CN115734969A - Purification of bispecific antibodies - Google Patents

Abstract

A method of purifying a bispecific antibody having a TCR constant domain from a liquid comprising the bispecific antibody and potential impurities, the method comprising subjecting the liquid to protein a chromatography followed by anion exchange chromatography and mixed mode chromatography in sequence, thereby obtaining a bispecific antibody purification product; wherein the anion exchange chromatography is performed in bind-elute mode.

Description

Purification of bispecific antibodies

Technical Field

The present invention relates to a method for the purification of bispecific antibodies (bsAb) containing TCR constant domains, in particular WuXiBody ^TM Purification of technical bispecific antibodies.

Background

Bispecific antibodies (bsabs) are artificial antibodies that are capable of binding two epitopes on the same or different targets. Their ability to bind two targets simultaneously enables new mechanisms of action (e.g., dual inhibition of signaling pathways and recruitment of effector cells). Bispecific antibodies are therefore expected to be tools for the treatment of numerous diseases such as cancer. bsabs can be broadly divided into two major classes based on molecular structure: igG-type molecules with an Fc region and smaller non-IgG-type molecules lacking an Fc region. Each of them has advantages and limitations. For example, fc-free bispecific antibodies are more permeable to solid tumors, while IgG-type bispecific antibodies have the advantage of having a longer half-life and supporting a secondary immune response. Generally, compared with the similar products without Fc, the IgG type bsAb is more complex and has higher technical difficulty in construction and production.

IgG-type bsabs can be further divided into two subclasses, symmetric and asymmetric. Asymmetric IgG-type bsabs are typically derived from two parent monoclonal antibodies (mabs) that bind with different specificities, and therefore comprise four different chains: two different Heavy Chains (HC) and two different Light Chains (LC). When these chains are co-expressed in the producer cell, the products of misassembly such as homodimers and molecules containing mismatched LC can be up to about 90% of the total mass if they are left to pair randomly [1]. To address this problem, scientists have developed various protein engineering approaches to improve the correct assembly of antibodies [2]. Among them, knob-into-holes (KiH) and CrossMab techniques are most commonly used to overcome HC homodimerization and LC mismatch [3,4], respectively. For the symmetric IgG class bsAb, bispecific specificity is usually achieved by fusing a second antigen binding unit to the N-or C-terminus of the HC or LC [1,2]. Since symmetric bsabs perform their function by chain extension rather than by introducing different chains, their associated byproducts are generally less than asymmetric bsabs. However, the tendency of symmetric bsAb to form aggregates is significantly increased [5,6], reported as high as 50% [6]. Due to the increased chain length and flexibility, aggregates can form as a result of intermolecular domain mismatches.

WuXiBody ^TM (hereinafter also referred to as "WuXiBody") is an innovative dual-antibody platform developed by Megming biology, inc. (Wuxi Biologics). The important feature is the replacement of one CH1/CL constant domain of the parent mAb with a constant domain of the T Cell Receptor (TCR) [7]。WuXiBody ^TM The design ensures homologous HC-LC pairing, with the same goal as CrossMab technology. The WuXiBody-based BsAb may take either an asymmetric or symmetric form (fig. 1). Asymmetric WuXiBody bispecific antibodies facilitate heterodimer formation using KiH technology.

Nevertheless, impurities are still present and need to be removed. Therefore, there is a need for new and/or better methods to separate the bispecific antibody of interest from impurities such that the final purified product can meet the requirements of the biotechnology industry for the production of diagnostic and therapeutic products.

Disclosure of Invention

In general, disclosed herein is a method of purifying a bispecific antibody having a TCR constant domain from a liquid comprising the bispecific antibody and potential impurities, the method comprising subjecting the liquid to protein a chromatography followed by anion exchange chromatography and mixed mode chromatography in sequence, thereby obtaining a bispecific antibody purification product; wherein the anion exchange chromatography is performed in bind-elute mode.

