Methods for producing controlled mixtures of two or more different antibodies

Abstract

The invention relates to a method for controlling the composition of a mixture of two or more different antibodies, such as two or more different monoclonal antibodies, using chromatography. The mixture is used as a pharmaceutical product, and the method comprises a controlled downstream process for producing a predetermined ratio of the two or more different antibodies.

Description

Invention Field

[0001] This invention relates to a method for controlling the composition of a mixture of two or more different antibodies using chromatography. The mixture is used as a pharmaceutical product, and the method includes a controlled downstream process for producing two or more different antibodies in a predetermined ratio. Background of the Invention

[0003] Many human diseases are now treated with therapeutic monoclonal antibodies. However, some diseases cannot be treated sufficiently and effectively with monoclonal antibodies, or treatment loses its effectiveness over time, for example, due to downregulation of the target or shift to a unique pathogenic pathway. Therefore, alternatives may be treatment with polyclonal antibodies or antibody mixtures, such as a mixture of different monoclonal antibodies. Such antibody mixtures may contain two or more antibodies targeting different epitopes on the same target, or alternatively, mixtures of antibodies targeting different targets, or combinations thereof.

[0004] To produce such antibody mixtures, two or more monoclonal antibodies can be generated and characterized separately, and then combined into a single pharmaceutical product. This requires controlled production and analysis of each separately produced monoclonal antibody, as well as analysis of the consistency of composition and potency of the final mixture. However, producing antibody mixtures using parallel production and purification processes can have higher manufacturing or development costs compared to producing mixtures of antibodies co-produced in a single bioreactor.

[0005] A mixture of antibodies can be produced from single-cell lines expressing two or more monoclonal antibodies. WO 2004 / 009618 describes a method for transfecting single cells with genes encoding antibodies that all use a single, identical light chain. This allows the single-cell line to produce at least three binding specificities. A drawback of this method is that it is limited to producing antibodies that all use the same light chain, which excludes the use of many available antibody sequences and common antibody identification platforms. Furthermore, without further engineering, the co-expression of a single light chain with multiple heavy chains during cell culture can result in both monospecific and bispecific antibodies, which may not be the desired components, due to the specific composition of the multiple heavy chains expressed in varying proportions. Another method described in WO 2010 / 0089387 involves producing at least two antibodies in single cells, where each gene encoding the antibody is under the control of a different eukaryotic promoter. The cell line is cultured under conditions that allow the sequential expression of the genes for each antibody. This method is expected to be highly sensitive to large-scale culture process parameters; for example, scale or feed variations will require strict control over promoter conversion and harvest timing, which may impair yield and only allow for the production of products with limited complexity. If the expression levels of individual antibody chains exhibit different sensitivities to changes in culture conditions, then neither of these co-expression methods can essentially control the composition of the product.

[0006] Alternatively, a mixture of antibodies can be produced by co-culturing cell lines, each expressing a single antibody. Recombinant antibody mixtures can be manufactured using adapted mammalian expression techniques based on site-specific integration of an antibody expression plasmid into the same genomic locus in each cell, as described in WO 2004 / 061104. WO 2008 / 145133 describes a method for preparing a recombinant antibody mixture by random integration, wherein host cells are transfected separately with a set of expression vectors while avoiding site-specific integration of the expression vector into the host cell genome. WO 2009 / 129814 describes various methods for producing recombinant antibody mixtures in multiple bioreactors, wherein cell lines or antibody preparations are combined at a later point in upstream processing or before or during downstream processing. WO 2012 / 068317 describes a method for expressing antibody mixtures using non-viral AAV-based preferential integration into multiple stable sites in the genome, and using stable cell conjugates instead of clonal cell lines. Although the composition of antibody mixtures can be controlled to some extent during production, this control is insufficient for producing recombinant antibody mixtures for clinical trials or pharmaceutical products.

[0007] Therapeutic antibodies are purified through various chromatographic steps to reduce contaminants such as DNA, host cell proteins, or product-related impurities to below predetermined specifications. Typically, antibody purification methods involve (1) grading based on the physicochemical characteristics of the antibody, such as size and charge; (2) grading based on class-specific affinity, using immobilized biological ligands with specific affinity for immunoglobulins to bind to specific antibody classes; or (3) grading based on antigen-specific affinity, as generally described in Current Protocols in Immunology, John Wiley & Sons, Coligan et al. (eds.).

[0008] In the case of bispecific antibodies, the purification step is used to separate the bispecific antibody from product-related impurities. EP2009101 describes a chromatographic purification method for antibodies based on the isoelectric point difference between the heavy chains of two types of antibodies, wherein the difference is introduced by modifying the amino acids present in the variable region of the antibody constituting the bispecific antibody.

[0009] Another method for separating bispecific antibodies based on differential binding to protein A is described in US 8,586,713. In this method, the Fc region of one of the heavy chains is engineered to have a reduced affinity for protein A, thereby allowing the separation of bispecific antibodies by differential binding of the IgG region to protein A. Another method described in US2015239991 is based on engineered antibodies with reduced affinity for protein G and separation using protein G affinity chromatography.

[0010] Various resins that specifically bind to the Kappa light chain of antibodies have been described, such as Protein L (GE Healthcare), KappaSelect (GE Healthcare), and KappaXL (Thermo Fisher). Their uses have been described in the context of bispecific antibodies. A method has been described in which a bispecific monoclonal antibody consisting of a single heavy chain and two distinct light chains (LCs) (one containing a Kappa constant domain and the other containing a Lambda constant domain) is purified using a light chain-specific resin (WO2013 / 088259). A method for purifying bispecific antibodies based on mutations in the CH1 domain has also been described (WO2013 / 136186). PCT / EP2016 / 065576 describes a method for purifying heterodimer-binding proteins, such as bispecific and multispecific antibodies containing two or more Kappa light chains, using a resin that binds to the Kappa light chain to prevent or reduce mutations in one or more binding resins of the Kappa light chain.

[0011] For recombinant antibody mixtures, methods for removing contaminating polymers using multi-peak chromatography, apatite chromatography, and hydrophobic interaction chromatography are described in WO 2014 / 209508.

[0012] There is a need for methods to control the composition of antibody mixtures produced downstream. This allows for the co-production of antibody mixtures while achieving the necessary control over the composition of the mixtures. The object of this invention is to provide methods for controlling the composition of recombinant antibody mixtures using chromatography. These methods include controlling the composition of the mixture using fractionation based on the physicochemical properties of the antibodies in the mixture. The methods also include protein engineering to alter the physicochemical properties of the antibodies in the mixture, thereby improving separation. These methods also include introducing mutations into the antibodies in the mixture to prevent or reduce antibody binding to affinity resins, and controlling the composition of the antibody mixture by purifying it using the resin that binds to the antibodies. Invention Overview

[0014] In a first aspect, the present invention relates to a method for producing an output mixture of two or more different antibodies, said different antibodies having differences in their amino acid sequences, said differences enabling the separation of said antibodies by chromatography, wherein

[0015] - The two or more different antibodies are present in the output mixture at a desired or predetermined concentration ratio or within their permissible deviations; and

[0016] -The method includes the following steps:

[0017] 1. Provide an input mixture, wherein the two or more different antibodies are not present, or substantially not present, at the desired or predetermined concentration ratio;

[0018] 2. Separate the two or more antibodies by chromatography;

[0019] 3. Recover the two or more antibodies in the amount required to produce the output mixture.

[0020] In a second aspect, the present invention relates to a mixture of two or more different antibodies, said mixture being obtained by the method of the present invention.

[0021] In a third aspect, the present invention relates to pharmaceutical compositions comprising the mixtures of the present invention.

[0022] In a fourth aspect, the present invention relates to antibody mixtures for methods of treating diseases. Brief description of the attached diagram

[0024] Figure 1 : Schematic diagram of the production and purification method of the present invention. (A) The method includes an upstream co-production process in which the antibody production level cannot be controlled to always meet release specifications. The antibody is purified in a downstream processing step that further controls the composition of the antibody mixture. Chromatography may include affinity chromatography based on differences in the binding properties of the antibody to the affinity resin, wherein (B) the resin is saturated with the antibody mixture in the correct ratio; (C) after the antibody ratio is predetermined using an analytical assay, the resin is bound with excess antibody to produce an antibody mixture in the correct ratio. Chromatography may also include chromatography based on differences in the physicochemical properties of the antibodies, wherein (D) after the antibody ratio is predetermined using an analytical assay, the design space of the chromatographic experiment has been pre-explored to remove excess antibody from the mixture to obtain an antibody mixture in the correct ratio. (E) Fractionation and fraction merging based on fraction chromatograms or concentration measurements.

[0025] Figure 2(A) Sequence alignment of the heavy chain variable region of human lineage and antibodies IgG1-1014-005, IgG1-2F8, and IgG1-1021-511. Amino acids are numbered according to IMGT numbers. An asterisk indicates the location of a point mutation introduced in any antibody. (B) Sequence alignment of the light chain variable region of human lineage and antibodies IgG1-1014-005, IgG1-2F8, and IgG1-1021-511. Amino acids are numbered according to IMGT numbers. An asterisk indicates the location of a point mutation introduced in any antibody. (CE) Alignment of charge-regulated variable domain variants of antibodies IgG1-2F8(C), IgG1-1014-005(D), and IgG1-1021-511(E). Amino acids are numbered sequentially (above the alignment) or according to the IMGT number of the human variable region (below the alignment); locations that pass the mutation test are indicated by highlighting.

[0026] HA1, HA2, and HA3 (more negative) and HB1, HB2, and HB3 (more positive) indicate heavy chain variable domains with progressively increasing charge differences relative to variant HC, which indicates a reference heavy chain variable domain sequence expressed as a fusion with a constant domain sequence without a C-terminal lysine. HP represents the sequence of the unmutated heavy chain variable domain of the parental antibody, expressed as a sequence containing a C-terminal lysine and an N-terminal pyroglutamic acid (where applicable). Similarly, LA1, LA2, LA3 (more negative) and LB1, LB2, and LB3 (more positive) indicate light chain variable domain sequences.

[0027] Figure 3 IgG titer assay for charge-regulated antibody variants. Scatter plots show antibody expression levels for each variant of IgG1-1014-005, IgG1-1021-511, and IgG1-2F8 as individual data points. The expression levels are sufficiently tightly clustered, indicating that the point mutation, except for the Q6E heavy chain mutation in IgG1-1014-005, has no major effect on protein expression and adversely affects antibody titers in all variants containing this mutation.

[0028] Figure 4Analysis of the charge properties of charge-regulated antibody variants. (A) Scatter plots show the theoretical isoelectric point (pI) for each variant of IgG1-1014-005, IgG1-1021-511, and IgG1-2F8. (B) Scatter plots show the retention times sampled according to antibody variants in analytical HPLC cation exchange (CEX) experiments. Control antibody variants are shown with hollow gray symbols, while antibody variants that eluted in the flow-through under these conditions and did not significantly interact with the column resin were excluded from the plots. (C) Correlation between analytical cation exchange retention time and pI for the charge variants IgG1-1014-005, IgG1-1021-511, and IgG1-2F8. These data show that charge-regulated point mutations have a significant impact on the charge properties of antibodies.

[0029] Figure 5 : From those containing (A) IgG1-1014-005-HCLC, IgG1-2F8-HCLC, IgG1-1021-511-HCLC and IgG1-1014-153; (B) IgG1-2F8-HB3LC, IgG1-1014-005-HB3LB1, IgG1-1021-511-HA3LB2 and IgG1-1014-153; (C) IgG1-2F8-HB3LB3, IgG1-1014-005-HB3LB1, IgG1-1021-511-HA3LB2 and IgG1-1014-153; (D) IgG1-2F8-V110D, IgG1-7D8-S12P and Exemplary protein A separation of a mixture of antibody variants from cell culture supernatants of IgG1-HepC,(E)IgG1-2F8-HB3LC,IgG1-1014-005-HB3LB1,IgG1-1021-511-HA3LB2, and IgG1-1014-153. Absorption at 280 nm (solid line) and conductivity or pH (grey dashed line) were monitored. All purifications showed increased absorption of unbound material from the cell culture supernatant during column loading. Specifically bound antibody variants were eluted at pH 3.0 and detected by an absorption peak at 280 nm. Small peaks at 280 nm during the washing step indicate less tightly bound material.

[0030] Figure 6: Purified (A) IgG1-1014-005-HCLC, IgG1-2F8-HCLC, IgG1-1021-511-HCLC and IgG1-1014-153 at similar concentrations; (B) IgG1-2F8-HB3LC, IgG1-1014-005-HB3LB1, IgG1-1021-511-HA3LB2 and IgG1-1014-153 at similar concentrations; (C) I purified from the supernatant containing antibody variants in an approximate ratio of 5:3:2:1 Exemplary preparative cation exchange chromatography separations of IgG1-2F8-HB3LB3, IgG1-1014-005-HB3LB1, IgG1-1021-511-HA3LB2, and IgG1-1014-153 from supernatant containing antibody variants in an approximate ratio of 1:3:2:5, and (D) IgG1-2F8-HB3LC, IgG1-1014-005-HB3LB1, IgG1-1021-511-HA3LB2, and IgG1-1014-153. Absorbance (solid line) and conductivity (grey dashed line) were monitored at 280 nm. The non-charge-regulated variant in (Fig. A) showed a defined peak at approximately 120 mL for IgG1-1021-511-HCLC, but the other three were poorly resolved. The charge-regulated variants were resolved (Figs. B, C) because four defined peaks were observed at each antibody ratio. The pooling schemes in each experiment were indicated by vertical markers and numbers (1–4). The chromatograms of the three antibody mixtures were quantified in Table 1. (E) Schematic diagram shows the resolution (Rs) calculated according to the equation Rs = 2(t2-t1) / (W1+W2), where t1 = retention time of the given antibody, t2 = retention time of the sequentially eluted antibody, and W1 and W2 are the corresponding peak widths of the antibody at the base of the peak obtained by extrapolating the relatively straight sides of the main peak to the baseline, in time.

[0031] Figure 7 : From loaded antibody mixture and from Figure 6The analytical cation exchange chromatography (CEC) chromatograms of the combined fractions collected in preparative cation exchange chromatography separation are shown. (A) Similar concentrations of IgG1-1014-005-HCLC, IgG1-2F8-HCLC, IgG1-1021-511-HCLC, and IgG1-1014-153. The dashed line is used to identify the main peak (ad). The antibody was not fully resolved on the high-resolution analytical column, and several of the combined fractions contain a mixture of antibodies. (B) Similar concentrations of IgG1-2F8-HB3LC, IgG1-1014-005-HB3LB1, IgG1-1021-511-HA3LB2, and IgG1-1014-153. The dashed line is used to identify the main peak (ad). The antibody was well resolved on the high-resolution analytical column, and the combined fractions contain >99% pure antibody, as inferred from the integral of the analytical cation exchange profile. (C) IgG1-2F8-HB3LB3, IgG1-1014-005-HB3LB1, IgG1-1021-511-HA3LB2, and IgG1-1014-153. Dashed lines are used to identify the main peak (ad). The antibodies resolve well on high-resolution analytical columns, and the combined fractions contain >99% pure antibody, as inferred from the integral analysis of the cation exchange profile. (D) Different concentrations of IgG1-2F8-HB3LC, IgG1-1014-005-HB3LB1, IgG1-1021-511-HA3LB2, and IgG1-1014-153. Dashed lines are used to identify the main peak (ad). The antibodies resolve well on high-resolution analytical columns, and the combined fractions contain >99% pure antibody, as inferred from the integral analysis of the cation exchange profile. (E) A non-equimolar mixture of IgG1-2F8-HB3LB3, IgG1-1014-005-HB3LB1, IgG1-1021-511-HA3LB2, and IgG1-1014-153 before preparative cation exchange chromatography (top panel), and the final product after preparative chromatography and recombination (bottom panel). Quantitative chromatography diagrams are shown in Table 2. (F) A non-equimolar mixture of IgG1-2F8-HB3LB3, IgG1-1014-005-HB3LB1, IgG1-1021-511-HA3LB2, and IgG1-1014-153 before preparative cation exchange chromatography (top panel), and the final product after preparative chromatography and recombination (bottom panel). Quantitative chromatography diagrams are shown in Table 2.

[0032] Figure 8 (A) Using FreeStyle TMPreparative cation exchange chromatography of materials produced transiently in 293-F cells, loaded with 2 g / L resin, of an equimolar mixture of IgG1-7D8, IgG1-224, IgG1-CD37-37-3, IgG1-CD19-21D4-K409R, and IgG1-CD52-Campath. Three distinct peaks could be identified in the chromatography of the separation from the five antibodies, indicating that the five antibodies were not sufficiently different in their charge properties to achieve separation in this experiment. The K409R mutation did not significantly affect the elution behavior of the IgG1-CD19-21D4 antibody because it is not on the antibody surface and does indeed result in a change in net charge. (B) Using FreeStyle TM Preparative cation exchange chromatography was performed on materials produced transiently in 293-F cells, loaded with 2 g / L resin, of an equimolar mixture of IgG1-7D8, IgG1-224, IgG1-CD37-37-3, IgG1-CD19-21D4-E345K, and IgG1-CD52-Campath-E345K. Introducing an E345K mutation into IgG1-CD19-21D4-E345K and IgG1-CD52-Campath-E345K altered the retention times of these antibodies, producing five resolved peaks in the chromatography under these chromatographic conditions. (C) Using FreeStyle TMPreparative cation exchange chromatography (CE) chromatograms of materials produced transiently in 293-F cells, loaded with 2 g / L resin, of an equimolar mixture of IgG1-7D8, IgG1-224, IgG1-CD37-37-3, IgG1-CD19-21D4-E345K, and IgG1-CD52-Campath-E345K. Eluted peaks were fractionated and combined as indicated by vertical labels and numbers (1–5). In each experiment, absorbance (solid line) and conductivity (grey dashed line) at 280 nm were monitored. (D) Individually collected peaks (labeled 1–5) were analyzed by cation exchange chromatography. Retention times designated peaks 1–5 as IgG1-7D8, IgG1-224, IgG1-CD37-37-3, IgG1-CD19-21D4-E345K, and IgG1-CD52-Campath-E345K, respectively. (E) Loading studies show the eluted portions of individually separated superimposed chromatograms, in which equimolar mixtures of IgG1-7D8, IgG1-224, IgG1-CD37-37-3, IgG1-CD19-21D4-E345K, and IgG1-CD52-Campath-E345K were separated using material produced from CHO cell lines at total loads of 0.2, 0.5, 1.0, 2.0, 5.0, 10, 20, or 50 g of antibody mixture per L of resin. Five resolved peaks were observed at all loads, detected by absorption at 280 nm, with some peak broadening detected at the highest load, as quantified in Table 3. The chromatograms were similar when using one or more transiently produced materials from CHO cell lines.

[0033] Figure 9(A) Preparative cation exchange chromatography, showing the separation of a mixture of IgG1-7D8, IgG1-224, IgG1-CD37-37-3, IgG1-CD19-21D4-E345K, and IgG1-CD52-Campath-E345K using a sequential elution step. The corresponding loading ratios of IgG1-7D8, IgG1-224, IgG1-CD37-37-3, IgG1-CD19-21D4-E345K, and IgG1-CD52-Campath-E345K are indicated in each figure based on calculations of the relative antibody mass in the mixture. Each separation was fractionated and combined as shown by vertical labels and numbers (1–5). Absorbance (solid line) and conductivity (grey dashed line) were monitored at 280 nm. Quantitative chromatography is shown in Table 4. (BE) Individual analysis of each of the five fractions from four fractionation experiments was performed by analytical cation exchange chromatography alongside the sample from the preparative chromatography loading material. Retention times designated the peaks ae as IgG1-7D8, IgG1-224, IgG1-CD37-37-3, IgG1-CD19-21D4-E345K, and IgG1-CD52-Campath-E345K, respectively. The ratios on each graph indicate the input ratios of IgG1-7D8, IgG1-224, IgG1-CD37-37-3, IgG1-CD19-21D4-E345K, and IgG1-CD52-Campath-E345K prior to the preparative chromatography experiments.

[0034] Figure 10 Sequence alignment of the CL domain of the kappa light chain. (allo) indicates allotype variation; CONSENSUS+ indicates conserved residues present in all cross-reactive species; CONSENSUS- indicates "CONSENSUS+" residues present in one of the non-cross-reactive species (and human lambda CL); PISA (INTERFACE) indicates residues (+) located at the CL-VL and CL-CH1 interfaces with <50% exposed surface area in the PDB 1HZH structure, as indicated by the PDBePISA tool ( http: / / pdbe.org / pisa / ) determination (Krissinel, E. and Henrick, K.; J Mol Biol (372):774-97, 2007); In this study, selected residues (*) were mutated to mouse equivalents. EU numbering convention is used to annotate amino acid residues (Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition Public Health Service, National Institutes of Health, Bethesda, MD. (1991)).

[0035] Figure 11 Sequence alignment of the VL domain of the kappa light chain. The PBD structure 1HEZ and 1MHH were analyzed using the PDBePISA tool (http: / / pdbe.org / pisa / ) (Krissinel, E. and Henrick, K.; J Mol Biol (372):774-97, 2007). Residues identified at the interface with protein L in all models were marked (+). In this study, selected residues (marked as *) were mutated to equivalent residues found in the Kappa V-II isotype (P01617) or Lambda V-1 isotype (P01699) sequences. IMGT numbers were used to annotate amino acid residues (Lefranc, M.-P. et al., Dev. Comp. Immunol., 2003, 27, 55-77).

[0036] Figure 12Exemplary KappaSelect purification was performed using purified protein (A) IgG1-2F8-F405L or modified IgG1-2F8-F405L variants from cell culture supernatants containing the generated (B) IgG1-2F8-F405L-mmF135L, (C) IgG1-2F8-F405L-V110D, (D) IgG1-2F8-F405L-E143D, and (E) IgG1-2F8-F405L-E165D. Absorption at 280 nm (solid line) and pH (grey dashed line) were monitored. Purification from the cell culture supernatant was shown as increased absorption from unbound material in the flowstream during column loading. Specifically bound IgG1-2F8-F405L variants were eluted at pH 3.0 and pH 2.0 and detected by an absorption peak at 280 nm. (F) Analyzed KappaSelect purified flow fractions from modified IgG1-2F8 variants using SDS-PAGE. Non-reducing SDS-PAGE gels showed bands of the complete IgG1 variant in the flow fractions of IgG1-2F8-F405L-mmF135L (lane 1) and IgG1-2F8-F405L-V110D (lane 2), but not in other IgG1-2F8-F405L variants (lanes 3-10). Other major bands were designated as antibody fragments.

[0037] Figure 13 Exemplary CaptureSelect KappaXL isolates of modified IgG1-7D8-K409R variants were isolated from cell culture supernatants containing (A) IgG1-7D8-K409R, (B) IgG1-7D8-K409R-V110R, (C) IgG1-7D8-K409R-V110K, (D) IgG1-7D8-K409R-V110D, (E) IgG1-7D8-K409R-V110E, and (F) IgG1-7D8-K409R-V110T. Absorption at 280 nm (solid line) and pH (grey dashed line) were monitored. All purifications showed increased absorption of unbound material from the cell culture supernatant during column loading. Specifically bound IgG1-7D8-K409R variants eluted at pH 3.5 and were detected by an absorption peak at 280 nm. The peak at 280 nm during pH 5.0 washing indicates less tightly bound material, while the peak at 280 nm during guanidine hydrochloride washing at approximately 30 mL is caused by incomplete elution of the antibody during the washing and elution phases.

[0038] Figure 14Fractions separated from CaptureSelect KappaXL containing modified IgG1-7D8-K409R variants were analyzed using Bio-Layer Interference Measurement and CE-SDS. (A) Concentrations of the IgG1-7D8-K409R variant in the loaded sample, pooled flow-through sample, and pooled eluent sample were inferred from the bio-layer interference measurement. Data from IgG1-7D8-K409R variants with different amino acids at position 110 (EU numbering convention) of the kappa light chain were grouped and labeled with the single amino acid code of that position. For variants that did not show detectable binding to the resin due to dilution of the flow-through sample during purification experiments, the protein concentration measured in the flow-through was lower than that in the loaded sample. (B) Fractions separated from CaptureSelect KappaXL containing modified IgG1-7D8-K409R variants were analyzed using CE-SDS. An exemplary non-reducing CE-SDS electrophoresis pattern, calibrated according to molecular weight standards, shows bands of intact IgG1 variants with a molecular weight of approximately 150 kDa in loadings of IgG1-7D8-K409R, IgG1-7D8-K409R-V110D, IgG1-7D8-K409R-V110E, IgG1-7D8-K409R-V110K, IgG1-7D8-K409R-V110R, and IgG1-7D8-K409R-V110T. The intact IgG1 variants can be detected in the flow-through and / or eluent, depending on the relative binding of the IgG1-7D8-K409R variant to CaptureSelect KappaXL resin. Other major bands with lower molecular weights are designated as antibody fragments, system calibration peaks, or other materials from transient production experiments.

[0039] Figure 15 : The purified HiTrap protein L of the IgG-2F8-F405L variant. The chromatography diagram shows the absorbance (solid line) at 280 nm and pH profile (grey dashed line) during the separation of (A) IgG1-2F8-F405L, (B) IgG1-2F8-F405L-S9L and (C) IgG1-2F8-F405L-S12P.

[0040] Figure 16Chromatographic experiments demonstrate the specificity of the antibody variants for three affinity chromatography resins. The purified (A) IgG1-2F8-V110D, (B) IgG1-7D8-S12P, and (C) IgG1-HepC were separated by HiTrap protein L. The purified (D) IgG1-2F8-V110D, (E) IgG1-7D8-S12P, and (F) IgG1-HepC were separated by HiTrap KappaSelect. (G) IgG1-2F8-V110D, (H) IgG1-7D8-S12P, and (I) IgG1-HepC were separated by HiTrap LambdaFabSelect. The chromatograms show the absorbance at 280 nm (solid line) and pH profile (grey dashed line) during separation.

[0041] Figure 17 Loading studies of purified IgG1-7D8-K409R onto HiTrap KappaSelect columns at residence times of (A) 4 min, (B) 2 min, and (C) 1 min. Chromatography plots show the absorbance at 280 nm (solid line) and pH profile (grey dashed line) during separation. The increase in absorbance at 280 nm during the loading phase indicates that the column loading is close to its binding capacity when the column is saturated.

[0042] Figure 18 Chromatographic images of KappaSelect purification of the mixture IgG1-2F8-V110D, IgG1-7D8-S12P, and IgG1-HepC under saturated conditions. Absorption at 280 nm (solid line) and pH (grey dashed line) were monitored on the chromatography during separation. An increase in absorption at 280 nm during the loading phase indicates that the column loading is close to its binding capacity when the column is saturated.

[0043] Figure 19 (A) Overlay analytical cation exchange chromatography of purified IgG1-2F8-V110D, IgG1-7D8-S12P, and IgG1-HepC. (B) Overlay analytical cation exchange chromatography of a mixture of IgG1-HepC (Note A), IgG1-7D8-S12P (Note B), and IgG1-2F8-V110D (Note C), before and after KappaSelect separation. Quantitative chromatography is shown in Table 10.

[0044] Figure 20(A) A superimposed preparative cation exchange chromatography plot showing the changes in peak shape at different salt concentrations during elution of IgG1-7D8, IgG1-224, IgG1-CD37-37-3, IgG1-CD19-21D4-E345K, and IgG1-CD52-Campath-E345K using a sequential elution step. Absorption at 280 nm (dashed line) and % buffer B (solid line) are monitored. Higher peaks at 280 nm correspond to conditions with higher ionic strength. (B) A preparative cation exchange chromatography plot showing the separation of a mixture of IgG1-7D8, IgG1-224, IgG1-CD37-37-3, IgG1-CD19-21D4-E345K, and IgG1-CD52-Campath-E345K using a sequential elution step. The five baseline separation peaks (black lines) in the A280 absorption profile correspond to the elution of IgG1-7D8, IgG1-224, IgG1-CD37-37-3, IgG1-CD19-21D4-E345K, and IgG1-CD52-Campath-E345K, respectively. % Buffer B (gray line) is monitored. (C) Exemplary preparative chromatograms collected during the designed space experiment, with a loading ratio of IgG1-7D8, IgG1-224, IgG1-CD37-37-3, IgG1-CD19-21D4-E345K, and IgG1-CD52-Campath-E345K of 2.5:1:2.5:1:2.5. The volumes (*) indicated within the 1st, 3rd, and 5th main peaks in the A280 absorption profile (solid line; number) are predefined variable volumes of 30 mL, removed from the pooling during elution of IgG1-7D8, IgG1-CD37-37-3, and IgG1-CD52-Campath-E345K. Monitor % Buffer B (grey line). (D) Overlay analytical cation exchange chromatography of IgG1-7D8, IgG1-224, IgG1-CD37-37-3, IgG1-CD19-21D4-E345K, and IgG1-CD52-Campath-E345K during a set of designed space experiments, with a loading ratio of 2.5:1:2.5:1:2.5 for IgG1-7D8, IgG1-224, IgG1-CD37-37-3, IgG1-CD19-21D4-E345K, and IgG1-CD52-Campath-E345K. Increasing the waste volume of IgG1-7D8, IgG1-CD37-37-3, and IgG1-CD52-Campath-E345K (0 mL, 10 mL, 20 mL, 30 mL, 40 mL from top to bottom) resulted in the consumption of these proteins from the mixture (peaks labeled 1, 3, and 5, respectively).The (EF) correlation plot shows the relationship between the amount of protein in the mixtures of IgG1-7D8, IgG1-CD37-37-3, and IgG1-CD52-Campath-E345K and the size of the waste fraction. (V=0) = mass obtained from the analytical cation exchange chromatography of purified protein, where waste volume = 0. Analytical cation exchange chromatography plots of HO output material and final product, and preparative cation exchange chromatography plots of the four mixtures of IgG1-7D8, IgG1-224, IgG1-CD37-37-3, IgG1-CD19-21D4-E345K, and IgG1-CD52-Campath-E345K are shown. Preparative cation exchange chromatography plots showing the separation of the mixtures by elution using sequential steps are shown in the G, I, K, M plots. Waste volume (*) is indicated in each chromatography experiment. Absorbance (black line) and conductivity (gray line) are monitored at 280 nm. The corresponding analytical cation exchange chromatography chromatograms of the input materials and final products are shown in the H, J, L, and N chromatograms, respectively, indicating the peak integration boundaries. Quantitative chromatography chromatograms are listed in Table 12.

