mM Tris-acetate, 150mM NaCl, pH7.4 buffer, 50mM Na-acetate/HAc, 1M NaCl, pH 5.5 buffer, and 50mM Na-acetate/HAc, pH 5.5 buffer, 5CV each time, in that order. Elution was performed with 50mM sodium acetate/HAc, pH 3.5 buffer.

Kappa aSelect affinity chromatography

The kappa electric affinity medium was packed in a 0.5cm diameter column with a bed height of 16.0 cm. The column volume was about 3ml (CV:. about.3 ml). Culture harvest clarified by depth filtration was loaded onto the column. The protein was loaded at 18mg per ml of medium (approximately the maximum dynamic binding capacity of the medium under the selected conditions) and run in bind-elute mode. The system flow rate was 193cm/hr (residence time: 5 minutes). After loading, the column was washed with 50mM Tris-acetate, 150mM NaCl, pH7.4 buffer, 50mM Na-acetate/HAc, 1M NaCl, pH 5.5 buffer and 50mM Na-acetate/HAc, pH 5.5 buffer, 5CV each time. Linear pH gradient elution the column elution was performed with a 20CV pH gradient from pH 5.5 to pH 3.0(50mM sodium acetate/HAc, pH 5.5 to 3.0). The stepwise elution was performed with 50mM sodium acetate/HAc at pH 3.0, pH3.2 and pH 3.5.

Anion Exchange (AEX) chromatography

AEX chromatography was performed in a bind and elute mode using 50HQ media from POROS, using a column with a diameter of 0.5cm and a bed height of 15.0cm (CV:. about.3 ml). The sample solution was protein A or KappaSelect eluate, pH adjusted to 7.0. 40mg protein per ml medium was loaded and run at 177cm/hr flow rate (residence time: 5 min). After loading, the column was washed with 50mM Tris-Hac, pH 7.0 buffer, and 50mM Tris-HAc, pH 8.0 buffer, 3CV each time, in that order. The column elution was performed with 50mM Tris-HAc, 130mM NaCl, pH 8.0.

Analytical grade Hydrophobic Interaction Chromatography (HIC)

HIC was analyzed using an Agilent system with MAbPac HIC-20 stainless steel columns (4.6X 100 mm). Mobile phase a consisted of 100mM sodium phosphate and 600mM ammonium sulfate, pH 7.0, and mobile phase B consisted of 10mM sodium phosphate, pH 7.0. The column elution was performed with a gradient of 0% to 100% B over 30 minutes (about 4.5CV) at a flow rate of 0.25 ml/min. Samples for HIC analysis were deglycosylated and diluted to 0.5mg/ml with mobile phase A. 50 μ g of sample was injected for each run.

Non-reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

8% Tris-glycine gel was prepared by standard method. A4 Xgel buffer (1.5M Tris-HCl, 0.4% SDS, pH 8.8), a 4 Xgel buffer (0.5M Tris-HCl, 0.4% SDS, pH 6.8), a 2 Xloading buffer (150mM Tris, 4% SDS, 20% v/v glycerol and 0.04% bromophenol blue, pH 6.8) and a running buffer consisting of 25mM Tris, 190mM glycine and 0.1% SDS were prepared in house. All samples were treated with 30mM iodoacetamide and heated at 75 ℃ for 5 minutes prior to analysis. 120V constant voltage electrophoresis for 1.5 hours. The gel was stained with coomassie blue and destained with a destaining solution containing 10% acetic acid, 20% ethanol and 70% water.

Deglycosylation

Mu.g of the purified bispecific antibody of interest (100. mu.l) buffer was replaced with 50mM sodium phosphate, pH 7.5. After buffer replacement, 1. mu.l of PNGase F (500U/. mu.l) was added and the reaction was allowed to proceed overnight at 37 ℃.

Comparative example: chromatographic separation of protein A

In this example, protein A chromatography was used to separate antibodies containing one kappa-type LC constant region from antibodies containing two kappa-type LC constant regions.

The culture harvest was clarified by depth filtration as described above. The resulting filtrate was loaded onto a MabSelect prism a column and subjected to separation chromatography as described above.