The method of the invention is particularly suitable for purification based on WuXiBody ^TM Bispecific antibodies of the art are capable of efficiently removing process-related impurities and product-related impurities, including host cell components, homodimers, aggregates and TCR constant domain-deleted by-products.

Drawings

FIG. 1: schematic representation of four different forms of WuXiBody bispecific antibodies (referred to as "molecule a" to "molecule D"), each with a respective pI marked below. As shown, molecule A and molecule B are asymmetric, and molecule C and molecule D are symmetric.

FIG. 2: protein a chromatograms of four WuXiBody bispecific antibodies. Illustration is shown: non-reducing SDS-PAGE analysis of relevant components, including M: protein molecular weight standards; l: loading a sample; e: eluting the sample; s: the sample was eluted vigorously.

FIG. 3: AEX chromatograms of four WuXiBody bispecific antibodies. Illustration is shown: non-reducing SDS-PAGE analysis of relevant components, including M: protein molecular weight standards; l: loading a sample; w: leaching the sample; e: eluting the sample; s: the sample was eluted vigorously.

FIG. 4: mixed mode chromatograms of four WuXiBody bispecific antibodies. The mixed mode resin specifically used for molecules A-C was Capto MMC Impres and for molecule D was Capto adhere Impres. Illustration is shown: non-reducing SDS-PAGE analysis of relevant components, including M: protein molecular weight standards; l: loading a sample; w: leaching the sample; e: eluting the sample; s: the sample was eluted vigorously.

FIG. 5: SEC-HPLC chromatograms of the final purified products of the four WuXiBody bispecific antibodies, molecule a to molecule D.

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.

For features defined as values or ranges of values, embodiments thereof include not only the indicated values and ranges, but also all integers and fractions within the indicated ranges and ranges formed by any two of the indicated values, and encompass understandable deviations.

All technical terms herein have the meanings commonly recognized as standard definitions and meanings by those skilled in the art, unless otherwise specified.

The following abbreviations are used herein: and (b) the antigen of the bsAb: a bispecific antibody; mAb: a monoclonal antibody; HC: a heavy chain; LC: a light chain; and (3) KiH: pestle-mortar (knobs-endo-holes); TCR: a T cell receptor; CH1: heavy chain constant region 1; CL: a light chain constant region; pI: isoelectric point; IEX: ion exchange; AEX: anion exchange; MMC: mixed mode chromatography; SEC-HPLC: size exclusion chromatography-high performance liquid chromatography; CV: column volume; HCP: a host cell protein; SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis.

WuXiBody ^TM Is an innovative double-antibody platform for the biological development of the pharmacomycin. An important feature is the replacement of the CH1/CL constant domain (e.g., the constant domain formed by CH1 and CL in the classical Fab fragment) of one parent mab with a TCR constant domain (e.g., the TCR constant domain formed by the C β and C α regions of the TCR). The purpose of this sequence engineering is to facilitate the corresponding HC-LC pairings. A new general feature of the WuXiBody bispecific antibodies thus obtained is a greatly reduced pI. This is because the pI of the TCR constant domain is relatively low, so bsAb containing TCR constant domain has a pI lower than that of normal mAb. The inventors have found that this additional feature helps to separate target bsabs (especially asymmetric bsabs) from TCR-free constant domain by-products (including half-antibodies and their corresponding homodimers) by IEX chromatography. Under selected conditions, the TCR-free constant domain by-product is charged differently than the target bsAb and can therefore be easily removed by AEX.

Presented herein is an ideal potential platform technology that facilitates the purification of various configurations of WuXiBody bispecific antibodies, including protein a chromatography, AEX chromatography, and mixed mode chromatography. In particular, AEX and mixed mode chromatography are very effective in removing TCR-free byproducts and aggregates, respectively. The platform greatly saves time and investment for the development of downstream purification processes of the WuXiBody project.