[0045] Figure 21(AB) Preparative chromatograms showing loading of tandem KappaSelect, LambdaFabSelect, and Protein L columns loaded with a 1:1:1 (A) or 1:1.5:2 (B) mixture of IgG1-2F8-V110D, IgG1-7D8-S12P, and IgG1-HepC. Absorption at 280 nm was monitored and indicated by a solid line in the chromatogram. During the loading step, the absorption at 280 nm reached a plateau, indicating column saturation. (CE): Exemplary elution from a 1:1:1 mixture of IgG1-2F8-V110D, IgG1-7D8-S12P, and IgG1-HepC from KappaSelect (C), Protein L (D), and LambdaFabSelect (E) columns. Absorption at 280 nm, pH, and conductivity were monitored. Specifically bound proteins were eluted at low pH and detected by an absorption peak at 280 nm. (F) Exemplary protein A separation of a mixture of antibody variants from a cell culture supernatant containing a 1:1:1 mixture of IgG1-2F8-V110D, IgG1-7D8-S12P, and IgG1-HepC. Monitor absorbance (solid line) at 280 nm and conductivity or pH (grey dashed line). Purification is shown by increased absorbance of unbound material from the cell culture supernatant during column loading. Elute specifically bound antibody variants at pH 3.0 and detect by an absorbance peak at 280 nm. (G) Fragments of analytical cation exchange chromatography plots of a 1:1:1 or 1:1.5:2 mixture of IgG1-2F8-V110D, IgG1-7D8-S12P, and IgG1-HepC supernatant, or of a mixture purified by protein A affinity chromatography or by tandem chromatography using KappaSelect, Protein L, and LambdaFabSelect columns. Peaks were identified by referring to individually purified reference proteins and indicated by dashed lines as IgG1-HepC(1), IgG1-7D8-S12P(2), and IgG1-2F8-V110D(3). Invention Details

[0047] definition

[0048] The term "immunoglobulin" refers to a class of structurally related glycoproteins composed of two pairs of polypeptide chains, a pair of light chains (LC) and a pair of heavy chains (HC), all four interconnected by disulfide bonds. The structure of immunoglobulins has been well characterized. See, for example, Fundamental Immunology Ch.7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989)). In short, each heavy chain typically consists of a heavy chain variable region (abbreviated VH in this paper) and a heavy chain constant region (CH). The heavy chain constant region typically consists of three domains: CH1, CH2, and CH3. The heavy chains are interconnected by disulfide bonds in so-called "hinge regions." Each light chain typically consists of a light chain variable region (abbreviated VL in this paper) and a light chain constant region. The light chain constant region typically consists of a domain CL. CL can be either κ (kappa) or λ (lambda) isoforms. Here, constant domain and constant region are used interchangeably.

[0049] Unless otherwise specified, the amino acid residues in the constant regions are numbered according to the EU index described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991). The VH and VL regions can be further subdivided into highly denatured regions (or hypervariable regions, which may be highly variable in the sequence and / or form of structurally defined loops), also known as complement-determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). Each VH and VL typically consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk J. Mol. Biol. 196, 901 917 (1987)). The amino acid residues in the variable region are numbered as described in Lefranc, M.-P. et al., Dev. Comp. Immunol., 2003, 27, 55-77, unless the context contradicts this.

[0050] In the context of this invention, substitutions that can alter the interaction between the antibody and the chromatography resin can be substitutions that change the amino acid to one of the different classes of amino acids reflected in the table below:

[0051] The categories of amino acid residues used for substitution:

[0052]

[0053] Histidine's imidazole group has a pKa of about 6.0, so it can be neutral or basic, depending on the pH of the solution and the local chemical environment of the histidine residues.

[0054] In the context of this invention, unless otherwise indicated, the following designations are used to describe amino acid modifications: the name of the modified amino acid, followed by the position number of the modification, and then the contents of the modification. Thus, if the modification is a substitution, it includes the name of the amino acid replacing the previous amino acid; if the amino acid is deleted, it includes *; and if the modification is an addition, it is followed by the added amino acid. Amino acid names can be one- or three-letter codes. Thus, for example, replacing lysine at position 409 with arginine is K409R, replacing lysine at position 409 with any amino acid is K409X, K409* indicates the deletion of lysine at position 409, and K409KP indicates the addition of P after lysine at position 409. How modifications can be introduced is well known to those skilled in the art.

[0055] When used herein, the terms “Fc region” and “Fc domain” are used interchangeably and refer to an antibody region that contains at least the hinge region, CH2 domain, and CH3 domain (see, for example, Kabat EA, in US Department of Health and Human Services, NIH publication no. 91-3242, Edn. 5). th Editions 662, 680, 689 (1991). The Fc region can be produced by digesting antibodies with papain, where the Fc region is the fragment obtained thereby, comprising two CH2-CH3 regions of the immunoglobulin and a hinge region. The constant domain of the antibody heavy chain defines the antibody isotype, such as IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgE. The Fc domain mediates the effector function of antibodies and proteins of the complement system that have cell surface receptors called Fc receptors.

[0056] The terms “CH1 region” or “CH1 domain” are used interchangeably and, as used herein, are intended to refer to the CH1 region of an immunoglobulin. Thus, for example, according to the EU numbering system, the CH1 region of a human IgG1 antibody corresponds to amino acids 118-215. However, the CH1 region can also be any other antibody isotype as described herein.

[0057] The terms “CH2 region” or “CH2 domain” are used interchangeably and, as used herein, are intended to refer to the CH2 region of an immunoglobulin. Thus, for example, according to the EU numbering system, the CH2 region of a human IgG1 antibody corresponds to amino acids 228-340. However, the CH2 region can also be any other antibody isotype as described herein.

[0058] The terms “CH3 region” or “CH3 domain” are used interchangeably and, as used herein, are intended to refer to the CH3 region of an immunoglobulin. Thus, for example, according to the EU numbering system, the CH3 region of a human IgG1 antibody corresponds to amino acids 341-447. However, the CH3 region can also be any other antibody isotype as described herein.

[0059] The term "antibody" includes immunoglobulin molecules, fragments of immunoglobulin molecules, or derivatives of any of them, which have a half-life under typical physiological conditions for a considerably long period of time, such as at least about 30 minutes, at least about 45 minutes, at least about 1 hour (h), at least about 2 hours, at least about 4 hours, at least about 8 hours, at least about 12 hours (h), about 24 hours or more, about 48 hours or more, about three, four, five, six, seven or more days, etc., or any other relevant functionally defined time period (e.g., sufficient to induce, promote, enhance, inhibit, and / or improve physiological responses associated with antibody-bound antigens and / or sufficient to recruit effector activity). The variable regions of the heavy and light chains of immunoglobulin molecules contain binding domains that interact with antigens. The constant regions of antibodies (Abs) can mediate the binding of immunoglobulin molecules to host tissues or factors, including various cells of the immune system (e.g., effector cells) and components of the complement system, such as C1q, the first component in the classical pathway of complement activation. Alternatively, constant regions can be inert or inactive, such that they cannot bind to any Fc gamma receptor (FcgR), induce Fc-mediated FcgR crosslinking, or induce FcgR-mediated target antigen crosslinking through the two Fc regions of an individual antibody, or cannot bind C1q. Antibodies can also be bispecific antibodies, biantibodies, multispecific antibodies, or similar molecules. Antibodies can have monospecific affinity because they bind to / are specific to only one epitope. Alternatively, antibodies can have multispecific affinity in the sense that an antibody molecule can bind to / are specific to epitopes on multiple antigens and / or multiple epitopes on the same antigen. The term "antibody" includes recombinant antibodies, biantibody molecules, and "multispecific antibodies," "bispecific antibodies," "humanized antibodies," "human antibodies," "chimeric antibodies," "full-length antibodies," and heavy-chain antibodies or similar molecules, as defined below.

[0060] As used herein, the term "monoclonal antibody" refers to a formulation of an antibody molecule recombined from a single primary amino acid sequence. Monoclonal antibody compositions exhibit single binding specificity and affinity for a specific epitope. Therefore, the term "human monoclonal antibody" refers to an antibody exhibiting single binding specificity, possessing variable and constant regions derived from human germline immunoglobulin sequences. Human monoclonal antibodies can be produced by hybridomas comprising B cells obtained from transgenic or transchromosomal nonhuman animals, such as transgenic mice, fused with immortalized cells and containing both human heavy-chain and light-chain transgenes.

[0061] As used herein, the term "humanized antibody" refers to a genetically engineered nonhuman antibody comprising a human antibody constant domain and a nonhuman variable domain modified to have a high level of sequence homology with the human variable domain. This can be achieved by grafting a nonhuman antibody complementarity-determining region (CDR) of six parts that together form the antigen-binding site onto a homologous human receptor frame region (FR) (see WO92 / 22653 and EP0629240). To fully reconstruct the binding affinity and specificity of the parent antibody, it may be necessary to replace the adult frame region with frame residues from the parent antibody (i.e., the nonhuman antibody) (reversion mutation). Structural homology modeling can help identify amino acid residues in the frame region that are important for the antibody's binding properties. Therefore, a humanized antibody may comprise a nonhuman CDR sequence, primarily a human frame region (which optionally contains one or more amino acid reversion mutations to a nonhuman amino acid sequence), and a fully human constant region. Optionally, other amino acid modifications, not necessarily reversion mutations, may be applied to obtain a humanized antibody with preferred properties such as affinity and biochemical properties.

[0062] As used herein, the term "chimeric antibody" refers to an antibody in which the variable region is derived from a non-human species (e.g., rodent-derived) and the constant region is derived from a different species, such as humans. Chimeric antibodies can be produced by antibody engineering. "Antibody engineering" is a general term for various types of modifications to antibodies and is a process well-known to those skilled in the art. In particular, chimeric antibodies can be produced using standard DNA techniques as described in Sambrook et al., 1989, Molecular Cloning: Alaboratory Manual, New York: Cold Spring Harbor Laboratory Press, Ch. 15. Thus, chimeric antibodies can be genetically or enzymatically engineered recombinant antibodies. The production of chimeric antibodies is within the knowledge of those skilled in the art, and therefore, chimeric antibodies according to the invention can be produced by methods other than those described herein. Chimeric monoclonal antibodies are developed for therapeutic applications to reduce the immunogenicity of antibodies. They may typically contain a non-human (e.g., mouse) variable region specific to the antigen of interest, as well as human constant antibody heavy and light chain domains. When used in the context of chimeric antibodies, the term "variable region" or "variable domain" refers to the region containing both the CDR and the framework region of immunoglobulins, including both the heavy and light chains.

[0063] The term "bispecific antibody" refers to an antibody that is specific to at least two distinct epitopes, typically non-overlapping epitopes, or an antibody containing two distinct antigen-binding sites. Bispecific antibodies may be described as heterodimeric proteins, while monospecific antibodies may be described as homodimeric proteins. As noted above, unless otherwise stated or clearly contradicted by the context, the term antibody as used herein includes antibody fragments that retain the ability to specifically bind antigens. Such fragments can be provided by any known technique, such as enzymatic cleavage, peptide synthesis, and recombinant expression techniques. It has been shown that the antigen-binding function of antibodies can be achieved by fragments of full-length antibodies, such as Fab or F(ab')2 fragments. It should also be understood that, unless otherwise stated, the term antibody also includes monoclonal antibodies (mAbs), antibody-like peptides, such as chimeric antibodies and humanized antibodies. The resulting antibodies can be of any isotype.

[0064] The term "multispecific antibody" refers to an antibody that is specific to more than two distinct epitopes, typically non-overlapping epitopes, or an antibody containing more than two distinct antigen-binding sites. As stated above, unless otherwise specified or clearly contradicted by the context, the term antibody as used herein includes antibody fragments that retain the ability to specifically bind antigens. Such fragments can be provided by any known technique, such as enzymatic cleavage, peptide synthesis, and recombinant expression. It has been shown that the antigen-binding function of antibodies can be achieved by fragments of full-length antibodies, such as Fab or F(ab')2 fragments. It should also be understood that, unless otherwise specified, the term antibody also includes monoclonal antibodies (mAbs), antibody-like peptides, such as chimeric antibodies and humanized antibodies. The resulting antibodies can be of any isotype.

[0065] When used herein, the term "full-length antibody" refers to an antibody that contains all the constant and variable domains of the heavy and light chains typically found in antibodies of that isotype.

[0066] As used in this article, “isotype” refers to the class of immunoglobulins (e.g., IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM) encoded by genes in the heavy chain constant region.

[0067] As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies of the present invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or somatic mutations in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which a CDR sequence derived from another mammalian species, such as a mouse, has been grafted onto a human frame sequence.

[0068] The term "epitope" refers to a protein determinant that specifically binds to an antibody. Epitopes are typically composed of surface groups of molecules, such as amino acids or sugar side chains, and usually possess specific three-dimensional structural features as well as specific charge characteristics. The difference between conformational and non-conformational epitopes is that, in the presence of denaturing solvents, binding to the former occurs, but binding to the latter is lost. An epitope can contain amino acid residues that directly participate in binding, as well as other amino acid residues that do not directly participate in binding, such as amino acid residues that are effectively blocked by antigen-binding peptides (in other words, the amino acid residues are within the footprint of the antigen-binding peptide).

[0069] As used herein, in the context of antibody binding to a predetermined antigen, the term "binding" is generally used to refer to affinity binding, which corresponds to approximately 10 when measured by, for example, the BioLayer Interference Measurement (BLI) technique in an Octet HTX instrument using the antibody as a ligand and the antigen as an analyte. -6 M or smaller, such as 10-7 M or smaller, such as about 10 -8 M or smaller, such as about 10 -9 M or smaller, approximately 10 -10 M or smaller, or about 10 -11 M or even smaller K D And wherein the antibody binds to a predetermined antigen with an affinity corresponding to a K0 higher affinity than it binds to non-specific antigens (e.g., BSA, casein) other than the predetermined antigen or closely related antigens. D At least ten times lower, for example, at least 100 times lower, for example, at least 1,000 times lower, for example, at least 10,000 times lower, for example, at least 100,000 times lower than K. D Combined with K D The lower amount depends on the antibody's K D Therefore, when the antibody's K D When the level is very low, the K+ that binds to the antigen... D Below the binding K of nonspecific antigens D The amount can be at least 10,000 times (i.e., the antibody is highly specific). As used herein, the term "K" refers to... D "(M)" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction. As used in this paper, affinity and K D There is an inverse correlation, meaning that higher affinity implies lower K. D Lower affinity is intended to refer to higher K D .

[0070] When used herein, the term “heterodimeric interaction between the first and second CH3 regions” refers to the interaction between the first CH3 region and the second CH3 region in a first-CH3 / second-CH3 heterodimeric protein.

[0071] When used herein, the term “homodimal interaction between first and second CH3 regions” refers to the interaction between a first CH3 region and another first CH3 region in a first-CH3 / first-CH3 homodimeric protein, and the interaction between a second CH3 region and another second CH3 region in a second CH3 / second-CH3 homodimeric protein.

[0072] As used herein, “isolated antibody” means that the material has been removed from its original environment (e.g., the natural environment if it is naturally occurring or derived from host cells, and a culture of host cells or its supernatant if it is recombinantly expressed). Antibodies in purified form are also advantageous. The term “purified” does not require absolute purity; rather, it is intended as a relative definition, indicating an increase in antibody concentration relative to the concentration of contaminants in the composition compared to the starting material.

[0073] The terms “antibody mixture,” “polyclonal mixture,” and “polyclonal antibody mixture” are used interchangeably to describe a mixture of two or more different recombinant antibodies having a predetermined molecular composition. A mixture of two or more different antibodies with a predetermined molecular composition is intended to refer to a mixture of antibodies whose molecular identity is known or can be known prior to the production of the mixture. The molecular identity of an antibody can be determined by identifying its amino acid sequence. Mixtures of the predetermined molecular composition can be collected from different expression or production systems, such as from recombinantly modified host cells, from hybridomas, or from cell extracts using in vitro transcription and / or translation supporting nucleic acid sequences. Mixtures of polyclonal antibodies isolated from, for example, blood, plasma, or serum of a human, animal, or transgenic animal immunized with a foreign antigen or combination of antigens are not considered, in the context of this application, to be mixtures of predetermined molecular compositions. If one or more antibodies in the mixture are produced using recombinantly modified host cells, then “antibody mixture” may be referred to as “recombinant antibody mixture.” As used herein, “recombinantly modified host cell” is intended to refer to a cell that has been introduced with an expression vector, such as an expression vector encoding an antibody. Recombinant host cells include, for example, transfected tumors, such as CHO cells, HEK293 cells, NS / O cells, and lymphocytes.

[0074] As used herein, the term "output mixture" is intended to refer to an antibody mixture in which two or more different antibodies are present at a desired or predetermined concentration ratio. However, as those skilled in the art will understand, some deviation from the desired or predetermined ratio in the output mixture may be permissible. This is especially true if the deviation has no measurable effect, or substantially no measurable effect, on the functionality of the output mixture or the pharmaceutical substance or pharmaceutical product produced from the output mixture. In particular, this is the case where it can be determined in relevant preclinical or clinical trials conducted to determine the profile of the pharmaceutical efficacy and safety of the output mixture, pharmaceutical substance, or pharmaceutical product. Therefore, the desired or predetermined concentration ratio of any two antibodies, as well as specifications for permissible deviations from the desired or predetermined concentration ratio and acceptable upper and lower limits for the antibody concentration ratio, can be defined.

[0075] Therefore, providing a method for generating an output mixture in which two or more different antibodies are present substantially at a desired or predetermined concentration ratio is also within the scope of this invention. Specifically, any two antibodies can be considered to be present substantially at the desired or predetermined concentration ratio if the ratio between the corresponding concentrations is within a permissible deviation from the desired or predetermined concentration ratio. For any two of the two or more different antibodies, the maximum permissible deviation from the desired or predetermined concentration ratio can be, for example, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 2.5%.

[0076] In one example, according to the invention, an output mixture is provided in which two antibodies are present at a desired or predetermined concentration ratio of 1 (1:1). An output mixture with a concentration ratio of 0.9 to 1.1 between the two antibodies, deviating by a 10% permissible deviation from the desired or predetermined concentration ratio, will be considered an output mixture in which the two antibodies are present substantially at the desired or predetermined concentration ratio. Similarly, for an output mixture in which two antibodies are present at a desired or predetermined concentration ratio of 0.5 (1:2), an output mixture with a concentration ratio of 0.45 to 0.55 between the two antibodies will be considered an output mixture in which the two antibodies are present substantially at the desired or predetermined concentration ratio if the permissible deviation from the desired or predetermined concentration ratio is 10%.

[0077] As used herein, the term “input mixture” is intended to refer to an antibody mixture in which at least two of two or more different antibodies mentioned in the context of “output mixture” are present in a concentration ratio that is not a desired or predetermined concentration ratio and / or is not within permissible deviations from a desired or predetermined concentration ratio.

[0078] When the term "purity" is applied to fractions collected in the method according to the invention, "purity" is preferably a measure of the amount of a specific antibody, such as a monoclonal antibody, relative to the amount of other proteins or protein substances, including one or more antibodies having a different amino acid sequence from the specific antibody. For example, when this disclosure teaches the collection of fractions containing a specific antibody with a purity of at least 80%, or teaches the merging of multiple such fractions, it is required that the amount of the specific antibody in each fraction is at least 80% of the total amount of other proteins or protein substances (including other antibodies, such as monoclonal antibodies) in that fraction. To determine "purity," all relative amounts of the antibody are determined or calculated on a weight / weight (w / w) basis.

[0079] As used herein, the term "host cell" is intended to refer to a cell that has been introduced with an expression vector, such as an expression vector encoding the antibody of the present invention. Recombinant host cells include, for example, transfected tumors, such as CHO cells, HEK293 cells, NS / O cells, and lymphocytes.

[0080] When used in this article, the term “co-expression” of two or more nucleic acid constructs refers to the expression of two constructs in a single host cell.

[0081] When used herein, the term “co-production” of two or more antibodies refers to the recombinant production of two or more antibodies in a single container, such as a bioreactor.

[0082] When used in this document, "antibody ratio" refers to the ratio of different antibodies in a mixture. This can be the mass ratio or molar ratio of the antibodies in the mixture. The antibody ratio can be derived from analytical methods such as analytical chromatography, mass spectrometry, or bioanalytical methods.

[0083] The terms “predetermined ratio,” “predetermined concentration ratio,” and “predetermined antibody ratio” are used interchangeably to describe the desired antibody ratio in a mixture for a given application. A predetermined ratio can be defined by a specification that specifies acceptable upper and lower limits for the relative ratio of each antibody.

[0084] As used herein, the term "effective cell" refers to an immune cell that participates in the effector phase of an immune response, in contrast to the cognitive and activation phases of the immune response. Exemplary immune cells include cells of myeloid or lymphoid origin, such as lymphocytes (e.g., B cells and T cells, including cytolytic T cells (CTLs)), killer cells, natural killer cells, macrophages, monocytes, eosinophils, and polymorphonuclear cells, such as neutrophils, granulocytes, mast cells, and basophils. Some effector cells express specific Fc receptors and perform specific immune functions. In some embodiments, effector cells are capable of inducing antibody-dependent cytotoxicity (ADCC), such as natural killer cells. In some embodiments, effector cells may phagocytose target antigens or target cells.

[0085] The term "reduction conditions" or "reduction environment" refers to conditions sufficient to allow the reduction of interchain disulfide bonds in the hinge region of an antibody.

[0086] The term "resin" refers to a matrix modified with ligands such as chemical groups or biomolecules to provide a matrix with binding properties for chromatographic applications. Chromatographic matrices include beads, monolithic supports, filters, membranes, and gels.

[0087] When used herein, the term "affinity reagent" refers to a resin containing a ligand immobilized on a matrix and specifically binding to surface groups of molecules, such as amino acid or sugar side chains, and typically possessing specific three-dimensional structural features and specific charge properties. Affinity reagents are tools in affinity chromatography, where purification is achieved through specific interactions between the ligand and the product.

[0088] When used herein, the term "protein L" refers to recombinant protein L immobilized on a matrix to form affinity ligands with affinity for a subset of the variable domains of the immunoglobulin kappa light chain. For example, protein L affinity reagents can be manufactured by GE Healthcare using HiTrap. TM Protein L and Capto TM L for sale.

[0089] When used herein, the term "LambdaFabSelect" refers to a recombinant 13kDa camel-derived single-chain antibody immobilized on a matrix to form an affinity ligand with affinity for the constant domain of the human immunoglobulin lambda light chain. For example, the LambdaFabSelect affinity reagent can be manufactured by GE Healthcare using LambdaFabSelect... TM sell.

[0090] When used herein, the term "KappaSelect" refers to a recombinant 13kDa camelid-derived single-chain antibody immobilized on a matrix to form an affinity ligand with affinity for the constant domain of the human immunoglobulin kappa light chain. For example, GE Healthcare may use KappaSelect... TM sell.

[0091] When used herein, the term "KappaXL" refers to a recombinant 13 kDa camelid-derived single-chain antibody immobilized on a matrix to form an affinity ligand with affinity for the constant domain of the human immunoglobulin kappa light chain. For example, the KappaXL affinity reagent can be manufactured by Thermo Fisher using CaptureSelect. TM KappaXL for sale.

[0092] When used herein, the term "IgG-CH1" refers to a recombinant 13kDa camelid-derived single-chain antibody immobilized on a matrix to form an affinity ligand with an affinity for the human CH1 domain. For example, the IgG1-CH1 affinity reagent can be manufactured by ThermoFisher using CaptureSelect. TM IgG-CH1 is for sale.

[0093] The term "treatment" refers to the application of an effective amount of the therapeutically active mixture of different antibodies of the present invention, such as monoclonal antibodies, to alleviate, improve, prevent or eliminate (cure) symptoms or disease states.

[0094] The term "effective amount" or "therapeutic effective amount" refers to the amount that effectively achieves the desired therapeutic outcome at the necessary dose and for the necessary duration. The therapeutically effective amount of the different antibodies, such as monoclonal antibodies, in the therapeutically active mixtures of the present invention can vary with factors such as disease state, age, sex, individual weight, and the ability of the binder to elicit the desired response in an individual. The therapeutically effective amount is also the amount by which any toxic or adverse effects of the different antibodies, such as monoclonal antibodies, in the mixtures of the present invention are outweighed by the beneficial therapeutic effects.

[0095] In the context of this invention, the term "active pharmaceutical ingredient" is defined as any substance or mixture of substances intended for use in the manufacture of a pharmaceutical (medicinal) product, and which, when used in the production of a pharmaceutical product, becomes an effective component of the pharmaceutical product. Such substances are intended to provide pharmacological activity or other direct effects in the diagnosis, cure, relief, treatment, or prevention of a disease, or to affect the structure and function of the body. This definition aligns with the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) (see “ICH HARMONISED TRIPARTITE GUIDELINE, GOODMANUFACTURING PRACTICE GUIDE FOR ACTIVE PHARMACEUTICAL INGREDIENTS, Q7; Current Step 4 version, dated November 10, 2000; available at http: / / www.ich.org / fileadmin / Public_Web_Site / ICH_Products / Guidelines / Quality / Q7 / Step4 / Q7_Guideline.pdf) and the U.S. Food and Drug Administration (FDA) (see Guidance for Industry CGMP for Phase 1 Investigational Drugs, U.S. Department of Health and Human Services FDA Center for Drug Evaluation and Research (CDER), Center for Biologics Evaluation and Research (CBER), Office of Regulatory Affairs). This definition of "active pharmaceutical ingredient" is consistent with that used in the Guidance Compliance Regulatory Information (ORA), July 2008, available at https: / / www.fda.gov / downloads / Drugs / GuidanceComplianceRegulatoryInformation / Guidances / UCM070273.pdf.

[0096] For the purposes of this invention, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol.Biol.48:443-453) was used to determine sequence identity between two amino acid sequences, as implemented in the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, TrendsGenet.16:276-277), preferably version 5.0.0 or higher of the Needle program. The parameters used were a nick opening penalty of 10, a nick extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The Needle output marked "Longest Identity" (obtained using the -nobrief option) was used as the percentage identity and calculated as follows:

[0097] (identical residues x 100) / (alignment length - total number of gaps in alignment).

[0098] The retention of similar residues can also be or alternatively determined by using similarity scores, such as by applying a BLAST procedure (e.g., using the standard setting BLOSUM62, open gap = 11 and extended gap = 1, BLAST 2.2.8 available from NCBI).

[0099] Other aspects and embodiments of the invention

[0100] In one embodiment, the present invention relates to a method for producing an output mixture of two or more different antibodies, said different antibodies having differences in their amino acid sequences, said differences enabling separation of the antibodies by chromatography, wherein...

[0101] - The two or more different antibodies are present in the output mixture at or substantially at a desired or predetermined concentration ratio; and

[0102] -The method includes the following steps:

[0103] a. Provide an input mixture, wherein the two or more different antibodies are not present, or substantially not present, at the desired or predetermined concentration ratio;

[0104] b. Separate the two or more antibodies by chromatography;

[0105] c. Recover the two or more antibodies in the amount required to produce the output mixture.