The results are shown in FIG. 3. The inset is a photograph of a non-reducing SDS-PAGE analysis of the relevant fractions, including: lane 1: isolated mortar-mortar homodimers as reference, lane 2: loading, lane 3: and (4) eluting the product. The mortar-mortar homodimer reference was generated by expressing only mortar half antibody and purified by protein a chromatography and size exclusion chromatography. The homodimer produced two bands (lane 1). Mass spectrometry analysis showed that, in addition to the intact antibody (data point), a truncated form was present, which was about 1500 daltons smaller. As shown in the electrophoresis gel, both the loading solution (lane 2) and the eluate (lane 3) contained the mortar-mortar homodimer, in which the band of the complete mortar-mortar homodimer overlapped with the band of the target bispecific antibody. Bands slightly lower than the main band suggest the presence of a mortar-mortar homodimer (visible in lanes 2 and 3), which corresponds to the truncated form in lane 1. As can be seen, protein a chromatography failed to separate the KiH heterodimer from the mortar-hole homodimer because they both contained Fc domains (fig. 3).

Notably, the band at the lower part of the gel was identified by mass spectrometry as a mortar half antibody (data omitted). It is clear and consistent that it is the socket half-antibody that is overexpressed that leads to the formation of socket-socket homodimers. Since homodimers are less stable than heterodimers, only partially overexpressed mortar half antibodies form mortar-mortar homodimers, while the remainder remain unpaired half antibodies.

Example 1: kappa-As-select separation using linear pH gradient elution

In this example, varying numbers of kappa LC constant regions were separated using KappaSelect and eluted using a linear pH gradient as described above.

The culture harvest was clarified by depth filtration as described above. The resulting filtrate was loaded onto a kappa-sec column and linear pH gradient elution was performed as described above.

The results are shown in FIG. 4. Inset is a photograph of a non-reducing SDS-PAGE analysis comprising: lane 1: isolated mortar-mortar homodimers as reference, lane 2: loading, lane 3: eluate 1(E1), lane 4: eluate 2(E2), lane 5: protein a eluate (for comparison) and lane 6: the eluate was strongly eluted. Isolated mortar-mortar homodimers and protein a eluates were obtained as described in the comparative examples. As shown by gel electrophoresis, neither of the eluates E1 and E2 (lane 3 and lane 4) had the hole-hole homodimer, which was mainly present in the strong eluate (lane 6). This indicates that the mortar-mortar homodimer containing two kappa-type LC constant regions binds to kappa-electric media much stronger than the target KiH heterodimer and mortar half antibody containing only one kappa-type LC constant region.

Example 2: kappa-As-select separation with staged pH gradient elution

Based on the results of the linear pH gradient elution, we further developed a stepwise pH gradient elution. To find the most suitable elution conditions, three pH values (3.0, 3.2 and 3.5) were evaluated (fig. 5A).

The culture harvest was clarified by depth filtration as described above. The obtained filtrate was applied to a kappa-As electrode column, and elution was performed at pH 3.0, pH3.2 and pH 3.5, respectively.

The results are shown in FIGS. 5A-5B. FIG. 5A is a chromatogram of three experiments at different pH values stacked. Figure 5B shows the non-reducing SDS-PAGE analysis photographs corresponding to these three experiments, each including: lane 1: isolated mortar-mortar homodimers as reference, lane 2: loading solution, lane 3: eluate 1(E1), lane 4: eluate 2(E2), lane 5: protein a eluate (for comparison) and lane 6: the eluate was strongly eluted. SDS-PAGE analysis suggested: in contrast, pH 3.5 elution was able to most completely remove the mortar-mortar homodimer (fig. 5B).

Further, removal of the mortar-mortar homodimer was confirmed by analysis of HIC (fig. 6). The peak of the hole-hole homodimer in the KappaSelect eluate was significantly reduced compared to the protein a eluate. The kappa electrolyte eluate used for this analysis was eluted at pH 3.5. As shown, kappaselelect removed the mortar-mortar homodimer, whereas protein a did not. This is consistent with the results of SDS-PAGE described above.

Since the migration rate of intact mortar-mortar homodimers was similar to that of the target bispecific antibody, it can be assumed that SDS-PAGE analysis only demonstrated the removal of truncated mortar-mortar homodimers, while the removal of intact homodimers remains to be confirmed.

Thus, the protein a eluate and the KappaSelect eluate were subjected to AEX chromatography, respectively, as described above. The kappa electrolyte eluate used for this analysis was eluted at pH 3.5. In this study, because the isoelectric points (pI) of the mortar half antibody and mortar-mortar homodimer were high (i.e., 7.5), they did not bind to the AEX column at pH 7.0. In contrast, the target KiH bispecific antibody has a much lower pI due to the TCR constant region replacement of low pI and therefore will bind to the AEX column under the same conditions.

