EA046335B1 - METHODS FOR DETERMINING CHARACTERISTICS OF PROTEIN COMPLEXES - Google Patents

Description

Cross reference to related applications

The invention claims benefit and priority under U.S. Provisional Application No. 62/724,700, filed August 30, 2018, and incorporated by reference in its entirety.

Technical field of the invention

The present invention generally relates to systems and methods for characterizing protein complexes.

BACKGROUND OF THE INVENTION

Monoclonal antibodies (mAbs) are an emerging therapeutic area, with more than 50 monoclonal antibodies currently on the market. Combination therapy with more than one monoclonal antibody has the potential to improve the effectiveness of existing monotherapies. Certain soluble substances, especially multimeric substances with multiple repeat epitopes, can bind to two or more antibodies, resulting in the formation of large complexes. The production of large heterogeneous antibody complexes is called modular diversity. Large antibody complexes can be rapidly eliminated by phagocytosis, resulting in reduced antibody effectiveness. Large protein complexes can also increase the immunogenicity of mAbs.

Protein complexes can vary in size from nanometer to visible particles, making it difficult to characterize them using a single analytical technique. One of the most common techniques for determining particle sizes from ~1 nm to ~1 μm is dynamic light scattering (DLS). DLS is an analytical technique used to determine the size distribution profile of proteins that is suitable for high-throughput applications (Zhou, M., et al., ChemMedChem, 11:738-756 (2016)). Brownian motion of proteins in solution causes light scattering, with variations in the intensity of the scattered light depending on the particle size. In this way, the average radius and width of the distribution with respect to polydispersity can be determined. However, DLS results are often biased toward larger particles, and groups of particles must differ by at least a factor of three to separate them. Therefore, DLS alone is not sufficient for the analysis of protein complexes.

To obtain more detailed information, due to the wide range of sizes of protein complexes and the high polydispersity of many aggregate samples, separation-based methods are required. Currently, size exclusion chromatography (SEC) is the most commonly used chromatography technique for protein separation (Brusotti, et al., Chromatographia, 81:3-23 (2018)). In SEC, separation occurs based on hydrodynamic volume or molecular size. Smaller molecules are retained longer because they can diffuse into the pores of the stationary phase, while larger molecules elute first because they pass by the pores. However, the disadvantages of SEC are its upper limits on molecular weight exclusion, sample adsorption to the stationary phase, shear failure at high pressures and flow rates, and the inability to separate analytes based on their composition.

Field-assisted flow fractionation (FFF) is a promising alternative to SEC when it comes to the separation of large proteins and high molar mass polymers. Sample separations using FFF use a flow separation and fractionation method in which analytes are separated along a ribbon-shaped channel due to differences in their diffusion coefficients (Fraunhofer, W. and Winter, G., Eur. J. Pharm. Biopharm, 58:369- 383 (2004)). FFF enables the separation of analytes over a wide range of sizes (from nanometers to microns). The open FFF channel helps reduce sample loss and allows the use of low pressures and low tangential velocities. Although FFF has been used in combination with other molecular techniques, such as light scattering, to identify protein aggregates, there is still a growing need to more fully characterize the heterogeneity and conformation of protein complexes, including mAb complexes and soluble ligands.

Therefore, it is an object of the present invention to provide methods for identifying protein drug products that have the ability to form large protein complexes.

Another object of the present invention is to provide methods for identifying and characterizing complexes of protein drug products and soluble ligands.

Brief description of the invention

Methods are provided for characterizing protein complexes in a sample. One embodiment provides a method for assessing the stoichiometry and size distribution of protein complexes in a sample by fractionating the sample via cross-field flow fractionation (A4F) and determining the molar mass, stoichiometry and size distribution of protein complexes in the sample using multi-angle laser light scattering (MALLS). . In some embodiments, the protein complexes comprise an antibody or fusion protein linked

- 1 046335 with its ligand. Such a ligand is usually a soluble ligand. The ligand may be monomeric or multimeric. In one embodiment, the ligand may be a homodimer or heterodimer. In another embodiment, the protein complexes comprise or consist of antibody:ligand complexes or fusion protein:ligand complexes.

Another embodiment provides a method for selecting a major protein drug product by adding a first protein drug product to its target or ligand to form protein:ligand complexes and adding a second protein drug product to the first complexing the protein drug product and the ligand to produce a plurality of protein:ligand complexes. This method involves the separation of protein:ligand complexes and determination of the molar mass, stoichiometry and size distribution of protein-ligand complexes using flow transverse field fractionation - MALLS. The method also involves selecting a protein drug product that forms fewer large protein:ligand complexes as the primary target protein drug. Typically, the protein drug product is an antibody or an antigen-binding fragment thereof, a fusion protein, or a recombinant protein. In some embodiments, the ligand is a soluble ligand. The ligand may be monomeric or multimeric. In some embodiments, the large protein complexes are heterometric.

Another embodiment provides a pharmaceutical composition containing a protein drug base product selected by the method described above.

In some embodiments, the disclosed methods can be used to determine whether two separate antibodies targeting the same ligand will form large heterogeneous complexes.

Another embodiment provides a method for characterizing protein complexes formed between protein drug products and soluble ligands by obtaining a sample containing the protein drug product and its ligand, thereby obtaining complexes of the protein drug product. drug, and ligand. This method involves fractionating drug:ligand protein complexes and analyzing the fractionated drug:ligand protein complexes using multi-angle laser scattering to determine the molar mass and heterogeneity of the protein complexes. In one embodiment, total protein fractionation is accomplished by flow-through cross-field fractionation. Protein concentration is determined in the UV/visible part of the spectrum.

Differences in the conformation of protein complexes formed by different protein drug products with the same ligand can be determined by comparing the elution profile/time of protein drug product-ligand complexes formed by different protein drug products. medicine. From the different profiles/elution times of complexes with the same molar mass, it is clear that the complexes can have different conformations or shapes. In one embodiment, each protein drug product and the same soluble ligand are analyzed separately to calculate the molar mass for each individual component, wherein the molar mass of each component is used to determine the estimated stoichiometry of said components in each protein complex.

Another embodiment provides a method for characterizing protein complexes formed between protein drug products and soluble ligands by fractionating the protein drug product and ligand complexes through cross-field flow fractionation, analyzing the fractionated product complexes, of the protein drug product and the ligand using multi-angle laser scattering to characterize the molar mass, stoichiometry, or both of the protein drug product and ligand complexes and determine the heterogeneity of the protein complexes by comparing the size of each protein complex with the estimated size of each individual component to identify the components that make up each complex.

Brief description of graphic materials

Fig. 1A is an illustration of a complex formed between one antibody and one ligand. Fig. 1B is an illustration of a complex formed between one antibody and two ligands. Fig. 1C is an illustration of a complex formed between four antibodies and five ligands, reflecting manual design.

Fig. 2A-2B are an illustration of a complex formed between Antibody 1 (black), two ligands and Antibody 2 (gray) in a non-linear conformation. Fig. 2C-2D represent

- 2 046335 is an illustration of a complex formed between antibody 1 (black), two ligands and antibody 2 (gray) in a linear conformation.

In fig. 3A shows a chromatogram obtained from SEC-MALLS analysis of Ab1, protein X, and combinations of Ab1 and protein X in molar ratios of 5:1, 1:1, and 1:5. The X-axis represents the elution time (minutes). The left Y-axis represents the molar mass (g/mol), and the right Y-axis represents the optical density at 280 nm (OD). Fig. 3B is a fractogram of the results of flow-through fractionation in a transverse field - MALLS (A4FMALLS) analysis of combinations of Ab1 and Ab2 in molar ratios of 5:1, 1:1 and 1:5. The X-axis represents the elution time (minutes). The left Y-axis represents molar mass (g/mol). The right Y-axis represents the optical density at 215 nm (OD).

Fig. 4A is a fractogram of the results of the A4F-MALLS analysis of core A (core A), core A+protein Y (1:3), and core B (core B)+protein Y (1:3). Fig. 4B is a fractogram of the results of the A4F-MALLS analysis of basic A, basic A+protein Y (1:1), and basic B+protein Y (1:1). Fig. 4C is a fractogram of the results of the A4F-MALLS analysis of basic A, basic A+protein Y (3:1), and basic B+protein Y (3:1). The X-axis represents time (minutes). The left Y-axis represents the molar mass (g/mol), and the right Y-axis represents the optical density at 215 nm (OD).

Fig. 5A and 5B are line graphs of mouse anti-human antibody titer in anti-Protein Y antibody complexes with major A (FIG. 5A) and major B (FIG. 5B). The X-axis represents time (days) and the Y-axis represents concentration (μg/mL).

Fig. 6A and 6B are line graphs showing the percentage of rabbit erythrocyte hemolysis with increasing concentrations of different Z protein mAbs (FIG. 6A) or combinations of Ab3 and different Z protein mAbs (FIG. 6B). The X-axis represents the mAb concentration (Log [M]). The Y-axis represents the percentage of hemolysis.

Fig. 7 is a fractogram of the results of the A4F-MALLS assay of free anti-Protein Z mAb1, Protein Z, and the combination of 1 μM:1 μM Anti-Protein Z mAb1 and Protein Z. The x-axis represents elution time (minutes). The left Y-axis represents the molar mass (g/mol), and the right Y-axis represents the optical density at 215 nm (OD).

Fig. 8 is a fractogram of the results of the A4F-MALLS assay of free mAb, the combination of 1 μM:1 μM mAb1 to protein Z and protein Z, the combination of 0.5 μM:0.5 μM:1 μM mAb1 to protein Z, mAb5 to protein Z and protein Z, as well as 0.5 µM:0.5 µM combinations: 1 µM mAb 1 to protein Z, mAb7 to protein Z and protein Z. The X-axis represents elution time (minutes). The left Y-axis represents the molar mass (g/mol), and the right Y-axis represents the optical density at 215 nm (OD).