[0106] Therefore, a novel method is provided for producing a controlled mixture of two or more different antibodies. The advantage of this method is that the same relative amount of each different antibody can be obtained in the output mixture in a manner independent of the concentration of the different antibodies provided in the input mixture. Thus, the method can compensate for or correct for the variability in the production yield of each different antibody in order to standardize the output mixture to obtain a desired predetermined ratio of antibodies.

[0107] In particular, the method according to the invention is intended for use in the manufacture of pharmaceutical products or drug products that are mixtures of antibodies comprising two or more different antibodies. In this case, the method of the invention can be used to produce a pharmaceutical substance in which the amount or concentration of the two or more antibodies and the ratio between the concentrations of the two or more antibodies are such that the pharmaceutical substance can be formulated into a composition conforming to the applicable pharmaceutical product specifications without any other means or measures to alter or substantially change the ratio between the antibody concentrations.

[0108] Drug Product Specifications (DPMS) list various standards that a batch of drug products must meet to be released. For drug products that are controlled mixtures of several antibodies, DPMS specifies the range of concentration ratios or relative amounts that each individual antibody must fall within. Typically, in early clinical development, such ranges listed in the DPMS are based on preclinical data obtained from in vitro and in vivo studies. The ranges listed at this stage of development are usually relatively broad. As clinical development progresses, the ranges can be modified based on data from clinical trials and the manufacturing process. As those skilled in the art will know, DPMS are dynamic throughout development and must be accepted by authorities before the drug can be marketed: for example, based on the manufacturer showing that the relative amounts of one or more antibodies are exactly within one or more corresponding ranges listed in the pending DPMS in several consecutive batches of drug products, the regulatory agency may require a narrowing of the acceptable ratio ranges listed in the DPMS.

[0109] In the context of this invention, the desired or predetermined concentration ratio of two or more different antibodies in the output mixture, and the permissible deviation from the desired or predetermined concentration ratio, can therefore correspond to the requirements of the pharmaceutical product specification. Alternatively, the predetermined concentration ratio can correspond to the concentration ratio of the pharmaceutical product specification, with a permissible deviation from that ratio being less than the concentration ratio allowed in the pharmaceutical product specification, because this will ensure that the relative amount of each antibody in the output mixture never approaches the limits of the range listed in the pharmaceutical product specification. It is also concluded that if a regulatory authority requires a change in the acceptable concentration ratio range, the predetermined concentration ratio established for the method of this invention and / or the permissible deviation therefrom can be changed accordingly.

[0110] In addition to the relative amount of each antibody, the pharmaceutical product specification may also specify the total amount of antibodies or the total protein concentration of the pharmaceutical product. Other criteria specified in the pharmaceutical product specification may include pH, as defined, for example by a target value and acceptable deviations therefrom; osmolality, as defined, for example by an acceptable range; host cell protein content, as defined, for example by an upper limit; the color and transparency of the product; and the content of visible and subvisible particles. Any of these criteria may be applied to the output mixtures or pharmaceutical substances provided according to the present invention and / or pharmaceutical products obtained by formulating output mixtures or pharmaceutical substances.

[0111] In the method according to the invention, the output mixture may be a pharmaceutical substance.

[0112] In some embodiments, the method of the present invention further includes processing the output mixture to produce a pharmaceutical substance, wherein two or more different antibodies are present at or substantially at the same concentration ratio as the desired or predetermined concentration ratio specified above. In further embodiments, the method of the present invention further includes processing the output mixture to produce a pharmaceutical product, wherein two or more different antibodies are present at or substantially at the same concentration ratio as the desired or predetermined concentration ratio specified above.

[0113] In other embodiments, the output mixture is processed to produce a pharmaceutical substance or pharmaceutical product without any other means or measures that alter or substantially change the ratio between antibody concentrations, wherein the relative amounts of two or more antibodies and the ratio between their concentrations conform to applicable pharmaceutical product specifications. Another advantage of the invention is that two or more different antibodies in the mixture can be produced, purified, and recovered in parallel, without having to produce and purify each antibody separately. This simplifies the manufacturing process compared to purifying each antibody separately and then mixing them in the correct ratio, thus saving costs associated with mixture production.

[0114] Therefore, the method of the present invention provides an efficient new way to produce a controlled output mixture of two or more different antibodies having a predetermined concentration ratio of various different antibodies, the method being carried out by downstream process control, wherein the concentrations of different antibodies provided from the input mixture from the upstream process are not adequately controlled or regulated.

[0115] In one embodiment of the invention, the different antibodies for the mixture are produced from separate host cells and then purified first by known methods, such as by using protein A or protein G, which capture the antibodies based on their affinity for the antibody constant region, thereby separating the antibodies from the cellular material. Thus, in one embodiment, antibodies for this method can be initially provided by purification without standardizing the ratio between the different antibodies. In this method, standardization is achieved by one or more chromatographic steps, wherein all the different antibodies in the mixture are recovered at a predetermined ratio. An important feature of this method is that the different antibodies in the mixture can be separated by a given chromatographic method because all the different antibodies will be recovered and standardized in the chromatographic step. Different antibodies can be separated using chromatography, and excess antibodies present in the desired composition can be subtracted from the product to produce the desired composition. Alternatively, different antibodies can be separated using chromatography, then fractionated, and recombined with the desired composition. Therefore, in cases where antibodies are initially found to be inseparable by chromatography, one or more antibodies must be modified to achieve separation. In a preferred embodiment, the method preferably uses a single chromatographic resin in a single step. In step c), two or more antibodies can be recovered by collecting a portion of the eluent or flow product containing two or more different antibodies produced in step b).

[0116] In the method according to the invention, it is preferred that each binding specificity and / or each antibody charge variant in the input mixture is also found in the output mixture.

[0117] In the method according to the invention, step (c) may include recovering two or more antibodies in the same merging or fraction to obtain an output mixture. Alternatively, step (c) may include recovering two or more antibodies from multiple mergings or fractions and combining the multiple mergings or fractions or portions thereof to obtain an output mixture.

[0118] In the method according to the invention, the chromatography in step (b) preferably produces an eluent and a flow product, and the output mixture can be produced as follows:

[0119] i) Collect the eluent and discard the flow-through; or

[0120] ii) Discard the eluent and collect the flow.

[0121] exist Figure 1 In a specific embodiment of the invention shown in B, step (b) includes adjusting the conditions of the chromatography step such that, under these conditions, the total binding capacity of a given antibody is sufficient to retain an amount of the antibody required to provide the output mixture.

[0122] exist Figure 1 In a particular embodiment of the invention shown in D, step (b) includes adjusting the conditions of the chromatography step such that the total binding capacity of a given antibody is sufficient to retain an amount of each antibody exceeding the amount required to provide the output mixture.

[0123] In one embodiment of the invention, the method includes separating two or more antibodies and reducing excess of one or more antibodies to recover a predetermined ratio of two or more different antibodies. The desired fraction of the chromatographic eluent is typically collected by controlling a valve directing the eluent flow to a waste container or collection container. To reduce excess of one or more antibodies, excess protein predetermined using an analytical assay can be introduced into the waste container. Each antibody is typically eluted as a collection of charge variants, the distribution of which is maintained between manufacturing batches through appropriate process control to ensure batch-to-batch consistency. Excess protein may be siphoned off during ion exchange or mixed-mode resin elution by redirecting antibody fractions separated by specific charges into the waste container. In one embodiment of the invention, the charge distribution of each individual antibody in the input mixture is recovered in the output mixture.

[0124] In one embodiment of the invention, the charge distribution of each individual antibody in the input mixture is recovered in the input mixture by alternating elution switching valves between waste and collection positions throughout the entire duration of the eluted antibody peak. The time delay allocated to the waste position relative to the time delay allocated to the elution position controls the relative amount of peak reduction throughout the entire duration. The frequency of subsequent waste / collection cycles defines the resolution for maintaining the charge distribution.

[0125] In another embodiment of the invention, the charge distribution of each individual antibody in the input mixture is recovered in the output mixture by using an adjustable flow divider or adjustable flow splitter that dynamically distributes the eluent between the waste and collection containers. The adjustable flow divider or adjustable flow splitter can be used to direct a predetermined fraction of the eluent stream to the waste container while directing the remaining fraction to the collection container. The relative fractions directed to the waste or collection container based on a predetermined ratio of two or more different antibodies in the output mixture can be inferred from the composition of the input mixture, measured using an analytical assay in series with or prior to step b). In one embodiment, the adjustable flow divider or adjustable flow splitter can be electronically controlled.

[0126] In one embodiment of the invention, dynamic control of the waste and collection streams is achieved by applying different diaphragm valves to the two liquid streams. In another embodiment, dynamic control of the waste and collection streams is achieved through feedback pressure control of the liquid streams using different diaphragm valves in combination with pressure relief valves. The diaphragm valves restrict the flow of material, eluent, or waste liquid to achieve a predetermined ratio of two or more different antibodies in the output mixture. In one embodiment, the diaphragm valves of the aforementioned embodiments are replaced by pinch valves, butterfly valves, or other valves suitable for controlling liquid flow in biological processes.

[0127] In the method according to the invention, each of the two or more different antibodies is preferably present in the output mixture in a therapeutically effective amount.

[0128] In a particular embodiment of the invention, the least abundant of the two or more different antibodies is present in an amount of at least 1% (w / w), 2% (w / w), 3% (w / w), 4% (w / w), 5% (w / w), 6% (w / w), 7% (w / w), 8% (w / w), 9% (w / w), or 10% (w / w) of the most abundant of the two or more different antibodies. Specifically, two or more antibodies may be present in amounts such that the ratio (w / w) between any two antibodies is between 1:5 and 5:1, such as 1:4 and 5:1, 1:3 and 5:1, 1:2 and 5:1, 1:1 and 5:1, 2:1 and 5:1, 3:1 and 5:1, 3:4 and 5:1, 1:5 and 4:1, 1:5 and 3:1, 1:5 and 2:1, 1:5 and 1:1, 1:5 and 1:2, 1:5 and 1:3. The ratios are between 1:5 and 1:4, 1:4 and 4:1, 1:4 and 3:1, 1:4 and 2:1, 1:4 and 1:1, 1:4 and 1:2, 1:4 and 1:3, 1:3 and 4:1, 1:3 and 3:1, 1:3 and 2:1, 1:3 and 1:1, 1:3 and 1:2, 1:2 and 4:1, 1:2 and 3:1, 1:2 and 2:1, 1:2 and 1:1, 1:1 and 4:1, 1:1 and 3:1, or for example, between 1:1 and 2:1. Each of the two or more different antibodies may be an active pharmaceutical ingredient.

[0129] In addition to requiring antibodies to be present in the output mixture produced according to the invention at a certain concentration ratio, there may also be minimum requirements for the absolute amount of each antibody present in the output mixture. In most commercial products, the amount of antibody is substantially higher than 1 g / L, whether intended for therapeutic or other purposes. Therefore, the method according to the invention may include recovering two or more antibodies in the amount required to provide the output mixture, wherein the total amount of antibodies (i.e., the combined amount of all antibodies present in the output mixture) is 0.5 g / L or more, for example 1 g / L or more, 1.5 g / L or more, 2 g / L or more, 3 g / L or more, 4 g / L or more, 5 g / L or more, 7 g / L or more, 8 g / L or more, 9 g / L or more, or for example 10 g / L or more.

[0130] Furthermore, the method according to the invention may include recovering two or more antibodies in the amount required to provide the output mixture, wherein the total amount of antibodies (i.e., the combined amount of all antibodies present in the output mixture) is 0.5-20 g / L, for example 1-20 g / L, 1.5-20 g / L, 2-20 g / L, 3-20 g / L, 4-20 g / L, 5-20 g / L, 7-20 g / L, 8-20 g / L, 9-20 g / L, or for example 10-20 g / L.

[0131] The method according to the invention is applicable to the production of high antibody titers, so the output mixture can be a pharmaceutical product for indications with high product demand, such as a pharmaceutical product for use in cancer therapy.

[0132] In some embodiments of the invention, at least one of the two or more antibodies is an antibody that binds to an antigen expressed on the surface of a tumor, such as a solid tumor, such as a metastatic solid tumor, or such as a metastatic locally advanced tumor, or such as a hematologic malignancy. Specifically, the solid tumor may be selected from: colorectal cancer, including colorectal carcinoma and colorectal adenocarcinoma, bladder cancer, osteosarcoma, chondrosarcoma, breast cancer, including triple-negative breast cancer, central nervous system cancers, including glioblastoma, astrocytoma, neuroblastoma, neurofibrosarcoma, neuroendocrine tumors, cervical cancer, endometrial cancer, gastric cancer, including gastric adenocarcinoma, head and neck cancer, kidney cancer, liver cancer, including hepatocellular carcinoma, lung cancer, including NSCLC and SCLC, ovarian cancer, pancreatic cancer, including pancreatic ductal carcinoma and pancreatic adenocarcinoma, sarcoma, or skin cancer, including malignant melanoma and non-melanoma skin cancer.

[0133] In other embodiments, at least one of the two or more antibodies is an antibody that binds to an antigen expressed in a blood malignancy, such as an antibody selected from the following blood malignancies: leukemia, including chronic lymphocytic leukemia and myeloid leukemia, including acute myeloid leukemia and chronic myeloid leukemia; lymphoma, including non-Hodgkin's lymphoma or multiple myeloma, including Hodgkin's lymphoma; and myelodysplastic syndromes.

[0134] According to other embodiments of the invention, at least one of the two or more antibodies is an antibody that binds to an antigen associated with an immune or autoimmune disease, an inflammatory disease, a cardiovascular disease, a disease of the central nervous system (CNS) or a musculoskeletal disease, or an antigen expressed during an immune or autoimmune disease, an inflammatory disease, a cardiovascular disease, a disease of the central nervous system (CNS) or a musculoskeletal disease.

[0135] In one embodiment of the method of the present invention, the mixture of different antibodies is a mixture of two different antibodies. In another embodiment of the present invention, the mixture of different antibodies is a mixture of three or more different antibodies, for example, a mixture of four or more, or five or more, or six or more, or seven or more, or eight or more, or nine or more, or even a mixture of ten or more different antibodies. In one embodiment of the present invention, the mixture of two or more different antibodies is a mixture of three different I antibodies. In another embodiment, it is a mixture of four different antibodies. In another embodiment, it is a mixture of five different antibodies. In another embodiment, it is a mixture of six different antibodies. In another embodiment, it is a mixture of seven different antibodies. In another embodiment, it is a mixture of eight different antibodies. In another embodiment, it is a mixture of nine different antibodies. In another embodiment, it is a mixture of ten different antibodies. In one embodiment of the present invention, the different antibodies in the mixture bind to the same target but different epitopes of the target. In one embodiment of the present invention, two or more different antibodies in the mixture bind to the same target but different epitopes of the target. In one embodiment of the invention, three or more different antibodies in the mixture bind to the same target but different epitopes of the target.

[0136] Specifically, a mixture of different antibodies may contain 2-10 different antibodies, such as 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, 5-6, 6-10, 6-9, 6-7, 7-10, 7-9, 7-8, 8-10, 8-9, or 9-10 different antibodies.

[0137] In one embodiment of the invention, all antibodies in the mixture bind to the same target but different epitopes of the target. The different epitopes may be overlapping epitopes. In another embodiment of the invention, the mixture of different antibodies is a mixture of antibodies in which two or more antibodies bind to the same target but different epitopes and one or more antibodies bind to different targets. In yet another embodiment, the different antibodies in the mixture bind to different targets.

[0138] In a particular embodiment of the invention, at least one of the two or more different antibodies is a monoclonal antibody. In a further embodiment, all of the two or more different antibodies are monoclonal antibodies. In one embodiment of the invention, the input mixture of two or more different antibodies in step a) is produced by co-expression from a clonal cell population. In one embodiment of the invention, the input mixture of two or more different antibodies in step a) is produced by co-culturing in a single bioreactor of different cells each expressing a single antibody species. In one embodiment of the invention, the input mixture of two or more different antibodies in step a) is produced by co-culturing different cells in a single bioreactor each expressing one or more antibody species. In one embodiment of the invention, the input mixture of two or more different antibodies in step a) is produced in more than one bioreactor, after which the cell culture supernatant is mixed and then downstream processed. In one embodiment of the invention, the input mixture of two or more different antibodies in step a) is produced by culturing different cells each expressing a single antibody species in separate bioreactors, after which the cell culture supernatant is mixed and then downstream processed.

[0139] In one embodiment of the invention, the two or more different antibodies in the mixture differ in their amino acid sequences, resulting in differences in the charge properties of the two or more antibodies, causing them to interact differentially with the chromatographic resin, such as an ion exchange resin. Therefore, the two or more different antibodies can be separated by using an ion exchange resin such as a cation exchange resin or an anion exchange resin, or a mixed-mode resin having interacting ionic components. In another embodiment of the invention, the two or more different antibodies in the mixture differ in their amino acid sequences, resulting in differences in the hydrophobic properties of the two or more antibodies, causing them to interact differentially with the chromatographic resin. Therefore, the two or more different antibodies can be separated, for example, by using a hydrophobically interacting resin or a mixed-mode resin having interacting hydrophobic components. In yet another embodiment of the invention, the two or more different antibodies in the mixture differ in their amino acid sequences, resulting in differences in the affinity of the two or more antibodies for the chromatographic resin. Therefore, the two or more different antibodies can be separated by using an affinity resin.

[0140] Those skilled in the art will be fully aware of the various methods that can be used to separate biomolecules from impurities, including precipitation, liquid-liquid extraction, and high-efficiency tangential flow filtration (Gagnon, PJ Chromatography A 1221 (2012) 57-70). Chromatography, as used in the context of this invention, is a primary method for the preparative separation of biomolecules such as antibodies, and the selection of a type of chromatography suitable for a particular purpose will be within the capabilities of those skilled in the art.

[0141] The chromatographic matrix can be applied in either fluidized bed or fixed bed chromatography, with the fixed bed form being predominant (Gagnon, 2012). Gagnon also classifies the stationary phase structure of chromatography as including diffusing microparticles, perfused microparticles, adsorbent microfiltration membranes, or monolithic materials. Therefore, following Gagnon's classification, the solid phase structure of the chromatography used in the method of this invention, including step b) of the method defined above, can include materials selected from the group consisting of diffusing microparticles, perfused microparticles, adsorbent microfiltration membranes, and monolithic materials.

[0142] In the method of the present invention, for example in step b) of the method as defined above, the material constituting the chromatography matrix may be selected from the group consisting of: natural polymers, such as cellulose, agarose, dextran and chitosan; synthetic polymers, such as hydrophobic vinyl polymers, polyacrylamide polymers and polyvinylstyrene; inorganic media, such as hydroxyapatite, silica or porous glass, or composite materials (Jungbauer, AJ Chromatography A, 1065 (2005) 3-12).

[0143] In the method of this invention, the chromatographic matrix used, for example, in step b) of the method defined above, can be modified with ligands to produce resins that can separate biomolecules using different mechanisms of action, depending on the properties of the ligands. These mechanisms of action include adsorption, ion exchange, size exclusion, affinity, hydrophobic interaction, metal chelation, normal-phase, reversed-phase chromatography, or mixed-mode chromatography utilizing more than one mechanism of action (Jungbauer, 2005; Gagnon, 2012). The resulting resins can separate mixtures of biomolecules based on their physical properties. The most important classes of such resins for antibody separation are anion exchange resins, cation exchange resins, hydrophobic interaction resins, or mixed-mode resins (Gagnon, 2012). Therefore, in a particular embodiment of the invention, the chromatographic resin used in this method, for example, in step b) of the method as defined above, can be selected from the group consisting of anion exchange resins, cation exchange resins, hydrophobic interaction resins, or mixed-mode resins. The resin can be selected based on the antibodies to be separated and how these differ in their charge, size, hydrophobicity, etc. That can be performed using standard assays known to those skilled in the art.

[0144] Because of the potential considerations in selecting a suitable purification strategy (e.g., Low, D et al. J Chromatography B; 848 (2007) 48-63; Clive Dennison. A Guide to Protein Isolation, Chromatography (Book Chapter), Chapter 4; p: 71-114; 2002; Springer Netherlands), some common ligands used in single-mode or mixed-mode protein chromatography have been reviewed (e.g., Kallberg, K et al. Biotechnol. J. 2012, 7, 1-11). The physical properties of the antibodies in the mixture can be sufficiently different on these resins to allow separation, thus controlling the composition of the antibody mixture. In such cases, separation can be improved by introducing one or more substitutions or point mutations, such as the substitutions disclosed below, to modulate the physical properties of one or more antibodies.

[0145] Affinity reagents are another important class of resins used to separate biomolecules such as antibodies. Here, biomolecules are immobilized on a matrix to form a resin that specifically binds to the antibody. The immobilized biomolecules can be selected from immunoglobulin ligands, such as naturally occurring immunoglobulin ligands including protein A, protein G, and protein L, which are often modified to increase their stability or other properties (Gagnon, 2012). The table below provides information on such biomolecules:

[0146] Biomolecular ligands / affinity reagents

[0147]

[0148]

[0149] Therefore, the chromatography in step b) of this invention can use an affinity reagent comprising a biomolecule immobilized on a matrix, the immobilized biomolecule containing an amino acid sequence selected from the group consisting of:

[0150] a) The amino acid sequence shown in any one of SEQ ID NO: 62, 63 and 64;

[0151] b) A subsequence of any of the sequences in a), which contains at least 200, for example at least 300, at least 400, at least 500, at least 600, at least 700 or at least 800 consecutive amino acid residues;

[0152] c) Amino acids that have at least 80%, for example 85%, 90%, 95%, 98%, or 99% sequence identity with any amino acid sequence defined in a) and b).

[0153] Alternatively, biomolecules can be affinity reagents, such as camelid VHH IgG analog ligands (Gagnon, 2012). To fully control the composition of an antibody mixture using affinity reagents, a sufficient number of specific affinity reagents need to be selected to provide specificity and control over all components in the mixture. Again, the specificity of the antibody to the affinity reagent can be engineered into the antibody by introducing substitutions and / or designing point mutants, which reduce and / or prevent the antibody from binding to the specific affinity resin.

[0154] Other affinity resins that may be useful in the method according to the invention are resins that bind to tags, including immobilized metal affinity chromatography (IMAC) resins that recognize His tags, and those that bind to Strep-tag II and C tags. Other potential affinity resins include those that bind to carbohydrates: lentil lectin resin and Con A resin. Other resins that can be used according to the invention include CaptureSelect FcXL.

[0155] If two or more antibodies are initially found to be non-separable in the chromatogram, at least one antibody can be modified in the amino acid sequence to achieve the difference in separation. In one embodiment, the modification may introduce one or more amino acids having a different charge than the wild-type amino acids that are substituted to introduce separability in the chromatogram. In one embodiment, two or more antibodies in the mixture are modified to achieve separability. In another embodiment, three or more antibodies in the mixture are modified to achieve separability. One advantage of the present invention is that two or more antibodies can be generated in the input mixture at an unknown concentration ratio and can be separated by chromatography and recovered at the correct predetermined concentration ratio. This means recovering each of the input antibodies in the chromatographic method and reducing excess one or more antibodies from the mixture to obtain the correct ratio, or fractionating the different antibodies in the chromatographic experiment and re-merging them in the desired composition. This can be obtained by analyzing the different peaks in the chromatogram and thus by tandem analysis of the concentrations of the different antibodies. Therefore, the correct ratio of the different antibodies can be recovered, and any excess one or more different antibodies can be discarded. Therefore, in one embodiment of the invention, two or more different I antibodies of the output mixture can be recovered in a single merge in step c). This can be achieved by tandem analysis of the concentrations of different antibodies, so that each antibody pool is recovered in the correct ratio to each other, and excess antibodies are discarded in the waste pool. Thus, the recovered mixture is recycled and collected in a single pool.

[0156] Therefore, in one embodiment of the invention, the method includes the isolation of two or more antibodies and the reduction of excess of one or more antibodies to recover a predetermined ratio of two or more different antibodies.

[0157] In another embodiment of the invention, two or more antibodies from step b) are separated into different fractions, and then fractions containing one of the antibodies with a purity of at least 80% are combined at predetermined concentration ratios of different compounds. Thus, a method is provided in which different antibodies are recovered into separate fractions by a chromatographic step. This is possible because different antibodies can be separated by chromatography. The collected fractions will contain different antibodies of different purities. It should be understood that only fractions containing one of the antibodies with a purity of at least about 80% will be used and combined with other fractions containing other antibodies with a purity of at least about 80%. The various pure fractions of each different antibody will be combined at predetermined ratios to obtain an output mixture. Therefore, the method according to the invention may include:

[0158] i) In step (b), two or more antibodies are separated into different fractions, and for each antibody, one or more fractions containing the antibody at a purity of at least 80% are selected; and

[0159] ii) The output mixture is provided by merging the volumes of the selected fractions, adjusting the volume size to provide a predetermined concentration ratio of the two or more antibodies.

[0160] In one embodiment, the method includes a further step of determining the antibody concentration in each fraction prior to antibody merging. This can be done by analytical or tandem analysis of the fractions. In other embodiments, only fractions containing one of the antibodies at a purity of at least about 85% will be used. In other embodiments where higher purity of the antibody in the fraction is required, only fractions containing the antibody at a purity of at least about 90%, or at least about 95%, or even at least about 97% or 98% will be used.

[0161] In yet another embodiment of the invention, the separation of two or more antibodies is accomplished using a single chromatography step with a single chromatography resin. In one embodiment, the single chromatography resin is a preparative chromatography resin.

[0162] In another embodiment of the invention, the separation of two or more antibodies is accomplished by using a mixture of chromatographic resins in a predetermined ratio or by using multiple resins in series. This can be advantageous when the resin has specific and known binding capacity, thus knowing how much antibody it will bind. Excess antibody is reduced by binding to the resin and saturating it so that the unbound fraction can be collected and the output mixture recovered at a predetermined concentration ratio, or by subsequently eluting the bound antibody at a predetermined ratio.

[0163] In another embodiment of the invention, the composition of the input mixture is measured using an analytical assay prior to step b). Therefore, the concentration of each antibody in the merged mixture is known prior to separation in step b), and this knowledge can be used to recover each antibody at a predetermined ratio.

[0164] In another embodiment of the invention, the composition of the input mixture is measured by an analytical assay in series with the chromatography step in step b. Therefore, the elution conditions of the chromatography are adjusted using the measurement of the composition of the input mixture to reduce excess antibody and produce a mixture with the desired ratio.

[0165] As discussed above, in one embodiment of the invention, the method includes an initial step of determining the separability of two or more antibodies by chromatography, and, if the different antibodies are not separable, then modifying the amino acid sequence of one or more antibodies to obtain separability by chromatography.

[0166] In one embodiment of the invention, the modification of the amino acid sequence of one or more antibodies is selected from: amino acid substitution, addition, or deletion, or a combination thereof, in one or more antibodies of the antibody. Thus, one or more antibodies in a mixture can be modified by one or more substitutions, while other antibodies in the mixture can be modified by the deletion of one or more amino acids and / or by the addition of one or more amino acids. In other embodiments, the only type of modification to the antibody is substitution.

[0167] In one embodiment of the invention, one or more modifications include modifications in the constant domain of one or more antibodies.

[0168] In another embodiment of the invention, one or more modifications include modifications in the variable domains of one or more antibodies. In a preferred embodiment, the modification is in the frame region rather than in the CDR region. Therefore, the specific affinity of the antibody is not altered, or is altered by less than 2-fold, or less than 3-fold, or less than 4-fold. Preferably, the modification is silent relative to antibody functionality.

[0169] In another embodiment of the invention, the modification includes modifications in the framework sequence of the light chain variable region and / or the framework sequence of the heavy chain variable region.

[0170] In another embodiment of the invention, the modification includes the substitution of one or more amino acids in one or more different antibodies.

[0171] In another embodiment of the invention, the modification is a single amino acid substitution in one or more antibodies. In another embodiment of the invention, the modification is a single amino acid substitution in only one antibody. In another embodiment of the invention, the modification is a single amino acid substitution in two antibodies. In another embodiment of the invention, the modification is a single amino acid substitution in a mixture of three different monoclonal antibodies. In another embodiment of the invention, the modification is a single amino acid substitution in a mixture of four different antibodies. In another embodiment of the invention, the modification is a single amino acid substitution in a mixture of five different antibodies.

[0172] In another embodiment of the invention, the modification is the substitution of two amino acids in one or more antibodies. In some embodiments, three, four, five, six or more substitutions may also be made in one or more antibodies to obtain separability.

[0173] In another embodiment of the invention, the modification does not alter the functional properties of one or more modified antibodies. The primary purpose of introducing modifications to the antibody is to enable its separation by chromatography, thereby allowing the antibody to be separated and recovered by chromatography at a selected ratio.

[0174] In another embodiment of the invention, the unchanged functional features are selected from: antibody binding affinity, effector functions such as CDC or ADCC, affinity and clustering.