The results are shown in FIGS. 7A-7B. In both figures, the inset is a photograph of a non-reducing SDS-PAGE analysis of the relevant fractions, including: lane 1: isolated mortar-mortar homodimers as reference, lane 2: sample loading, lanes 3-5: flow through fractions 1-3 (F1-F3). As shown, the flow-through run with the protein A eluate contained both the mortar half antibody and the mortar-mortar homodimer (FIG. 7A, lanes 3-5), whereas the flow-through run with the KappaSelect eluate contained only half antibody (FIG. 7B, lanes 3-5). This indicates that kappaselelect completely removed the mortar-mortar homodimer by-product.

This also confirmed the presence of both intact mortar-mortar homodimers and truncated homodimers, and more importantly, the complete clearance of kappa-select as determined by SDS-PAGE based on the presence of the truncated homodimer band (FIG. 7B).

In view of the foregoing it will be evident that equivalent changes and modifications may be made, which are intended to be included within the scope of the appended claims.

Reference to the literature

[1]WO 2019/057122 A1

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[4] GE Healthcare Life Sciences platform enumeration for the purification of antibody fragments (Fab), Application note,29-0655-41, AA,2013.

[5] Moore, m.bernet, r.rasid, j.desjarlais. Novel heterodimeric proteins (Novel heterodimeric proteins) US patent application publication US 2014/0294835 a1.

[6] Fischer, g.elson, g.magistelli, e.dheilly, n.fouque, a.laurendon, f.guineau, u.ravn, j.f.depoisier, v.moine, s.raimondi, p.malinge, l.di Grazia, f.rousseau, y.pointevin, s.caloud, p.a.cayatte, m.alcoz, g.pontini, s.fag. gene, l.broyer, m.biocorer, d.schrag, g.diedelot, n.bosson, n.costes, l.cons, v.buatotis, z.johnson, w.ferlin, k.mastership, m.kosco-virginic, developed for the generation of natural IgG (purified IgG) and purification of human IgG (export) light chain, and native IgG (origin).

Claims (14)

1. A method of purifying a bispecific antibody of interest having only one kappa light chain constant region from a liquid comprising the bispecific antibody of interest and at least one by-product having two kappa Light Chain (LC) constant regions, the method comprising the step of separating the bispecific antibody of interest from the at least one by-product by kappa electric affinity chromatography; the separation step comprises

(a) Loading a liquid comprising the bispecific antibody of interest and the at least one byproduct onto a KappaSelect affinity media packed column;

(b) leaching the medium; and

(c) eluting the bispecific antibody of interest under conditions wherein the at least one byproduct remains bound to the column.

2. The method of claim 1, wherein the bispecific antibody of interest is an IgG-like bispecific antibody or a bispecific F (ab) ₂ And (3) fragment.

3. The method of claim 1, wherein the bispecific antibody is an IgG-like bispecific antibody based on a knob-hole (KiH) model.

4. The method of claim 3, wherein one of the Fab arms of the bispecific antibody of interest comprises a kappa-type LC constant region and the CH1 and CL regions in the other Fab arm are replaced by TCR constant regions, and wherein the chain in which CH1 is replaced has a "knob" mutation and the chain comprising CH1 has a "hole" mutation.

5. The method of claim 1, wherein the byproduct is a homodimer byproduct in bispecific antibody production.

6. The method according to claim 3, wherein the byproduct is a hole-hole homodimer byproduct in the production of KiH bispecific antibody.

7. The method of claim 1, wherein the bispecific antibody of interest is a kappa lambda antibody and the at least one byproduct comprises a mono lambda homodimer and a mono kappa homodimer.

8. The method of claim 1, wherein the eluting step (c) comprises a linear pH gradient elution.

9. The method of claim 8, wherein the linear pH gradient is from pH 5.5 to pH 3.0.

10. The method of claim 1, wherein the eluting step (c) is a step pH gradient elution.

11. The method of claim 10, wherein the staged pH gradient elution comprises a pH 3.0, pH3.2, or pH 3.5 stage.

12. The method of claim 11, wherein the staged pH gradient elution comprises a pH 3.5 stage.

13. The method of claim 1, wherein the washing step (b) sequentially washes the column with buffers of different pH values.

14. The method of claim 13, wherein the washing step (b) sequentially washes the column with buffers having pH values of pH7.4 and pH 5.5, respectively.

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