Fig. 9 is a fractogram of the results of the A4F-MALLS analysis of the combination of free mAb, the combination of 1 μM:1 μM mAb1 to protein Z and protein Z, the combination of 0.5 μM:0.5 μM:1 μM mAb1 to protein Z, the combination of mAb3 to protein Z and protein Z and combinations 0.5 µM:0.5 µM:1 µM mAb1 to protein Z, combinations of mAb6 to protein Z and protein Z, and combinations 0.5 µM:0.5 µM:1 µM mAb6 to protein Z , mAb7 to protein and protein Z. The x-axis represents the elution time (minutes). The left Y-axis represents the molar mass (g/mol), and the right Y-axis represents the optical density at 215 nm (OD).

Fig. 10 is a fractogram of the results of the A4F-MALLS analysis of the combination of free mAb, combination of 1 μM: 1 μM mAb1 to protein Z and protein Z, combination of 0.5 μM: 0.5 μM: 1 μM mAb1 to protein Z, mAb2 to protein Z and protein Z, and combinations of 0.5 μM:0.5 μM:1 μM mAb1 to protein Z, mAb COMP1 and protein Z. The x-axis represents elution time (minutes). The left Y-axis represents the molar mass (g/mol), and the right Y-axis represents the optical density at 215 nm (OD).

Fig. 11 is a fractogram of the results of the A4F-MALLS analysis of a combination of free mAb, a combination of 1 μM: 1 μM mAb1 to protein Z and protein Z, a combination of 0.5 μM: 0.5 μM: 1 μM mAb1 to protein Z, mAb4 to protein Z and protein Z. The X-axis represents the elution time (minutes). The left Y-axis represents the molar mass (g/mol), and the right Y-axis represents the optical density at 215 nm (OD).

Fig. 12A and 12B are a fractogram of the results of the A4F-MALLS assay, or simultaneous addition (Fig. 12A), or sequential addition (Fig. 12B) of 0.3 μM:1 μM:1 μM mAb1 to Z protein: COMP1 mAb: Z protein. 1 μM: 1 μM: 1 μM mAb1 to protein Z: mAb COMP1: protein Z and 3 μM: 1 μM: 1 μM mAb1 to protein Z: mAb COMP1: protein Z. The X-axis represents the elution time (minutes). The left Y-axis represents the molar mass (g/mol) and the right Y-axis represents the optical density at 215 nm (OD).

Fig. 13 is a fractogram of the results of the A4F-MALLS assay of representative mAbs, W protein, and combinations of mAbs and W protein in ratios of 1 μM: 1 μM. The X-axis represents the elution time (minutes). The left Y-axis represents the molar mass (g/mol), and the right Y-axis represents the optical density at 215 nm (OD).

Fig. 14 is a fractogram of the results of the A4F-MALLS analysis of representative mAbs, W protein and combinations of mAb2 and W protein, mAb3 and W protein, and COMP1 and W protein in ratios of 1 μM: 1 μM. The X-axis represents the elution time (minutes). The left Y axis represents molar mass

- 3 046335 (g/mol), and the right Y-axis represents the optical density at 215 nm (EOD).

Fig. 15 is a fractogram of the results of the A4F-MALLS analysis of COMP2, W protein, and combinations of COMP2 and W protein in ratios of 1 μM: 1 μM. The X-axis represents the elution time (minutes). The left Y-axis represents the molar mass (g/mol), and the right Y-axis represents the optical density at 215 nm (OD).

Fig. 16 is a fractogram of the results of the A4F-MALLS analysis of representative mAbs, protein W and combinations of mAb4 and protein W, and COMP3 and protein W in ratios of 1 μM: 1 μM. The X-axis represents the elution time (minutes). The left Y-axis represents the molar mass (g/mol) and the right Y-axis represents the optical density at 215 nm (OD).

Detailed Description of the Invention

I. Definitions.

It should be understood that the present invention is not limited to the compositions and methods described herein, nor to the experimental conditions described, as these may vary. It should also be understood that the terminology used herein is for purposes of describing certain embodiments only and is not intended to be limiting, as the scope of the present invention will be limited only by the appended claims.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. However, in the practice or testing of the present invention, any compositions, methods and materials similar or equivalent to those described herein can be used. All publications mentioned are incorporated herein by reference in their entirety.

The use of singular terms and similar definitions in the context of the description of the present invention (especially in the context of the claims) should be construed to include both the singular and the plural, unless otherwise stated herein or otherwise clearly inconsistent with the context.

It is intended that the presentation of ranges of values herein serve solely as a means of abbreviating the individual indication of each individual value included within a given range, unless otherwise stated herein, and each individual value is included herein as if it were individually mentioned in this document.

The use of the term approximate is intended to describe values either above or below the stated value within a range of approximately +/- 10%; in other embodiments, the values may range either above or below the stated value within a range of about +/- 5%; in other embodiments, the values may range either above or below the stated value within a range of about +/- 2%; in other embodiments, the values may range either above or below the stated value within a range of approximately +/- 1%. The preceding ranges are intended to be clear from the context and no further limitations are intended. All methods described herein can be performed in any suitable order, unless otherwise indicated herein or otherwise clearly inconsistent with the context. The use of any kind of examples or illustrative phrases (eg, such) provided herein is intended solely to better illustrate the present invention and not to state a limitation on the scope of the present invention, unless otherwise stated. Nothing in this specification should be construed as indicating that any unstated element is essential to the practice of the present invention.

Protein refers to a molecule containing two or more amino acid residues linked to each other through a peptide bond. Protein includes polypeptides and peptides and may also include products of modifications such as glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, alkylation, hydroxylation and ADP-ribosylation. Proteins may be of scientific or commercial interest, including protein-based drugs, and proteins include, but are not limited to, enzymes, ligands, receptors, antibodies, and chimeric or fusion proteins. Proteins are produced by various types of recombinant cells using well-known cell culture techniques and are typically introduced into the cell using genetic engineering techniques (e.g., such as through a chimeric protein coding sequence or codon-optimized sequence, intronless sequence, etc.). etc.), and in the cell the genetic material can be in the form of an episome or can be integrated into the cell genome.

An antibody refers to an immunoglobulin molecule containing four polypeptide chains, two heavy (H) chains and two light (L) chains, linked by disulfide bonds. Each heavy chain contains a heavy chain variable region (HCVR or VH) and a heavy chain constant region. The heavy chain constant region contains three domains - CH1, CH2 and CH3. Each lay down

- 4 046335 This chain contains a light chain variable region and a light chain constant region. The light chain constant region consists of a single domain (CL). The VH and VL regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs), interspersed with regions that are more conserved called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, located from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The term antibody includes reference to both glycosylated and non-glycosylated immunoglobulins of any isotype or subclass. The term antibody includes antibody molecules produced, expressed, generated or isolated by recombinant methods, such as antibodies isolated from a host cell transfected to express the antibody. The term antibody also includes a bispecific antibody that includes a heterotetrameric immunoglobulin that can bind to more than one different epitope. Bispecific antibodies are generally described in US Pat. No. 8,586,713, which is incorporated herein by reference.

Fusion proteins containing an Fc fragment include part or all of a protein of two or more proteins that are not otherwise found in nature together, one of which is the Fc portion of an immunoglobulin molecule. The production of fusion proteins containing certain heterologous polypeptides fused to various portions of antibody-derived polypeptides (including the Fc domain) has been described, for example, by Ashkenazi et al., Proc. Natl. Acad. ScL USA 88: 10535, 1991; Byrn et al., Nature 344:677, 1990; and Hollenbaugh et al., Construction of Immunoglobulin Fusion Proteins, in Current Protocols in Immunology, Suppl. 4, pp. 10.19.1-10.19.11, 1992. Receptor Fc fusion proteins include one or more extracellular receptor domains linked to an Fc moiety, which in some embodiments comprises a hinge region followed by a CH2- and CH3 domains of immunoglobulin. In some embodiments, the Fc fusion protein includes two or more different receptor chains that bind to one or more ligand(s). For example, the Fc fusion protein is a Trap protein, such as, for example, IL-1 Trap or VEGF Trap.

As used herein, the term soluble ligand refers to polar or charged ligands that do not readily cross the plasma membrane of a cell. Most soluble ligands bind to the extracellular domains of receptors on the cell surface.

A4F is transverse field flow fractionation, which is a fractionation technique in which the separation of analytes is achieved by interaction of the sample with an external perpendicular physical field.

The term multi-angle light scattering (MALS) describes a technique for measuring light scattered by a sample at multiple angles. It is used to determine both the absolute molar mass and the average size of molecules in a solution by identifying how they scatter light. The most common method used is directed light from a laser source, and in this case the technique can be called multi-angle laser scattering (MALLS). In practice, the terms MALS and MALLS are used interchangeably.

As used herein, the term Brownian motion refers to the continuous motion of particles suspended in a liquid.

II. Methods for determining the characteristics of protein complexes.

Combination therapy with monoclonal antibodies has emerged as a promising therapeutic strategy for the treatment of diseases such as cancer and inflammatory conditions in which multiple signaling pathways are involved. In addition, in the event that monotherapy alone does not completely inhibit this pathway, administration of more than one monoclonal antibody targeting the same pathway could be useful in completely blocking pathways involved in disease pathogenesis. However, binding of therapeutic mAbs to soluble multimeric targets can result in the formation of large heterogeneous complexes. Among other factors, the immunogenicity and clearance time of antibodies can be influenced by the size, shape, and conformation of the protein complex. Analysis of protein complexes is important to gain insight into the pharmacokinetics of mAbs during drug development.

Therefore, methods and systems are provided for characterizing protein complexes. One embodiment provides a method for assessing the stoichiometry and size distribution of heterogeneous complexes of a protein drug product and a ligand in a sample by fractionating the sample by flow-through cross-field fractionation and determining the molar mass, stoichiometry, and size distribution of the heterogeneous protein complexes in the sample with using multi-angle laser radiation scattering. Typically, protein complexes consist of a protein that specifically binds to a protein of interest, also called a target or ligand. In one embodiment, the protein that specifically binds to the target is an antibody or fusion protein.