[0175] In yet another embodiment of the invention, one or more amino acids replace modifications in the heavy chain variable region and / or light chain variable region comprising one or more antibodies, wherein the substitution is located at one or more positions selected from the group consisting of: 1, 6, 17, 24, 48, 75, 90, 93, 96, 97 in the heavy chain variable region and / or 1, 4, 47, 48, 51, 68, 74, 80, 90, 93, and 95 in the light chain variable region, wherein the numbering is based on the IMGT number of the IgG1 variable region.

[0176] In another embodiment of the invention, one or more amino acids replace modifications in the heavy chain variable region and / or light chain variable region of one or more antibodies, wherein the substitution is at one or more positions selected from the group consisting of: 1, 6, 17, 24, 48, 75, 90, 93, 96, 97 in the heavy chain variable region and / or 1, 4, 47, 48, 51, 68, 74, 80, 90, 93 and 95 in the light chain variable region, wherein the numbering is based on the IMGT number of the IgG variable region.

[0177] Therefore, amino acid positions are provided that, for example, if two or more different antibodies are not separated in the chromatogram, they can be modified by substitution. The inventors have discovered that these amino acid positions are suitable for substitution to enable the separation of antibodies by chromatography. It has been found that these amino acids affect resin binding, such that, for example, changes in the charge, size, or hydrophobic interactions of the substituted amino acids compared to the wild type will result in differential interactions between the antibody and the chromatographic resin. Thus, separability can be obtained. It should be understood that amino acid substitution should preferably not alter the functional properties of the antibody. Therefore, variant antibodies can be screened for functionality in standard assays, and variants with unchanged functionality are preferred.

[0178] In another implementation, the mutation is a substitution of an amino acid present in the entirety of the human antibody lineage and is not obviously immunogenic because the mutation in the intact antibody or the processed peptide is not recognized as non-self by the human immune system. Therefore, one or more substitutions can be selected based on naturally occurring lineage variation, allowing for substitutions that are not immunogenic but introduce separability in the chromatogram.

[0179] In one embodiment of the invention, one or more substitutions are introduced to introduce an amino acid having a different charge than the wild-type amino acid at the corresponding position.

[0180] In one particular implementation, two or more different antibodies comprise first and second antibodies that are mutated in the CH3 region of the heavy chain to allow Fab arm exchange, as described in WO 11 / 131746.

[0181] In one embodiment, conditions allowing the formation of bispecific antibodies are described in WO 11 / 131746. Preferably, these conditions are reducing conditions that allow the reduction of interchain disulfide bonds in the hinge region. In one embodiment, the first and second antibodies contain one or more mutations in the CH3 region that are different and cause a stronger heterodimeric interaction between the first and second CH3 regions than a homodimeric interaction between each of the first and second CH3 regions. In one embodiment, the first antibody has amino acid substitutions at positions selected from the group consisting of 366, 368, 370, 399, 405, 407, and 409, and the second antibody has amino acid substitutions at positions selected from the group consisting of 366, 368, 370, 399, 405, 407, and 409, and the first and second antibodies are not substituted at the same positions. In one embodiment, the first antibody is substituted at position 405, and the second antibody is substituted at position 409. In a particular embodiment, the first antibody has an F405L substitution. In another embodiment, the second antibody has a K409R substitution. In a preferred embodiment, the first antibody has an F405L substitution in the CH3 region, and the second antibody has a K409R mutation.

[0182] In a further embodiment, two or more different antibodies comprise monoclonal antibodies with mutations in the CH3 region of the heavy chain discussed above, or bispecific antibodies generated by Fab arm exchange as described in WO 11 / 131746.

[0183] In one embodiment, one or more antibodies comprise the Fc region of human immunoglobulin IgG, wherein, according to EU designations, the Fc region contains a mutation at an amino acid position corresponding to E430, E345, or S440 in human IgG1. According to EU designations, the positions corresponding to E430, E345, and S440 in human IgG1 are located within the CH3 domain of the Fc region. In the context of this invention, these mutations are considered "hexamerization-enhancing mutations".

[0184] The basic principle behind introducing mutations at these sites is based on the finding that when each antibody has a mutation at the E430, E345, or S440 site, a combination of two antibodies binding to the first and second epitopes on cell surface antigens can form a heterohexamer. This heterohexamer formation significantly enhances antibody binding compared to a combination of two antibodies without mutations in the Fc region. Therefore, when binding to the corresponding target on the cell surface, hexamerization enhances the Fc-Fc interaction between the mutated antibodies, while the antibody molecule remains a monomer in solution (WO2013 / 004842; WO2014 / 108198).

[0185] In one embodiment of the invention, the Fc region of one or more antibodies contains a mutation corresponding to E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y, or S440W in human IgG1 (EU number). More specifically, each of the one or more antibodies contains an Fc region comprising a first heavy chain and a second heavy chain, wherein one of the aforementioned hexamerization-enhancing mutations may be present in the first and / or second heavy chain.

[0186] In one embodiment of the invention, one or more antibodies contain a mutation at the amino acid position corresponding to E430 in human IgG1 according to EU numbering, wherein said mutation is selected from the group consisting of E430G, E430S, E430F, and E430T. In one embodiment of the invention, one or more antibodies contain a mutation corresponding to E430G.

[0187] One or more antibodies may contain a mutation at the amino acid position corresponding to E345 in human IgG1 according to the EU number, wherein the mutation is selected from the group consisting of E345K, E345Q, E345R, and E345Y. Preferably, the mutation corresponds to E345K.

[0188] In one specific embodiment of the invention, using the EU numbering system, one or more amino acid substitutions include E345K substitution in the heavy chain constant region.

[0189] In one embodiment of the invention, one or more antibodies may contain a "hexamerization-inhibiting mutation," such as K439E or S440K in human IgG1, EU number. The hexamerization-inhibiting mutation (e.g., K439E or S440K) prevents Fc-Fc interaction with antibodies containing the same hexamerization-inhibiting mutation; however, by combining an antibody with the K439E mutation and an antibody with the S440K mutation, the inhibition is neutralized and Fc-Fc interaction is restored. In one embodiment of the invention, the antibody contains a further mutation at an amino acid position corresponding to one of S440 or K439 in human IgG1, EU number, provided that the mutation at position S440 is not S440Y or S440W. In one embodiment of the invention, the Fc region contains a further mutation at a position corresponding to S440 or K439, provided that if a hexamerization-enhancing mutation is in S440, the further mutation is not in position S440. Antibodies containing mutations at positions corresponding to E430, E345, or S440 according to the invention and further mutations at amino acid positions corresponding to K439, such as the K439E mutation, do not form oligomers with antibodies containing further mutations at amino acid positions corresponding to K439, such as the K439E mutation. However, antibodies containing hexamer-enhancing mutations in E430, E345, or S440 and further mutations in K439, such as the K439E mutation, do form oligomers with antibodies containing hexamer-enhancing mutations in E430 or E345 and further mutations in S440, such as the S440K mutation. Antibodies containing mutations at positions corresponding to E430 or E345 according to the invention and further mutations at amino acid positions corresponding to S440, such as the S440K mutation, do not form oligomers with antibodies containing further mutations at amino acid positions corresponding to S440, such as the S440K mutation. However, antibodies containing hexamer-enhancing mutations in E430 or E345 and further mutations in S440, such as S440K, do indeed form oligomers with antibodies containing hexamer-enhancing mutations in E430 or E345 and further mutations in K439, such as K439. In one embodiment of the invention, the Fc region contains hexamer-enhancing mutations such as E430G and hexamer-inhibiting mutations such as K439E. In one embodiment of the invention, the Fc region contains hexamer-enhancing mutations such as E345K and hexamer-inhibiting mutations such as K439E. In another embodiment of the invention, the Fc region contains hexamer-enhancing mutations, such as E430G, and hexamer-inhibiting mutations, such as S440K. In one embodiment of the invention, the Fc region contains hexamer-enhancing mutations, such as E345K, and hexamer-inhibiting mutations, such as S440K. In one embodiment of the invention, the Fc region contains hexamer-enhancing mutations, such as S440Y, and hexamer-inhibiting mutations, such as K439E.Therefore, an implementation scheme is provided that allows for exclusionary hexamerization between a combination of antibodies containing the K439E mutation and antibodies containing the S440K mutation.

[0190] In one embodiment of the invention, the Fc region contains a mutation at the amino acid position corresponding to S440 in human IgG1 according to EU number, wherein the mutation is selected from the group consisting of S440W and S440Y.

[0191] In another embodiment of the invention, modifying one or more antibodies includes introducing at least one amino acid substitution into the light chain of one or more antibodies, wherein, using the IMGT numbering system, the substitution introduces a proline (P) at position 12 in the variable region of the light chain. Preferably, the substitution eliminates binding to the affinity resin, and chromatography is performed using the affinity resin targeted by the substitution elimination. Thus, the modified antibody will have a different affinity for the affinity resin, which can then be used to separate two or more antibodies. In one embodiment, the affinity reagent is protein L, which has an affinity for the kappa light chain. HiTrap TM Protein L and Capto TM L can be obtained from GE Healthcare.

[0192] In another embodiment of the invention, modifying one or more antibodies includes introducing at least one amino acid substitution into the light chain of one or more antibodies, wherein the substitution eliminates binding to the affinity resin, and wherein the substitution is selected from V110D, V110R, V110E, V110H, V110K, V110N, V110P, V110Q, V110W, and E143D using the EU numbering system, wherein chromatography is performed using the affinity resin targeted by the substitution-eliminated binding. Thus, the modified antibody will have a different affinity for the affinity resin, which can then be used to separate two or more antibodies. In one embodiment, the affinity reagent is CaptureSelect, which has an affinity for the constant region of the kappa light chain. TM Or KappaXL. CaptureSelect can be obtained from ThermoFisher. TM And KappaXL. In another embodiment, the affinity agent is KappaSelect, which has an affinity for the constant region of the kappa light chain. TM KappaSelect is available from GE Healthcare. TM .

[0193] In another embodiment of the invention, modifying one or more antibodies comprises introducing at least one amino acid substitution in the one or more antibodies, wherein the substitution is in the CH1 domain, wherein the substitution eliminates binding to the affinity resin, and the substitution comprises an S157T and / or T164S mutation using the EU numbering system, and wherein chromatography uses the affinity resin targeted by the substitution-eliminated binding. Thus, the modified antibody will have a different affinity for the affinity resin, which can then be used to separate two or more antibodies. In one embodiment, the affinity reagent is an IgG-CH1 affinity reagent, such as CaptureSelect, which is available, for example, from ThermoFisher. TM IgG-CH1.

[0194] In another embodiment of the invention, modifying one or more antibodies comprises introducing at least one amino acid substitution in the heavy chain constant region of the one or more antibodies, wherein the substitution is selected from: M252A, S254M, E380A, E380M, E382A, E382L, S426M, M428G, M428T, M428V, H433D, N434A, N434G, N434S, and M428A using the EU numbering system, wherein the substitution eliminates binding to the affinity resin, and chromatography is performed using the affinity resin targeted by the eliminated binding. Therefore, the modified antibody will have a different affinity for the affinity resin, which can then be used to separate two or more antibodies. In one embodiment, the affinity reagent is protein G.

[0195] In another embodiment of the invention, if the resolution (Rs) is Rs≥0.3 as determined by cation exchange chromatography, then two or more antibodies are determined to be separable; this is done using an ionic strength gradient with Rs≥0.3 according to the formula Rs=2(t2-t1) / (W1+W2), where t1=the retention time of a given antibody, t2=the retention time of the sequentially eluted antibody, and W1 and W2 are the corresponding peak widths of the antibodies at the base of the peaks obtained by extrapolating the relative straight sides of the main peak to the baseline. Thus, the separability of different antibodies in the mixture can be determined. This determination can be performed as an initial step prior to step a) of the method disclosed herein. If two or more different antibodies are not separable in such a chromatographic assay, one or more different antibodies can be modified as described above to allow for chromatographic separation. However, the antibodies in the mixture may be separable in different chromatographic analyses using different resins or different elution conditions. Therefore, in another embodiment, if the resolution (Rs) is Rs≥0.3 as determined by hydrophobic interaction chromatography, then two or more antibodies are determined to be separable; the ionic intensity gradient is used with Rs≥0.3 according to the formula Rs=2(t2-t1) / (W1+W2), where t1=the retention time of the given antibody, t2=the retention time of the sequentially eluted antibody, and W1 and W2 are the corresponding peak widths of the antibodies at the base of the peaks obtained by extrapolating the relatively straight sides of the main peak to the baseline. In another embodiment, if the resolution (Rs) is Rs≥0.3 as determined in a mixed-mode chromatography assay, then two or more antibodies are determined to be separable; the assay is performed using an ionic intensity gradient with Rs≥0.3 according to the formula Rs=2(t2-t1) / (W1+W2), where t1=the retention time of the given antibody, t2=the retention time of the sequentially eluted antibody, and W1 and W2 are the corresponding peak widths of the antibodies at the base of the peaks obtained by extrapolating the relatively straight sides of the main peak to the baseline. In another embodiment, if baseline separation is achieved between the unbound fraction in the unbound column and the antibody in the fraction eluted from the column, or if the resolution (Rs) is Rs≥0.3 as determined in an affinity chromatography assay using a pH gradient according to the formula Rs=2(t2-t1) / (W1+W2) with Rs≥0.3, where t1=retention time of a given antibody, t2=retention time of sequentially eluted antibody, and W1 and W2 are the corresponding peak widths of the antibody at the base of the peak obtained by extrapolating the relatively straight sides of the main peak to the baseline, then, as determined in an affinity chromatography assay, two or more antibodies are determined to be separable.

[0196] Therefore, various tests suitable for determining whether different antibodies can be separated by chromatography are provided, and it can be determined which chromatographic resin is best suited for separating different antibodies. As mentioned above, in the case where antibodies are initially found to be separable by chromatography, it is desirable to modify one or more antibodies in the mixture of amino acid sequences so that the antibodies become separable when tested as described above. When the antibodies are separable by chromatography, different antibodies can be recovered from a single chromatographic step at a desired and predetermined ratio.

[0197] In one embodiment, two or more different antibodies of the input mixture are expressed and provided in different production host cells. In another embodiment, two or more different antibodies are expressed and provided in different production host cells co-cultured in a single container. In yet another embodiment, two or more different antibodies are co-expressed in a single production host cell. Therefore, the present invention is universal in terms of the production of different antibodies in the mixture. An important element of the present invention is that it is not necessary to completely control the concentrations of the different antibodies in the input mixture within the required specifications for the output mixture; therefore, complete control of the upstream process is not required in terms of the relative concentration of each antibody in the mixture. Complete control and standardization of the different antibody ratios are achieved via a downstream process using chromatography.

[0198] It should also be understood that the method according to the invention can be used downstream of a production process in which measures have been taken to standardize different antibody ratios, but such standardization has failed. In that case, there is no suitable downstream process, and the only option is to discard the entire production batch.

[0199] In one embodiment of the invention, two or more different antibodies are selected from the group consisting of IgG1, IgG2, IgG3, or IgG4 antibodies or combinations thereof. In one embodiment, all different antibodies in the mixture are from the same isotype. Thus, in one embodiment, all different antibodies in the mixture are IgG1 antibodies. In another embodiment, all different antibodies in the mixture are IgG2 antibodies. In another embodiment, all different antibodies in the mixture are IgG3 antibodies. In another embodiment, all different antibodies in the mixture are IgG4 antibodies. In another embodiment, the different antibodies in the mixture are a combination of IgG1, IgG2, IgG3, and IgG4 antibodies. In another embodiment, the different antibodies in the mixture are a combination of IgG1 and IgG4 antibodies. In another embodiment, the different antibodies in the mixture include bispecific antibodies.

[0200] On one hand, the method of the present invention is used to produce pharmaceutical products, which are mixtures of different antibodies. On the other hand, the method of the present invention is used to manufacture pharmaceutical products for treating diseases, for clinical trials, for toxicological studies, or for determining batch-to-batch consistency.

[0201] A key element of this invention is that the method results in reproducible outcomes between different batches of output mixtures, such that two or more different antibodies are present at or substantially at a desired or predetermined concentration ratio.

[0202] In another aspect, the present invention relates to mixtures of two or more different antibodies, wherein the mixtures can be obtained by the methods of the present invention. In the mixtures according to the invention, the two or more different antibodies are present at or substantially at a desired or predetermined concentration ratio.

[0203] On the other hand, the present invention relates to a mixture of two or more different antibodies having a predetermined ratio of two or more different antibodies, said antibodies being different in size, charge, hydrophobicity or affinity for chromatographic resins.

[0204] In one embodiment of the invention, the mixture of different antibodies is a mixture of three different antibodies. In another embodiment, it is a mixture of four different antibodies. In another embodiment, it is a mixture of five different antibodies. In another embodiment, it is a mixture of six different antibodies. In another embodiment, it is a mixture of seven different antibodies. In another embodiment, it is a mixture of eight different antibodies. In another embodiment, it is a mixture of nine different antibodies. In another embodiment, it is a mixture of ten different antibodies.

[0205] The antibody mixtures of the present invention can be used to treat diseases. Using mixtures of different antibodies can be an advantage in treating various diseases where conventional monoclonal antibodies as a single therapy are insufficient. This may be due to the downregulation or redirection of the target to a unique pathogenic pathway. By using mixtures of different antibodies, multiple cell surface receptor antigens can be targeted, which can prevent the downregulation or redirection of the target to a unique pathogenic pathway. Using mixtures of antibodies to target multiple epitopes on a single target can be a further advantage, as different antibodies can have different mechanisms of action or different therapeutic efficiencies.

[0206] On one hand, the mixture of two or more different antibodies according to the invention comprises at least one modified antibody containing at least one amino acid substitution in the heavy chain variable region and / or the light chain variable region, wherein the substitution is located at one or more positions selected from the group consisting of: 1, 6, 17, 24, 48, 75, 90, 93, 96, 97 in the heavy chain variable region, and / or 1, 4, 47, 48, 51, 68, 74, 80, 90, 93, and 95 in the light chain variable region, wherein the numbering is based on the IMGT number of the IgG variable region.

[0207] On the other hand, a mixture of two or more different antibodies according to the invention comprises at least one modified antibody, which contains at least an E345K substitution in the heavy chain constant region using the EU numbering system. Thus, the antibodies are modified to have different charges, which can aid in the separation of the antibodies by, for example, ion exchange chromatography.

[0208] In another aspect, the mixture of two or more different antibodies of the present invention comprises at least one modified antibody containing at least one amino acid substitution in the constant region of the kappa light chain of one or more antibodies, wherein the substitution is selected from V110D, V110R, V110E, V110H, V110K, V110N, V110P, V110Q, V110W, and E143D using the EU numbering system. Therefore, the modified antibody will have different affinities to the affinity resin, which can then be used to separate two or more antibodies. In one embodiment, the affinity resin is KappaSelect or KappaXL resin.

[0209] In another aspect, a mixture of two or more different antibodies according to the invention comprises at least one modified antibody containing substitutions of S157T and / or T164S in the CH1 domain using the EU numbering system. Antibodies with such substitutions can have improved affinity for IgG-CH1 affinity resins, such as CaptureSelect affinity resin. In another aspect, a mixture of two or more different antibodies according to the invention comprises at least one modified antibody containing one or more substitutions selected from the group consisting of: M252A, S254M, E380A, E380M, E382A, E382L, S426M, M428G, M428T, M428V, H433D, N434A, N434G, N434S, M428A, using EU numbers. Antibodies with such modifications can have reduced binding to affinity resins such as, for example, Protein G resin.

[0210] In another embodiment, the present invention provides a pharmaceutical composition comprising a mixture of different antibodies as described above as an active ingredient.

[0211] In particular, the pharmaceutical compositions of the present invention may be sterile and have one or more of the following characteristics:

[0212] I. Physiologically acceptable pH, such as pH between 5 and 8, or pH between 6 and 8;

[0213] II. Osmotic pressure concentration by weight moles, which is 600 mOsm / kg or lower, for example, between 600 and 100 mOsm / kg, or for example, between 600 and 200 mOsm / kg; and

[0214] III. Aggregate level, which results in 10% by weight or less of the antibody in the composition being present in aggregate form, such as 9%, 8%, 7%, 6%, 5%, 4%, 3%, or 2% by weight or less.

[0215] The pharmaceutical composition may be particularly isotonic or substantially isotonic, for example having a weight molar osmotic concentration of 290-300 mOsm / kg, such as 295 mOsm / kg.

[0216] In another embodiment, the present invention relates to a mixture of two or more different antibodies described above, used as a medicament. In a preferred embodiment, the mixture is used in a method of treating and / or preventing a disease. In one embodiment, the disease is cancer. In another embodiment, the disease is an infectious disease. The pharmaceutical composition can be formulated using conventional techniques with a pharmaceutically acceptable carrier or diluent and any other known adjuvants and excipients. The pharmaceutical compositions of the present invention may include diluents, fillers, salts, buffers, detergents (e.g., nonionic detergents, such as Tween-20 or Tween-80), stabilizers (e.g., sugars or protein-free amino acids), preservatives, tissue fixatives, solubilizers, and / or other materials suitable for inclusion in the pharmaceutical composition.

[0217] The actual dose level of the active ingredient in the pharmaceutical composition of the present invention can be varied to obtain an amount of active ingredient that effectively achieves the desired therapeutic response for a specific patient, composition, and administration method, without toxicity to the patient. The selected dose level will depend on a variety of pharmacokinetic factors, including the activity of the specific composition of the present invention or its amide, the route of administration, the time of administration, the excretion rate of the specific compound used, the duration of treatment, other drugs, compounds, and / or materials used in combination with the specific composition used, the age, sex, weight, condition, general health status, and medical history of the patient being treated, and similar factors known in the medical field.

[0218] The pharmaceutical composition can be administered via any suitable route and manner. Suitable routes for administering the compounds of the present invention in vivo and in vitro are well known in the art and can be selected by those skilled in the art.

[0219] In one embodiment, the pharmaceutical composition of the present invention is administered parenterally.

[0220] As used herein, the terms "parenteral administration" and "of which parenteral" refer to administration methods other than intestinal and local administration, typically by injection, including intradermal, intravenous, intramuscular, intraarterial, intrasheath, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratendinous, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, intracranial, intrathoracic, epidural, and intrasternal injections and infusions. In one embodiment, the pharmaceutical composition of the invention is administered by intravenous or subcutaneous injection or infusion.

[0221] As those skilled in the art will recognize, the utility of this invention is not limited to antibodies against any particular target or antigen. Exemplary targets for antibodies treated according to this invention include antigens selected from the group consisting of: 5T4; ADAM-10; ADAM-12; ADAM17; AFP; AXL; ANGPT2 anthrax antigen; BSG; CAIX; CAXII; CA72-4; cancer-associated antigen CTAA16.88; CCL11; CCL2; CCR4; CCR5; CCR6; CD2; CD3E; CD4; CD5; CD6; CD15; CD18; CD19; CD20; CD22; CD24; CD25; CD29; CD30; CD32B; CD33; CD37; CD38; CD40; CD40LG; CD44 CD47; CD52; CD56; CD66E; CD72; CD74; CD79a; CD79b; CD80; CD86; CD98; CD137; CD147; CD138; CD168; CD200; CD248; CD254; CD257; CDH3; CEA; CEACAM5; CEACAM6; CEACAM8; Claudin4; CS-1; CSF2RA; CSPG-4; CTLA4; Cripto; DLL4; ED-B; EFNA2; EGFR; Endothelial angiotensin B receptor; ENPP3; EPCAM; ERBB2; ERBB3; FAP alpha; Fc gamma RI; FCER2; FGFR3; fibrin II beta chain; FLT1; FOLH1; FOLR1; FRP-1; GD3 ganglioside; GDF2; GLP1R; Glypican-3; GPNMB; HBV (Hepatitis B virus); HCMV (Human Cytomegalovirus); Heat shock protein 90 homolog [Candida albicans]; Herpes simplex virus gD glycoprotein; HGF; HIV-1; HIV-1IIIB gp120 V3 loop; HLA-DRB (HLA-DR beta); Human respiratory syncytial virus, glycoprotein F; ICAM1; IFNA1; IFNA1; IFNB1; IgE Fc; IGF1R; IGE junction region; IL12B; IL13; IL15; IL17A; IL1A; IL1B; IL2RA; IL4; IL5; IL5RA; IL6; IL6R; IL9; interleukin-2 receptor beta subunit; ITGA2; ITGA2B; ITGB3; ITGA4; ITGB7; ITGA5; ITGAL; ITGAV_ITGB3; ITGB2; KDR; L1CAM; Lewis-y; lipid A, lipopolysaccharide (LPS) domain; LTA; MET; MMP14; MMP15; MST1R; MSTN;MUC1; MUC4; MUC16; MUC5AC; NCA-90 granulocyte cell antigen; Nectin 4; NGF; NRP; NY-ESO-1; OX40L; PLAC-1; PLGF; PDGFRA; PD1; PDL1; PSCA; phosphatidylserine; PTK-7; Pseudomonas aeruginosa serotype IATS O11; RSV (human respiratory syncytial virus, glycoprotein F); ROR1; RTN4; SELL; SELP; STEAP1; Shiga-like toxin II B subunit [E. coli]; SLAM7; SLC44A4; SOST; Staphylococcus epidermidis lipoteichoic acid; T cell receptor alpha_beta; TF; TGFB1; TGFB2; TMEFF2; TNC; TNF; TNFRSF10A; TNFRSF10B; TNFRSF12A; TNFSF13; TNFSF14; TNFSF2; TNFSF7; TRAILR2; TROP2; TYRP1; VAP-1; and vimentin.

[0222] In some embodiments, at least one of two or more different antibodies treated according to the present invention can be specific for targets on tumor cells, such as targets selected from the group consisting of: erbB1 (EGFR), erbB2 (HER2), erbB3, erbB4, MUC-1, CD19, CD20, CD4, CD38, CD138, CXCR5, c-Met, HERV-capsule protein, periosteal protein, Biggs3, SPARC, BCR, CD79, CD37, EGFrvIII, U-CAM, AXL, tissue factor (TF), CD74, EpCAM, and MRP3.

[0223] Alternatively, at least one of two or more different antibodies is specific to a target on effector cells, such as CD1, CD3, CD4, CD8, FcgammaRIII (CDI6), CD25, CD89, CD32, CD32a, FCεRI, CD40, or FcgammaRI (CD64). In other embodiments, at least one of two or more different antibodies is specific to a death receptor, such as death receptors selected from the group consisting of FAS, DR1, DR2, DR3, DR4, DR5, DR6, TNFR1, EDAR, or NGFR.

[0224] In other embodiments, at least one of two or more different antibodies is specific for an immune checkpoint target, such as immune checkpoint targets selected from the group consisting of: CTLA4, PD-1, PD-L1, LAG-3, TIM-3, OX40, Nectin-2, Nectin-3, PVR, HVEM, CD80, PD-L2, CD86, ICOSL, 4-1BBL, GITRL, CD27L, CD30L, CD40, OX40L, LIGHT, TL1A, CD3 ,TIGIT,BTLA,CD160,CD28,ICOS,4-1BB,GITR,CD27,CD30,CD40L,OX40,DR3,GAL9,TNF-R3,RANK,TACI,BAFFR,BC M,RELT,CD120b,TWEAKR,TAJ-alpha,EDA2R,KIR2DL1,KIR2DL2,KIR2DL3,LY49,CD94,NKG2D,NKG2A,VISTA,CD96.

[0225] In other embodiments, at least one of two or more different antibodies is specific for a blood-brain barrier protein, such as blood-brain barrier proteins selected from the group consisting of: TfR, insulin receptor, MTfR, LfR, ApoER2, LRP1, LRP2, RAGE, DTR (=HB-EGF), or gp190.

[0226] In yet another aspect, the present invention relates to a mixture of two or more different monoclonal antibodies as described above, and a method for targeting tumors in a subject, the method comprising administering the mixture to the subject.

[0227] Preferably, each of the two or more different antibodies is present in the mixture at a therapeutically effective amount; that is, the presence or concentration of each of the two or more different antibodies is such that the mixture can be processed into a pharmaceutical product without additional steps to increase the amount or concentration of each of the two or more different antibodies relative to the amount or concentration of other antibodies, wherein the pharmaceutical product contains each of the two or more different antibodies as an active pharmaceutical ingredient. In the context of this invention, the term "pharmaceutical product" refers to a finished dosage form (e.g., tablets, capsules, solutions) containing an active pharmaceutical ingredient or an active pharmaceutical ingredient that is typically, but not necessarily, combined with an inactive ingredient.

[0228] In the mixture provided according to the invention, the least abundant of the two or more different antibodies is present in an amount of at least 1% (w / w), 2% (w / w), 3% (w / w), 4% (w / w), 5% (w / w), 6% (w / w), 7% (w / w), 8% (w / w), 9% (w / w), or 10% (w / w) of the most abundant of the two or more different antibodies.

