NZ728700B2 - Purification platform for bispecific antibodies - Google Patents

Abstract

High resolution protein A chromatography employing a chaotropic agent and pH gradient or pH step elution buffer results in improved peak resolution between closely related molecular species. Bispecific antibodies containing a protein A-binding-ablating substitution CH3 domain paired with a protein A-binding CH3 domain are separated with high peak resolution from monospecific antibodies containing a protein A-binding-ablating substituted CH3 domain paired with the protein A-binding-ablating substituted CH3 domain and monospecific antibodies containing a protein A-binding CH3 domain paired with the protein A-binding CH3 domain. Useful chaotropic agents include magnesium chloride and calcium chloride.

Description

PURIFICATION RM FOR BISPECIFIC DIES FIELD A method for purifying a speci?c multimeric protein from a complex mixture of proteins via y chromatography is ed. Speci?cally a method for isolating a heterodimer ding bispeci?c antibody) from a complex mixture of monomers and homodimers via af?nity chromatography (including protein A chromatography) using a chaotropic agent is provided.

BACKGROUND Multiple bispeci?c antibody formats have been proposed and are currently under development. One such format is based upon a standard fully human IgG antibody having an improved pharmacokinetic pro?le and minimal immunogenicity (see US Patent No. 8,586,713, which is incorporated herein in its entirety). A single common light chain and two distinct heavy chains combine to form the bispeci?c. One of the heavy chains contains a substituted Fc sequence (hereinafter "Fc*") that reduces or eliminates binding of the Fc* to n A. For e, one such Fc* sequence contains H435R/Y43 6F (by EU numbering system; H95R/Y96F by IMGT exon numbering system) substitutions in the CH3 domain. As a result of co-expression of the two heavy chains and the common light chain, three products are created: two of which are homodimeric for the heavy chains and one of which is the desired heterodimeric bispeci?c product. The Fc* sequence allows selective puri?cation of the Fch* bispeci?c product on commercially available y columns, due to ediate binding af?nity for Protein A compared to the high avidity Fch heavy chain homodimer, or the weakly binding Fc*Fc* homodimer.

To achieve commercial scale puri?cation of the bispeci?c heterodimer, good resolution between the Fch homodimer, the Fc*Fc heterodimer, and the Fc*Fc* homodimer is required. Here, Applicants describe an improved tion process that optimizes resolution of these three molecular forms.

SUMMARY In one or more aspects and embodiments thereof, the invention is directed to methods of purifying a dimeric n, such as for e a bispec?c antibody, from a complex mixture of proteins that include homodimers and dimers, by employing an af?nity capture and elution process. In one aspect, the invention is directed to the puri?ed heterodimeric protein produced by any one of these methods.

In a ?rst aspect, the invention is ed to a method of making a protein comprising the steps of loading a mixture of multimeric ns onto an af?nity matrix, and then eluting and collecting a heterodimeric protein from that matrix at a particular pH range and in a buffer containing a opic agent. In one embodiment, the af?nity matrix is lly loaded with 5 to 50 grams of protein per liter of af?nity matrix. In some cases, the mixture of multimeric proteins is produced by a plurality of eukaryotic cells, such as for example Chinese hamster ovary (CHO) cells in a cell culture.

In one embodiment, the e of multimeric proteins contains (i) a ?rst homodimer comprising two copies of a ?rst polypeptide, (ii) a heterodimer comprising the ?rst polypeptide and a second polypeptide, and optionally (iii) a second homodimer comprising two copies of the second polypeptide. Here, the ?rst and second polypeptides have different af?nities for the af?nity matrix, such that the ?rst homodimer, the heterodimer and the second homodimer can be separated on the basis of differential binding to the af?nity matrix.

Differential binding to an affinity matrix can be manipulated by changing inter alia the pH and/or ionic strength of a on passed over the affinity matrix. The addition of the opic agent to the solution enhances the elution each dimer species from the affinity matrix thereby sing the purity of each individual dimer species. In one embodiment, the dimer is eluted from the affinity matrix in a buffer having a first pH range, and the first homodimer is eluted from the af?nity matrix in a buffer having a second pH range. The heterodimer is collected. In an optional embodiment, in which the second homodimer is included in the e of multimers, the second homodimer either flows through the column t binding or is eluted from the af?nity matrix in a wash buffer having a third pH range. The third pH range comprises a higher pH than the ?rst pH range, which comprises a higher pH than the second pH range.