When an antibody or fusion protein is combined with its target or ligand in vivo or in vitro, a heterogeneous mixture of antibody:ligand or fusion protein:ligand can be formed. In one embodiment, binding therapeutic proteins such as monoclonal antibodies (mAbs)

- 5 046335 or fusion proteins with soluble multimeric targets leads to the formation of large heterogeneous complexes or large heteromeric complexes. For example, large protein complexes can be characterized as protein complexes characterized by a protein:ligand ratio selected from the group consisting of 3:2, 2:3, 4:4, 6:6, or [2:2]n. Large heterogeneous complexes refer to complexes formed between several multimeric ligand molecules and several molecules of the protein drug product. The term large heteromeric complex refers to a ligand bound by two different protein drug products, such as two different antibodies, two different fusion proteins, or an antibody and a fusion protein that bind to the same ligand but at different sites.

In another embodiment, a method is provided for identifying protein drug products that form large heterogeneous complexes with soluble targets in vivo, in vitro, or both in vivo and in vitro. The method involves obtaining a sample containing a protein drug product and its soluble ligand to obtain protein drug product-ligand complexes, fractionating the sample to separate the protein drug product-ligand complexes, and analysis of fractionated protein drug product-ligand complexes using multi-angle laser scattering to determine the size and heterogeneity of protein complexes. In one embodiment, the protein sample is fractionated by flow-through cross-field fractionation.

Additional details of the disclosed methods and systems are provided below.

A. Systems for characterizing protein complexes.

In one embodiment, the system includes a cross-field flow fractionation (A4F) system and a multi-angle laser light scattering (MALLS) system. An example of a commercially available A4F system is the Eclipse™ 3+ A4F separation system. An example of a commercially available MALLS system is the HELEOS® II laser scatter meter from Wyatt Technology Dawn. The system typically includes a UV/visible detector and/or a refractive index detector. An exemplary commercially available UV/visible detector is the Agilent 1260 Infinity UV detector. An exemplary commercially available refractive index detector is the Optilab® T-rEX refractive index detector. In one embodiment, the A4F system includes a short A4F channel equipped with a 350W separator and a hydrophilic NADIR® PES (PESH) membrane with a 4 kDa MWCO. In another embodiment, the short channel for A4F is equipped with a 490W spacer and a Nadir® regenerated cellulose membrane with a 10 kDa MWCO. Exemplary mobile phases include 10 mM phosphate and 500 mM NaCl at pH 7.0. However, the advantage of A4F separation over column chromatography separation is that there are no restrictions on the type of mobile phase or carrier liquid that could be used. In one embodiment, samples are separated using a linear gradient over 60 minutes. In one embodiment, the program for monitoring the flow and cross-flows in the channel is specifically optimized to achieve the required resolution in each specific case. It should be understood that one of ordinary skill in the art will be able to modify and optimize the elution profile according to the resolution required for the particular sample being separated by the A4F.

Typically, the sample is introduced into the sample inlet of the A4F channel. The sample is then focused by allowing a carrier fluid to flow into the channel from both the inlet and outlet ports, meeting at a specific point in the channel, typically adjacent to the sample inlet, to form a focusing zone. During the focusing period, particles from the injected sample are retained in this focusing zone, allowing relaxation before fractionation. At the final stage, particle fractionation occurs. As particles move through the channel, the perpendicular separation field of the cross-flow pushes the molecules towards the bottom of the channel. As they accumulate at the bottom of the channel, they are subject to counter-diffusion back into the channel against the cross-flow field. The extent to which molecules can diffuse back into the channel is determined by their natural Brownian motion, a characteristic determined by the translational diffusion coefficient, which in turn is related to the size and shape that are unique to each individual particle. Typically, smaller particles have a higher diffusion coefficient than larger particles and can diffuse into the channel above by passing through the cross-flow field. The speed of laminar flow in the channel is non-uniform. It moves along a parabolic trajectory, with the flow velocity increasing towards the center of the channel and decreasing towards the upper and lower walls of the channel. Consequently, the speed at which the particles will travel will depend on their position in the channel. Particles with higher diffusion, located near the center of the channel, will move with greater

- 6 046335 speed. Larger particles in a shallow, slow-moving flow near the bottom wall of the channel accumulator travel at a lower flow rate and elute later than smaller particles. This results in a neat separation of particles based on their mass, with elution order from smallest to largest.

As the sample flows through the A4F channel, the leading part of the sample exits the channel through the outlet. A multi-angle laser scattering (MALLS) detector is in fluid communication with the A4F system and receives a sample from the A4F outlet. In some embodiments, the sample is first passed through a UV/visible detector to measure sample concentration as a function of absorbance. The MALLS system focuses a beam of polarized light (or laser) onto a sample molecule, and the scattered light is detected using a photodetector.

Multi-angle light scattering (MALS) measures the light scattered by a sample containing molecules, particles, or protein complexes. This scattering depends on the optical configuration of the setup, and in a typical experimental implementation, light is then detected from one or more different angles. For a single scattering angle solution, the three most popular designs are 90° (also called rectangular light scattering or RALS), 7° (also called small angle light scattering or LALS) or 173° (also called non-invasive backscattering or MBS). In the case of a multi-angle setup, there are, in principle, those with fixed angles (usually called a MALS or MALLS setup) and those with variable angles (usually called a goniometer or light scattering spectrometer). MALS typically refers to a multiple fixed angle system used as part of a particle separation facility such as the A4F. The most common application of MALS is as an absolute molar mass detector combined with a concentration detector (RI or single wavelength UV type).

MALS can be used to measure the following: M _w is the weighted average molar mass of the protein complex; R _g is the average radius of the protein complex and A2 is the solubility of the protein in solution.

B. Methods for determining the characteristics of protein complexes.

The disclosed systems and methods can be used to characterize protein complexes, for example, complexes of a protein drug product and a ligand in a sample. One embodiment provides a method for assessing the stoichiometry and size distribution of heterogeneous protein complexes in a sample by fractionating the sample through cross-field flow fractionation (A4F) and determining the molar mass, stoichiometry, and size distribution of protein complexes in the sample using multi-angle laser scattering (MALLS). ), wherein the complexes contain or consist of antibody:ligand complexes or fusion protein:ligand complexes. In some embodiments, the ligand is a soluble ligand. Typically, the antibody is a monoclonal antibody. In one embodiment, the protein complex is characterized by the ratio of its antibody or fusion protein to its ligand. In a non-limiting example, the ratio of antibody or fusion protein to ligand may be selected from the group consisting of 1:0, 0:1, 1:1, 1:2, 2:1, 2:2, 3:2, 2:3, 4:4, 6:6 or [2:2]n. It should be understood that the ratio of antibody or fusion protein to ligand will depend on the particular antibody or fusion protein to ligand being tested.

1. Mixtures of protein complexes.

To characterize protein complexes, molar mass can be determined experimentally for each component of the complex to calculate the expected molar mass of complexes with different stoichiometries. In one embodiment, each protein and ligand in the mixture is analyzed separately to determine the molar mass for each component. In one embodiment, the protein drug product and its ligand are mixed to form complexes of the protein drug product and the ligand, and the complexes are then characterized. Fractionated protein drug product-ligand complexes are subjected to A4F-MALLS to determine the molar mass of the complexes. The calculated values of the fractionated complexes are then compared with the experimentally determined molar masses of the individual components to determine the likely stoichiometric ratio of the individual components present in each complex. In one embodiment, the methods enable the detection of a 1:1 complex of a protein drug product and a ligand. In another embodiment, the methods allow the detection of any ratio of protein drug product to ligand. In a non-limiting example, the methods are capable of detecting a complex of protein drug product and ligand in ratios of 1:0, 0:1, 2:1, 1:2, 2:2, 3:2, 2:3, 4: 4, 6:6 or [2:2] _n . It should be understood that the ratio of antibody or fusion protein to ligand will depend on the particular antibody or fusion protein to ligand being tested. In some embodiments, the complex contains several different protein drug products complexed with a common soluble

- 7 046335 ligand.

A. Ligands.

The ligand in the complex of the protein drug product and the ligand may be a monomeric or multimeric ligand. In one embodiment, the ligand is a soluble ligand. In some embodiments, the soluble ligands correspond to extracellular portions of transmembrane proteins, including, but not limited to, transmembrane receptor proteins.

Monomeric ligands contain only one protein or one protein unit. Multimeric ligands may be, for example, dimeric, trimeric, etc., containing multiple proteins or protein subunits. For example, the ligands may be homodimers or heterodimers. In some embodiments, multimeric ligands bind to more than one molecule of the protein drug product. In fig. 1A shows an exemplary 1:1 antibody:ligand complex. In fig. 1B shows an exemplary 1:2 antibody:ligand complex, and FIG. Figure 1C shows an example of a hand-designed effect in which each arm of an internal antibody binds to a different ligand, creating a large heterogeneous complex.

In one embodiment, the large heterogeneous complex of protein drug product and ligand is 500 kDa in size or larger. In another embodiment, the heterogeneous complex of the protein drug product and the ligand has a size of 500-4000 kDa. In another embodiment, the large heterogeneous protein drug product-ligand complex has a protein drug product to ligand ratio of 3:2, 2:3, 4:4, or 6:6.

In one embodiment, the disclosed methods are used to determine whether a major protein drug product designed to target a multimeric ligand will form large heterogeneous complexes of the protein drug product and the ligand.

In one embodiment, the disclosed methods can be used to determine whether a multimeric ligand will form complexes with more than one protein drug product or fusion protein. The complexes that can be formed are characterized by, without limitation, protein:ligand ratios of 1:0, 0:1, 2:1, 1:2, 2:2, 3:2, 2:3, 4:4, 6:6, or [2:2]n. In fig. 1A-1C show exemplary complexes that can be formed between a multimeric ligand and a monoclonal antibody.

b. Combination of several mAbs.