[0229] Two or more antibodies are preferably present in the mixture in amounts such that the ratio (w / w) between any two antibodies is between 1:5 and 5:1, for example, 1:4 and 5:1, 1:3 and 5:1, 1:2 and 5:1, 1:1 and 5:1, 2:1 and 5:1, 3:1 and 5:1, 3:4 and 5:1, 1:5 and 4:1, 1:5 and 3:1, 1:5 and 2:1, 1:5 and 1:1, 1:5 and 1:2, 1:5 and 1:3. Between 1:5 and 1:4, 1:4 and 4:1, 1:4 and 3:1, 1:4 and 2:1, 1:4 and 1:1, 1:4 and 1:2, 1:4 and 1:3, 1:3 and 4:1, 1:3 and 3:1, 1:3 and 2:1, 1:3 and 1:1, 1:3 and 1:2, 1:2 and 4:1, 1:2 and 3:1, 1:2 and 2:1, 1:2 and 1:1, 1:1 and 4:1, 1:1 and 3:1, or for example, between 1:1 and 2:1.

[0230] In a preferred embodiment, the mixture provided according to the invention is a mixture in which each of the two or more different antibodies is an active pharmaceutical ingredient.

[0231] The mixture provided by the present invention may contain 2-10 different antibodies, such as 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, 5-6, 6-10, 6-9, 6-7, 7-10, 7-9, 7-8, 8-10, 8-9, or 9-10 different antibodies.

[0232] In the mixture according to the invention, at least one of the two or more different antibodies may be a monoclonal antibody. Furthermore, in the mixture according to the invention, all of the two or more different antibodies may be monoclonal antibodies.

[0233] In other embodiments, at least one of the one or more antibodies is a bispecific or multispecific antibody.

[0234] In the mixture according to the invention, determination is performed using one or more chromatographic assays selected from the group consisting of hydrophobic interaction chromatography, cation exchange chromatography, and / or mixed-mode chromatography, wherein the resolution (Rs) of the two or more different antibodies is preferably Rs ≥ 0.3; using an ionic strength gradient, pH gradient, or salt gradient having Rs ≥ 0.3, according to the formula Rs = 2(t2 - t1) / (W1 + W2), where t1 = retention time of a given antibody, t2 = retention time of sequentially eluted antibodies, and W1 and W2 are the corresponding peak widths of the antibodies at the base of the peaks obtained by extrapolating the relatively straight sides of the main peak to the baseline.

[0235] In the mixture according to the invention, if baseline separation is achieved between the unbound fraction in the unbound column and the antibody in the fraction eluted from the column, or if the resolution (Rs) is Rs≥0.3 as determined by affinity chromatography using a pH gradient according to the formula Rs=2(t2-t1) / (W1+W2), where t1=retention time of a given antibody, t2=retention time of sequentially eluted antibody, and W1 and W2 are the corresponding peak widths of the antibody at the base of the peak obtained by extrapolating the relatively straight sides of the main peak to the baseline, then, as determined by affinity chromatography, the two or more antibodies are preferably separable, and the antibodies are separable.

[0236] In yet another aspect, the present invention relates to a method of treating a disease, the method comprising administering a mixture of two or more different antibodies as described above or a pharmaceutical composition as described above to a subject in need of such treatment.

[0237] In yet another aspect, the present invention relates to the use of a mixture of two or more different antibodies as described above in the preparation of a medicament for treating a disease.

[0238] The disease to be treated can be cancer, tumor, immune or autoimmune disease, inflammatory disease, cardiovascular disease, disease of the central nervous system (CNS), musculoskeletal disease, or infectious disease.

[0239] In particular, treatment can be for solid tumors, such as those selected from the following group: colorectal cancer, including colorectal carcinoma and colorectal adenocarcinoma; bladder cancer; osteosarcoma; chondrosarcoma; breast cancer, including triple-negative breast cancer; central nervous system cancers, including glioblastoma, astrocytoma, neuroblastoma, neurofibrosarcoma; neuroendocrine tumors; cervical cancer; endometrial cancer; gastric cancer, including gastric adenocarcinoma; head and neck cancer; kidney cancer; liver cancer, including hepatocellular carcinoma; lung cancer, including NSCLC and SCLC; ovarian cancer; pancreatic cancer, including pancreatic ductal carcinoma and pancreatic adenocarcinoma; sarcoma or skin cancer, including malignant melanoma and non-melanoma skin cancer.

[0240] In particular, treatment can be for hematologic malignancies, such as those selected from the following group: leukemia, including chronic lymphocytic leukemia and myeloid leukemia, including acute myeloid leukemia and chronic myeloid leukemia; lymphoma, including non-Hodgkin's lymphoma or multiple myeloma, including Hodgkin's lymphoma; and myelodysplastic syndromes. Example

[0241] Example 1: Expression vectors for expressing human IgG1-2F8, human IgG1-7D8, human IgG1-1014-005, IgG1-1021-511 or human IgG1-HepC and their variants.

[0242] For antibody expression of isolated immunoglobulin proteins, variable heavy (VH) and variable light (VL) chain sequences were prepared via gene synthesis (GeneArt GeneSynthesis; ThermoFisher Scientific, Germany) and cloned into a pcDNA3.3 expression vector (ThermoFisher Scientific, US) containing the constant regions of the IgG1m(f) type 1 heavy chain (HC) and light chain (LC). The amino acid sequences of the heavy chain constant regions used are identified in the sequence reference table below.

[0243] The desired mutation is introduced through gene synthesis or site-directed mutagenesis. The antibodies mentioned in this application have VH and VL sequences derived from the previously described IgG1-1014-005 (WO11 / 147982), IgG1-2F8 (WO 02 / 100348), and IgG1-1021-511 (WO16 / 005593), IgG1-7D8 (WO 04 / 035607), IgG1-1014-153 (WO2012 / 143523), IgG1-CD37-37-3 (WO11 / 112978), IgG1-CD19-21D4 (WO / 2009 / 054863), Camppath (Crowe et al., Immunology 87(1):105–110(1992)), IgG1-HepC (WO 00 / 05266), and IgG1-224. Sequences are also provided herein.

[0244] Sequence reference:

[0245]

[0246]

[0247] Example 2: Introducing normally non-immunogenic charge-regulating mutations into IgG1-1014-005, IgG1-1021-511, and IgG1-2F8

[0248] The heavy and light chain DNA sequences of IgG1-1014-005, IgG1-2F8, and IgG1-1021-511, selected for co-production and purification as a mixture, were compared with a set of human germline sequences. Figure 2 A shows the alignment of the hominid heavy chain variable region, and Figure 2 B shows the alignment of the variable regions of the human kappa light chain, numbered according to the IMGT numbering scheme for human variable domains. To regulate the pI of parental antibodies IgG1-1014-005, IgG1-2F8, and IgG1-1021-511 and minimize potential immunogenicity, charge-regulating mutations were introduced at amino acid positions in the parental antibody sequences where charge changes were observed in the native human set or compared to the human variable domain. For each parental light or heavy chain sequence, seven variant variable domains were designed: a reference sequence lacking an N-terminal pyroglutamic acid when present in the parental antibody, three sequence variants with progressively decreasing pI, and three sequence variants with progressively increasing pI. Each of these seven heavy chain variable domains was expressed as the complete heavy chain by fusing the sequence with the human IgG1 heavy chain constant domain lacking a C-terminal lysine (SEQ ID: 61). Each of the seven light chain variable domains was expressed as the complete kappa light chain by fusing the sequence with the human kappa constant domain (SEQ ID: 47). For comparison, the parental antibody is expressed, which has a sequence encoding an N-terminal pyroglutamic acid (if present in the parental sequence) and a C-terminal lysine.

[0249] Figure 2 C shows the alignment of antibody chain sequence variable domain variants designed for antibodies IgG1-1014-005, IgG1-2F8, and IgG1-1021-511. The sequence variants are named as follows: HA1 indicates a more acidic heavy chain variant with one additional negative charge compared to the reference sequence HC, while variants HA2 and HA3 contain two and three additional negative charges, respectively, compared to the reference sequence HC. Similarly, the more basic charged variants HB1, HB2, and HB3 contain one, two, and three additional positive charges, respectively, compared to the reference sequence HC. HP indicates the sequence of the unmutated parental heavy chain variable domain, expressed as a fusion with a constant domain encoding a C-terminal lysine. Light chain variants are named similarly; therefore, LA1 indicates a more acidic light chain variant with one additional negative charge compared to the reference sequences LC, etc.

[0250] Antibody chain DNA sequence variants were generated via gene synthesis as described in Example 1. As described in Example 3, antibodies were generated by co-transfection of a vector encoding a heavy chain variant and a vector encoding a light chain variant, and named as follows: IgG1-1014-005-HA1LA1 comprises a heavy chain having a variable domain sequence 1014-005HA1 (SEQ ID 1: heavy chain variable domain 1014-005HA1) and a light chain having a variable domain 1014-005LA1 (SEQ ID 9: light chain variable region 1014-005LA1). Table A summarizes the composition of the antibodies generated by co-transfection of heavy and light chain charge variants.

[0251] Table A:

[0252]

[0253]

[0254]

[0255]

[0256]

[0257]

[0258]

[0259]

[0260]

[0261] Example 3: Antibody Production

[0262] Using 293fectin TM (LifeTechnologies) According to the manufacturer's instructions, through FreeStyle TM 293-F cells (Life Technologies) were transfected with relevant heavy and light chain expression vectors to generate antibodies under serum-free conditions. Alternatively, ExpiFectamine was used. TM 293 (Life Technologies) According to the manufacturer's instructions, via Expi293F TM Cells (Life Technologies) were co-transfected with relevant heavy and light chain expression vectors to generate antibodies under serum-free conditions. The culture supernatant was filtered through a 0.2 μm dead-end filter prior to analysis and purification.

[0263] Alternatively, in Example 1, DNA sequences encoding the full-length heavy and light chain open reading frames (ORFs) of IgG1-7D8, IgG1-224, IgG1-CD37-37-3, IgG1-CD19-21D4-E345K, and IgG1-CD52-Campath-E345K were prepared. The sequences were subcloned from the pcDNA 3.3 expression vector into the internally developed expression vector pGENpr6DGV, which expresses both ORFs from the same vector. The expression vector contains both antibody ORFs regulated by the upstream CMV promoter and the downstream TK poly-A transcription termination signal, as well as a glutamine synthetase selection marker expressed under the control of the SV40 promoter fragment and the SV40 poly-A transcription termination signal. The vector was transferred to CHO-K1 cell lines (ECACC catalog number: 85051005) suitable for suspension at 1 μg / 1.0E+06 cells on chemiluminescent medium using the Amaxa Solution V kit, essentially following the manufacturer's instructions (Lonza Nucleofector 2b). Cells containing the expression vector were cultured in 96-well plates in CD-CHO medium (Life Technologies / ThermoScientific) supplemented with GS EM (Sigma) for 4 weeks under MSX selection (Sigma), after which a group of parental cultures exhibiting growth and IgG expression were expanded to larger volumes. IgG expression of the highest-yielding clones was tested on the ambr15 platform (TAP Biosystems), and the highest-yielding parental clones were then seeded at 500 mL up to a 3L bioreactor to provide IgG material. Cell cultures were harvested after 10–12 days, and the IgG-containing supernatant was collected by filtration. Alternatively, as described in Gramer et al., MAbs2013,5:962-973, IgG1-7D8-K409R is generated.

[0264] Example 4: Antibody quantification in cell culture samples or chromatographic fractions using the Bio-layer interference metric.

[0265] IgG concentrations in cell culture samples were quantified using a Bio-Layer interference metric with a Protein A biosensor and Octet QK (FortéBio). Samples were diluted 4-fold and 20-fold in sample dilution buffer (FortéBio). The initial binding rate of each sample was measured using a 60-second readout time and a shaking speed of 200 rpm, and the concentration was inferred from a reference standard curve. 10 mM glycine at pH 1.0 was used as the regeneration solution.

[0266] Example 5: Purification of antibodies from cell culture supernatant using protein A chromatography

[0267] Protein A purification is used to purify antibodies or antibody mixtures from cellular material for biochemical or subsequent chromatographic experiments. Isolated antibodies are purified in large quantities using protein A affinity chromatography. Briefly, the culture supernatant is loaded onto a 5 mL LAbSelect SuRe column (GE Healthcare), washed, and eluted with 0.02 M sodium citrate-NaOH, pH 3. The eluent is immediately loaded onto a HiPrep desalting column (GE Healthcare) after purification, and the antibody is exchanged for 12.6 mM NaH₂PO₄, 140 mM NaCl, pH 7.4 buffer (PBS, B. Braun, or Thermo Fisher). Alternatively, the eluted fractions are combined and dialyzed into PBS using an appropriately sized 10 kDa molecular weight cutoff Slide-A-Lyzer cartridge (Thermo Fisher). After buffer replacement, the sample is aseptically filtered through a 0.2 μm dead-end filter. Purity is determined by SDS-PAGE / CE-SDS, and concentration is measured by absorbance at 280 nm. Store the purified antibodies at 2-8°C. Alternatively, purify a large quantity of the antibody mixture from the cellular material using the same protocol. This aims to produce a pure mixture of antibodies without controlling the antibody ratio.

[0268] Alternatively, perform small-scale purification to purify the isolated antibodies for biochemical assays. Basically, follow the product manual and use a PreDictor MabSelect SuRe plate (GE-Healthcare) pre-loaded with 50 μL of MabSelect SuRe resin in a 96-well format. Mount the plate on a Multi Screen HTS vacuum manifold connected to a pressure vacuum station. Remove the stock solution and wash the resin with PBS (12.6 mM NaH2PO4, 140 mM NaCl, pH 7.4; B. Braun or Thermo Fisher). Incubate the resin with 0.33 mL of cell culture supernatant at 900 rpm with orbital stirring for 5 minutes, and remove the supernatant using the vacuum manifold. Repeat this process until 2 mL of supernatant is loaded. Wash the resin with PBS. Elute the bound antibody per well with 150 μL of elution buffer (20 mM citrate, pH 3.0) and collect by centrifugation. Neutralize the elution to approximately pH 6.0 in each well by adding neutralization buffer (2 M Tris-HCl, pH 9.0). The protein concentration of the elution buffer in each well was determined by measuring the absorbance at 280 nm.

[0269] Example 6: Preparative cation exchange chromatography of a mixture of monoclonal antibodies using a HiScreen Capto S ImpAct column with an ionic intensity gradient

[0270] Preparative cation exchange chromatography was used to resolve a mixture of purified antibodies against protein A to investigate whether the antibody sequences contained differences in their charge properties. This allows for the separation of monoclonal antibodies by chromatography and ultimately, control over their composition. The Capto S ImpAct (GE Healthcare) column was selected as the high-resolution cation exchange column suitable for manufacturing applications. The HiScreen chromatography column format (GE Healthcare) has a bed height of 10 cm and is suitable for screening applications such as production-scale purification of models.

[0271] The input mixture of antibodies purified from protein A was buffer-exchanged to loading buffer (20 mM NaHPO4, pH 6.75 or 20 mM NaHPO4, pH 6.5) to reduce ionic strength, allowing them to bind to the column. This can be achieved by dialysis using a 10 kDa molecular weight cutoff Slide-A-Lyzer (ThermoFisher) of appropriate size or by diluting 20-fold in the loading buffer. The antibody mixture was loaded onto a 5 mL Capto S ImpAct column (GE Healthcare) at 2.3 mL / min and washed with 5 column volumes of loading buffer. The antibody mixture was separated using a linear gradient from loading buffer to elution buffer. The loading and elution buffers and elution gradient were selected according to the properties of the antibody mixture, and are described for different antibody mixtures in Examples 7, 9, 23, 25, and 26. The column was sequentially washed with 1M tris(hydroxymethyl)aminomethane buffer at pH 9.0 or 20mM tris(hydroxymethyl)aminomethane, 1000mM NaCl at pH 8.5 or 20mM tris(hydroxymethyl)aminomethane, 50mM NaCl at pH 8.0; and 0.2 or 0.5M NaOH, and then reequilibrated with loading buffer.

[0272] The resolution of adjacent peaks is calculated using the Peak Integrate function in Unicorn software version 6.32 (GE Healthcare). The Peak Window is manually selected, and peaks are manually allocated between two minimums by visually inspecting the contours. The resolution is calculated using the height difference line using the resolution algorithm (Ret2–Ret1) / ((Width2+Width1) / 2). Alternatively, as shown in the various embodiments, the resolution is calculated using the Peak Integrate function in Unicorn software version 6.32 (GE Healthcare) with a skim ratio of 10. For simplicity, the height difference line is the preferred method. Figure 6 E summarizes the principle of resolution calculation. The protein content in each peak is estimated as follows: the chromatogram is integrated, and the extinction coefficient of each antibody is corrected, which is calculated based on the primary amino acid sequence of the antibody.

[0273] Example 7: Using charge-regulated antibodies, an antibody mixture was separated by gradient elution on a preparative cation exchange column and recovered to produce an antibody mixture of a predetermined composition.

[0274] This embodiment describes the following procedure: using a mixture of variable composition, performing a chromatographic step to provide resolution between the individual components of the polyclonal mixture, and fractionating the eluted antibodies so that they can be combined using concentration measurements of the fractions to produce a mixture of a predetermined composition. Figure 1 E).

[0275] Example 2 describes the design and generation of charge-regulated variants of human antibodies IgG1-1014-005, IgG1-1021-511, and IgG1-2F8. In short, mutations are introduced at framework amino acid positions in the natural germline ensemble that exhibit charge changes to minimize the impact on potential immunogenicity. In specific cases, a peptide environment with charged amino acids is introduced in the transfer germline, as illustrated by the light chain variant with a neutral mutation at position 4.

[0276] Each of the seven light chain vectors was paired with each of the seven heavy chain vectors to produce 49 unique combinations of IgG1-1014-005, IgG1-1021-511, and IgG1-2F8. Sequence summaries are provided in... Figure 2 Antibodies were generated by transfecting heavy and light chain DNA sequences as described in Example 3, and antibody titers were determined as described in Example 4. Figure 3The results showed that most charge variants were well tolerated in terms of production levels, except for the heavy chain mutation Q6E in antibody IgG1-1014-005, which adversely affected the expression of all variants containing this mutation.

[0277] Using the pepstats module of EMBOSS (Jemboss version 1.5; Carver, T and Bleasby A. Bioinformatics. 2003 Sep 22; 19(14):1837-43), the theoretical isoelectric point of antibody charge variants was analyzed using tandem sequences of heavy and light chains. A range of isoelectric points could be sampled by combining possible heavy and light chain variants. Figure 4 A).

[0278] The antibody charge variants were purified as described in Example 5 and analyzed by analytical cation exchange chromatography as described in Example 8 to serve as a model system to describe the diversity of charge properties of the antibody variants. A variety of retention times were observed for each sample of IgG1-1014-005, IgG1-1021-511, and IgG1-2F8. Figure 4 (BE) indicates that the mutation is sufficient to affect the behavior of antibody variants in cation exchange chromatography assays.

[0279] To mimic the upstream process of generating an antibody mixture in a single co-generation event, antibody supernatants were generated separately and mixed. In the first case, the antibody supernatants were mixed together so that the highest and lowest antibody concentrations were within twice each other to evaluate the separation behavior of the mixture on a preparative cation exchange resin. Antibody supernatants were generated separately as described in Example 3, immunoglobulin titers were determined as described in Example 4, and the supernatants were mixed using the antibody titers to generate a mixture of approximately 10 mg of immunoglobulin in a total volume of approximately 150 mL. An input mixture of antibody variants IgG1-1014-005-HCLC, IgG1-2F8-HCLC, IgG1-1021-511-HCLC, and IgG1-1014-153 was generated as a control antibody mixture without charge regulation. Alternatively, charge variants IgG1-2F8-HB3LC, IgG1-1014-005-HB3LB1, and IgG1-1021-511-HA3LB2 can be selected together with IgG1-1014-153 to be included in the input mixture to improve separation behavior by cation exchange chromatography. These charge variants contain the following mutants compared to HCLC variants: IgG1-2F8-HB3LC: E6Q, A24K, E97G; IgG1-1014-005-HB3LB1: E17A, Q75A, S93R (HC) and E95Q (LC); IgG1-1021-511-HA3LB2: K48Q, Q90E, A96D (HC) and E48K (LC). Figure 2 As described in Example 5, the cell culture supernatant mixture was purified by protein A affinity chromatography. The antibody mixture was captured and purified from a large amount of contaminants. Figure 5 ).

[0280] The behavior of a mixture of two purified protein A antibodies was analyzed on a preparative cation exchange chromatography column. The mixture buffer was exchanged to loading buffer (20 mM NaHPO4, pH 6.75) by dialysis and loaded onto a 5 mL Capto S ImpAct column (GE Healthcare) in separate experiments, as described in Example 6. Antibodies were eluted using a linear gradient of elution buffer (20 mM NaHPO4, 1 M NaCl, pH 6.75) from 0% to 38% (v / v) between 30 column volumes, and the eluent was collected in 2 mL fractions. Antibodies in the charge-regulated antibody mixture were adequately separated, while antibodies in the non-charge-regulated mixture were not adequately separated. Figure 6(A, B). As described in Example 6, the resolution of the separated peaks from the charge-regulated mixture was calculated using height difference lines. The non-charge-regulated antibodies were not sufficiently separated, and only two peaks were observed from the separations of the four antibodies; therefore, only a single resolution could be calculated (Table 1).

[0281] Fractions from the preparative cation exchange assay and the loaded fractions were analyzed by analytical cation exchange chromatography to determine their identity and relative purity. Based on the examination of the pooled fractions with a chromatogram showing detection at 280 nm, concentration was performed using a Sartorius Stedim Biotech Vivaspin 6, 10000MWCO PES (product number VS060L). The composition of the pooled fractions was confirmed using analytical cation exchange chromatography as described in Example 8. The pooled fractions separated from the charge-regulated antibody mixture were substantially pure, indicating the resolution of antibody species (…). Figure 7 B). Conversely, fractions from non-charge-regulated variants were not substantially pure, confirming that the species were not adequately separated by preparative cation exchange chromatography. Figure 7 A).

[0282] The examples illustrate a method for determining the separability of antibody mixtures by chromatography: uncharge-modified variants were not separated by chromatography because the peaks could not be resolved, and the fractions contained predominantly (>80%) pure protein. The examples further demonstrate that the amino acid sequence of the antibody can be modified to achieve separability by chromatography. For example, basic residues can be introduced through point mutations in E6Q, A24K, and E97G, or acidic residues can be removed from IgG1-2F8-HCLC to produce IgG1-2F8-HB3LC (…). Figure 2 A) In preparative cation exchange assays, it resulted in an increased retention time of IgG1-2F8-HB3LC compared to IgG1-2F8-HCLC. Figure 6 The combination of amino acid variations allows for the resolution of antibody mixtures via preparative cation exchange chromatography. Figure 6 B, Table 1).

[0283] Alternative charge-regulated antibody mixtures were prepared to demonstrate that the input antibody mixture could be separated using preparative cation exchange chromatography so that the mixture could be recovered at a predetermined ratio. Antibody supernatants containing recombinantly expressed IgG1-2F8-HB3LB3 (with HC mutants E6Q, A24K, E97G and LC mutants E68Q, E80G, and T90K compared to IgG1-2F8-HCLC), IgG1-1014-005-HB3LB1, IgG1-1021-511-HA3LB2, and IgG1-1014-153 were prepared as described in Example 3. Immunoglobulin titers were determined as described in Example 4, and the supernatants were mixed using the antibody titers to produce a final amount of 21 mg of an input mixture of recombinant antibodies in a 5:3:2:1 ratio. Alternatively, antibody supernatants containing recombinantly expressed IgG1-2F8-HB3LC, IgG1-1014-005-HB3LB1, IgG1-1021-511-HA3LB2, and IgG1-1014-153 were prepared as described in Example 3. Immunoglobulin titers were determined as described in Example 4, and the supernatants were mixed with antibody titers to produce a final amount of 24 mg of an input mixture of recombinant antibodies in a ratio of 1:3:2:5. These mixtures were intended to simulate a co-production process with a release specification of 1:1:1:1, but the upstream process was not under sufficient control to provide the desired composition, thus requiring a chromatographic step to normalize the ratios. The antibody mixture was purified by protein A affinity chromatography as described in Example 5. The antibody mixture was captured and purified from a large amount of contaminants. Figure 5 ).

[0284] The protein A purified input antibody mixture was separated on a preparative cation exchange chromatography column. The mixture buffer was exchanged for loading buffer (20 mM NaHPO4, pH 6.75) by dialysis and loaded onto a 5 mL Capto S ImpAct column (GE Healthcare) as described in Example 6. The antibody was eluted using a linear gradient of elution buffer (20 mM NaHPO4, 1 M NaCl, pH 6.75) from 0% to 38% (v / v) over 30 column volumes, and the eluent was collected in 2 mL fractions. The chromatogram shows four resolved peaks (…). Figure 6 (C and D). The resolution of the separated peaks from the charge-controlled mixture was calculated as described in Example 6, and the antibodies resolved at a resolution of >0.3 are shown in Table 1.

[0285] Fractions from the preparative cation exchange assay and the loaded fractions were analyzed by analytical cation exchange chromatography to determine their identity and relative purity. Based on the examination of the pooled fractions with a chromatogram showing detection at 280 nm, concentration was achieved using a Sartorius Stedim Biotech Vivaspin 6, 10000MWCO PES (product number VS060L). The composition of the pooled fractions was confirmed using analytical cation exchange chromatography as described in Example 8. The pooled fractions isolated from the charge-regulated antibody mixture were sufficiently pure to provide complete control over the relative composition of the mixture components. Figure 7 CF). Individual peaks were combined, and their concentrations were analyzed using a Nanodrop ND-1000 spectrophotometer (Isogen Life Science, Maarssen, The Netherlands) and by calculating extinction coefficients from the primary amino acid sequence of the pure antibody. The fractions were remixed using concentration measurements of the combined fractions to produce a mixture of antibodies with approximately equal mass concentrations. The composition of the mixture was analyzed using analytical cation exchange chromatography as described in Example 8 to produce an antibody mixture with an approximately predetermined 1:1:1:1 composition. Figure 7 E, F; Table 2). Purity analysis of intermediate merges is not required prior to merging for the established process, in which the purity of each merge has been shown to be consistently similar during robustness testing experiments.

[0286] Table 1: Preparative cation exchange chromatography chromatograms of the quantitative antibody mixtures, analyzing the integrated peak area and resolution relative to previous peaks. ND – Undetermined. The concentrations of IgG1-2F8-HB3LC, IgG1-1014-005-HB3LB1, IgG1-1021-511-HA3LB2 and IgG1-1014-153 in the input mixture (*) are within twice each other, and the ratio (**) is 1:3:2:5.

[0287]

[0288] Table 2: Quantitative analysis of the analytical cation exchange chromatography profile of the input and standardized antibody mixtures.

[0289]

[0290]

[0291] Example 8: Analysis of purified antibodies and antibody mixtures using analytical cation exchange chromatography

[0292] Retention times of different antibodies and charge-regulating antibody mutants were compared using high-performance liquid chromatography (HPLC)-analytical cation exchange chromatography (CIEX), and the relative amounts of antibodies in the antibody input and output mixtures were quantified. A sodium phosphate buffer stock solution at pH 7.0 was prepared in MilliQ water from Na₂HPO₄·2H₂O and NaH₂PO₄ (anhydrous). A 2 mg / mL antibody sample in mobile phase A (10 mM phosphate buffer, pH 7.0) was injected into the HPLC. Alternatively, the small-scale purified product described in Example 5 was directly injected into the HPLC. Differently charged IgG molecules were separated using a ProPac WCX-10 4 mm x 250 mm analytical column at a flow rate of 1 mL / min. 25 μL of sample was injected, and elution was performed with a gradient from mobile phase A (10 mM phosphate buffer, pH 7.0) to mobile phase B (10 mM phosphate buffer, pH 7.0, 0.25 M NaCl) under detection at 280 nm. Empower 3 (Waters) software was used to analyze the chromatograms and report the retention time and total peak area of ​​specific antibodies. This was further corrected using extinction coefficients calculated from the primary amino acid sequence of the antibodies to determine the relative abundance of each component in the input and output mixtures and chromatographic fractions. Individual antibody chromatograms were used as references to identify their positions in the final product and to define the integral boundaries of the antibodies.

[0293] Example 9: Using charge-regulated antibodies, an antibody mixture was separated by a sequential elution step on a preparative cation exchange column and recovered to produce an antibody mixture of a predetermined composition.

[0294] This embodiment describes the following procedure: using a mixture of variable composition, performing a chromatographic step to provide resolution between the individual components of the polyclonal mixture, and fractionating the eluted antibodies so that they can be combined using concentration measurements of the fractions to produce a mixture of a predetermined composition. Figure 1 E).