In one embodiment, the affinity matrix comprises a n A ligand affixed to a substrate. In some cases, the substrate is a bead or particle, such that the affinity matrix is a plurality of particles d with Protein A. The Protein A may be a naturally occurring or modified Staphylococcal Protein A, or it may be an engineered Protein A. Engineered Protein A may be for example a Z-domain tetramer, a Y-domain tetramer, or an engineered Protein A that lacks D and E domains. These engineered Protein A exemplars are unable to bind (or bind with very low affinity if at all) to the VH3 domain of an immunoglobulin, but can still bind to the CH3 domains of IgG1, IgG2 and IgG4.

In some cases, the affinity matrix substrate contains or is made of agarose, poly(styrene divinylbenzene), polymethacrylate, controlled pore glass, spherical silica, cellulose and the like. In the embodiments in which the substrate is shaped as a bead or particle, the mean er of the particles is from 25 pm to 100 pm. In some embodiments, the particles have a mean diameter of 35 um, 45 um, 60 um, 75 pm, or 85 pm. In a particular embodiment, the particles have a mean diameter of 45 um and contain pores having a mean diameter of 1100 A.

In some embodiments, after the initial loading of the af?nity matrix with the mixture of proteins, the matrix is washed with a buffer having a pH that is greater than pH 5. In some cases the buffer comprises 20 mM sodium phosphate at pH 7.2. When the second homodimer is included in the e of proteins, the second homodimer is washed from the af?nity matrix in the wash buffer. Thus, here the wash buffer is of the third pH range.

In some ments, a buffered pH gradient is applied to the loaded af?nity matrix, or in the alternative, tial elution buffers, each having a different pHs are applied to the loaded af?nity matrix. In one embodiment, the pH gradient is 11111 from pH 6 to pH 3. The ?rst pH range, within which the heterodimer is eluted from the af?nity matrix, is about pH .5 to about pH 3.6. In some cases, the elution buffer and/or buffered pH gradient contains a suitable buffer, such as citrate, acetate, 4-Morpholineethanesulfonate (MES), citrate- phosphate, succinate, and the like, which in one embodiment is 40 mM acetate, and a chaotropic agent. The chaotropic agent can be a salt, having a cation selected from lithium, ium, calcium, and guanidinium, and an anion selected from de, nitrate, bromide, te, iodide, perchlorate, and thiocyanate. In one particular embodiment, the opic agent is CaClz, for example 250 - 500 mM CaClz. In r particular embodiment, the chaotropic agent is MgClz, for e 250 - 500 mM MgC12.

In one embodiment, the heterodimer is a bispeci?c antibody. Here, the ?rst polypeptide comprises a CH3 domain that is capable of binding to Protein A ("PC") and the second polypeptide comprises a CH3 domain that is not capable of binding to Protein A ("Fc*"). In some cases, the second polypeptide comprises a H43 5R/Y436F (by EU numbering system; 96F by IMGT exon numbering system) substitution in its CH3 domain (a.k.a "Fc*" or "star substitution"). Thus, in some embodiments, the ?rst homodimer is a monospeci?c antibody having two unsubstituted CH3 domains (i.e., Fch); the second homodimer is a monospeci?c dy having two H435R/Y436F tuted CH3 domains (i.e., Fc*Fc*); and the heterodimer is a bispeci?c antibody having one unsubstituted CH3 domain and one H435R/Y436F tuted CH3 domain (i.e., Fc*Fc).

In one embodiment, the heterodimer that is collected from the af?nity matrix is subsequently loaded onto a chromatography medium at a more acidic pH, and eluted from that medium in a more alkaline buffer which lacks the chaotropic agent (or has a lower amount or trace amount of chaotrope). In one case, the chromatography medium is a multimodal chromatography resin. The heterodimer may be further puri?ed.

] In a second aspect, the second homodimer is ?rst removed from the mixture of proteins by applying the mixture to a ?rst af?nity matrix such that the ?rst homodimer and the heterodimer remain bound to the matrix while the second homodimer ?ows through and is discarded. The ?rst homodimer and the heterodimer are subsequently eluted from the ?rst af?nity matrix and then subsequently applied to a second af?nity . In one embodiment, the ?rst af?nity matrix is initially loaded with 5 to 50 grams of protein per liter of af?nity matrix. The mixture of multimeric proteins is produced in some cases by a plurality of eukaryotic cells, such as for example Chinese hamster ovary (CHO) cells in a cell culture.