Accordingly, provided herein is a method of purifying a bispecific antibody having a TCR constant domain (also known as "TCR-containing bsAb") from a liquid comprising the bispecific antibody and potential impurities, the method comprising subjecting the liquid to protein a chromatography followed by anion exchange chromatography and mixed mode chromatography in sequence, thereby obtaining a bispecific antibody purification product; wherein the anion exchange chromatography is performed in bind-elute mode.

Protein a chromatography may employ standardized platform protocols to capture the product. Elution can be performed by either gradient pH or step pH. In some protocols, protein a chromatography employs a staged pH elution. In some cases, the staged pH elution includes a pH of about 3.0 to about 4.0, e.g., about pH 3.6.

AEX chromatography for intermediate purification is usually run in flow-through mode. The pI of most mAbs is relatively high (6.5-9.0). Thus, under appropriate pH (i.e., below the pI of the target protein) and conductivity conditions, the target product stream passes through the chromatography column while negatively charged impurities (e.g., HCPs) bind to the chromatography column. In the present invention, AEX chromatography is performed on low pI TCR-containing bsAb in bind-elute mode. In this mode, AEX is able to remove not only TCR-free high pI by-products, including TCR-free homodimers in asymmetric bsabs represented by molecule a and molecule B, but also lower pI by-products, including TCR-containing homodimers in asymmetric bsabs represented by molecule a and molecule B. In some embodiments, the AEX chromatography column elutes at a pH of about 8.0. In some embodiments, the AEX chromatography column elutes at an ionic strength of about 130 to about 190mM, preferably about 130 to about 165 mM. In some embodiments, the AEX chromatography column is eluted with a buffer comprising about 130 to about 190mM, preferably about 130 to about 165mM NaCl, about pH 8.0 (e.g., 50mM Tris-HAc buffer, pH 8.0).

In some embodiments, optionally, the methods of the invention may include depth filtration as an intermediate step after protein a chromatography and before AEX chromatography. In general, post-protein a chromatography neutralization followed by depth filtration enables robust and efficient clearance of HCPs. The target pH for neutralization is generally from about 5.0 to about 7.5. The principle is that most CHO host cell proteins have pI in the range of about 4.5-7.0 and show a decrease in solubility upon titration to this pH range, whereas mAbs with higher pI are generally unaffected. However, in some embodiments of the invention, a lower pH may be used for TCR-containing bsAb with a pI lower than that of the normal mAb. In some embodiments, in this step, the eluted sample of protein a chromatography is titrated to about pH 4-5, and then filtered to obtain a filtrate for subsequent processing. In some cases, the filtrate was subsequently subjected to AEX chromatography.

In some embodiments, the mixed mode chromatography may be Capto MMC ImpRes or Capto adhere ImpRes mixed mode chromatography. Also, mixed mode chromatography can be performed in bind-elute mode. Both Capto MMC imprres and Capto adhere imprres are multimodal ion exchange media aimed at best removing aggregates [8-10]. Their ligands contain groups capable of ion exchange (MMC and adhere are cation exchange and anion exchange, respectively), hydrophobic interaction and hydrogen bonding. We have previously found that both resins are very effective at removing antibody aggregates [11].

In some cases, the liquid comprising bsAb and potential impurities may be a cell culture harvested in recombinant bsAb production. In one embodiment, the harvested culture is subjected to a pretreatment, including, for example, clarification. For example, the harvested culture may be clarified (e.g., by centrifugation or filtration) to provide a liquid or "sample load" for chromatography.

In this application, the TCR-containing bsAb can be WuXiBody ^TM Technical IgG type bsAb, e.g. WO2019/057122 A1[7 ]]The method as described in (1). For example, the bsAb may comprise a TCR constant domain in one or more Fab units or Fab arms thereof. It will be appreciated that, as also shown in the example of figure 1, the bsAb may have more than two Fab arms or an extended Fab arm, i.e. an extended Fab arm comprising a plurality of Fab units. Herein, the terms "Fab arm" and "Fab unit" both refer to a moiety that is structurally and/or functionally equivalent to a classical Fab fragment as commonly understood in the art of engineering antibodies.