[0295] A mixture of five recombinant antibodies in equal mass ratios was generated using either charge-regulated or non-charge-regulated antibodies, and its separability was assessed by preparative cation exchange chromatography. The non-charge-regulated mixture comprised IgG1-7D8, IgG1-224, IgG1-CD37-37-3, IgG1-CD19-21D4-K409R, and IgG1-CD52-Campath (Example 1). Alternatively, as described in Example 1, IgG1-CD19-21D4-E345K and IgG1-CD52-Campath-E345K were subjected to an E345K point mutation to generate a charge-regulated mixture of IgG1-7D8, IgG1-224, IgG1-CD37-37-3, IgG1-CD19-21D4-E345K, and IgG1-CD52-Campath-E345K.

[0296] As described in Example 3, antibody mixtures were prepared by using transiently generated or recombinantly expressed individual antibodies via a CHO-K1-based expression system. Individual antibodies were purified by protein A affinity chromatography as described in Example 5. The concentrations of individual antibodies were measured using a Nanodrop ND-1000 spectrophotometer (Isogen Life Science, Maarssen, The Netherlands) and by calculating the extinction coefficient from the primary amino acid sequence of the purified antibodies. Antibody mixtures were prepared in PBS buffer (12.6 mM NaH2PO4, 140 mM NaCl, pH 7.4 buffer, B. Braun or Thermo Fisher) by mixing antibodies at equal mass ratios of concentration.

[0297] The behavior of charge-regulated and non-charge-regulated mixtures was analyzed on a preparative cation exchange chromatography column. In separate experiments, the mixture was diluted 20-fold to loading buffer (20 mM NaHPO4, pH 6.5) and loaded onto a 5 mL Capto S ImpAct column (GE Healthcare) as described in Example 6. Antibodies were eluted using a linear 40-column volume gradient of elution buffer (20 mM NaHPO4, 1000 mM NaCl, pH 6.5) from 0% to 25% (v / v) using 1 g / L resin loading. Alternatively, antibodies were eluted using a linear 40-column volume gradient of elution buffer (20 mM NaHPO4, 1000 mM NaCl, pH 6.5) from 0% to 75% (v / v). Figure 8A shows that five non-charge-regulated antibodies failed to resolve in the chromatography assay because the charge properties of the antibodies were not sufficiently different to achieve separation. The K409R mutation did not significantly affect the elution behavior of the IgG1-CD19-21D4 antibody because it is not on the antibody surface and does indeed cause a change in net charge. Figure 8 B shows the separation of five charge-regulated antibodies, producing five distinct peaks. The identity of each of the five main peaks was assessed using analytical cation exchange chromatography, as described in Example 8, by combining the peaks and using purified protein as a reference standard. Each peak in the preparative chromatogram corresponds to a single antibody ( Figure 8 C, D), and the peaks assigned in order of increasing retention time were IgG1-7D8, IgG1-224, IgG1-CD37-37-3, IgG1-CD19-21D4-E345K and IgG1-CD52-Campath-E345K, respectively. Figure 8 The comparison between A and 8B shows that the introduction of the E345K point mutation results in antibody elution with an increased retention time, which allows for the resolution of the antibody's chromatographic peaks. Therefore, these data demonstrate that the charge-regulated antibody mixture does indeed contain differences in monoclonal antibodies that can be separated by chromatography.

[0298] Cost-effective production benefits from high antibody loading on the chromatography resin, reducing the resin volume required to purify a given mass of antibody mixture. Changes in the chromatographic properties of charge-regulated antibodies with increasing loading were investigated to control peak broadening that occurs with increasing antibody loading. Equal masses of mixtures of IgG1-7D8, IgG1-224, IgG1-CD37-37-3, IgG1-CD19-21D4-E345K, and IgG1-CD52-Campath-E345K were prepared. Individual antibodies were recombinantly expressed from CHO-K1 cells as described in Example 3. Individual antibodies were purified by protein A affinity chromatography as described in Example 5. The concentrations of individual antibodies were measured using a Nanodrop ND-1000 spectrophotometer (Isogen Life Science, Maarssen, The Netherlands) and by calculating extinction coefficients from the primary amino acid sequences of the purified antibodies. Antibody mixtures were prepared in PBS buffer (12.6 mM NaH2PO4, 140 mM NaCl, pH 7.4 buffer, B. Braun or Thermo Fisher) by mixing antibodies at equal mass ratios. Loading studies were performed on preparative cation exchange chromatography columns. In individual experiments, the mixture was diluted 20-fold to loading buffer (20 mM NaHPO4, pH 6.5) and loaded onto 5 mL Capto SImpAct columns (GE Healthcare) as described in Example 6. Antibodies were eluted using a linear 40 column volume gradient from 0% to 25% (v / v) elution buffer (20 mM NaHPO4, 1000 mM NaCl, pH 6.5) with a final total loading of 0.2, 0.5, 1.0, 2.0, 5.0, 10, 20, or 50 g / L.

[0299] Five distinct peaks were detected for all antibody loadings. Figure 8 E). The height difference line quantization resolution was used as described in Example 6. A degree of broadening was detected under the highest column load, but peaks were resolved in all cases (resolution > 0.3) (Table 3), demonstrating that separation can be performed under loads relevant to manufacturing applications.

[0300] Antibody mixtures were generated to simulate the process, including co-production and capture purification steps, with a release specification of 1:1:1:1:1. However, the upstream process was not under sufficient control to provide the desired composition, thus requiring additional chromatographic steps to normalize the antibody ratios. Three non-normalized mixtures and a 1:1:1:1:1 mixture of IgG1-7D8, IgG1-224, IgG1-CD37-37-3, IgG1-CD19-21D4-E345K, and IgG1-CD52-Campath-E345K were prepared. Individual antibodies were recombinantly expressed from CHO-K1 cells as described in Example 3. Individual antibodies were purified by protein A affinity chromatography as described in Example 5. The concentrations of individual antibodies were measured using a Nanodrop ND-1000 spectrophotometer (Isogen Life Science, Maarssen, The Netherlands) and extinction coefficients calculated from the primary amino acid sequences of the purified antibodies. Antibody mixtures were prepared by mixing individually purified antibodies in PBS buffer (12.6 mM NaH2PO4, 140 mM NaCl, pH 7.4 buffer, B. Braun or Thermo Fisher) at a mass ratio of 1:1:1:1:1, 0.30:0.50:1.0:0.38:0.38, 1.0:0.25:0.38:1.0:0.50, or 0.50:1.0:0.40:1.0:0.83.

[0301] The gradient-based separation protocol was converted to sequential elution as an alternative that could simplify the manufacturing process. In the final chromatography protocol, the mixture was diluted 20-fold in loading buffer (20 mM NaHPO4, pH 6.5). Separation was performed according to Example 6, except that the antibody was eluted in 5 sequential elution steps with 8 column volumes of loading buffer containing 19.5%, 29.4%, 38.6%, 44.6%, and 61.2% (v / v) of elution buffer (20 mM NaHPO4, 250 mM NaCl, pH 6.5). 30 mL fractions were collected, and fractionation was initiated at the beginning of each elution step.

[0302] This elution and fractionation scheme is challenged by the different compositions of the charge-regulated antibody mixtures, and the total loading of each antibody is summarized in Table 4. Figure 9A shows the peaks of each mixture as a separate fraction of 5 individual fractions. Concentrations were derived from the integral of the chromatograms using the merge function of Unicorn software version 6.32 (GE Healthcare), following the manufacturer's guidelines. Alternatively, fraction concentrations were analyzed using absorbance at 280 nm measured using a Nanodrop ND-1000 spectrophotometer (Isogen Life Science, Maarssen, The Netherlands) and extinction coefficients calculated from the primary amino acid sequence of the pure antibody. Analytical cation exchange chromatography (Example 8) was used to confirm the composition of the loaded sample and each individual fraction.

[0303] Chromatographic analysis showed a good correlation between the loading of each antibody and the amount inferred from the chromatographic integral (Table 4). Analytical cation exchange chromatography was used to analyze each fraction, and the data confirmed that each fraction contained high-purity (>98%) antibody. Figure 9 (BE). Analytical cation exchange analysis of fractions is not necessarily required before merging fractions during the optimization process, as process design and / or robustness testing can demonstrate that fraction purity is under control.

[0304] An approximately equimolar output mixture of antibodies is prepared by mixing fractions using the concentration of each antibody fraction inferred from the integral of the chromatography to calculate the volume required to produce approximately equal mass concentrations of antibody. Alternatively, an approximately equimolar output mixture of antibodies is prepared by mixing fractions using the concentration of each antibody fraction inferred from the concentration of the separated fractions to calculate the volume required to produce approximately equal mass concentrations of antibody. As described in Example 8, the composition of the final product is analyzed using analytical cation exchange chromatography. In both cases, antibody products conforming to a predetermined composition are combined within certain tolerances (Table 5). This example demonstrates that the composition of antibody loading with variable composition can be controlled using chromatography under relevant loading and by using a series of stepwise elutions.

[0305] Table 3: Quantitative analysis of preparative cation exchange chromatography of antibody mixtures, using height difference lines to analyze the integrated peak area and resolution relative to previous peaks.

[0306]

[0307]

[0308]

[0309] Table 4: Quantitative analysis of preparative cation exchange chromatography for antibody mixtures with different compositions, based on the analysis of the integrated peak area.

[0310]

[0311]

[0312] Table 5: Quantitative analysis of the input and standardized antibody mixtures using cation exchange chromatography. ND – Not determined.

[0313]

[0314] Example 10: Kappa Select separation of modified IgG1-2F8-F405L variant using purified protein or cell culture supernatant.

[0315] Four 1 mL KappaSelect (GE Healthcare) columns were connected in series. The columns were pre-equilibrated with phosphate-buffered saline (PBS; 12.6 mM sodium phosphate, 140 mM sodium chloride, pH 7.4, B. Braun or Thermo Fisher). Antibody cell culture supernatant was filtered through a 0.2 μm dead-end filter and IgG1 expression levels were quantified using the bio-Layer interference metric as described in Example 4. 40 mL to 80 mL of cell culture supernatant containing 10 mg to 30 mg of the unpurified IgG1-2F8-F405L variant was loaded onto the KappaSelect column. Alternatively, 16 mg of purified IgG-2F8-F405L was diluted to a total volume of 80 mL with PBS (B. Braun) and loaded onto the column. The column was washed with PBS and eluted sequentially with 0.1 M glycine HCl pH 3.0 and 0.1 M glycine HCl pH 2.0. Neutralize the eluted fraction with a few drops of 2M Tris HCl at pH 9.0 and dialyze into PBS (B. Braun) using a suitable-sized Slide-A-Lyzer cartridge with a molecular weight cutoff of 10 kDa. Wash the column with 6M guanidine hydrochloride. Combine the eluent fractions with the PBS wash buffer and analyze by SDS-PAGE as described in Example 13.

[0316] Example 11: Isolation of modified IgG1-7D8-K409R variant from CaptureSelect KappaXL in cell culture supernatant or purified immunoglobulin solution

[0317] Following the manufacturer's instructions, a column containing approximately 1 mL of resin was manually packed into a 6.6 mm pore size HiT column (Omnifit) from homogenized CaptureSelect KappaXL (ThermoFisher) slurry. The column was pre-equilibrated with phosphate-buffered saline (PBS; 12.6 mM sodium phosphate, 140 mM sodium chloride, pH 7.4, ThermoFisher). The antibody cell culture supernatant was filtered through a 0.2 μm dead-end filter, and IgG1 expression levels were quantified using the Bio-Layer Interference Measurement method as described in Example 4. 10 mL of supernatant (containing 1–10 mg of unpurified IgG1-7D8-K409R variant) was loaded onto the CaptureSelect KappaXL column. The column was sequentially washed with approximately five column volumes of PBS and three column volumes of 0.1 M citrate NaOH pH 5.0. The bound material was eluted with 0.1 M citrate NaOH pH 3.5. Neutralize 1 mL fraction with a few drops of 2M Tris HCl at pH 9.0. Wash the column with 6M guanidine hydrochloride. Combine the flow-through with PBS wash buffer. Fractions with significant absorption at the 280 nm peak from either pH 5.0 wash buffer or pH 3.5 elution buffer are combined. The load, combined flow-through, and combined fractions are analyzed using the Bio-Layer Interference Measurement and CE-SDS method as described in Examples 4 and 14.

[0318] Example 12: Protein L isolates modified IgG1-2F8-F405L variant with modified Kappa light chain variable domain.

[0319] A 5 mL HiTrap protein L column (GE Healthcare) was pre-equilibrated with phosphate-buffered saline (PBS; 12.6 mM sodium phosphate, 140 mM sodium chloride, pH 7.4). Antibody cell culture supernatant containing IgG-2F8-F405L-R18P, IgG-2F8-F405L-T20S-T22S, IgG-2F8-F405L-R24S, or IgG-2F8-F405L-K107L was filtered through a 0.2 μm dead-end filter, and IgG1 expression levels were quantified using the Bio-Layer interference metric as described in Example 4. 10 mL of the supernatant was loaded onto a HiTrap protein L column. Alternatively, the antibody culture supernatant was purified by protein A chromatography as described in Example 5. 0.8 mg to 2.8 mg of purified IgG-2F8-F405L, IgG-2F8-F405L-S9L, or IgG-2F8-F405L-S12P were mixed into a total volume of 10 mL of PBS and loaded onto a HiTrap protein L column. The column was washed with PBS, and the specifically bound material was sequentially eluted with 0.1 M glycine-HCl at pH 3.5, 3.0, and 2.5, and neutralized with a few drops of 2 M Tris at pH 9.0. The column was washed with 15 mM sodium hydroxide. The material in the flow-through was analyzed using the Bio-Layer Interference Measurement and CE-SDS assays as described in Examples 4 and 14. Example 13: Analysis of Chromatographic Flow-Through Samples by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)

[0320] The sample was mixed with an equal volume of NuPAGE LDS sample buffer (Invitrogen) and heated at 70°C for 10 minutes. SDS-PAGE was performed on 4–12% NuPAGE Bis-Tris gels (Invitrogen) under non-reducing conditions using the modified Laemmli method (Laemmli 1970 Nature 227(5259):680-5) with 1x NuPAGE MOPSSDS running buffer (Invitrogen). The SDS-PAGE gels were stained with Coomassie stain and digitally imaged using the OptiGo imaging system (Isogen Life Sciences). SeeBlue Plus2 pre-stained standards were used as molecular weight standards (Invitrogen).

[0321] Example 14: Analysis of chromatographic fractions using capillary electrophoresis-sodium dodecyl sulfate (CE-SDS)

[0322] Prior to analysis, the sample was filtered through a 0.2 μm dead-end filter. Concentration was measured using the Bio-Layer interference metric method described in Example 4, or based on absorbance at 280 nm, and the sample concentration was adjusted by dilution in PBS to ensure a concentration not exceeding 250 μg / mL. CE-SDS was performed on the HT Protein Express LabChip (Caliper Life Sciences, MA) using LabChip GXII (Caliper Life Sciences, MA) under non-reducing conditions, according to the manufacturer's instructions. Data were analyzed using LabChipGX software V3.1 (Caliper Life Sciences, MA).

[0323] Example 15: Identification of knockout mutations for CaptureSelect LC-kappa(Hu) affinity matrix

[0324] As stated by the manufacturer, both CaptureSelect LC-kappa(Hu) affinity matrices KappaSelect and CaptureSelect KappaXL (GE-Healthcare, BAC) contain a 13kDa Llama antibody fragment that recognizes a unique epitope on a constant moiety of the human kappa L chain (CL). Furthermore, according to the manufacturer, this fragment is cross-reactive with non-human primate species and non-cross-reactive with the L chains of mice, rabbits, cattle, and rats, or with the human lambda L chain.

[0325] Sequence alignments of the kappa CL domain from these different species revealed several conserved amino acid residues in human and primate kappa sequences, but which differed in other sequences. Specifically, residues exposed in both the light and heavy chain complexes were selected for analysis, and human kappa L chains containing either the mouse (mm) CL domain or single-point mutations corresponding to their mouse-specific counterparts were engineered. Figure 10 Another point mutation (F135L) was introduced into the mmCL domain to facilitate efficient pairing with the human H chain.

[0326] Nine kappa L-chain mutants were expressed with suitable H-chain combinations (Table 6), and their binding affinity to KappaSelect resin was evaluated (as described in Example 10). Purified IgG1-2F8-F405L was used as a positive control for affinity purification using KappaSelect resin. Figure 12 As expected, IgG1-2F8-F405L containing the mmCL(F135L)L chain could not be purified by KappaSelect resin. Figure 12B). Except for V110D, all mutants were still purifiable, suggesting that V110 in Kappa LC is directly or indirectly part of the KappaSelect binding site. Figure 12 IgG1-2F8-F405L-E143D eluted significantly at higher pH than other mutants, indicating weaker interaction with the column resin. Figure 12 Table 7 summarizes the effect of single-point mutations on binding to KappaSelect resin, where (+++) indicates similar binding characteristics to the positive control; (++) indicates a greater proportion of IgG1 eluted at higher pH compared to the control; (+) indicates significant IgG1 protein detected in the flow-through and PBS wash buffer, and (-) indicates no binding to the resin detected.

[0327] The tolerance of the CaptureSelect KappaXL affinity matrix to substitutions at residue V110 of the kappa L chain was further evaluated by purifying (as described in Example 11) individually expressed kappa L chain mutants in combination with appropriate H chains. The appropriate H chain contained substitutions at the V110 position for all native amino acids (except C). As expected, IgG1-7D8-K409R bound to the resin, as evidenced by an elution peak at 280 nm detected during elution at pH 3.5. V110D substitution eliminated binding to KappaSelect resin and also prevented binding to CaptureSelect KappaXL, indicating that both matrices bind to the same or similar epitopes. V110R was the only other mutation that did not show detectable interaction with the resin under these conditions. Figure 13 and 14 Other IgG1-7D8-K409R variants showed reduced affinity for the resin. For example, IgG1-7D8-K409R-V110E was detected in the pH 3.5 elution buffer and fraction, IgG1-7D8-K409R-V110K was detected in the pH 5.0 wash buffer and fraction, and IgG1-7D8-K409R-V110T eluted during both pH 5.0 wash and pH 3.5 elution. Figure 13 and 14 Table 8 summarizes the effect of single-point mutations on binding to CaptureSelect KappaXL resin, where (++) indicates similar binding characteristics to the positive control; (++) indicates a greater proportion of IgG1 eluted at a higher pH compared to the control; and (+) indicates significant IgG1 protein detected in the flow-through and PBS wash, while (-) indicates no binding to the resin was detected.

[0328]

[0329]

[0330]

[0331]

[0332] Example 16: Identification of knockout mutations for protein L affinity matrix

[0333] Protein L has also been described as binding variable portions of kappa isoforms I, III, and IV, but not kappa isoform II or most lambda isoforms (Nilson et al. J Biol Chem. 1992; 267(4):2234-9). Furthermore, epitopes of protein L on the light chain of human and mouse kappa have been identified by X-ray crystallography (Graille et al. Structure. 2001 9(8):679-87; Graille et al. Biol Chem. 2002 277(49):47500-6). Analysis of these crystal structures identified 17 residues as important contact residues between the two structures (…). Figure 11 Of these, seven residues were selected based on structural and sequence alignment analysis, and single- or double-point mutations were used to mutate them to residues typically found in kappa type II or most lambda type I sequences. Figure 11 (Table 6).

[0334] These kappa L-chain mutants were expressed in appropriate H-chain combinations, and their ability to bind protein L resin was evaluated (as described in Example 12). The resin bound purified IgG1-2F8-F405L positive control and most mutant proteins. Conversely, IgG1-2F8-S12P did not bind to the resin under these conditions. Figure 15 Table 9 summarizes the effect of point mutations on binding to HiTrap protein L-columns, where (+++) indicates a similar binding profile to the positive control; (++) indicates a greater proportion of IgG1 eluted at higher pH compared to the control; (+) indicates significant IgG1 protein detected in the flow-through and PBS wash, and (-) indicates no binding to the resin detected.

[0335]

[0336] Example 17: Binding specificity of IgG1 variant to HiTrap protein L

[0337] A 5 mL HiTrap protein L column (GE Healthcare) was pre-equilibrated with phosphate-buffered saline (PBS; 12.6 mM sodium phosphate, 140 mM sodium chloride, pH 7.4). Antibody culture supernatant was purified by protein A chromatography as described in Example 5. Approximately 250 μg of purified IgG1-2F8-V110D, IgG1-7D8-S12P, or IgG1-HepC was mixed with PBS to a total volume of 5 mL and loaded onto a HiTrap protein L column in a separate experiment. The column was washed with PBS, then with 0.02 M sodium citrate-NaOH, pH 5.0, and specifically bound material was eluted with 0.1 M glycine-HCl, pH 3.0. The column was washed with 0.015 M NaOH.

[0338] Example 18. Binding specificity of IgG1 variant to HiTrap KappaSelect

[0339] A 5 mL HiTrap KappaSelect column (GE Healthcare) was pre-equilibrated with phosphate-buffered saline (PBS; 12.6 mM sodium phosphate, 140 mM sodium chloride, pH 7.4). Antibody culture supernatant was purified by protein A chromatography as described in Example 5. Approximately 500 μg of purified IgG1-2F8-V110D, IgG1-7D8-S12P, or IgG1-HepC was mixed with PBS to a total volume of 5 mL and loaded onto the HiTrap KappaSelect column in separate experiments. The column was washed with PBS and 0.1 M glycine-HCl pH 3.0. Specifically bound material was eluted with 0.1 M glycine-HCl pH 2.5. The column was washed with 6 M guanidine hydrochloride.

[0340] Example 19: Binding specificity of IgG1 variant to HiTrap LambdaFabSelect

[0341] A 1 mL HiTrap LambdaFabSelect column (GE Healthcare) was pre-equilibrated with phosphate-buffered saline (PBS; 12.6 mM sodium phosphate, 140 mM sodium chloride, pH 7.4). Antibody culture supernatant was purified by protein A chromatography as described in Example 5. Approximately 500 μg of purified IgG1-2F8-V110D, IgG1-7D8-S12P, or IgG1-HepC was mixed with PBS to a total volume of 5 mL and loaded onto the HiTrap LambdaFabSelect column in separate experiments. The column was washed with PBS and eluted with 0.1 M glycine-HCl pH 2.0, followed by elution with 0.5 M acetic acid. The column was washed with 0.025 M NaOH.

[0342] Example 20: Specificity of antibody variants to affinity chromatography resins

[0343] As described by the manufacturer, CaptureSelect LC-lambda(Hu) affinity matrix LambdaFabSelect (GE-Healthcare) contains a 13kDa Llama antibody fragment that recognizes a unique epitope on the constant moiety of the human kappa L chain. KappaSelect binds to the epitope on the constant moiety of the human kappa L chain, and the V110D mutation prevents interaction with resin (Example 15), while protein L binds to an isotype of the kappa light chain, and the introduction of the S12P mutation into antibodies containing the kappa light chain prevents interaction with resin (Example 16). IgG1-HepC with the lambda L chain and IgG1-2F8 and IgG1-7D8 with the kappa L chain were selected as components of the recombinant antibody mixture. As described in Example 1, point mutations of V110D and S12P were introduced into IgG1-2F8-V110D and IgG1-7D8-S12P, and the specificity of the individually generated and purified antibodies was tested for binding to proteins L, KappaSelect, and LambdaFabSelect, as described in Examples 17, 18, and 19, respectively. Figure 16 The resins show that IgG1-2F8-V110D, IgG1-7D8-S12P, and IgG1-HepC specifically bind to proteins L, KappaSelect, and LambdaFabSelect, respectively.

[0344] Example 21: Binding capacity determination of HiTrap KappaSelect

[0345] Pre-equilibrate a 1 mL KappaSelect (GE Healthcare) column with phosphate-buffered saline (PBS; 12.6 mM sodium phosphate, 140 mM sodium chloride, pH 7.4, B. Braun or Thermo Fisher). Load approximately 75 mg of purified IgG1-7D8-K409R in a total volume of 40 mL PBS onto the KappaSelect (GE Healthcare) column at a flow rate of 0.25, 0.5, or 1 mL / min. Wash the column with PBS, elute specifically bound proteins with 0.1 M glycine HCl pH 2.5, and clean the column with 6 M guanidine hydrochloride. Combine the flow products, aseptically filter, and analyze protein concentration using a Nanodrop ND-1000 spectrophotometer (IsogenLife Science, Maarssen, The Netherlands) at 280 nm. Analysis of protein concentration in the flow products allows inference of column capacity between 37 and 41 mg based on the difference in protein mass between the loaded and flow products. The combined capacity is largely independent of the flow rate, which confirms that the column is saturated. Figure 17 The chromatogram of the combined volume determination experiment is shown.

[0346] Example 22: Composition of recombinant antibody mixtures controlling IgG1 variants using KappaSelect affinity chromatography

[0347] This embodiment describes the following procedure: using a mixture of variable composition and performing chromatography with an affinity chromatography resin specific to different components, thereby eliminating excess antibody to produce a mixture of predetermined composition. Figure 1 C).

[0348] As described in Example 3, IgG1-2F8-V110D, IgG1-7D8-S12P, or IgG1-HepC were recombined, and antibody titers were calculated as described in Example 4. The culture supernatant was mixed to produce a theoretical antibody concentration ratio of 1:2.4:1, measured using the biolayer interference metric, to simulate the upstream process of co-production of the antibody mixture. This ratio was chosen to simulate an upstream co-production process with a 1:1:1 mass ratio of the three antibodies, but with an overproduction of IgG1-7D8-S12P, the composition of the mixture was not under control and required chromatographic separation to achieve the desired ratio. The mixture was purified by protein A affinity chromatography as described in Example 5. Figure 5D shows the chromatogram of purified protein A from the antibody mixture. Protein concentration was analyzed by absorption at 280 nm using a Nanodrop ND-1000 spectrophotometer (Isogen Life Science, Maarssen, The Netherlands), with extinction coefficients calculated using the average of the three antibodies based on their primary amino acid sequences. Composition was analyzed using analytical cation exchange chromatography as described in Example 8. Figure 19 The image shows IgG1-2F8-V110D, IgG1-7D8-S12P, and IgG1-HepC purified by analytical cation exchange chromatography. (Analytical cation exchange chromatography diagram) Figure 19 Concentration measurements and volume allow for estimation of the mass of the three components of the mixture (Table 10). IgG1-7D8-S12P is appropriately in excess because, based on the binding capacity assay of IgG1-7D8-K409R in Example 21, it can be removed by a 1 mL KappaSelect column, which assumes that the capacities of the IgG1-7D8 variants are similar because the mutation and the epitope of the KappaSelect resin are not in the same domain. This experimental setup is to simulate the process in which the column size or number of cycles is adjusted to make the column capacity suitable for the specific removal of excess antibody from the mixture.

[0349] A 1 mL KappaSelect (GE Healthcare) column was pre-equilibrated with phosphate-buffered saline (PBS; 12.6 mM sodium phosphate, 140 mM sodium chloride, pH 7.4, B. Braun or Thermo Fisher). 116.9 mg of the recombinant antibody mixture in a total volume of 56.7 mL of PBS was loaded onto the KappaSelect column using a 50 mL superloop (GE Healthcare) eluted. The column was washed with PBS, eluted with 0.1 M glycine-HCl pH 2.5, and washed with 6 M guanidine hydrochloride. The fractions were combined, dialyzed into PBS using a suitable 10 kDa molecular weight cutoff Slide-A-Lyzer cartridge (Thermo Fisher), and the combined fractions were aseptically filtered. The concentration of the combined fractions was measured by absorbance at 280 nm using a Nanodrop ND-1000 spectrophotometer, using the extinction coefficient calculated as the average of the three antibodies using the primary amino acid sequences of the antibodies (Isogen Life Science, Maarssen, The Netherlands). like Figure 19As shown, the composition was analyzed using analytical cation exchange chromatography as described in Example 8. Quantitative analysis of the composition of the antibody mixture showed that chromatography produced antibody products conforming to a predetermined 1:1:1 ratio within tolerance limits (Table 10), demonstrating that the composition of antibody loading with variable composition can be controlled using chromatography.

[0350] Table 10: Quantification of analytical cation exchange chromatography profiles of input and standardized antibody mixtures.

[0351]

[0352]

[0353] Example 23: Using charge-regulated antibodies, an antibody mixture was separated by a sequential elution step on a preparative cation exchange column and recovered to produce an antibody mixture of a predetermined composition.

[0354] This embodiment describes the following procedure: using a mixture with variable composition, performing an analytical assay to determine the composition and a chromatography step in which the design space has been adequately pre-analyzed, thereby allowing the extraction of waste fractions containing excess antibodies to produce a mixture of the predetermined composition. Figure 1 D).