Combination therapies using multiple protein drug products to target the same pathway or the same ligand are becoming increasingly popular. In some embodiments, the disclosed methods can be used to differentiate different combinations of antibodies targeting the same ligand based on the stoichiometry and size distributions of protein complexes formed by protein drug products. When two protein drug products are mixed together with a monomeric ligand, the protein drug products are capable of forming a heteromeric complex. A heteromeric complex, as defined herein, refers to two different protein drug products that bind the same target molecule. In addition, each of the two arms of the same antibody has the ability to bind two ligands, which can also be bound by a second antibody to form a heteromeric complex. In fig. 2A-2D show typical heteromeric complexes. In fig. 2A and 2C show complexes formed when one protein drug product (black) or antibody binds two ligands, and one of the ligands is also bound by a second unique protein drug product (gray). If the gray protein is bound to another ligand, which then binds to the black protein, larger heterogeneous complexes can form, as shown in FIG. 2B and 2D.

2. Selection of the main product, which is a protein drug.

Another embodiment provides a method for selecting a major protein drug product by adding a first protein drug product to a first sample containing the target of the first protein drug product to produce heterogeneous protein:ligand complexes and adding a second protein drug product to a second sample containing the target to form protein:ligand complexes. The method involves the separation of heterogeneous protein:ligand complexes and determination of the size distribution and stoichiometry of heterogeneous protein:ligand complexes by means of flow fractionation in the transverse field of multi-angle scattering of laser radiation. The methods also involve selection

- 8 046335 product, which is a protein drug that forms a smaller number of heterogeneous protein:ligand complexes, as the main target protein drug. In some embodiments, the ligand is a soluble ligand. The soluble ligand may be a monomeric ligand or a multimeric ligand. Typically, the protein drug product is an antibody or an antigen-binding fragment thereof, a fusion protein, or a recombinant protein. A protein complex can be characterized as a protein complex with a ratio of antibody or fusion protein to ligand that is selected from the group consisting of, but not limited to, 1:0, 0:1, 1:1, 1:2, 2:1, 2:2, 3 :2, 2:3, 4:4, 6:6 or [2:2]n. Another embodiment provides a pharmaceutical composition containing a protein drug base product selected by the above method.

3. Determination of the size and shape of protein complexes.

In one embodiment, the disclosed methods can be used to determine the size of protein complexes. The mixture of protein drug products and optionally soluble ligands is separated using A4F fractionation. The size and stoichiometry of protein complexes can then be determined using MALLS analysis. In MALLS analysis, a beam of polarized light (or laser) is focused onto a sample molecule and the scattered light is detected using a photodetector. Scattered light is simultaneously detected from different angles. The intensity of the scattered light at each angle is proportional to the molar mass of the complex. In one embodiment, UV/visible spectrometry is used to determine the concentration of each protein complex.

In another embodiment, the shape/conformation of the protein complex formed between different protein drug products and a common ligand can be differentiated using the disclosed methods. Differences in elution time or elution profile between complexes of the same molar mass indicate differences in the shape or conformation of the protein complexes. Complexes with the same or similar molar mass but different elution times indicate that the complex with the slower elution time has an increased hydrodynamic volume due to a difference in the shape or conformation of the complex.

Heterogeneity of molar mass and size among protein complexes can be used to predict the clearance of a protein drug product. In one embodiment, the larger the protein complex, the faster the protein drug product is cleared from the body.

c. Proteins in protein complexes.

In one embodiment, one of the proteins in the protein complex is a protein drug product or is a protein of interest suitable for expression in prokaryotic or eukaryotic cells. For example, the protein in protein complexes may be an antibody or an antigen-binding fragment thereof, a chimeric antibody or an antigen-binding fragment thereof, a ScFv or a fragment thereof, an Fc fusion protein or a fragment thereof, a growth factor or a fragment thereof, a cytokine or a fragment thereof, or an extracellular domain of a cell receptor. surface or its fragment. The proteins in these complexes may be simple polypeptides consisting of a single subunit or complex multisubunit proteins containing two or more subunits. The protein of interest may be a biopharmaceutical product, a food additive or preservative, or any protein product subject to purification and quality standards.

In some embodiments, the protein in the protein complexes is an antibody, a human antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a multispecific antibody, a bispecific antibody, an antigen binding fragment of an antibody, a single chain antibody, a diabody, a tribody or a tetrabody, a bispecific quadrivalent immunoglobulin G-like molecule , called dual variable domain immunoglobulin (DVD-IG), IgD antibody, IgE antibody, IgM antibody, IgG antibody, IgG1 antibody, IgG2 antibody, IgG3 antibody, or IgG4 antibody. In one embodiment, the antibody is an IgG1 antibody. In one embodiment, the antibody is an IgG2 antibody. In one embodiment, the antibody is an IgG4 antibody. In another embodiment, the antibody comprises a chimeric hinge. In yet other embodiments, the antibody comprises a chimeric Fc. In one embodiment, the antibody is an IgG2/IgG4 chimeric antibody. In one embodiment, the antibody is an IgG2/IgG1 chimeric antibody. In one embodiment, the antibody is an IgG2/IgG1/IgG4 chimeric antibody.

In some embodiments, the antibody is selected from the group consisting of an anti-programmed cell death receptor 1 antibody (e.g., an anti-PD1 antibody as described in U.S. Patent Application Publication No. US 2015/0203579 A1), an anti-programmed cell death ligand-1 antibody ( for example, an anti-PD-L1 antibody as described in US Patent Application Publication No. US

- 9 046335

2015/0203580 A1), anti-D114 antibodies, anti-angiopoietin-2 antibodies (e.g. anti-ANG2 antibody as described in US Pat. No. 9402898), anti-angiopoietin-like protein 3 antibodies (e.g. anti-AngPtl3 antibody as described in US Pat. No. 9018356), anti-platelet growth factor receptor antibodies (e.g., anti-PDGFR antibody, as described in US Pat. No. 9,265,827), anti-Erb3 antibodies, anti-prolactin receptor antibodies (e.g., anti-PRLR antibody, as described in US Pat. No. 9302015), anti-complement component 5 antibodies (eg anti-C5 as described in US Patent Application Publication No. US2015/0313194A1), anti-TNF antibodies, anti-epidermal growth factor receptor antibodies (eg anti-EGFR as described in US Patent No. 9132192, or anti-EGFRvIII antibody as described in US Patent Application Publication No. US 2015/0259423 A1), anti-proprotein convertase subtilisin/kexin-9 antibody (e.g. anti-PCSK9 antibody as described in US Patent No. 8062640 or US Patent No. 9540449 ), growth and differentiation factor-8 antibodies (e.g., anti-GDF8 antibody, also known as anti-myostatin antibody, as described in US Pat. Nos. 8,871,209 or 9,260,515), anti-glucagon receptor antibodies (e.g., anti-GCGR antibody, as described in US Patent Application Publication Nos. US2015/0337045A1 or US2016/0075778A1), anti-VEGF antibodies, anti-IL1R antibodies, anti-interleukin 4 receptor antibodies (e.g., anti-IL4R antibody as described in US Patent Application Publication No. US 2014/0271681 A1 or US Pat. Nos. 8,735,095 or 8,945,559), anti-interleukin 6 receptor antibodies (e.g., anti-IL6R antibody as described in US Pat. Nos. 7,582,298, 8,043,617, or 9,173,880), anti-IL1 antibodies, anti-IL2 antibodies, anti-IL3 antibodies, IL4, anti-IL5 antibodies, anti-IL6 antibodies, anti-IL7 antibodies, anti-interleukin 33 antibodies (e.g., anti-IL33 antibody as described in US Pat. Nos. 9,453,072 or 9,637,535), anti-respiratory syncytial virus antibodies (e.g., anti-RSV antibody, as described in US Patent Application Publication No. 9,447,173), anti-cluster of differentiation 3 antibodies (e.g., anti-CD3 antibody, as described in US Pat. Nos. 9,447,173 and 9,447,173 and US Patent Application No. 62/222605), anti-cluster of differentiation antibodies 20 (e.g., anti-CD20 as described in US Pat. No. 9657102 and US 20150266966 A1 and US Pat. No. 7879984), anti-CD19 antibodies, anti-CD28 antibodies, anti-cluster of differentiation 48 antibodies (e.g., anti-CD48 antibody as described in U.S. Patent No. 9,228,014), anti-Fel d1 antibodies (e.g., as described in U.S. Patent No. 9,079,948), anti-Middle East Respiratory Syndrome virus antibodies (e.g., anti-MERS antibody, as described in U.S. Patent Application Publication No. US 2015/0337029 A1) , anti-Ebola virus antibodies (e.g., as described in US Patent Application Publication No. US 2016/0215040), anti-Zika virus antibodies, anti-lymphocyte activation gene 3 antibodies (e.g., anti-LAG3 antibody or anti-CD223 antibody), anti-growth factor antibodies nervous tissue (e.g., an anti-NGF antibody as described in U.S. Patent Application Publication No. US 2016/0017029 and U.S. Patent Nos. 8,309,088 and 9,353,176) and an anti-protein Y antibody. In some embodiments, the bispecific antibody is selected from the group consisting of a bispecific anti-CD3 and anti-CD20 antibodies (as described in US Patent Application Publications Nos. US 2014/0088295 A1 and US 20150266966 A1), anti-CD3 and anti-mucin-16 bispecific antibodies (e.g., anti-CD3 and anti-Muc16 bispecific antibodies) and bispecific antibodies to CD3 and to prostate-specific membrane antigen (for example, bispecific antibody to CD3 and to PSMA). In some embodiments, the protein of interest is selected from the group consisting of abciximab, adalimumab, adalimumab-atto, ado-trastuzumab, alemtuzumab, alirocumab, atezolizumab, avelumab, basiliximab, belimumab, benralizumab, bevacizumab, bezlotoxumab, blinatumomab, brentuximab vedotin, bro dalumaba, canakinumab, capromab pendetide, certolizumab pegola, cemiplimab, cetuximab, denosumab, dinutuximab, dupilumab, durvalumab, eculizumab, elotuzumab, emicizumab-kxwh, emtansine alirocumab, evinacumab, evolocumab, fasinumab, golimumab, guselkumab, and britumomab tiuxetan, idarucizumab, infliximab, infliximab- abda, infliximab-diib, ipilimumab, ixekizumab, mepolizumab, necitumumab, nesvacumab, nivolumab, obiltoxaximab, obinutuzumab, ocrelizumab, ofatumumab, olaratumab, omalizumab, panitumumab, pembrolizumab, pertuzumab, ramucirumab, ranibizumab ba, raxibacumab, reslizumab, rinucumab, rituximab, sarilumab, secukinumab, siltuximab, tocilizumab, tocilizumab, trastuzumab, alarmrumab, ustekinumab and vedolizumab.