TCR constant domains are typically formed by constant regions of the cognate counterpart chains in the TCR. In some embodiments, the TCR constant domain is formed from the C β region and the C α region of the TCR linked by an interchain disulfide bond.

The TCR-containing bsAb may be in an asymmetric or symmetric form. Typically, the symmetric form will use a KiH design to promote heterodimerization.

In some embodiments, the TCR-containing bsAb is asymmetric, having a TCR constant domain substituted for the CH1/CL constant domain in one of its Fab arms, wherein the CH1 replacement chain (i.e., the chain in which CH1 is substituted) has a "knob" mutation, and the chain in which CH1 is located has a "hole" mutation. For example, the bsAb may have an asymmetric form as molecule a in figure 1, in which the CH1 domain of the "knob" mutant heavy chain is replaced by the C β region of the TCR and the CL domain in the corresponding light chain is replaced by the C α region.

Herein, the term "CH1/CL constant domain" refers to a domain formed by pairing between a corresponding CH1 region and a CL region.

In some embodiments, the TCR-containing bsAb is asymmetric, having a TCR constant domain substituted for the CH1/CL constant domain in one of its Fab arms, wherein the CH1 replacement chain has a "hole" mutation and the chain in which CH1 is located has a "knob" mutation. For example, the bsAb may have an asymmetric form as molecule B in fig. 1, in which the CH1 domain of the "hole" mutant heavy chain is substituted by the C β region of the TCR and the CL domain in the corresponding light chain is substituted by the C α region.

In other embodiments, the TCR-containing bsAb is symmetrical, having attached to the N-terminus of the Fc region two identical extended Fab arms, each comprising a first Fab unit and a second Fab unit, the first and second Fab units having different specificities and wherein at least one of the CH1/CL constant domains is replaced by a TCR constant domain. For example, the bsAb may have a symmetrical form as molecule C in fig. 1, with an extended Fab arm, in which the first Fab unit comprises the C β region of the TCR replacing the CH1 domain and the C α region replacing the corresponding light chain CL domain, and the C-terminus of the C β region of the first Fab unit in the heavy chain is fused to the N-terminus of the second Fab unit.

In another embodiment, the TCR-containing bsAb is symmetric, having a first pair of Fab arms (i.e., two identical first Fab arms) linked to the N-terminus of the Fc region and a second pair of Fab arms (i.e., two identical second Fab arms) linked to the C-terminus of the Fc region, wherein the first and second Fab arms have different specificities, and wherein at least one of the CH1/CL constant domains is replaced with a TCR constant domain. For example, the bsAb may have a symmetrical form as molecule D in figure 1, in which the first Fab arm comprises a CH1 domain substituted with the C β region of the TCR, the CL domain of the corresponding light chain is substituted with the C α region, and the C-terminus of the C β region of the first Fab arm is fused to the N-terminus of the Fc region in the heavy chain.

It will be appreciated that the above descriptions of "substitutions" and "substitutions" of the TCR constant domain for the CH1 and CL regions do not imply that the bsAB has been prepared by particular methods such as deletion, insertion or substitution of the regions, but merely indicate the reference to the location of typical IgG antibodies and typical Fab fragments.

In other embodiments, the TCR-containing bsAb has a pI of about 5.5-6.5, e.g., a pI of about 6.0-6.5, e.g., a pI of about 6.1-6.5.

Impurities in bsAb production may include process-related impurities (e.g., HCP and host DNA) and product-related impurities (e.g., homodimers, low molecular weight impurities such as half antibodies, 3/4 antibodies, HC dimers, free HC, and free LC, and high molecular impurities such as polymers). For TCR-containing bsAb, e.g. WuXiBody ^TM Bispecific antibodies, a significant portion of the impurities would be those designed without the TCR constant domain, also referred to herein as "TCR-free by-products," such as TCR-free half-antibodies and TCR-free homodimers. Meanwhile, for the symmetric bsAb, the product-related impurities are mainly aggregates. As shown in the examples below, in the methods of the invention, protein a chromatography is effective to remove process-related impurities (e.g., HCP and DNA), then AEX is effective to remove TCR-free byproducts, and mixed mode chromatography is effective to remove aggregates.