[0355] Mixtures of IgG1-7D8, IgG1-224, IgG1-CD37-37-3, IgG1-CD19-21D4-E345K, and IgG1-CD52-Campath-E345K (Example 1) were prepared using transient generation or recombinant expression of individual antibodies based on a CHO-K1 expression system, as described in Example 3, and were individually purified by protein A affinity chromatography as described in Example 5. The concentrations of the individual antibodies were measured using a Nanodrop ND-1000 spectrophotometer (Isogen Life Science, Maarssen, The Netherlands) and the extinction coefficients calculated from the primary amino acid sequences of the purified antibodies. Antibody mixtures were prepared by mixing the antibodies at a final concentration of 15.6 mg / mL in equal mass ratios in PBS buffer (12.6 mM NaH2PO4, 140 mM NaCl, pH 7.4 buffer, B. Braun or Thermo Fisher).

[0356] Example 9 describes an elution protocol that allows for the individual elution of each antibody via a sequential step. Each of the five steps in the elution protocol is converted into two steps, with the first having variable ionic strength and the second being identical to the one used in Example 9. The first step is designed to elute a broad peak containing single antibody specificity and similar in height and width for each of the five antibodies. The second step is designed to elute all remaining proteins and provide baseline separation before eluting the next protein.

[0357] The antibody mixture was diluted 20-fold in loading buffer (20 mM NaHPO4, pH 6.5). During salt concentration screening, separation was performed with 10 g / L resin loading according to Example 6, except that in a separate experiment, the antibody was eluted with 10 sequential steps, alternating between 10 and 7 column volumes, containing 13%, 19.5%, 20.7%, 29.4%, 31.5%, 39.5%, 39.5%, 44.6%, 47%, 61.2%; 13.5%, 19.5%, 21.2%, ... 29.4%, 32%, 39.5%, 39.6%, 44.6%, 47.5%, 61.2%; 14%, 19.5%, 21.7%, 29.4%, 32.5%, 39.5%, 39.8%, 44.6%, 48%, 61.2%; 14.5%, 19.5%, 22.2%, 29.4%, 33%, 39.5%, 40%, 44.6%, 48.5%, 61.2% or 15%, 19.5%, 22.7%, 29.4%, 33.5%, 39.5%, 40.2%, 44.6%, 49%, 61.2% (v / v) elution buffer (20mM NaHPO4, 250mM NaCl pH 6.5) loading buffer. The final conditions were to elute the antibody using loading buffers mixed with 14%, 19.5%, 21.7%, 29.4%, 32.5%, 39.5%, 39.8%, 44.6%, 48%, and 61.2% (v / v) elution buffers (20 mM NaHPO4, 250 mM NaCl, pH 6.5). Figure 20 A shows the peak shape of each of the five antibodies as a function of ionic intensity; higher % buffer B corresponds to increased peak height, and... Figure 20 B shows the elution protocol selected based on the chromatography diagram.

[0358] To investigate the design space, different ratios of five antibodies were applied to the column in separate experiments. For each ratio, the systematic change in the volume of eluted protein reduced from the mixture was described below. The output mixture pool was analyzed by analytical cation exchange chromatography to understand the relationship between the amount of each protein in the pool and the volume of the reduced fraction. In this example, the design space of three proteins (IgG1-7D8, IgG1-CD37-37-3, and IgG1-CD52-Campath-E345K; peaks 1, 3, and 5) was investigated, while the design space of two proteins (IgG1-224 and IgG1-CD19-21D4-E345K; peaks 2 and 4) was not reduced and therefore could be used as controls in the analytical cation exchange experiments.

[0359] Five mixtures of IgG1-7D8, IgG1-224, IgG1-CD37-37-3, IgG1-CD19-21D4-E345K, and IgG1-CD52-Campath-E345K with different antibody ratios were prepared in PBS buffer (12.6 mM NaH2PO4, 140 mM NaCl, pH 7.4 buffer, B. Braun or Thermo Fisher) at final concentrations ranging from 2.6 mg / mL to 3.0 mg / mL. The mixtures had mass ratios of 1.5:1:1.5:1:1.5, 2.5:1:2.5:1:2.5, 1.5:1:1:1.5:1.5, 2:1:1:2:2, and 2.5:1:1:2.5:2.5, respectively.

[0360] Each mixture was diluted 20-fold in loading buffer (20 mM NaHPO4, pH 6.5). Separation was performed with a 10 g / L resin loading, as per Example 6, except that the antibody was eluted with 10 sequential steps, each step alternating with 10 and 7 column volumes of loading buffer (20 mM NaHPO4, 250 mM NaCl, pH 6.5) mixed with elution buffers of 14%, 19.5%, 21.7%, 29.4%, 32.5%, 39.5%, 39.8%, 44.6%, 48%, and 61.2% v / v). The final product protein mixture was eluted into individual output containers, except for a predetermined waste volume, which was eluted into separate containers by switching their respective outlet valves. Two column volumes were started after the elution of IgG1-7D8, IgG1-CD37-37-3, and IgG1-CD52-Campath-E345K; peaks 1, 3, and 5 ( Figure 20C). Each mixture was purified in a design space experiment consisting of five essentially identical different chromatography experiments, except that the waste volumes were set to 0 mL, 10 mL, 20 mL, 30 mL or 40 mL (mixtures of 1.5:1:1.5:1:1.5 and 2.5:1:2.5:1:2.5), or 0 mL, 12.5 mL, 25 mL, 37.5 mL or 50 mL (mixtures of 1.5:1:1:1.5:1.5, 2:1:1:2:2, 2.5:1:1:2.5:2.5). Figure 20 C shows an example tomographic diagram from the design space experiment.

[0361] As described in Example 8, the final product pools collected during the design space experiment were analyzed by analytical cation exchange chromatography. Figure 20 Figure D shows a set of analytical cation exchange chromatography plots for a design space experiment (2.5:1:2.5:1:2.5 mixture), demonstrating the effect of reducing the increased volume during the first elution step of IgG1-7D8, IgG1-CD37-37-3, and IgG1-CD52-Campath-E345K.

[0362] By correcting for the specific extinction coefficients of individual proteins, the area of ​​each of the five peaks in each analytical cation exchange chromatography pattern was converted to concentration and normalized relative to the concentrations of IgG1-224 (peak 2; mixtures of 1.5:1:1:1.5:1.5, 2:1:1:2:2, and 2.5:1:1:2.5:2.5) or IgG1-224 and IgG1-CD19-21D4-E345K (peaks 2 and 4; mixtures of 1.5:1:1.5:1:1.5 and 2.5:1:2.5:1:2.5). For each set of experiments corresponding to single antibody mixtures with different waste volumes, the mass of each protein was normalized relative to the amount of purified protein from the volume from which waste was not removed to calculate the fraction of retained protein (Table 11). Figure 20 EF).

[0363] In this embodiment, a linear correlation is assumed between protein mass and waste fraction volume in the pooled fraction, and data for IgG1-7D8, IgG1-CD37-37-3, and IgG1-CD52-Campath-E345K are fitted for loading levels normalized for each protein. This is a simplified model of chromatographic behavior because it assumes no peak shape complexity and that peak fraction does not change with column loading. Ri for the three proteins 2 The correlation coefficients were 0.97, 0.95, and 0.93, respectively. Figure 20E). Finally, all data standardized for the loading amount of each protein were simultaneously fitted into a simple model to describe the relative reduction of all five proteins as waste volume increases. Figure 20 F). This model assumes that all proteins behave identically under these conditions.

[0364] The model was applied to four different mixtures of antibodies with different antibody compositions: IgG1-7D8, IgG1-224, IgG1-CD37-37-3, IgG1-CD19-21D4-E345K, and IgG1-CD52-Campath-E345K, with release specifications of 20% + / - 2% for each component. First, the mixture was prepared as follows: components purified from individual protein A were mixed in PBS buffer (12.6 mM NaH2PO4, 140 mM NaCl, pH 7.4 buffer, B. Braun or Thermo Fisher) to simulate an upstream process with insufficient control over composition, followed by a capture chromatography step. Next, the mixture was analyzed by analytical cation exchange chromatography as described in Example 8. The area of ​​each of the five peaks in the analytical cation exchange chromatography was converted to mass concentration by correcting for the specific extinction coefficients of individual proteins, and the amount of each protein to be loaded onto the preparative cation exchange column was inferred by assuming a total protein loading of 10 g / L resin. The waste volume for each protein was calculated according to the following formula:

[0365] V=(m min -m) / mk

[0366] Where V = the volume of waste liquid producing an equal mass mixture, m = the mass of protein on the column, m min = The mass of the protein restricted on the column, k = the first-order approximation of the deceleration rate. Figure 20 F(-0.0178mL -1 ).

[0367] Next, preparative cation exchange chromatography was performed to recover an equal mass mixture of the five antibodies. Separation was carried out with a 10 g / L resin loading, following Example 6, by eluting the antibodies with 10 sequential steps, alternating between 10 and 7 column volumes containing loading buffers mixed with elution buffers (20 mM NaHPO4, 250 mM NaCl, pH 6.5) of 14%, 19.5%, 21.7%, 29.4%, 32.5%, 39.5%, 39.8%, 44.6%, 48%, and 61.2% v / v. The final product protein mixture was eluted into a single output container, except for a predetermined waste volume specified for each of the four non-restrictive antibodies, starting two column volumes after elution, which was eluted into separate containers. Finally, the final product protein mixture was analyzed by analytical cation exchange chromatography, according to Example 8. Figure 20 GN summarizes the preparative cation exchange chromatography (PET) chromatograms of four mixtures, as well as the analytical PET chromatograms of the input materials and the final products. Waste quantities and results are summarized in Table 12.

[0368] The results showed that although the input mixture AC exceeded the specifications (20% + / - 2% of each component), the output mixture was within the specifications, demonstrating that the method can be used to control the composition of polyclonal mixtures.

[0369] Input mixture D is not within the specification but was not included in the specification by application method. This mixture has the maximum excess of IgG1-CD52-Campath-E345K, which is least adequately described by design space experiments, as evidenced by the low correlation coefficient. Figure 20 E), and with a large excess of IgG1-224, which was not altered in the design space experiment. This method can be improved for more challenging mixtures by refining the model to better describe the relationship between the output of each antibody and the waste volume. For example, improved models can be built by applying a different model to each component of the mixture, by using more data points in the model, by applying interpolation between experimental points instead of relying on a first-order approximation, or by using a more complex model, such as fitting the shape of a Gaussian peak.

[0370] Table 11: During preparative cation exchange chromatography of a 2.5:1:1:2.5:2.5 mixture of IgG1-7D8, IgG1-224, IgG1-CD37-37-3, IgG1-CD19-21D4-E345K, and IgG1-CD52-Campath-E345K, the reduction rate of individual antibody components from the input mixture was quantified as a function of the reduction volume. For each test antibody, the analytical cation exchange chromatography peak area (Area_rel) was normalized relative to the area (Area_ref) observed as an internal control of unreduced IgG1-224, after correcting for its corresponding extinction coefficient (ext.coeff.; ε). The retained protein fraction (prot_ret) was then normalized relative to the amount present in the input mixture measured at a reduction volume of 0.0 mL. The reduction rate k was then determined as the first derivative (slope) of the retained protein fraction (prot_ret) relative to the reduction volume (reduction of V).

[0371]

[0372] Calculation of relative area: Area_rel = (Area_sample / Area_ref) * (_ref / _sample)

[0373]

[0374] Calculation of retained protein fraction: prot_ret = Area_rel(V) / Area_rel (V = 0.0)

[0375]

[0376] Calculate the deceleration rate k (1 / mL): k = d(prot ret) / dV (the slope of the first-order approximation)

[0377]

[0378] Table 12:

[0379] Overview of analytical cation exchange chromatography for quantitative input and output of antibody mixtures, standardization of specific extinction coefficients for individual components, and preparative chromatography waste volume.

[0380]

[0381]

[0382] Example 24: Composition of recombinant antibody mixtures controlling IgG1 variants using affinity chromatography

[0383] This embodiment describes the following procedure: using a mixture of variable composition, and performing chromatography with an affinity chromatography resin that is specific to the different components being eluted to produce a mixture of predetermined composition. Figure 1 B).

[0384] Recombinant IgG1-2F8-V110D, IgG1-7D8-S12P, or IgG1-HepC were produced as described in Example 3, and antibody titers were calculated as described in Example 4. These proteins were selected or engineered to specifically bind to Protein L (GE Healthcare), KappaSelect (GE Healthcare), or LambdaFabSelect (GE Healthcare) resins, as described in Examples 15, 16, and 20. The culture supernatants were mixed to produce theoretical antibody concentration ratios of approximately 1:1:1 or 1:1.5:2 and total amounts of approximately 185 mg or 275 mg, respectively, as measured by the biolayer interference metric, to simulate the upstream process for co-producing the antibody mixture.

[0385] 1 mL connected in series KappaSelect (GE Healthcare), LambdaFabSelect (GE Healthcare), and Protein L (GE Healthcare) columns were pre-equilibrated with phosphate-buffered saline (PBS; 12.6 mM sodium phosphate, 140 mM sodium chloride, pH 7.4, B. Braun or Thermo Fisher). The supernatant containing a 1:1:1 or 1:1.5:2 mixture of approximately 180 mg or 270 mg of IgG1-2F8-V110D, IgG1-7D8-S12P, and IgG1-HepC was loaded onto the column at a flow rate of 0.5 mL / min and washed with PBS. Figure 21 AB).

[0386] Due to differences in elution and washing buffers between columns, individual elution was performed. The HiTrap KappaSelect column was eluted with 0.1M glycine-HCl at pH 3.0. Specifically bound material was eluted with 0.1M glycine-HCl at pH 2.5. The column was washed with 6M guanidine hydrochloride. The HiTrap Protein L column was washed with 0.02M sodium citrate-NaOH at pH 5.0, and specifically bound material was eluted with 0.1M glycine-HCl at pH 3.0. The column was washed with 0.015M NaOH. The HiTrap LambdaFabSelect column (GE Healthcare) was eluted with 0.1M glycine-HCl at pH 2.0, followed by elution with 0.5M acetic acid. The column was washed with 0.025M NaOH. An exemplary chromatography plot is shown in [link to chromatography]. Figure 21 As shown in CE. Elution fractions with significant absorption at 280 nM in each case were combined, dialyzed into PBS using an appropriately sized Slide-A-Lyzer tube (ThermoFisher) with a molecular weight cutoff of 30 kDa, and the combined fractions were aseptically filtered.

[0387] Alternatively, as described in Example 5, approximately 5 mg of the supernatant containing a 1:1:1 or 1:1.5:2 mixture of IgG1-2F8-V110D, IgG1-7D8-S12P and IgG1-HepC was purified by protein A affinity chromatography. Figure 21 F shows an exemplary chromatography diagram of protein A purification from an antibody mixture.

[0388] The composition of the output mixtures from different purification experiments was analyzed using analytical cation exchange chromatography as described in Example 8, and a mixture of sterile filtered cell culture supernatant was loaded onto an analytical cation exchange column and analyzed using a method similar to that described in Example 8. Chromatography results are shown in... Figure 21 The results are shown in G and quantified in Table 13. The data show that although the ratios of the input mixtures differed significantly and the protein A purification step did not significantly alter the composition of the mixtures, tandem purification of proteins L, KappaSelect, and LambdaFabSelect yielded output mixtures with similar compositions for both mixtures. This demonstrates that this method can be used to control the composition of polyclonal antibody mixtures. The output ratios can be adjusted by varying the relative amounts of resin in the three orthogonal affinity columns under relevant loading conditions, based on the experimentally determined dynamic binding capacity of the resin. If light chain-specific affinity resins are used, an orthogonal step may be required to remove co-purified free light chains or light chain dimers.

[0389] Table 13: Quantification of analytical cation exchange chromatography profiles of input and standardized antibody mixtures.