In one ment, the e of eric proteins applied to the ?rst af?nity matrix contains (i) a ?rst homodimer comprising two copies of a ?rst polypeptide, (ii) a dimer comprising the ?rst polypeptide and a second polypeptide, and (iii) a second homodimer comprising two copies of the second polypeptide. Here, the ?rst and second polypeptides have different af?nities for the ?rst af?nity matrix as well as for the second af?nity matrix, such that the ?rst homodimer, the heterodimer and the second mer can be separated on the basis of differential binding to the first and/or second af?nity matrix.

In one embodiment, the first af?nity matrix comprises an ered Protein A ligand, which lacks the y to bind the VH3 domain of an immunoglobulin, af?xed to a substrate. In some cases, the protein A lacks a D—domain and an E-domain, such as the engineered proteins A that comprise a Z-tetramer or a Y—tetramer. ential binding of the ?rst homodimer and the heterodimer to the second af?nity matrix can be manipulated by changing inter diet the pH and/or ionic strength of a solution that is passed over the af?nity matrix. The addition of a chaotropic agent to the solution enhances the elution each dimer species from the second af?nity matrix in erlapping fractions, thereby increasing to purity of each dimer species. In one embodiment, the heterodimer is eluted from the second af?nity matrix in a buffer haVing a ?rst pH range, and the ?rst homodimer is eluted from the second af?nity matrix in a buffer haVing a second pH range. The heterodimer is collected. Here, the ?rst pH range comprises a higher pH than does the second pH range.

In one embodiment, the second af?nity matrix comprises a Protein A ligand af?xed to a ate. In some cases, the substrate is a bead or particle, such that the second af?nity matrix is a plurality of particles af?xed with Protein A ligand. The Protein A may be a naturally occurring or modi?ed Staphylococcal Protein A, or it may be an engineered n A. Engineered protein A may be for example a Z-domain er, a Y-domain tetramer, or another engineered Protein A that lacks D and E s. These engineered n A molecules are unable to bind (or bind with very low af?nity if at all) to the VH3 domain of an immunoglobulin, but remain able to bind to the CH3 domains of IgG1, IgG2 and IgG4.

In some cases, the substrate contains or is made of agarose, poly(styrene divinylbenzene), polymethacrylate, controlled pore glass, spherical silica, cellulose (e.g., HYPERCEL) the like. In those embodiments in which the substrate is shaped as a bead or particle, the mean diameter of the particles is from 30 pm to 90 pm. In some ments, the particles have a mean diameter of 35 um, 45 um, 60 um, 75 pm, or 85 pm. In a particular embodiment, the particles have a mean diameter of 45 um and contain pores having a mean diameter of 1100 A.

In some embodiments, after the initial loading of the second affinity matrix with the mixture containing the ?rst homodimer and the dimer, the matrix is washed with a buffer having a pH that is greater than pH 5. In some cases the buffer comprises 20 mM sodium phosphate at pH 5 — 8.5, for example pH 7.2. In some embodiments, a ed pH nt is applied to the loaded affinity matrix, or in the alternative, sequential elution buffers, each having a different pHs are applied to the loaded second affinity matrix. In one embodiment, the pH gradient is run from pH 6 to pH 2.5. The first pH range, within which the heterodimer is eluted from the affinity matrix, is about pH 5.5 to about pH 3.6. In some cases, the elution buffer and/or buffered pH nt contains acetate, which in one ment is 40 mM acetate, and a chaotropic agent. The chaotropic agent can be a salt, having a cation selected from lithium, magnesium, calcium, and guanidinium, and an anion selected from chloride, nitrate, bromide, chlorate, iodide, perchlorate, and thiocyanate. In one particular embodiment, the chaotropic agent is CaClz, for example 500 mM CaClz. In another particular embodiment, the chaotropic agent is MgClz, for example 500 mM MgC12.