In some embodiments, the protein in the complexes is a recombinant protein that contains an Fc moiety and another domain (eg, an Fc fusion protein). In some embodiments, the Fc fusion protein is a receptor Fc fusion protein that contains one or more extracellular receptor domains linked to an Fc moiety. In some embodiments, the Fc fragment comprises a hinge region followed by the CH2 and CH3 domains of the IgG. In some embodiments, the receptor Fc fusion protein contains two or more different receptor chains that bind to either a single ligand or multiple ligands. For example, the Fc fusion protein is a TRAP protein, such as, for example, IL-1 Trap (eg, rilonacept, which contains the IL-1RAcP ligand binding region fused to the extracellular region of IL-1R1 fused to the Fc fragment of hIgG1; see US Pat. No. 6,927,004, which is incorporated herein by reference in its entirety) or VEGF Trap (e.g., aflibercept or zivaflibercept, which contain VEGF Flt1 receptor Ig domain 2 fused to VEGF Flk1 receptor Ig domain 3 fused to Fc- fragment of hIgG1; see US patent Nos. 7087411 and 7279159). In other countries

- 10 046335 an embodiment of an Fc fusion protein is a ScFv-Fc fusion protein that contains one or more of one or more antigen binding domain(s), such as a heavy chain variable fragment and an antibody light chain variable fragment, coupled to Fc fragment.

Examples

Example 1: A4F analysis, compared to SEC fractionation, provides superior resolution for samples containing large heterogeneous complexes.

Ways.

Mobile phase buffer for SEC-MALLS.

For the SEC-MALLS assay, the SEC mobile phase buffer composition was 10 mM sodium phosphate, 500 mM sodium chloride, pH 7.0, and filtered (through a 0.2 μm filter) before use.

SEC-MALLS analysis.

The SEC-MALLS system consisted of a Superose 6 GL column (10x300 mm; GE Healthcare, cat. no. 175172-01) coupled to an Agilent 1200 Series HPLC system equipped with an ultraviolet (UV) diode array detector. for measuring laser light scattering (LS) miniDawn TREOS® from Wyatt Technology and differential refractometer (RI) detector Optilab® T-rEX. The detectors were connected in series in the following order: UV-LS-RI. The LS and RI detectors were calibrated according to instructions provided by Wyatt Technology.

Each determined amount of Protein X mAb was combined with recombinant Protein X and diluted in 1X DPBS, pH 7.4, resulting in the following molar ratios: 5 μM Protein X mAb: 1 μM Protein X, 1 μM Protein X mAb: 1 μM protein X and 1 μM mAb to protein X: 5 μM protein X. All samples were incubated at ambient temperature for 2 h and stored unfiltered at 4°C until insertion into the column. Before injection of each sample, the column was pre-equilibrated with mobile phase buffer (10 mM sodium phosphate, 500 mM sodium chloride, pH 7.0 ± 0.1) at a flow rate of 0.3 mL/min. Bovine serum albumin (BSA; 2 mg/ml; sample weight 150 μg) was separately administered and included as a system suitability control.

SECMALLS mobile phase buffer (10 mM sodium phosphate, 500 mM sodium chloride, pH 7.0 ± 0.1) was used throughout fractionation. Each sample (100~200 μg) was injected and eluted at a flow rate of 0.3 mL/min. BSA was fractionated using the same parameters.

Mobile phase buffer for A4F-MALLS.

For the SEC-MALLS assay, the SEC mobile phase buffer composition was 10 mM sodium phosphate, 500 mM sodium chloride, pH 7.0, and filtered (through a 0.2 μm filter) before use.

A4F-MALLS.

The A4F-MALLS system consisted of an Eclipse™ 3+ A4F separation system coupled to an Agilent 1200 Series HPLC system equipped with an ultraviolet (UV) diode array detector and a Dawn HELEOS laser scattering (LS) meter. ® II from Wyatt Technology and the Optilab® T-rEX differential refractometer (RI) detector. The detectors were connected in series in the following order: UV-LS-RI. The LS and RI detectors were calibrated according to instructions provided by Wyatt Technology.

Each determined amount of Protein X mAb was combined with recombinant Protein X and diluted in 1X DPBS, pH 7.4, resulting in the following molar ratios: 5 μM Protein X mAb: 1 μM Protein X, 1 μM Protein X mAb: 1 μM Protein X and 1 µM mAb to Protein X: 5 µM Protein X. All samples were incubated at ambient temperature for 2 h and stored unfiltered at 4°C until insertion into a short Eclipse™ channel equipped with W350 separator foil (separator thickness 350 µm, separator width 2.2 cm) and using Nadir regenerated cellulose membrane with 10 kDa MWCO. Before injection of each sample, the channel was pre-equilibrated with mobile phase buffer (10 mM sodium phosphate, 500 mM sodium chloride, pH 7.0 ± 0.1). Bovine serum albumin (BSA; 2 mg/mL; sample weight 10 μg) was separately administered and included as a system suitability control.

The fractionation method consisted of four stages: injection, focusing, elution and channel washing stage. Mobile phase buffer for A4F-MALLS (10 mM sodium phosphate, 500 mM sodium chloride, pH 7.0 ± 0.1) was used throughout fractionation. Each sample (7 μg) was injected at 0.2 mL/min for 1 min and then focused for 2 min at a focal flow rate of 1.5 mL/min. The sample was eluted at a channel flow rate of 1.0 mL/min with a linear cross-flow gradient from 3.0 to 0 mL/min over 45 min. Finally, the crossflow was held at 0 mL/min for an additional 5 min. for cleaning the channel. BSA was fractionated using the same parameters.

Analysis of MALLS data.

- 11 046335

Data were analyzed using ASTRA V software (version 5.3.4.14 from Wyatt Technology). The data were converted into an equation that related excess scattered light radiation to solute concentration and weighted average molar mass Mw (Wyatt, 1993; Kendrick, 2001).

K*s ¹

----------н 2 AcEquation 1: MwP(&) , where c denotes the solute concentration, R^, c) denotes the Rayleigh coefficient of solute excess as a function of scattering angle and concentration, Mw denotes molar mass , Ρ(Θ) describes the angular dependence of the scattered light radiation (~1 for particles with a radius of rotation < 50 nm), A ₂ denotes the second virial coefficient in the factorization of osmotic pressure (which can be neglected, since the measurements were carried out on dilute solutions) and άηΫ equation!: -kD \dcj where n ₀ denotes the refractive index of the solvent, N _A denotes Avogadro's number, λ ₀ denotes the wavelength of incident light radiation in a vacuum and dn/dc denotes the specific increment of the refractive index for the solute.

The molar mass of the BSA monomer served to estimate the calibration constants of the light scattering detectors and the differential refractive index during data acquisition (system suitability check). The relative standard deviation (%RSD) of the average molar mass of BSA determined using the UV and RI detectors was <5.0%.

Normalization factors for light scatter detectors, interdetector delay volume, and zone blur conditions were calculated from BSA chromatograms obtained for the A4F-MALLS conditions used. These values were applied to the data files collected for all other samples to adjust for these conditions.

The dn/dc value and extinction coefficient at 215 nm or 280 nm (corrected for glycosylation) were determined experimentally using a protein conjugate assay implemented in Astra software. The corrected extinction coefficient and dn/dc value were used to analyze all protein-protein complex samples.

Results.

The results of SEC-MALLS analysis of the samples showed poor resolution of higher order complexes (elution volume = 8-14 ml) and the inability to differentiate intermediate complexes (Fig. 3A, Table 1). In contrast, the results of A4F-MALLS analysis of the samples showed excellent resolution of higher order complexes (elution time = 11-30 min) and clear differentiation of intermediate complexes (Fig. 3B, Table 2).

Table 1

Approximate molar mass and retention volume for mAb:protein X complexes

Sample Molar ratio (mol:mol) Peak 1 Peak 2 Peak 3 Peak 4 Higher order complexes [Intact Antibody] 2: [Protein Χ]ι Complex [Intact antibody] g [Protein Χ]ι Complex [Intact antibody] g [Protein X] ₂ Complex EV, min. _Mw , kDa EV, min. Mw? kDa EV, min _Mw , kDa EV, min. _Mw , kDa PAS: protein X 5:1 9.0- 12.6 -390- 1000 13.1 331 N.A. N.A. ΝΑ N.A. PAS: protein X 1:1 8.0- 13.4 -400- 3000 N.A. N.A. 15.4 167 ΝΑ N.A. PAS: protein X 1:5 8.5- 13.8 -430- 2000 N.A. N.A. N.A. N.A. 16.0 216

EV: elution volume; _Mw : weighted average molar mass; NA: no data; min: minutes; kDa: kilodalton.

- 12 046335

table 2

Approximate molar mass and retention times for mAb:protein X complexes

Sample Molar ratio (mol:mol) Peak 1 Peak 2 PikZ Peak 4 Peak 5 [Intact antibody] p [Protein X]1-2 Complex [Intact antibody] 2'. [Protein Χ]ι Complex [Intact antibody] h: [Protein X] ₂ Complex [Intact antibody] 4'. [Protein X]z Complex Higher order complexes K, min M _w kDa RT, min _Mw , kDa RT, min Mw? kDa RT, min _Mw , kDa RT, min _Mw , kDa PAS: protein X 5:1 N.A. N.A. 10.8 321 12.4 498 13.7 674 14.6 830119 0 PAS: protein X 1:1 N.A. N.A. 10.7 333 12.5 515 13.7 671 14.6 780162 0 mAb 1: white kX 1:5 9.4 191 N.A. N.A. 11.8 461 13.3 600 14.4 720118 0

Rt: retention time; _Mw : weighted average molar mass; NA: no data; min: minutes; kDa: kilodalton.