Thus, the method of the invention enables the obtaining of a purified bispecific antibody substantially free of impurities. Herein, "substantially free of impurities" refers to a purity of at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or higher, including 100% purity.

Examples

The present invention will be explained in further detail 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 performed as described herein or according to conventional methods, e.g. as taught in the technical manual, such as molecular cloning: a laboratory manual, or according to the manufacturer's instructions.

Materials and methods

Material

Sodium dihydrogen phosphate monohydrate, disodium hydrogen phosphate dihydrate, ammonium sulfate, ethanol, sodium acetate trihydrate, sodium chloride, sodium hydroxide, glycine and Tris (hydroxymethyl) aminomethane were purchased from Merck (darmstadt, germany). Acetic acid, L-arginine hydrochloride, histidine and histidine monohydrochloride were purchased from JT Baker, inc. (Philippisburg, N.J.). MabSelect SureLX, capto MMC ImpRes, capto adhere ImpRes and HisCaleA26/40 column (internal diameter 26mm, length 40 cm) and an XK16/40 column (internal diameter 16mm, length 40 cm) were purchased from GE Healthcare (Uppsala, sweden). Poros 50HQ and MOPS SDS running buffer (20X) were purchased from Thermo Fisher Scientific, inc (waltham, massachusetts, usa). A1HC deep filter (MA 1HC23CL3, 23 cm) ² ) And VL11/25 columns (11 mm ID, 25cm length) were purchased from Millipore corporation (Billerica, mass.). TSKgel G3000SWxl stainless steel columns (7.8X 300 mm) were purchased from Tosoh corporation (Tokyo, japan). 30% acrylamide/bisacrylamide solution (37.5. Ammonium persulfate, coomassie blue R-250, glycerol, sodium lauryl sulfate, iodoacetamide, bis-Tris, and bromophenol blue were purchased from Sigma-Aldrich (St. Louis, mo.). The following four WuXiBody bispecific antibodies, "molecule A" to molecule D ", were expressed from CHO-K1 cells using HyClone ActiPro medium and Cell Boost 7a and 7b feed medium (both purchased from GE Healthcare). Harvest cultures were clarified by centrifugation to give clear harvest.

Device for measuring the position of a moving object

Column chromatography was performed using the AKTA pure 150 system (GE Healthcare, uppsala, sweden) with Unicorn software version 7.3 installed. 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, waltham, mass.). Size exclusion chromatography-high performance liquid chromatography (SEC-HPLC) was performed using an Agilent 1260 liquid chromatograph (Agilent Technologies, inc., santa Clara, calif.). Cell culture was carried out using a bioreactor system from Applikon Biotechnology, inc. (Dutch Delfti). Harvesting cell cultures were clarified using a Sorvall LYNX 6000 ultra-high speed centrifuge from Thermo Fisher Scientific.

Method

Protein a chromatography

MabSelect SureLX was loaded onto a 2.6cm diameter column with a bed height of 20.2cm. The Column Volume (CV) was about 107.2ml. The centrifugally clarified harvest culture was loaded onto a column. Each loading was 30mg protein per ml resin. The flow rate of the system was 242 cm/hr (residence time: 5 minutes). Each time in a bind-elute mode, a stepwise pH elution is used to elute bound proteins. Specific information on the protein A chromatography process is summarized in Table 1. The chromatograms recorded are UV absorbance monitored at 280 nm. The Host Cell Protein (HCP) content, purity and concentration of the eluted samples were measured for each run.