[0390] sequence list <110> Jianma Bao <120> Method for producing a controlled mixture of two or more different antibodies <130> P / 0132-WO-PCT <160> 64 <170> PatentIn version 3.5 <210> 1 <211> 122 <212> PRT <213> Homo sapiens <400> 1 Glu Val Gln Leu Val Glu Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe His Phe Tyr 20 25 30 Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Ser Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Arg Pro Ser Phe 50 55 60 Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr 65 70 75 80 Leu Gln Trp Thr Ser Leu Lys Ala Ser Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Ala Arg Gln Arg Gly Asp Tyr Tyr Tyr Phe Tyr Gly Met Asp Val Trp 100 105 110 Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 2 <211> 122 <212> PRT <213> Homo sapiens <400> 2 Glu Val Gln Leu Val Glu Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe His Phe Tyr 20 25 30 Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Ser Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Arg Pro Ser Phe 50 55 60 Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr 65 70 75 80 Leu Gln Trp Thr Ser Leu Lys Ala Glu Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Ala Arg Gln Arg Gly Asp Tyr Tyr Tyr Phe Tyr Gly Met Asp Val Trp 100 105 110 Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 3 <211> 122 <212> PRT <213> Homo sapiens <400> 3 Glu Val Gln Leu Val Glu Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu Lys Ile Ser Cys Ala Ala Ser Gly Tyr Ser Phe His Phe Tyr 20 25 30 Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Ser Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Arg Pro Ser Phe 50 55 60 Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr 65 70 75 80 Leu Gln Trp Thr Ser Leu Lys Ala Glu Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Ala Arg Gln Arg Gly Asp Tyr Tyr Tyr Phe Tyr Gly Met Asp Val Trp 100 105 110 Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 4 <211> 122 <212> PRT <213> Homo sapiens <400> 4 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe His Phe Tyr 20 25 30 Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Ser Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Arg Pro Ser Phe 50 55 60 Gln Gly Arg Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr 65 70 75 80 Leu Gln Trp Thr Ser Leu Lys Ala Ser Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Ala Arg Gln Arg Gly Asp Tyr Tyr Tyr Phe Tyr Gly Met Asp Val Trp 100 105 110 Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 5 <211> 122 <212> PRT <213> Homo sapiens <400> 5 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Leu Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe His Phe Tyr 20 25 30 Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Ser Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Arg Pro Ser Phe 50 55 60 Gln Gly Arg Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr 65 70 75 80 Leu Gln Trp Thr Ser Leu Lys Ala Ser Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Ala Arg Gln Arg Gly Asp Tyr Tyr Tyr Phe Tyr Gly Met Asp Val Trp 100 105 110 Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 6 <211> 122 <212> PRT <213> Homo sapiens <400> 6 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Leu Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe His Phe Tyr 20 25 30 Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Ser Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Arg Pro Ser Phe 50 55 60 Gln Gly Arg Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr 65 70 75 80 Leu Gln Trp Thr Arg Leu Lys Ala Ser Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Ala Arg Gln Arg Gly Asp Tyr Tyr Tyr Phe Tyr Gly Met Asp Val Trp 100 105 110 Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 7 <211> 122 <212> PRT <213> Homo sapiens <400> 7 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe His Phe Tyr 20 25 30 Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Ser Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Arg Pro Ser Phe 50 55 60 Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr 65 70 75 80 Leu Gln Trp Thr Ser Leu Lys Ala Ser Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Ala Arg Gln Arg Gly Asp Tyr Tyr Tyr Phe Tyr Gly Met Asp Val Trp 100 105 110 Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 8 <211> 122 <212> PRT <213> Homo sapiens <400> 8 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe His Phe Tyr 20 25 30 Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Ser Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Arg Pro Ser Phe 50 55 60 Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr 65 70 75 80 Leu Gln Trp Thr Ser Leu Lys Ala Ser Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Ala Arg Gln Arg Gly Asp Tyr Tyr Tyr Phe Tyr Gly Met Asp Val Trp 100 105 110 Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 9 <211> 107 <212> PRT <213> Homo sapiens <400> 9 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Val Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 10 <211> 107 <212> PRT <213> Homo sapiens <400> 10 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Asp Gln Val Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 11 <211> 107 <212> PRT <213> Homo sapiens <400> 11 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Asp Gln Val Pro Gln Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 12 <211> 107 <212> PRT <213> Homo sapiens <400> 12 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Val Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 13 <211> 107 <212> PRT <213> Homo sapiens <400> 13 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Val Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 14 <211> 107 <212> PRT <213> Homo sapiens <400> 14 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 35 40 45 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Arg Leu Leu 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 15 <211> 107 <212> PRT <213> Homo sapiens <400> 15 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Val Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 16 <211> 124 <212> PRT <213> Homo sapiens <400> 16 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Gly Ser Gly Gly His Thr Tyr His Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Arg Tyr Asp Ile Leu Thr Gly Tyr Tyr Asn Leu Leu Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 17 <211> 124 <212> PRT <213> Homo sapiens <400> 17 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Gly Ser Gly Gly His Thr Tyr His Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Arg Tyr Asp Ile Leu Thr Gly Tyr Tyr Asn Leu Leu Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 18 <211> 124 <212> PRT <213> Homo sapiens <400> 18 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Gly Ser Gly Gly His Thr Tyr His Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Glu Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Arg Tyr Asp Ile Leu Thr Gly Tyr Tyr Asn Leu Leu Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 19 <211> 124 <212> PRT <213> Homo sapiens <400> 19 Glu Val Gln Leu Leu Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Gly Ser Gly Gly His Thr Tyr His Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Arg Tyr Asp Ile Leu Thr Gly Tyr Tyr Asn Leu Leu Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 20 <211> 124 <212> PRT <213> Homo sapiens <400> 20 Glu Val Gln Leu Leu Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Gly Ser Gly Gly His Thr Tyr His Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Gly Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Arg Tyr Asp Ile Leu Thr Gly Tyr Tyr Asn Leu Leu Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 21 <211> 124 <212> PRT <213> Homo sapiens <400> 21 Glu Val Gln Leu Leu Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Lys Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Gly Ser Gly Gly His Thr Tyr His Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Gly Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Arg Tyr Asp Ile Leu Thr Gly Tyr Tyr Asn Leu Leu Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 22 <211> 124 <212> PRT <213> Homo sapiens <400> 22 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Gly Ser Gly Gly His Thr Tyr His Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Arg Tyr Asp Ile Leu Thr Gly Tyr Tyr Asn Leu Leu Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 23 <211> 124 <212> PRT <213> Homo sapiens <400> 23 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Gly Ser Gly Gly His Thr Tyr His Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Arg Tyr Asp Ile Leu Thr Gly Tyr Tyr Asn Leu Leu Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 24 <211> 107 <212> PRT <213> Homo sapiens <400> 24 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Glu Glu Ala Pro Lys Ser Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Ala Lys Val Glu Ile Lys 100 105 <210> 25 <211> 107 <212> PRT <213> Homo sapiens <400> 25 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Glu Glu Ala Pro Lys Ser Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Glu Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Ala Lys Val Glu Ile Lys 100 105 <210> 26 <211> 107 <212> PRT <213> Homo sapiens <400> 26 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Glu Glu Ala Pro Lys Ser Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Glu Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Ala Lys Val Glu Ile Lys 100 105 <210> 27 <211> 107 <212> PRT <213> Homo sapiens <400> 27 Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Glu Glu Ala Pro Lys Ser Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Ala Lys Val Glu Ile Lys 100 105 <210> 28 <211> 107 <212> PRT <213> Homo sapiens <400> 28 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Ala Lys Val Glu Ile Lys 100 105 <210> 29 <211> 107 <212> PRT <213> Homo sapiens <400> 29 Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Ala Lys Val Glu Ile Lys 100 105 <210> 30 <211> 107 <212> PRT <213> Homo sapiens <400> 30 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Glu Glu Ala Pro Lys Ser Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Ala Lys Val Glu Ile Lys 100 105 <210> 31 <211> 125 <212> PRT <213> Homo sapiens <400> 31 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Asp Asp Gly Ser Tyr Lys Tyr Tyr Gly Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Gly Ile Thr Met Val Arg Gly Val Met Lys Asp Tyr Phe 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 32 <211> 125 <212> PRT <213> Homo sapiens <400> 32 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Asp Asp Gly Ser Tyr Lys Tyr Tyr Gly Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Gly Ile Thr Met Val Arg Gly Val Met Lys Asp Tyr Phe 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 33 <211> 125 <212> PRT <213> Homo sapiens <400> 33 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Asp Asp Gly Ser Tyr Lys Tyr Tyr Gly Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Gly Ile Thr Met Val Arg Gly Val Met Lys Asp Tyr Phe 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 34 <211> 125 <212> PRT <213> Homo sapiens <400> 34 Glu Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Asp Asp Gly Ser Tyr Lys Tyr Tyr Gly Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Gly Ile Thr Met Val Arg Gly Val Met Lys Asp Tyr Phe 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 35 <211> 125 <212> PRT <213> Homo sapiens <400> 35 Glu Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Asp Asp Gly Ser Tyr Lys Tyr Tyr Gly Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Gly Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Gly Ile Thr Met Val Arg Gly Val Met Lys Asp Tyr Phe 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 36 <211> 125 <212> PRT <213> Homo sapiens <400> 36 Glu Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Lys Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Asp Asp Gly Ser Tyr Lys Tyr Tyr Gly Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Gly Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Gly Ile Thr Met Val Arg Gly Val Met Lys Asp Tyr Phe 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 37 <211> 125 <212> PRT <213> Homo sapiens <400> 37 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30 Ala Val Ile Trp Asp Asp Gly Ser Tyr Lys Tyr Tyr Gly Asp Ser Val 35 40 45 Ala Val Ile Trp Asp Asp Gly Ser Tyr Lys Tyr Tyr Gly Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Gly Ile Thr Met Val Arg Gly Val Met Lys Asp Tyr Phe 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 38 <211> 125 <212> PRT <213> Homo sapiens <400> 38 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Asp Asp Gly Ser Tyr Lys Tyr Tyr Gly Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Gly Ile Thr Met Val Arg Gly Val Met Lys Asp Tyr Phe 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 39 <211> 107 <212> PRT <213> Homo sapiens <400> 39 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Ala 20 25 30 Leu Val Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Glu Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 40 <211> 107 <212> PRT <213> Homo sapiens <400> 40 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Ala 20 25 30 Leu Val Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Glu Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 41 <211> 107 <212> PRT <213> Homo sapiens <400> 41 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Ala 20 25 30 Leu Val Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Glu Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Glu Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 42 <211> 107 <212> PRT <213> Homo sapiens <400> 42 Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Ala 20 25 30 Leu Val Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 43 <211> 107 <212> PRT <213> Homo sapiens <400> 43 Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Ala 20 25 30 Leu Val Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 44 <211> 107 <212> PRT <213> Homo sapiens <400> 44 Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Ala 20 25 30 Leu Val Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 45 <211> 107 <212> PRT <213> Homo sapiens <400> 45 Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Ala 20 25 30 Leu Val Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Glu Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 46 <211> 330 <212> PRT <213> Homo sapiens <400> 46 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 47 <211> 107 <212> PRT <213> Homo sapiens <400> 47 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 48 <211> 105 <212> PRT <213> Homo sapiens <400> 48 Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu 1 5 10 15 Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe 20 25 30 Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val 35 40 45 Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys 50 55 60 Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser 65 70 75 80 His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu 85 90 95 Lys Thr Val Ala Pro Thr Glu Cys Ser 100 105 <210> 49 <211> 122 <212> PRT <213> Homo sapiens <400> 49 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Asp Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe His Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Thr Ile Ser Trp Asn Ser Gly Thr Ile Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Lys Asp Ile Gln Tyr Gly Asn Tyr Tyr Tyr Gly Met Asp Val Trp 100 105 110 Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 50 <211> 107 <212> PRT <213> Homo sapiens <400> 50 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105 <210> 51 <211> 121 <212> PRT <213> Homo sapiens <400> 51 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Val Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Thr Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Ser Ala Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Ile Thr Gly Thr Thr Gly Val Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 52 <211> 107 <212> PRT <213> Homo sapiens <400> 52 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile 35 40 45 Tyr Asp Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Tyr Gly Thr Asp Phe Ser Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Ile Tyr Tyr Cys Gln Gln Tyr Lys Ser Tyr Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105 <210> 53 <211> 115 <212> PRT <213> Homo sapiens <400> 53 Gln Val Gln Val Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Thr Ser 20 25 30 Gly Val Ser Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Ile Trp Gly Asp Gly Ser Thr Asn Tyr His Ser Ala Leu Lys 50 55 60 Ser Arg Leu Ser Ile Lys Lys Asp His Ser Lys Ser Gln Val Phe Leu 65 70 75 80 Lys Leu Asn Ser Leu Gln Thr Asp Asp Thr Ala Thr Tyr Tyr Cys Ala 85 90 95 Lys Gly Gly Tyr Ser Leu Ala His Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ala 115 <210> 54 <211> 107 <212> PRT <213> Homo sapiens <400> 54 Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Val Ser Val Gly 1 5 10 15 Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Arg Ser Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val 35 40 45 Asn Val Ala Thr Asn Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Gly Thr Tyr Tyr Cys Gln His Tyr Trp Gly Thr Thr Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 55 <211> 121 <212> PRT <213> Homo sapiens <400> 55 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Ser Ser Ser 20 25 30 Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Ile Ile Tyr Pro Asp Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe 50 55 60 Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Arg Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg His Val Thr Met Ile Trp Gly Val Ile Ile Asp Phe Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 56 <211> 107 <212> PRT <213> Homo sapiens <400> 56 Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Ala 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 57 <211> 121 <212> PRT <213> Homo sapiens <400> 57 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Thr Phe Thr Asp Phe 20 25 30 Tyr Met Asn Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp Ile 35 40 45 Gly Phe Ile Arg Asp Lys Ala Lys Gly Tyr Thr Thr Glu Tyr Asn Pro 50 55 60 Ser Val Lys Gly Arg Val Thr Met Leu Val Asp Thr Ser Lys Asn Gln 65 70 75 80 Phe Ser Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Glu Gly His Thr Ala Ala Pro Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Ser Leu Val Thr Val Ser Ser 115 120 <210> 58 <211> 107 <212> PRT <213> Homo sapiens <400> 58 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Ile Asp Lys Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asn Thr Asn Asn Leu Gln Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln His Ile Ser Arg Pro Arg 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 59 <211> 117 <212> PRT <213> Homo sapiens <400> 59 Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Leu Ser Gly Tyr 20 25 30 Phe Trp Thr Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ser Asn Tyr Ala Gly Ser Thr Arg Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Gln Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Trp Ala Val Asp Gly Met Asp Val Trp Gly Gln Gly Thr Thr 100 105 110 Val Thr Val Ser Ser 115 <210> 60 <211> 109 <212> PRT <213> Homo sapiens <400> 60 Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln 1 5 10 15 Thr Ala Arg Ile Thr Cys Ser Gly Asp Ala Leu Pro Lys Gln Tyr Ala 20 25 30 Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45 Lys Asp Asn Glu Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Arg Ser Gly Thr Thr Val Thr Leu Thr Ile Ser Gly Val Gln Ala Glu 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Ala Asp Ser Ser Gly Ser Ser 85 90 95 Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 <210> 61 <211> 329 <212> PRT <213> Homo sapiens <400> 61 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly 325 <210> 62 <211> 251 <212> PRT <213> Staphylococcus aureus <400> 62 Ala Ala Gln His Asp Glu Ala Gln Gln Asn Ala Phe Tyr Gln Val Leu 1 5 10 15 Asn Met Pro Asn Leu Asn Ala Asp Gln Arg Asn Gly Phe Ile Gln Ser 20 25 30 Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Val Leu Gly Glu Ala Lys 35 40 45 Lys Leu Asn Glu Ser Gln Ala Pro Lys Ala Asp Asn Asn Phe Asn Lys 50 55 60 Glu Gln Gln Asn Ala Phe Tyr Glu Ile Leu Asn Met Pro Asn Leu Asn 65 70 75 80 Glu Glu Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser 85 90 95 Gln Ser Ala Asn Leu Leu Ser Glu Ala Lys Lys Leu Asn Glu Ser Gln 100 105 110 Ala Pro Lys Ala Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe 115 120 125 Glu Ile Leu His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Gly Phe 130 135 140 Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu Ala 145 150 155 160 Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Ala Asp Asn Lys 165 170 175 Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile Leu His Leu Pro 180 185 190 Asn Leu Thr Glu Glu Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys Asp 195 200 205 Asp Pro Ser Val Ser Lys Glu Ile Leu Ala Glu Ala Lys Lys Leu Asn 210 215 220 Asp Ala Gln Ala Pro Lys Glu Glu Asp Asn Asn Lys Pro Gly Lys Glu 225 230 235 240 Asp Gly Asn Lys Pro Gly Lys Glu Asp Gly Asn 245 250 <210> 63 <211> 384 <212> PRT <213> Streptococcus sp. group <400> 63 Val Asp Ser Pro Ile Glu Asp Thr Pro Ile Ile Arg Asn Gly Gly Glu 1 5 10 15 Leu Thr Asn Leu Leu Gly Asn Ser Glu Thr Thr Leu Ala Leu Arg Asn 20 25 30 Glu Glu Ser Ala Thr Ala Asp Leu Thr Ala Ala Ala Val Ala Asp Thr 35 40 45 Val Ala Ala Ala Ala Ala Glu Asn Ala Gly Ala Ala Ala Trp Glu Ala 50 55 60 Ala Ala Ala Ala Asp Ala Leu Ala Lys Ala Lys Ala Asp Ala Leu Lys 65 70 75 80 Glu Phe Asn Lys Tyr Gly Val Ser Asp Tyr Tyr Lys Asn Leu Ile Asn 85 90 95 Asn Ala Lys Thr Val Glu Gly Ile Lys Asp Leu Gln Ala Gln Val Val 100 105 110 Glu Ser Ala Lys Lys Ala Arg Ile Ser Glu Ala Thr Asp Gly Leu Ser 115 120 125 Asp Phe Leu Lys Ser Gln Thr Pro Ala Glu Asp Thr Val Lys Ser Ile 130 135 140 Glu Leu Ala Glu Ala Lys Val Leu Ala Asn Arg Glu Leu Asp Lys Tyr 145 150 155 160 Gly Val Ser Asp Tyr His Lys Asn Leu Ile Asn Asn Ala Lys Thr Val 165 170 175 Glu Gly Val Lys Glu Leu Ile Asp Glu Ile Leu Ala Ala Leu Pro Lys 180 185 190 Thr Asp Thr Tyr Lys Leu Ile Leu Asn Gly Lys Thr Leu Lys Gly Glu 195 200 205 Thr Thr Thr Glu Ala Val Asp Ala Ala Thr Ala Glu Lys Val Phe Lys 210 215 220 Gln Tyr Ala Asn Asp Asn Gly Val Asp Gly Glu Trp Thr Tyr Asp Asp 225 230 235 240 Ala Thr Lys Thr Phe Thr Val Thr Glu Lys Pro Glu Val Ile Asp Ala 245 250 255 Ser Glu Leu Thr Pro Ala Val Thr Thr Tyr Lys Leu Val Ile Asn Gly 260 265 270 Lys Thr Leu Lys Gly Glu Thr Thr Thr Lys Ala Val Asp Ala Glu Thr 275 280 285 Ala Glu Lys Ala Phe Lys Gln Tyr Ala Asn Asp Asn Gly Val Asp Gly 290 295 300 Val Trp Thr Tyr Asp Asp Ala Thr Lys Thr Phe Thr Val Thr Glu Met 305 310 315 320 Val Thr Glu Val Pro Gly Asp Ala Pro Thr Glu Pro Glu Lys Pro Glu 325 330 335 Ala Ser Ile Pro Leu Val Pro Leu Thr Pro Ala Thr Pro Ile Ala Lys 340 345 350 Asp Asp Ala Lys Lys Asp Asp Thr Lys Lys Glu Asp Pro Glu Ala Lys 355 360 365 Lys Asp Asp Ala Lys Lys Ala Glu Thr Leu Pro Thr Thr Gly Glu Gly 370 375 380 <210> 64 <211> 938 <212> PRT <213> Finegoldia magna <400> 64 Ala Glu Glu Asp Asn Thr Asp Asn Asn Leu Ser Met Asp Glu Ile Ser 1 5 10 15 Asp Ala Tyr Phe Asp Tyr His Gly Asp Val Ser Asp Ser Val Asp Pro 20 25 30 Val Glu Glu Glu Ile Asp Glu Ala Leu Ala Lys Ala Leu Ala Glu Ala 35 40 45 Lys Glu Thr Ala Lys Lys His Ile Asp Ser Leu Asn His Leu Ser Glu 50 55 60 Thr Ala Lys Lys Leu Ala Lys Asn Asp Ile Asp Ser Ala Thr Thr Ile 65 70 75 80 Asn Ala Ile Asn Asp Ile Val Ala Arg Ala Asp Val Met Glu Arg Lys 85 90 95 Thr Path Glu Lys Glu Glu Path Glu Lys Leu Path Path Path Lys Glu Thr 100 105 110 Ala Lys Lys His Ile Asp Glu Leu Lys His Leu Ala Asp Lys Thr Lys 115 120 125 Glu Leu Ala Lys Arg Asp Ile Asp Ser Ala Thr Thr Ile Asn Ala Ile 130 135 140 Asn Asp Ile Val Ala Arg Ala Asp Val Met Glu Arg Lys Thr Ala Glu 145 150 155 160 Lys Glu Glu Ala Glu Lys Leu Ala Ala Ala Lys Glu Thr Ala Lys Lys 165 170 175 His Ile Asp Glu Leu Lys His Leu Ala Asp Lys Thr Lys Glu Leu Ala 180 185 190 Lys Arg Asp Ile Asp Ser Ala Thr Thr Ile Asp Ala Ile Asn Asp Ile 195 200 205 Val Ala Arg Ala Asp Val Met Glu Arg Lys Leu Ser Glu Lys Glu Thr 210 215 220 Pro Glu Pro Glu Glu Glu Val Thr Ile Lys Ala Asn Leu Ile Phe Ala 225 230 235 240 Asp Gly Ser Thr Gln Asn Ala Glu Phe Lys Gly Thr Phe Ala Lys Ala 245 250 255 Val Ser Asp Ala Tyr Ala Tyr Ala Asp Ala Leu Lys Lys Asp Asn Gly 260 265 270 Glu Tyr Thr Val Asp Val Ala Asp Lys Gly Leu Thr Leu Asn Ile Lys 275 280 285 Phe Ala Gly Lys Lys Glu Lys Pro Glu Glu Pro Lys Glu Glu Val Thr 290 295 300 Ile Lys Val Asn Leu Ile Phe Ala Asp Gly Lys Thr Gln Thr Ala Glu 305 310 315 320 Phe Lys Gly Thr Phe Glu Glu Ala Thr Ala Lys Ala Tyr Ala Tyr Ala 325 330 335 Asp Leu Leu Ala Lys Glu Asn Gly Glu Tyr Thr Ala Asp Leu Glu Asp 340 345 350 Gly Gly Asn Thr Ile Asn Ile Lys Phe Ala Gly Lys Glu Thr Pro Glu 355 360 365 Thr Pro Glu Glu Pro Lys Glu Glu Val Thr Ile Lys Val Asn Leu Ile 370 375 380 Phe Ala Asp Gly Lys Ile Gln Thr Ala Glu Phe Lys Gly Thr Phe Glu 385 390 395 400 Glu Ala Thr Ala Lys Ala Tyr Ala Tyr Ala Asn Leu Leu Ala Lys Glu 405 410 415 Asn Gly Glu Tyr Thr Ala Asp Leu Glu Asp Gly Gly Asn Thr Ile Asn 420 425 430 Ile Lys Phe Ala Gly Lys Glu Thr Pro Glu Thr Pro Glu Glu Pro Lys 435 440 445 Glu Glu Val Thr Ile Lys Val Asn Leu Ile Phe Ala Asp Gly Lys Thr 450 455 460 Gln Thr Ala Glu Phe Lys Gly Thr Phe Glu Glu Ala Thr Ala Glu Ala 465 470 475 480 Tyr Arg Tyr Ala Asp Leu Leu Ala Lys Val Asn Gly Glu Tyr Thr Ala 485 490 495 Asp Leu Glu Asp Gly Gly Tyr Thr Ile Asn Ile Lys Phe Ala Gly Lys 500 505 510 Glu Gln Pro Gly Glu Asn Pro Gly Ile Thr Ile Asp Glu Trp Leu Leu 515 520 525 Lys Asn Ala Lys Glu Glu Ala Ile Lys Glu Leu Lys Glu Ala Gly Ile 530 535 540 Thr Ser Asp Leu Tyr Phe Ser Leu Ile Asn Lys Ala Lys Thr Val Glu 545 550 555 560 Gly Val Glu Ala Leu Lys Asn Glu Ile Leu Lys Ala His Ala Gly Glu 565 570 575 Glu Thr Pro Glu Leu Lys Asp Gly Tyr Ala Thr Tyr Glu Glu Ala Glu 580 585 590 Ala Ala Ala Lys Glu Ala Leu Lys Asn Asp Asp Val Asn Asn Ala Tyr 595 600 605 Glu Ile Val Gln Gly Ala Asp Gly Arg Tyr Tyr Tyr Val Leu Lys Ile 610 615 620 Glu Val Ala Asp Glu Glu Glu Pro Gly Glu Asp Thr Pro Glu Val Gln 625 630 635 640 Glu Gly Tyr Ala Thr Tyr Glu Glu Ala Glu Ala Ala Ala Lys Glu Ala 645 650 655 Leu Lys Glu Asp Lys Val Asn Asn Ala Tyr Glu Val Val Gln Gly Ala 660 665 670 Asp Gly Arg Tyr Tyr Tyr Val Leu Lys Ile Glu Asp Lys Glu Asp Glu 675 680 685 Gln Pro Gly Glu Glu Pro Gly Glu Asn Pro Gly Ile Thr Ile Asp Glu 690 695 700 Trp Leu Leu Lys Asn Ala Lys Glu Asp Ala Ile Lys Glu Leu Lys Glu 705 710 715 720 Ala Gly Ile Ser Ser Asp Ile Tyr Phe Asp Ala Ile Asn Lys Ala Lys 725 730 735 Thr Val Glu Gly Val Glu Ala Leu Lys Asn Glu Ile Leu Lys Ala His 740 745 750 Ala Glu Lys Pro Gly Glu Asn Pro Gly Ile Thr Ile Asp Glu Trp Leu 755 760 765 Leu Lys Asn Ala Lys Glu Ala Ala Ile Lys Glu Leu Lys Glu Ala Gly 770 775 780 Ile Thr Ala Glu Tyr Leu Phe Asn Leu Ile Asn Lys Ala Lys Thr Val 785 790 795 800 Glu Gly Val Glu Ser Leu Lys Asn Glu Ile Leu Lys Ala His Ala Glu 805 810 815 Lys Pro Gly Glu Asn Pro Gly Ile Thr Ile Asp Glu Trp Leu Leu Lys 820 825 830 Asn Ala Lys Glu Asp Ala Ile Lys Glu Leu Lys Glu Ala Gly Ile Thr 835 840 845 Ser Asp Ile Tyr Phe Asp Ala Ile Asn Lys Ala Lys Thr Ile Glu Gly 850 855 860 Val Glu Ala Leu Lys Asn Glu Ile Leu Lys Ala His Lys Lys Asp Glu 865 870 875 880 Glu Pro Gly Lys Lys Pro Gly Glu Asp Lys Lys Pro Glu Asp Lys Lys 885 890 895 Pro Gly Glu Asp Lys Lys Pro Glu Asp Lys Lys Pro Gly Asp Lys Lys 900 905 910 Pro Glu Asp Lys Lys Pro Gly Lys Thr Asp Lys Asp Ser Pro Asn Lys 915 920 925 Lys Lys Lys Ala Lys Leu Pro Lys Ala Gly 930 935

Claims

1. A method for producing an output mixture of two or more different antibodies, said different antibodies differing in their amino acid sequences, said differences enabling separation of said antibodies by chromatography, wherein... - The two or more different antibodies are present in the output mixture at a desired or predetermined concentration ratio; and - The method includes the following steps: a. Provide an input mixture, wherein the two or more different antibodies are not present at the desired or predetermined concentration ratio; b. Separation of the two or more antibodies by means of chromatography based on a mechanism of action selected from the group consisting of: adsorption chromatography, ion exchange chromatography, size exclusion chromatography, affinity chromatography, hydrophobic interaction chromatography, metal chelate chromatography, normal-phase chromatography, reversed-phase chromatography, or mixed-mode chromatography, and optionally... i. Adjust the conditions of the chromatography such that, under these conditions, the total binding capacity of a given antibody is sufficient to retain an amount of the antibody required to produce the output mixture, or ii. Adjust the chromatography conditions such that the total binding capacity of a given antibody under these conditions is sufficient to retain an amount of each antibody exceeding the amount required to produce the output mixture. The chromatography produces an eluent and a flow product; and c. Recover the two or more antibodies in the amount required to provide the output mixture, wherein i) Recover the two or more antibodies in the same merge or fraction as follows: i. Collect the eluent and discard the flow-through after adjusting the chromatography conditions such that the total binding capacity of a given antibody under these conditions is sufficient to retain the amount of antibody required to provide the output mixture; or ii. Discard the eluent and collect the flow-through after adjusting the chromatography conditions such that the total binding capacity of a given antibody under these conditions is sufficient to retain more of each antibody than is required to provide the output mixture; or ii) Recovering the two or more antibodies from multiple mergings or fractions, and combining the multiple mergings or fractions or portions thereof; Thus, the output mixture is obtained; wherein the differences in the amino acid sequences of the two or more different antibodies result in: (i) differences in the charge properties of the two or more antibodies, such that the two or more antibodies interact differently with the chromatographic resin; (ii) differences in the hydrophobic properties of the two or more antibodies, such that the two or more antibodies interact differently with the chromatographic resin; or (iii) differences in affinity for the chromatographic resin. The differences in the amino acid sequences mentioned above are selected from: 1) Modification comprising substitution of one or more amino acids in one or more heavy chain variable regions and / or light chain variable regions of the antibody, wherein the substitution is at a position selected from the group consisting of: 1, 6, 17, 24, 48, 75, 90, 93, 96, 97 in the heavy chain variable region, and / or at a position selected from the group consisting of: 1, 4, 47, 48, 51, 68, 74, 80, 90, 93, and 95 in the light chain variable region, wherein the numbering is based on the IMGT number of the IgG1 variable region, wherein the one or more substitutions introduce an amino acid having a different charge than the wild-type amino acid at the corresponding position; 2) At least one amino acid substitution in the constant region of one or more kappa light chains in the antibody, wherein the substitution eliminates binding to the affinity resin and wherein the substitution is selected from the group consisting of: V110D, V110R, V110E, V110H, V110K, V110N, V110P, V110Q, V110W and E143D, using the EU numbering system, and wherein the chromatography uses an affinity resin for which the substitution eliminates binding; 3) At least one amino acid substitution, which is an S12P substitution in the light chain variable region when numbered using IMGT, wherein the substitution eliminates binding to the affinity resin, and wherein the chromatography uses an affinity resin for which the substitution eliminates binding.

2. The method of claim 1, wherein the output mixture is a pharmaceutical substance.

3. The method of claim 1, further comprising processing the output mixture to produce a pharmaceutical substance, wherein the two or more different antibodies are present at the concentration ratio specified in claim 1.

4. The method of claim 1, further comprising processing the output mixture to produce a pharmaceutical product, wherein the two or more different antibodies are present at the concentration ratio specified in claim 1.

5. The method of claim 1, wherein the output mixture is processed to produce a pharmaceutical substance or pharmaceutical product without any other means or measures for altering or substantially changing the ratio between the concentrations of the two or more antibodies, wherein the ratio between the relative amounts of the two or more antibodies and the concentrations of the two or more antibodies conforms to applicable pharmaceutical product specifications.

6. The method of claim 1, wherein each of the two or more different antibodies is present in a therapeutically effective amount.

7. The method of claim 1, wherein the least abundant of the two or more different antibodies is present in an amount of at least 1% (w / w), 2% (w / w), 3% (w / w), 4% (w / w), 5% (w / w), 6% (w / w), 7% (w / w), 8% (w / w), 9% (w / w), or 10% (w / w) of the most abundant of the two or more different antibodies.

8. The method of claim 1, wherein the two or more antibodies are present in amounts such that the ratio (w / w) between the amounts of any two antibodies is between 1:5 and 5:1, or between 1:4 and 5:1, 1:3 and 5:1, 1:2 and 5:1, 1:1 and 5:1, 2:1 and 5:1, 3:1 and 5:1, 3:4 and 5:1, 1:5 and 4:1, 1:5 and 3:1, 1:5 and 2:1, 1:5 and 1:1, 1:5 and 1:2, 1:5 and 1:3, 1:5 and 1:4, 1:4 Between 4:1, 1:4 and 3:1, 1:4 and 2:1, 1:4 and 1:1, 1:4 and 1:2, 1:4 and 1:3, 1:3 and 4:1, 1:3 and 3:1, 1:3 and 2:1, 1:3 and 1:1, 1:3 and 1:2, 1:2 and 4:1, 1:2 and 3:1, 1:2 and 2:1, 1:2 and 1:1, 1:1 and 4:1, 1:1 and 3:1, or between 1:1 and 2:

1.

9. The method of claim 1, wherein each of the two or more different antibodies is an active pharmaceutical ingredient.

10. The method of claim 1, comprising 2-10 different antibodies.

11. The method of claim 1, wherein at least one of the two or more antibodies is an antibody that binds to an antigen expressed on the surface of a tumor.

12. The method of claim 11, wherein the tumor is a metastatic solid tumor, a metastatic locally advanced tumor, or a hematologic malignancy.

13. The method of claim 1, wherein at least one of the two or more antibodies is an antibody that binds to an antigen, said antigen being associated with or expressed during an immune or autoimmune disease, inflammatory disease, cardiovascular disease, disease of the central nervous system (CNS), or musculoskeletal disease.

14. The method of claim 1, wherein the difference in the amino acid sequences of the two or more different antibodies results in a difference in the charge properties of the two or more antibodies, such that the two or more antibodies interact differently with the chromatography resin.

15. The method of claim 1, wherein the difference in the amino acid sequences of the two or more different antibodies results in a difference in the hydrophobic properties of the two or more antibodies, such that the two or more antibodies interact differently with the chromatography resin.

16. The method of claim 1, wherein the difference in the amino acid sequences of the two or more different antibodies results in a difference in affinity for the chromatography resin.

17. The method of claim 1, wherein the chromatography resin is selected from the group consisting of: affinity resins, ion exchange resins, hydrophobically interacting resins, or mixed-mode resins.

18. The method of claim 1, wherein the method comprises separating the two or more antibodies and reducing excess of one or more of the antibodies to recover a predetermined ratio of the two or more different antibodies.

19. The method of claim 1, wherein in step c, the two or more different antibodies of the output mixture are recovered in a single merge.

20. The method of claim 1, wherein the two or more antibodies in step b are separated into different fractions, and wherein the fractions containing one of the antibodies at a purity of at least 80% are subsequently combined at predetermined concentration ratios of the different antibodies to recover the output mixture.

21. The method of claim 1, comprising: i) In step (b), the two or more antibodies are separated into different fractions, and for each antibody, one or more fractions containing the antibody at a purity of at least 80% are selected; and ii) Provide the output mixture by combining the volumes of selected fractions, adjusting the volume to provide the two or more antibodies at the predetermined concentration ratio.

22. The method of claim 1, further comprising the step of determining the concentration of the antibody in each fraction prior to merging the antibodies.

23. The method of claim 1, wherein the separation of the two or more antibodies is performed by a single chromatography step using a single chromatography resin.

24. The method of claim 23, wherein the single chromatographic resin is a preparative chromatographic resin.

25. The method of claim 1, wherein the separation of the two or more antibodies is carried out by using a mixture of chromatographic resins in a predetermined ratio.

26. The method of claim 1, wherein the composition of the input mixture is measured using an analytical assay prior to step b.

27. The method of claim 1, wherein the composition of the input mixture is measured by an analytical determination in series with the chromatography step in step b.

28. The method of claim 1, wherein the method comprises an initial step of determining the separability of the two or more antibodies by chromatography, and, in the case that the different antibodies are not separable, modifying one or more amino acid sequences of the antibodies to obtain separability by chromatography.

29. The method of claim 28, wherein the modification of the amino acid sequence of one or more of the antibodies is selected from: substitution, addition or deletion of one or more amino acids in the antibodies or combinations thereof.

30. The method of claim 28, wherein the modification comprises modification in one or more constant domains of the antibody.

31. The method of claim 28, wherein the modification comprises modification in one or more variable domains of the antibody.

32. The method of claim 28, wherein the modification comprises modifications to the framework sequence of the light chain variable region and / or the framework sequence of the heavy chain variable region.

33. The method of claim 28, wherein the modification comprises substitution of one or more amino acids of one or more of the different antibodies.

34. The method of claim 28, wherein the modification does not alter the functional characteristics of one or more modified antibodies.

35. The method of claim 34, wherein the unchanged functional feature is selected from the group consisting of antibody binding affinity, effector function, affinity, and clustering.

36. The method of claim 29, wherein the modification comprises one or more amino acid substitutions in one or more heavy chain variable regions and / or light chain variable regions of the antibody, wherein the substitutions are at positions selected from the group consisting of: The numbers 1, 6, 17, 24, 48, 75, 90, 93, 96, 97 in the heavy chain variable region and / or 1, 4, 47, 48, 51, 68, 74, 80, 90, 93, and 95 in the light chain variable region are based on the IMGT number of the IgG1 variable region.

37. The method of claim 36, wherein the one or more substitutions introduce amino acids having a different charge than the wild-type amino acid at the corresponding position.

38. The method of claim 29, wherein the one or more amino acid substitutions comprise E345K substitution in the heavy chain constant region using the EU numbering system.

39. The method of claim 28, wherein modifying the one or more antibodies comprises introducing at least one amino acid substitution in the constant region of the kappa light chain of one or more of the antibodies, wherein the substitution eliminates binding to the affinity resin, and wherein the substitution is selected from the group consisting of: The EU numbering system is used for V110D, V110R, V110E, V110H, V110K, V110N, V110P, V110Q, V110W and E143D, and the chromatography uses the affinity resin targeted by the substitution elimination binding.

40. The method of claim 39, wherein the affinity agent targeted by the eliminated binding is a resin that binds the light chain of kappa.

41. The method of claim 40, wherein the resin is KappaSelect or KappaXL resin.

42. The method of claim 28, wherein modifying the one or more antibodies comprises introducing at least one amino acid substitution, said amino acid substitution being an S12P substitution in the light chain variable region when numbered using IMGT, wherein said substitution eliminates binding to the affinity resin, and wherein said chromatography uses the affinity resin targeted by the binding eliminated by said substitution.

43. The method of claim 42, wherein the affinity resin is a protein L resin.

44. The method of claim 1, wherein the affinity resin is a protein G resin.

45. The method of claim 1, wherein when determined by one or more chromatographic assays selected from the group consisting of hydrophobic interaction chromatography, cation exchange chromatography, and / or mixed-mode chromatography, a resolution (Rs) of Rs ≥ 0.3 is determined; the assay is performed using an ionic intensity gradient with Rs ≥ 0.3 according to the formula Rs = 2(t2 - t1) / (W1 + W2), where t1 = retention time of a given antibody, t2 = retention time of sequentially eluted antibodies, and W1 and W2 are the corresponding peak widths of antibodies at the base of the peaks obtained by extrapolating the relatively straight sides of the main peak to the baseline.

46. ​​The method of claim 1, wherein baseline separation is achieved between the unbound fraction in the unbound column and the antibody in the fraction eluted from said column, or when determined as in an affinity chromatography assay using a pH gradient with a resolution (Rs) of Rs ≥ 0.3, as determined according to the formula Rs = 2(t2 - t1) / (W1 + W2), where t1 = retention time of a given antibody, t2 = retention time of sequentially eluted antibody, and W1 and W2 are the corresponding peak widths of the antibody at the base of the peak obtained by extrapolating the relatively straight sides of the main peak to the baseline, then, as determined in the affinity chromatography assay, two or more antibodies are determined to be separable.

47. The method of claim 1, wherein the two or more different antibodies are expressed and provided from the different production host cells.

48. The method of claim 1, wherein the two or more different antibodies are expressed and provided from the different production host cells in different production host cells co-cultured in a single container.

49. The method of claim 1, wherein the two or more different antibodies are co-expressed in a single production host cell.

50. The method of claim 1, wherein the method results in reproducible results between different batches of the output mixture, such that the two or more different antibodies are present at a desired or predetermined concentration ratio.

51. The method of claim 1, wherein the two or more different antibodies are selected from the group consisting of antibodies comprising IgG1, IgG2, IgG3 or IgG4 or combinations thereof.

52. The method of claim 1, wherein the two or more different antibodies are full-length antibodies.

53. The method of claim 1, wherein the two or more different antibodies are humanized antibodies, chimeric antibodies, human antibodies, or combinations thereof.

54. The method of claim 1, wherein the two or more different antibodies are all humanized antibodies.

55. The method of claim 1, wherein the two or more different antibodies are all human antibodies.

56. The method of claim 1, wherein at least one of the two or more different antibodies is a monoclonal antibody.

57. The method of claim 1, wherein all of the two or more different antibodies are monoclonal antibodies.

58. The method of claim 1, wherein the method is used to produce antibody batches for the preparation of a medicament for treating a disease, for clinical trials, for toxicological studies, or for determining batch-to-batch consistency.

59. The method of claim 1, wherein at least one of the two or more different antibodies is specific to the target on tumor cells.

60. The method of claim 59, wherein the target is selected from the group consisting of: erbB1 (EGFR), erbB2 (HER2), erbB3, erbB4, MUC-1, CD19, CD20, CD4, CD38, CD138, CXCR5, c-Met, HERV-enveloping protein, peristin, Big3, SPARC, BCR, CD79, CD37, EGFrvIII, U-CAM, AXL, tissue factor (TF), CD74, EpCAM, and MRP3.

61. The method according to any one of the preceding claims, wherein at least one of the two or more different antibodies is specific to the target on the effector cells.

62. The method of claim 61, wherein the target is CD1, CD3, CD4, CD8, FcgammaRIII (CDI6), CD25, CD89, CD32, CD32a, FCεRI, CD40, or FcgammaRI (CD64).

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