Example 2. Complexes with antibodies to protein Y.

Ways.

Mobile phase buffer for A4F-MALLS.

1X DPBS, pH 7.4, was prepared by diluting 500 ml of 10X DPBS with HPLC grade water to a total volume of 4.9 L. A solution of 0.0025% (w/v) sodium azide was added as an antimicrobial agent. Hydrochloric acid (12 M) was added slowly in small volume increments to adjust the pH to 7.4 before bringing the final volume to 5.0 L. The final buffer pH measured was 7.4. A fresh buffer solution was prepared and filtered (through a 0.2 μm filter) before use.

A4F-MALL S analysis.

Each of the specified amounts of protein Y mAb (core A and core B) was combined with recombinant human protein Y and diluted in 1X DPBS, pH 7.4, to give the following molar ratios: 1 μM mAb and protein Y: 3 μM hActA, 1 µM mAb and protein Y: 1 µM protein Y and 3 µM mAb and protein Y: 1 µM protein Y. All samples were incubated at ambient temperature for 2 hours and stored unfiltered at 4°C until injection into the Eclipse™ short channel , equipped with W490 separator foil (separator thickness 490 µm, separator width 2.2 cm) and using Nadir regenerated cellulose membrane with 10 kDa MWCO. Before each sample was introduced, the channel was pre-equilibrated with 1X DPBS buffer, pH 7.4. Bovine serum albumin (BSA; 2 mg/mL; sample weight 10 μg) was separately administered and included as a system suitability control.

The fractionation method consisted of four stages: injection, focusing, elution and channel washing stage. Mobile phase buffer for A4F-MALLS (1X DPBS, pH 7.4) was used throughout fractionation. Each sample (10 μg) was injected at 0.2 mL/min for 1 min and then focused for 2 min at a focal flow rate of 1.5 mL/min. The sample was eluted at a channel flow rate of 1.0 mL/min with a linear cross-flow gradient from 1.2 to 0 mL/min over 20 min. Finally, the crossflow was held at 0 ml/min for an additional 5 min to wash the channel. BSA was fractionated using the same parameters.

Titer of mouse antibodies to human antibodies.

Mouse anti-human antibody (MAHA) titers were determined using a sandwich ELISA specific for the detection of mouse IgG to mAb A or mAb B. Briefly: mAb A or mAb B at a concentration of 1 μg/ml in phosphate-buffered saline (PBS) passively adsorbed onto a microtiter plate overnight at 4°C, followed by blocking of nonspecific binding with 5% bovine serum albumin (BSA) in PBS. Serial dilutions of serum samples were prepared in dilution buffer (0.5% BSA in PBS), starting at 1:100. Therefore, the corresponding dilution factor (100) was taken as the lower limit of detection (LOD) of the assay. Samples were then added to a plate coated with mAb A or mAb B (100 μl/well) and incubated for 16–18 h at 4°C. To determine background signal, wells containing only dilution buffer were included in the assay. Then captured on a MANA plate specific to mAb A or mAb B, you

- 13 046335 was demonstrated using horseradish peroxidase (HRP)-conjugated anti-mouse Fcy antibody in an amount of 40 ng/ml. To detect HRP activity, a chromogenic substrate for HRP was used, i.e. 3,3',5,5'-tetramethylbenzidine (TMB); and the resulting optical density at 450 nm (OD450) was read on a Perkin Elmer Victor X4 multi-mode plate reader. Binding signal data versus dilution factor were analyzed by nonlinear regression using GraphPad Prism software and titers were calculated. The MAHA titer was defined as the calculated dilution factor of the serum sample corresponding to a binding signal equivalent to twice the background signal of the assay.

Results.

The two major mAbs formed distinctly different complexes with protein Y. Under all conditions tested, major-A mAb formed smaller, less heterogeneous complexes with protein Y than did major-B mAb (Figures 4A-4C, Tables 3-5).

Table 3

Approximate molar mass and retention times for mAb complexes: Protein Y

Sample Molar ratio (mol:mol) Peak 1 Peak 2 PikZ [Intact Antibody] ι: [Protein Y] ₂ Complex [Intact Antibody] 2: [B ate ok From Complex Higher order complexes _Rt , min. Mw? kDa _Rt , min. _Mw , kDa K, min. _Mw , kDa Basics -A: protein Y 1:3 10.1 216 N.A. N.A. 13.1 -500- 1000 Basics -B: protein 1:3 10.1 219 12.5 390 14.3 -550- 2000

R _t : retention time; _Mw : weighted average molar mass; NA: no data; min: minutes; kDa: kilodalton.

Table 4

Approximate molar mass and retention times for mAb complexes: Protein Y

Sample Molar ratio (mol:mol) Peak 1 Peak 2 PikZ Peak 4 [Intact Antibody] 2: [Protein Υ]ι.2 Complex [Intact antibody] 2-h: [Protein Υ] ₃ - ₄ Complex [Intact Antibody^: [Protein Y] ₃ Complex Higher order complexes K, min. _Mw , kDa RT, min. _Mw , kDa RT, min. _Mw , kDa RT, min. _Mw , kDa Main- A: protein 1:1 11.9 348 13.5 589 N.A. N.A. 16.5 -850- 1400 Main- B: protein 1:1 N.A. N.A. 14.0 449 15.3 636 16.4 -815- 2300

R _t : retention time; _Mw : weighted average molar mass; NA: no data; min: minutes; kDa: kilodalton.

Table 5

Approximate molar mass and retention time for mAb:protein Y complexes

Sample Molar ratio (mol:mol) Peak 1 Peak 2 [Intact Antibody] 2: [Protein Υ]ι Complex Higher order complexes _Rt , min. _Mw , kDa _Rt , min. _Mw , kDa Basic-A: Protein Y 3:1 11.3 265 13.0 -400-900 Core-B: Protein Y 3:1 11.6 307 12.9 -480-1600

R _t : retention time; _Mw : weighted average molar mass; NA: no data; min: minutes; kDa: kilodalton.

The size and heterogeneity of protein Y antibody complexes correlated well with the PK observations in mice (Fig. 5A, 5B). The larger complexes observed in the case of basic-B with protein Y were correlated with faster clearance (Fig. 5B).

Example 3. Complexes with antibodies to human protein Z.

Ways.

Receiving samples.

Samples were prepared in 1X DPBS, pH 7.4, and incubated at room temperature for 2 hours before total protein fractionation using A4F-MALLS. The following samples were used: 1 mM mAb1 to protein Z + secondary mAb (0.5 μM + 0.5 μM) + 1 μM additional protein Z (7

- 14 046335 combinations) or 1 mM mAb6 to protein Z + mAb7 to protein Z (0.5 μM + 0.5 μM) + 1 μM additional protein Z. The list of secondary antibodies can be found in table. 6.

Table 6

Sample nomenclature

Secondary mAbs tested Nomenclature of combination with mAbl to protein Z mAL2 to protein Z Combination with shAL2 shalz to protein Z Combination with shabz mAL4 to protein Z Combination with shAL4 mAL5 to protein Z Combination with shAL5 mAb to protein Z Combination with shab mAL7 to protein Z Combination with shAL7 mAb COMP1 Combination with COMP1

A4F-MALLS analysis.

All samples were incubated at ambient temperature for a total of 2 hours and stored unfiltered at 4°C until insertion into an Eclipse™ short channel equipped with W350 separator foil (350 µm separator thickness, 2.2 cm separator width) and using a membrane from hydrophilic PES (PESH) with MWCO 4 kDa. Before injection of each sample, the channel was pre-equilibrated with mobile phase buffer (10 mM sodium phosphate, 500 mM sodium chloride, pH 7.0 ± 0.1). BSA (2 mg/ml; sample weight 10 μg) was added separately and included as a control for the suitability of the system.

The fractionation method consisted of four stages: injection, focusing, elution and channel washing stage. Mobile phase buffer for A4F-MALLS (10 mM sodium phosphate, 500 mM sodium chloride, pH 7.0 ± 0.1) was used throughout fractionation. Each sample (7 μg of complex or 4 μg of individual component) was injected at a rate of 0.2 ml/min and focused for 5 min with a focal flow rate of 1 ml/min. The sample was eluted at a channel flow rate of 1 ml/min with a linear cross-flow gradient from 2 to 0 ml/min over 45 min. Finally, the cross-flow was held at 0 mL/min for an additional 5 min to clean the channel. BSA was fractionated using the same parameters.

RBC hemolysis assay.

Alternative pathway (AP) hemolysis assay was used as an indicator of complement activation to evaluate the ability of protein Z mAb to block lysis of rabbit red blood cells (RbRBC). The basis of the assay is the lysis of rabbit erythrocytes using a membrane attack complex, with which complement activation is measured experimentally.

The required number of RbRBCs were washed in GVB-Mg ²⁺ /EGTA buffer and resuspended at 2x10 ⁸ cells/ml. To test the efficacy of either a single C5 mAb or a combination of multiple C5 mAbs, normal human serum was diluted to 50-96% in GVB-Mg ²⁺ /EGTA buffer to achieve a final concentration of 25-48% when added to RBC. Round-bottomed 96-well plates were used to measure hemolysis activity. A total of 100 μl of RbRBC (2 x 10 ⁸ cells/ml) was seeded into a 96-well plate at 37°C, followed by the addition of 100 μl of diluted serum. The cells were gently mixed and incubated at 37°C for 30–120 min. After the incubation time, the cells were pelleted by centrifugation at 1250 xg at 4°C. A total of 100 μL of supernatant was transferred to a fresh flat-bottomed 96-inch plate and read at 412 nm on a Spectramax microplate reader. The percentage of hemolysis was calculated as described below.

The percentage of hemolysis was calculated from the optical density values using the following equation:

„ (experimental cell lysis - background cell lysis) equation 3: %hemolysis = 100 X —-— ------ _: ^£ _: ---------—.