Table 1: specific information on protein A chromatography

Step (ii) of	Volume (CV)	Composition of
			Balancing	5	50mM Tris-HAc，150mM NaCl，pH 7.4
Sample loading	NA a	Clarified harvest
			Leaching 1:	5	50mM Tris-HAc，150mM NaCl，pH 7.4
leaching 2:	5	50mM NaAc/HAc，250mM NaCl，pH 5.5
			leaching 3:	5	50mM NaAc/HAc，pH 5.5
elution is carried out	NA	50mM NaAc/HAc，pH 3.6
			Strong elution	3	1M HAc
Disinfection
		3	0.1M NaOH

^a Not applicable to

Intermediate depth filtration

Intermediate depth filtration following protein A chromatography Using Millipore A1HC (MA 1HC23CL3, 23 cm) ² ). Protein A eluted samples were first neutralized to pH5.0 (for molecules A, C and D) or pH 4.8 (for molecule B) and then at 1000g/m ² Is added to the filter. The filtrates from each run were analyzed for HCP levels, monomer purity and product recovery was measured.

AEX chromatography

AEX chromatography was performed in bind-elute mode using POROS 50HQ resin, using a column 1.6cm in diameter and a bed height of 24.0cm. CV was about 48.2ml. The specific information for each step is summarized in Table 2. Each loading was 40mg protein per ml resin. The system flow rate for each chromatography was 288 cm/h (residence time: 5 minutes). The chromatograms recorded are UV absorbance monitored at 280 nm. The eluted samples were analyzed for HCP levels and monomer purity for each run. Step recoveries were calculated from the measured concentrations.

Table 2: detailed information of AEX chromatography

^a For molecule A, the column was equilibrated at pH 7.0, for molecules B-D, the column was equilibrated at pH 8.0

^b Is not applicable to

^c For molecule A, the filtrate was adjusted to pH 7.0, and for molecules B-D, the filtrate was adjusted to pH 8.0

^d For molecule A only

^e For molecules B to D, respectively

^f For molecules A-D, respectively

Capto MMC/adhere Impres chromatography

Capto MMC Impres chromatography and Capto adhere Impres chromatography were run in bind-elute mode using columns of 1.1cm diameter and 20.0cm bed height (CV:. About.19.0 mL) and columns of 1.1cm diameter and 18.0cm bed height (CV:. About.17.1 mL), respectively. MMC and adhere were loaded at 40mg and 30mg protein per ml resin, respectively. The information about each step is shown in Table 3. The flow rates of the Capto MMC and Capto adhere systems were 240 cm/h and 216 cm/h, respectively (residence time: 5 minutes). The chromatograms recorded are UV absorbance monitored at 280 nm. The eluted samples were analyzed for HCP levels and monomer purity for each run. Step recoveries were calculated from the measured concentrations.

Table 3: MMC imprmes/adhere imprmes ^a Specific information on chromatography

^a Molecule A-C was refined twice using MMC Impp Res as second refining (po)lishing), molecule D was then used as adhere ImpRes

^b For MMC and adhere ImpRes respectively

^c Not applicable to

^d For MMC and adhere ImpRes respectively

^e For molecules A-D, respectively

^f For molecule A and molecule B only (molecule C and molecule D without rinsing 3)

^g For molecule a and molecule B, respectively.

^h For molecules A-D, respectively

ⁱ For molecules A-D, respectively

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

SDS-PAGE was performed using an 8% non-gradient Bis-Tris gel prepared by the standard method. 3.5 Xgel buffer (1.25M bis-Tris, pH 6.5-6.8) was prepared in-house. This buffer was used for both the isolation gel (8%) and the concentrate gel (4%) preparations. 80V constant voltage electrophoresis for 2 hours. The gel was stained with coomassie blue and destained with a destaining solution containing 10% acetic acid, 20% ethanol and 70% water.

SEC-HPLC

SEC-HPLC analysis was carried out on an Agilent 1260 liquid chromatograph using Tosoh TSKgel G3000SWxl stainless steel columns (7.8X 300 mm). 100 μ g of sample was injected for each run. The mobile phase was a 50mM sodium phosphate, 300mM sodium chloride solution, pH 6.8. Each sample was eluted isocratically at a flow rate of 1.0 ml/min for 20 minutes. The protein eluted sample was monitored for absorbance at 280nm in the UV.