In one embodiment, the heterodimer is a bispecif1c antibody. Here, the first ptide ses a CH3 domain that is capable of binding to Protein A ("PC") and the second polypeptide comprises a CH3 domain that is not capable of binding to Protein A ). In some cases, the second polypeptide comprises a H43 5R/Y43 6F (a.k.a. "star") substitution in its CH3 domain ("Fc*"). Thus, in some embodiments, the ?rst homodimer is a monospeci?c antibody having two unsubstituted CH3 domains (i.e., Fch); the second mer is a monospeci?c antibody having two H435R/Y436F tuted CH3 s (i.e., ); and the heterodimer is a bispeci?c antibody having one unsubstituted CH3 domain and one Y436F substituted CH3 domain (i.e., Fc*Fc).

In one embodiment, heterodimer that is collected from the second af?nity matrix is subsequently loaded onto a chromatography medium at an acidic pH, and eluted from that medium in a more alkaline buffer and without the chaotropic agent (or reduced levels or trace amounts of chaotrope). In one case, the chromatography medium is a multimodal chromatography resin. The dimer may be further puri?ed.

In a third aspect, the invention is directed to a puri?ed heterodimer made according to the methods of the aspects described above. In one embodiment, the heterodimer is a bispeci?c antibody.

DESCRIPTION OF THE DRAWINGS Figure 1 depicts chromatograms rating pH (dotted) and absorbance at 280 nm (solid) during elution step for puri?cation of bsAb E with a recombinant Protein A resin LECT XTRATM, panel A), and an engineering Protein A-based resin which lacks VH binding (MABSELECT SURETM, panel B).

Figure 2 depicts the peak resolutions (Rs) obtained between the bispeci?c (Fc*Fc) and non—CH3—substituted homodimer (Fch) peaks as a function of residence time during a column volume ("CV") gradient elution in 40 mM acetate, 500 mM calcium chloride with either MABSELECT SURETM (open circles), or POROS MABCAPTURE ATM (closed squares) as the stationary phase.

Figure 3 depicts chromatograms illustrating pH (dotted) and absorbance at 280 nm (solid) during elution step for puri?cation of bsAb A with either sodium citrate (panel A), sodium chloride (panel B), magnesium chloride (panel C), or calcium chloride (panel D) added as modi?ers to the n mobile phase. Bispeci?c peak fractionation is marked by vertical dotted lines.

Figure 4 depicts the ?ow scheme of puri?cation processes for star substitution- containing (CH3—substituted, Fc*) bispeci?c antibodies (Fc*Fc) exhibiting (panel A) and not ting (panel B) VH domain SpA binding.

DETAILED DESCRIPTION This ion is not d to particular methods and experimental ions described, as such methods and conditions may vary. It is also to be understood that the ology used herein is for the purpose of describing ular embodiments only, and is not intended to be limiting, since the scope of the present invention is de?ned by the claims.

Unless de?ned otherwise, all technical and scienti?c terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention s. Although any methods and materials similar or equivalent to those described herein can be used in the practice or g of the present invention, particular methods and materials are now described. All publications mentioned are hereby incorporated by reference.

] The term "antibody", as used herein, includes immunoglobulin molecules comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain ses a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region ses one domain, CL. The VH and VL regions can be further subdivided into regions of ariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino—terminus to carboxy—terminus in the following order: FRI, CDRl, FR2, CDR2, FR3, CDR3, FR4 (heavy chain CDRs may be abbreviated as HCDRl, HCDR2 and HCDR3; light chain CDRs may be abbreviated as LCDRl, LCDR2 and LCDR3. The term "high affinity" antibody refers to those antibodies having a binding affinity to their target of at least 10’9 M, at least 10’1 M; at least 10’11 M; or at least 10’12 M, as measured by surface plasmon resonance, e. g., BIACORETM or solution—affinity ELISA.

The phrase "bispecific antibody" includes an antibody e of selectively binding two or more epitopes. Bispecific antibodies generally comprise two ent heavy chains, with each heavy chain specifically binding a different epitope—either on two different molecules (e. g., ns) or on the same molecule (e. g., on the same antigen). If a ific antibody is capable of selectively binding two different es (a first epitope and a second epitope), the affinity of the first heavy chain for the first epitope will generally be at least one to two or three or four orders of magnitude lower than the affinity of the first heavy chain for the second epitope, and Vice versa. The epitopes ized by the bispecific dy can be on the same or a different target (e. g., on the same or a different protein).