(maximum cell lysis - background cell lysis)

In this equation, background cell lysis means an OD of A412 nm from cells incubated in serum-free GVB-Mg ²⁺ /EGTA buffer alone. Maximum cell lysis means an OD of A412 nm from water-treated cells. Maximum lysis inhibition was calculated as the difference between the lower and upper values on the curve, expressed as a percentage of the upper value. Data are presented as mean + standard error of the mean.

Results.

Anti-Z protein mAb1 (primary Z protein mAb) in combination with COMP1 mAb or other anti-Protein Z mAbs completely blocked rabbit RBC hemolysis through activation of the alternative pathway (Fig. 6B, Table 8) compared with monotherapies that did not completely block hemolysis (Fig. 6A, Table 7). Because all combinations of anti-Z protein mAb1:anti-Z protein mAb completely blocked rabbit RBC hemolysis, it was important to determine whether there were differences in complex formation, such as size, shape, and orientation, that could provide insight into the pharmacokinetics (PK) of the mAb during drug development, such as immunogenicity and/or target-mediated clearance.

- 15 046335

Table 7

Effect of antibodies to protein Z on rabbit RBC hemolysis

Squirrel ID AP, IC50 [M] AR, IC80 [M] % of maximum lysis inhibition mAb 1 to protein Z 8.896е-008 1.970е-007 81.25 mAL2 to protein Z 8.366е-008 1.81 Ze-007 88.66 shalz to protein Z 5.252е-008 9.390е-008 59.24 mAL4 to protein Z 4.942е-008 5.963е-008 36.63 mAL5 to protein Z 7.419е-008 1.467е-007 77.83 mAb to protein Z 9.346е-008 2.260е-007 62.80 mAL7 to protein Z 7.424e-008 1.414e-007 76.21 mAb COMP1 6.291е-008 1.185e-007 61.14 mAb COMP2 4.568е-008 5,600е-008 88.83

Table 8

Effect of combinations of antibodies to protein Z on rabbit RBC hemolysis

Squirrel ID AP, IC50 [M] AR, IC80 [M] % maximum lysis inhibition ShAY to protein Z+mAb2 to protein Z 7.182е-008 7.528е-008 97.59 mAbl to protein Z + mAbl to protein Z 7.709е-008 8.749е-008 98.11 mAbl to protein Z+mAb4 to protein Z 9.751е-008 1.064e-007 98.16 mAbl to protein Z+mAb5 to protein Z 9.571е-008 1.051е-007 98.10 mAbl to protein Z+mAb6 to protein Z 7.737е-008 8.461е-008 97.29 mAbl to protein Z+mAb7 to protein Z 8.353е-008 9.432е-008 98.14 mAb 1 to protein Z+mAb COMP1 7.372е-008 7.776е-008 98.26 mAb 1 to protein Z+mAb COMP2 7.306e-008 8.017e-008 98.07

In the absence of secondary mAbs, mAb1 to protein Z formed canonical 1:1 and 1:2 complexes with protein Z when mixed in equimolar amounts (Fig. 7, Table 9).

Table 9

Approximate molar mass and retention times for mAb:protein Z complexes

Sample Molar ratio (mol:mol) Peak 1 Peak 2 [Intact Antibody]ι: [Protein Ζ]ι Complex [Intact Antibody] ι: [Protein Z] ₂ Complex _Rt , min. _Mw , kDa _Rt , min. _Mw , kDa mAbl: protein Z 1:1 13.7 341 15.3 499

Rt: retention time; _Mw : weighted average molar mass; min: minutes; kDa: kilodalton.

Most combinations of secondary mAbs with mAb1 to protein Z favored the formation of well-defined complexes of smaller sizes, which corresponded to a heteromeric 2:2 mAb:protein Z complex (Figs. 8 and 9 and Tables 10-12).

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Table 10

Molar mass of tAL:protein Z complex

mAb complex: protein Z Theoretical molar mass (kDa) 1:0 150 0:1 195 1:1 345 2:1 495 1:2 540 2:2 690 3:2 840 2:3 885 4:4 1380 6:6 2070

Table 11

Approximate molar mass and retention time for tAL:protein Z complexes

Sample Molar ratio (mol:mol) Peak 1 Peak 2 Peak 3 Peak 4 [Intact Antibody] 2: [Protein Z]2 Complex [Intact Antibody^: [Protein Z]4 Complex [Intact Antibody] 6: [Protein Ζ]ό Complex Higher order complexes K, min. _Mw , kDa K, min. _Mw , kDa K, min. _Mw , kDa RT, min. _Mw , kDa mAL1:mAL5: protein Z 0.5:0.5:1 16.0 684 18.5 1342 20.1 1876 21.5 -2250- 3560 mAL1:mAL7: protein Z 0.5:0.5:1 16.1 688 18.5 1327 20.1 1866 21.5 -2380- 4250

Rt: retention time; _Mw : weighted average molar mass; min: minutes; kDa: kilodalton.

Table 12

Approximate molar mass and retention times for mAb:protein Z complexes

Sample Molar ratio (mol: mol) Peak 1 Peak 2 PikZ Peak 4 [Intact Antibody] g: [Protein Z] ₂ Complex [Intact Antibody]4: [Protein Z] ₄ Complex [Intact Antibody] in: [Protein Z] ₆ Complex Higher order complexes RT, min. _Mw , kDa RT, min. _Mw , kDa RT, min. _Mw , kDa RT, min. _Mw , kDa wb1:bw3:white κΖ wа1: wа6: white κΖ 0.5:0.5:1 0.5:0.5:1 16.4 15.7 685 686 18.4 17.7 1262 1320 N.A. 19.4 N.A. 1850 20.2 20.6 -1700 -2700 -2300 -3800 wа6: wа7: white κΖ 0.5:0.5:1 15.8 688 17.8 1334 19.3 1872 20.6 -2300 -3600

Although the combination mAb1 to protein ZmAb3 to protein Z formed complexes of similar size with protein Z, differences in elution time/profile suggested that the complexes formed had differences in shape/orientation compared to the other combinations (Fig. 9). Combinations of mAb1 to protein Z with mAb2 to protein Z and mAb COMP1 promoted the formation of larger heterogeneous complexes with protein Z, which indicated modular diversity (Fig. 10, Table 13).

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Table 13

Approximate molar mass and. retention time values for mAb: protein Z complexes

Sample Molar ratio (mol:mol) Peak 1 Peak 2 Peak 3 Peak 4 [Intact Antibody] g: [Protein Z] ₂ Complex [Intact Antibody: [Protein Z] ₄ Complex [Intact Antibody] in: [Protein Z] ₆ Complex Higher order complexes K, min. Mw? kDa K, min. _Mw , kDa K, min. _Mw , kDa K, min. _Mw , kDa taY: mAL2: protein Z 0.5:0.5:1 15.5 665 19.2 1304 21.9 1901 23.6 -2300- 4100 taY: COMP1: protein Z 0.5:0.5:1 15.4 714 18.8 1346 21.5 2002 24.2 -2500- 5000

Rt: retention time; _Mw : weighted average molar mass; min: minutes; kDa: kilodalton.

The combination of mAb4 to protein Z and mAbl to protein Z showed a reduced tendency to form heteromeric complexes with protein Z (Fig. 11, Table 14). The presence of free mAbs and 1:1 mAb:protein Z particles indicated incomplete formation of heteromeric complexes with protein Z. Mixtures of homomeric and heteromeric complexes with protein Z were also noticeable.

Table 14

Approximate molar mass and retention time for tAL:protein Z complexes

Sample Molar ratio (mol:mol) Peak 1 Peak 2 Peak 3 [Intact Antibody] 2: [Protein Z] ₂ Complex [Intact Antibody: [Protein Z] ₄ Complex Higher order complexes RT, min. _Mw , kDa RT, min. _Mw , kDa RT, min. _Mw , kDa mAL1:mAL4: protein Z 0.5:0.5:1 15.9 650 19.1 1288 20.6 -1700- 2300

R _t : retention time; _Mw : weighted average molar mass; min: minutes; kDa: kilodaltons.

Example 4: The order of addition did not significantly affect the molar mass and distribution of complexes formed between mAb1 to protein Z, mAb COMP1 and protein Z.

Ways.

To determine whether the order of addition affected complex formation, equimolar combinations of mAb COMP1 and protein Z were prepared in 1X DPBS, pH 7.4, resulting in a molar ratio of 1 μM mAb COMP1:1 μM protein Z and allowed to incubate at ambient temperature. for 1 hour. After incubation, various amounts of mAb1 to protein Z were added to the preformed mAb COMP1:protein Z complexes and diluted in 1X DPBS, pH 7.4, to obtain the following molar ratios: 0.3 μM mAb1 to protein Z: 1 μM mAb COMP1:1 μM protein Z, 1 μM mAb1 to protein Z: 1 μM mAb to protein Z: 1 μM protein Z, and 3 μM mAb1 to protein Z: 1 μM mAb COMP1: 1 μM protein Z, and incubated them in for an additional hour before insertion into the device. The A4F-MALLS assay methods from Example 3 were then used.

Results.

Complexes of similar molar mass and distribution were formed between mAb1 to protein Z, mAb COMP1, and protein Z, regardless of whether mAb1 to protein Z was added simultaneously (Fig. 12A, Table 15) or sequentially (Fig. 12B, Table 15). to other components. The slight shift in elution time observed between the two data sets was indicative of variability inherent to the method and was not considered unusual.

Table 15

Approximate molar mass and retention times for mAb:protein Z complexes

Sample taY: COMP1: Protein Z Molar ratio (mol: mol) Peak 1 Peak 2 Peak 3 Peak 4 [Intact Antibody]ι: [Protein Ζ]ι Complex [Intact Antibody] ι: [Protein Z] ₂ Complex [Intact Antibody] 2-z: [Protein Z] ₂ -3 Complex Higher order complexes RT, min. _MW , kDa RT, min. _Mw , kDa RT, min. _Mw , kDa RT, min Mw? kDa Simultaneous 3:1:1 N.A. N.A. 14.5 537 16.7 89 18.0 -1200- 1950 addition 1:1:1 N.A. N.A. 14.6 523 16.7 793 18.6 -1400-

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2300 0.3:1:1 13.3 344 15.0 541 16.6 750 18.4 -1000- 1800 Sequential addition 3:1:1 N.A. N.A. 15.6 500 18.0 817 20.0 -1200- 1800 1:1:1 N.A. N.A. 15.7 496 18.0 806 20.0 -1500- 2400 0.3:1:1 14.2 380 16.4 663 17.9 818 19.9 -1500- 2600

Example 5. Complexes with antibody to protein W.