HCP measurement

HCP levels of samples at various stages were measured using a third generation universal ELISA kit from Cygnus Technologies (shapsort, north carolina, usa) according to the manufacturer's instructions. The detection range is 3-100ng/ml. Serial dilutions were made to the samples so that the measurements were always within the calibration range. Absorbance at 450nm (absorbance) and 650nm (reference) was measured using an Infinite 200PRO plate reader (Tecan corporation, doffy in Switzerland).

Procedure

Chromatographic capture of protein A products

The clarified harvest was loaded onto a MabSelect SuRe LX column. Protein a chromatography for capture of the product was performed as described above, eluting the column with staged pH elution (fig. 2).

In general, protein a chromatography is effective in removing process-related impurities (e.g., HCPs and DNA), but has limited ability to remove product-related impurities (e.g., half antibodies and aggregates). The relevant mass data and protein a chromatography step yields for the protein a eluted samples are shown in table 4.

Table 4: protein a elution sample quality data and protein a chromatography step yield

Molecule	HCP(ppm)	SEC-HPLC(％)	Yield (%)
				A	946	85.7/7.6/6.7 a	100.0
B	1945	86.3/11.1/2.6	100.0
				C	3655	89.6/9.4/0.9	94.2
D	3145	90.4/9.6/ND b	100.0

^a Respectively as a percentage of monomer, high molecular weight material and low molecular weight material

^b Not detected

It can be seen that for all four bsabs, HCP decreased to levels of approximately 1000-4000 ppm, which is generally the average of the levels detected at this stage to low values. The SEC-HPLC purity of the protein A eluted samples ranged from about 86% to 90%. Protein a post-chromatographic byproducts of the asymmetric molecules (a and B) and symmetric molecules (C and D) were low molecular weight impurities and aggregates, respectively, according to SDS-PAGE results (fig. 2, inset) and SEC chromatograms (data not shown). The distinct characteristic impurity profiles of these two groups of bsabs were in agreement with expectations and consistent with previous studies [4-6].

Neutralization and intermediate depth filtration

Protein a eluted samples were neutralized and filtered as described above. We chose a relatively low pH for neutralization (5.0 for molecules A, C and D and 4.8 for molecule B) because the pI of WuXiBody bispecific antibodies is significantly lower than that of the normal mAb. Of the four bsabs studied, the pI of molecule B was the lowest and relatively more precipitation was observed when its protein a eluting sample was adjusted to ph 5.0. Thus, the protein a eluted sample of the molecule was titrated to pH 4.8. Even at this pH, the step yield of molecule B is still lower than the other three molecules. The data in table 5 show that the mean HCP levels of these bsabs decreased moderately after this step.

Table 5: quality data and filtration recovery rate of middle deep layer filtration filtrate

Molecule	HCP(ppm)	SEC-HPLC(％)	Yield (%)
				A	272	86.2/6.8/6.9 a	98.0
B	1458	86.6/10.7/2.5	88.9
				C	2121	91.2/7.8/0.9	94.9
D	1537	90.4/9.6/0.1	97.6

^a Respectively as a percentage of monomer, high molecular weight material and low molecular weight material

AEX chromatography

The intermediate depth filtered filtrate was subjected to AEX chromatography as described above. The four bsabs of lower pI (6.1-6.5) were all AEX chromatographed in bind-elute mode (figure 3). The mass data of the AEX eluted samples are summarized in table 6.

Table 6: quality data and AEX step yield of AEX elution samples

Molecule	HCP(ppm)	SEC-HPLC(％)	Yield (%)
				A	116	93.8/5.5/0.7 a	89.2
B	306	92.9/5.5/1.6	74.7
				C	147	93.0/6.0/1.0	88.1
D	126	90.4/9.5/0.1	88.7

^a Respectively as a percentage of monomer, high molecular weight material and low molecular weight material

HCP levels of each type of molecule were further reduced by this step. In addition, for the asymmetric form of bsAb (molecules a and B), this step greatly improved SEC purity because those TCR-free byproducts of high pI did not bind under the selected conditions. Notably, the TCR-free byproducts removed include potential homodimers. In the case of the asymmetric versions (molecules a and B), this step also effectively separated the target bsAb from the unwanted lower pI TCR-containing homodimers. Although the homodimer cannot be monitored by SEC-HPLC because its size is similar to the target product, we previously demonstrated that its presence and removal can be characterized by analytical hydrophobic interaction chromatography [12-14]. For the symmetric form of bsAb (molecules C and D), this procedure did not improve or improved very limited the SEC purity. This is not completely unexpected. Product related impurities of symmetric bsAb are mainly aggregates, and previous studies showed that IEX chromatography has limited ability to remove aggregates [11].

Capto MMC/adhere Impres chromatography

For molecules A-C, MMC ImpRes were used for the final purification step, and for molecule D, adhere ImpRes were used. MMC chromatography was performed as described above. The corresponding chromatograms are shown in fig. 4, and the relevant quality data of the final purified product are summarized in table 7. In all cases, the final polishing step further reduced the HCP to below the normally allowable standard (i.e., 100 ppm). And, the SEC purity of molecules a to D increased to 99.6%, 97.1%, 96.5% and 95.3%, respectively (fig. 5).

Table 7: MMC/adhere ^a Elution sample quality data and MMC/adhere step yield

^a The molecules A-C adopt MMC imprres as secondary refining, and the molecule D adopts adhere imprres

^b The percentages of the monomers, the high molecular weight substances and the low molecular weight substances respectively

^c Not detected

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

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Claims (18)

1. A method of purifying a bispecific antibody from a liquid comprising the bispecific antibody with TCR constant domains and potential impurities, the method comprising subjecting the liquid to protein a chromatography followed by anion exchange chromatography and mixed mode chromatography in sequence, thereby obtaining a bispecific antibody purification product; wherein the anion exchange chromatography is performed in bind-elute mode.

2. The method of claim 1, wherein the elution of the protein a chromatogram employs a gradient pH elution or a step pH elution.

3. The method of claim 1, wherein the elution of the protein a chromatogram employs a staged pH elution.

4. The method of claim 3, wherein the stage pH elution comprises a stage at a pH of about 3.0-4.0.

5. The method of claim 4, wherein the stage pH elution comprises a stage at a pH of about 3.6.

6. The method of claim 1, wherein the anion exchange chromatography column elutes at about pH 8.0.

7. The method of claim 1, wherein the anion exchange chromatography column elutes at an ionic strength of about 130-190 mM.

8. The method of claim 7, wherein the anion exchange chromatography column elutes at an ionic strength of about 130-165 mM.

9. The method of claim 1, further comprising an intermediate depth filtration after the protein a chromatography and before the anion exchange chromatography, wherein the filtrate is obtained after filtration after titrating an eluted sample of the protein a chromatography to a pH of about 4-5.

10. The method of claim 1, wherein the mixed mode chromatography is Capto MMC imprmes or Capto adhere imprmes and is performed in a bind-elute mode.

11. The method of claim 1, wherein the bispecific antibody has a pI of about 5.5-6.5.

12. The method of claim 11, wherein the bispecific antibody has a pI of about 6.0-6.5.

13. The method of claim 1, wherein the bispecific antibody is WuXiBody ^TM Bispecific antibodies are of the art in which one or more Fab units comprise a TCR constant domain.

14. The method of claim 1, wherein the bispecific antibody is asymmetric.

15. The method of claim 14, wherein the bispecific antibody is of the KiH type.

16. The method of claim 1, wherein the bispecific antibody is symmetric.

17. The method of claim 1, wherein the impurities comprise one or more of: host cell proteins, DNA, by-products without TCR constant domains, homodimers containing TCR constant domains, and aggregates.

18. The method of claim 1, wherein the purified product of the bispecific antibody is substantially free of impurities.

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