Ways.

Mobile phase buffer for A4F-MALLS.

Mobile phase buffer (10 mM sodium phosphate, 500 mM sodium chloride, pH 7.0 ± 0.1) was prepared by combining 1.4 g of sodium phosphate monohydrate, 10.7 g of sodium phosphate dibasic heptahydrate and 500 ml of 5 M chloride sodium; the solution volume was then adjusted to 5.0 L with HPLC grade water. The final buffer pH measured was 7.0. Before use, the mobile phase buffer was filtered (through a 0.2 μm filter).

A4F-MALLS analysis.

The A4F-MALLS system consisted of an Eclipse™ 3+ A4F separation system coupled to an Agilent 1200 Series HPLC system equipped with an ultraviolet (UV) diode array detector and a Dawn HELEOS laser scattering (LS) meter. ® II from Wyatt Technology and the Optilab® T-rEX differential refractometer (RI) detector. The detectors were connected in series in the following order: UV-LS-RI. The LS and RI detectors were calibrated according to instructions provided by Wyatt Technology.

Each determined amount of W protein mAb was combined with W protein and diluted in 1X DPBS, pH 7.4, to give an equimolar ratio of 1 μM W protein mAb:1 μM W protein. All samples were incubated at ambient temperature for 2 h and stored unfiltered at 4°C until insertion into an Eclipse™ short channel equipped with W350 separator foil (350 µm separator thickness, 2.2 cm separator width) and using a 10 kDa MWCO regenerated cellulose membrane. Before injection of each sample, the channel was pre-equilibrated with mobile phase buffer (10 mM sodium phosphate, 500 mM sodium chloride, pH 7.0 ± 0.1). Bovine serum albumin (BSA; 2 mg/mL; sample weight 10 μg) was separately administered and included as a system suitability control.

The fractionation method consisted of four stages: injection, focusing, elution and channel washing stage. Mobile phase buffer for A4F-MALLS (10 mM sodium phosphate, 500 mM sodium chloride, pH 7.0 ± 0.1) was used throughout fractionation. Each sample (7 μg) was injected at 0.2 mL/min for 1 min and then focused for 3 min at a focal flow rate of 1.0 mL/min. The sample was eluted at a channel flow rate of 1.0 mL/min with a linear cross-flow gradient from 3.0 to 0 mL/min over 25 min. Finally, the cross-flow was held at 0 ml/min for an additional 5 min to clean the channel. BSA was fractionated using the same parameters.

Results.

A4F-MALLS was used to estimate the relative size distribution of complexes formed between the W protein, a dimeric multidomain ligand, and several anti-W protein antibodies that specifically bind to different domains within the ligand. The theoretical molar mass and predicted stoichiometry of potential antibody complexes with protein W are presented in Table. 16.

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Table 16

Theoretical molar mass of tAL:protein W complexes

Unequal ratios (eg 1:2 and 2:1) cannot be differentiated as they will have the same MW.

Overall, of the panel of different mAbs, mAb1 targeting domain A formed the highest proportion of lower order complexes, with a predominance of species presenting a discrete 1:1 and 2:2 complex with the protein when combined in equimolar ratios W (peak 2: ~289 kDa and peak 3: ~562 kDa, Fig. 13, Table 17).

Table 17

Molar masses and retention times of human complexes

Each of the mAbs targeting the B domain (mAb2, mAb3 and COMP1) predominantly formed a discrete 2:2 complex with the W protein (peak 3: ~563-580 kDa, Fig. 14, Table 18), with mAb2 and COMP1 gave the most homogeneous distribution of complexes relative to other mAbs tested. While COMP2 targeting the A domain predominantly favored the formation of a mixture of 1:2 and 2:2 complexes with protein W (peak 3: ~550 kDa, Fig. 15, Table 19), a moderate degree of large heterogeneous complexes was also observed. This suggested that, unlike mAb1, which also targets the A domain, COMP2 bound to a unique epitope on the W protein, allowing it to form extended antibody-antigen lattices in a process called modular diversification. This sample exhibited a distinct peak (peak 4) with a molar mass of approximately 835 kDa, followed by a series of particles that produced a broad, low-resolution peak (peak 5) with a broad molar mass distribution ranging from ~1000-1900 kDa (Figure 15, Table 19).

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Table 18

Molar masses and retention times of human W protein complexes with various mAbs targeting the B domain

Sample Molar Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Ratio (mol:mol) Free mAb/protein W [mAb]i: [Protein W]i Complex [mAb] ₂ : [Protein W] ₂ Complex [mAb] ₃ : [Protein W] ₃ Complex Heteromeric complexes of higher order RT, min _Mw , kDa RT, min _Mw , kDa RT, min _Mw , kDa RT, min Mw? kDa RT, min. _Mw , kDa Protein W - 9.6 147 ND ND ND ND ND ND ND ND wAL2 - 9.6 152 ND ND ND ND ND ND ND ND wb2:white to W 1:1 9.4 144 10.9 283 13.2 574 13.9 731 15.4 -1000 -1900 WALZ:white to W 1:1 9.3 144 10.7 26.8 12.9 563 14.0 831 14.6 -1000 -1900 SOMRKbel okW 1:1 9.4 143 10.8 298 13.0 580 13.9 765 15.0 -1000 -1900

Rt: retention time; _Mw : weighted average molar mass; NA: no data; min: minutes; kYes:

kilodalton.

Table 19

Molar masses and retention times of complexes of human protein W with COMP2 targeting domain A

Sample Molar ratio (mol:mol) Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Free protein W Free Sh [mAb]i- ₂ : [Protein W] ₂ Complex [mAb] ₂ _ ₃ : [Protein W] ₃ Complex Heteromeric complexes of higher order RT, min M W? kD a Rt, mi and. _Mw , kDa _Rt , min _Mw , kDa _Rt , min _Mw , kDa RT, min _Mw , kDa Protein W - 9.6 14 7 ND ND ND ND ND ND ND ND COMP2 - 10.5 12 2 ND ND ND ND ND ND ND ND COMP2:white to W 1:1 ND ND 9.4 147 13.1 550 15.2 835 16.9 -1000 -1900

R _t : retention time; _Mw : weighted average molar mass; NA: no data; min: minutes; kDa: kilodalton.

Based on the calculated molar masses of the individual components, peak 4 likely represented complexes containing at least 3 mAb molecules coordinating 2–3 W protein molecules, whereas peak 5 corresponded to a heterogeneous distribution of higher order heteromeric complexes consisting of >3 mAb molecules coordinating > 4 W protein molecules (Table 18). In contrast, mAbs targeting the C domain (mAb4 and COMP3) produced a broad distribution of large heterogeneous complexes (with molar masses ranging from ~700-8000 kDa) with mAb4, indicating the most extensive modular diversity among the panel of mAbs tested (Fig. 16 , table 20).

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Claims (13)

Table 20 Molar masses and retention times of human complexes Although numerous embodiments have been provided for purposes of illustration, it will be apparent to those skilled in the art that the present invention is capable of additional embodiments and that some of the details described herein may vary significantly without departing from the basic principles of the present invention. All references cited herein are incorporated by reference in their entirety. The present invention may be embodied in other specific forms without departing from its spirit or essential attributes, and accordingly reference should be made to the appended claims rather than to the preceding description, which indicate the scope of the present invention. CLAIM 1. A method for assessing the stoichiometry and size distribution of heterogeneous protein complexes in a sample, involving the stages: sample fractionation by flow-through cross-field fractionation (A4F); and determining the molar mass, stoichiometry, and size distribution of heterogeneous protein complexes in a sample using multi-angle laser light scattering (MALLS), where the heterogeneous protein complexes contain heterogeneous protein:ligand complexes containing more than one antibody coupled to the ligand, or heterogeneous fusion protein complexes :ligand containing more than one fusion protein linked to a ligand, wherein said protein in said heterogeneous protein:ligand complexes is a monoclonal antibody or an antigen-binding fragment thereof. 2. The method of claim 1, wherein the ligand is a soluble ligand. 3. The method according to claim 1, wherein said monoclonal antibody is a bispecific antibody or an antigen-binding fragment thereof. 4. The method according to claim 1, wherein said antibody and said antigen-binding fragment thereof in said heterogeneous protein:ligand complexes are an antibody of the same type. 5. The method of claim 1, wherein said heterogeneous protein:ligand complex is an antibody:ligand complex having a molar ratio of monoclonal antibody to ligand of 2:1. 6. The method of claim 1, wherein said heterogeneous protein:ligand complex is an antibody:ligand complex having an antibody to ligand molar ratio of 3:2. 7. The method of claim 1, wherein said heterogeneous protein:ligand complex is an antibody:ligand complex having a molar ratio of antibody to ligand of 2:3. 8. The method of claim 1, wherein said heterogeneous protein:ligand complex is an antibody:ligand complex having an antibody to ligand molar ratio of 3:3. 9. The method of claim 1, wherein said heterogeneous protein:ligand complex is an antibody:ligand complex having an antibody to ligand molar ratio of 3:4. 10. The method of claim 1, wherein said heterogeneous protein:ligand complex is an antibody:ligand complex having an antibody to ligand molar ratio of 4:4. 11. The method of claim 1, wherein said heterogeneous protein:ligand complex is an antibody:ligand complex having an antibody to ligand molar ratio of 6:6. 12. The method of claim 1, wherein said heterogeneous fusion protein:ligand complexes comprise more than two fusion proteins bound to a ligand. 13. The method according to claim 1, wherein said heterogeneous antibody:ligand complex has a molecular weight of 700 to 8000 kDa. -