Bispecific antibodies can be made, for example, by combining heavy chains that recognize different epitopes of the same antigen. For example, nucleic acid sequences encoding heavy chain variable sequences that recognize different epitopes of the same antigen can be fused to nucleic acid ces encoding different heavy chain constant regions, and such sequences can be expressed in a cell that expresses an immunoglobulin light chain. A typical bispecif1c antibody has two heavy chains each having three heavy chain CDRs, followed by (N—terminal to C-terminal) a CHl domain, a hinge, a CH2 domain, and a CH3 domain, and an immunoglobulin light chain that either does not confer antigen-binding specificity but that can associate with each heavy chain, or that can associate with each heavy chain and that can bind one or more of the epitopes bound by the heavy chain antigen—binding s, or that can associate with each heavy chain and enable binding or one or both of the heavy chains to one or both epitopes.

The phrase "heavy chain," or "immunoglobulin heavy chain" includes an immunoglobulin heavy chain constant region ce from any organism, and unless otherwise specified includes a heavy chain variable domain. Heavy chain variable domains e three heavy chain CDRs and four FR regions, unless otherwise specified. Fragments of heavy chains include CDRs, CDRs and FRs, and ations thereof A typical heavy chain has, following the variable domain (from N—terminal to inal), a CHl domain, a hinge, a CH2 domain, and a CH3 domain. A functional fragment of a heavy chain includes a fragment that is capable of specifically recognizing an antigen (e.g., recognizing the antigen with a KD in the olar, lar, or picomolar range), that is capable of expressing and secreting from a cell, and that comprises at least one CDR.

The phrase "light chain" includes an immunoglobulin light chain constant region sequence from any sm, and unless otherwise specified includes human kappa and lambda light chains. Light chain variable (VL) domains typically include three light chain CDRs and four framework (FR) regions, unless otherwise specified. Generally, a full-length light chain includes, from amino terminus to carboxyl us, a VL domain that includes FRl-CDRl-FRZ-CDRZ-FR3-CDR3 -FR4, and a light chain nt domain. Light chains that can be used with this invention include those, e. g., that do not selectively bind either the first or second antigen selectively bound by the antigen—binding protein. Suitable light chains include those that can be identified by screening for the most commonly ed light chains in existing antibody libraries (wet ies or in silico), where the light chains do not substantially interfere with the affinity and/or ivity of the antigen—binding domains of the antigen-binding proteins. Suitable light chains include those that can bind one or both es that are bound by the antigen—binding regions of the antigen-binding n.

The phrase "variable domain" includes an amino acid sequence of an immunoglobulin light or heavy chain ied as desired) that comprises the following amino acid regions, in sequence from N—terminal to C—terminal (unless otheiwise indicated): FRl, CDRl, FR2, CDR2, FR3, CDR3, FR4. A "variable domain" includes an amino acid sequence capable of folding into a canonical domain (VH or VL) having a dual beta sheet structure wherein the beta sheets are connected by a disulfide bond between a residue of a first beta sheet and a second beta sheet.

The phrase "complementarity determining region," or the term "CDR," includes an amino acid sequence encoded by a nucleic acid sequence of an organism's immunoglobulin genes that normally (i.e., in a wild-type animal) appears n two ork regions in a variable region of a light or a heavy chain of an immunoglobulin molecule (e. g., an antibody or a T cell receptor). A CDR can be encoded by, for example, a germline sequence or a rearranged or unrearranged sequence, and, for example, by a naive or a mature B cell or a T cell. In some circumstances (e.g., for a CDR3), CDRs can be encoded by two or more sequences (e. g., germline sequences) that are not contiguous (e. g., in an unrearranged c acid sequence) but are uous in a B cell nucleic acid sequence, e.g., as the result of splicing or connecting the sequences (e.g., V-D—J recombination to form a heavy chain CDR3).

The phrase "Fc—containing protein" includes antibodies, bispecific antibodies, immunoadhesins, and other binding proteins that comprise at least a functional portion of an immunoglobulin CH2 and CH3 region. A "functional portion" refers to a CH2 and CH3 region that can bind a PC receptor (e.g., an FcyR; or an FcRn, i.e., a neonatal Fc receptor), and/or that can participate in the activation of complement. If the CH2 and CH3 region contains deletions, substitutions, and/or insertions or other modi?cations that render it unable to bind any Fc or and also unable to activate complement, the CH2 and CH3 region is not functional.

Fc—containing proteins can comprise cations in immunoglobulin domains, including where the ations affect one or more or function of the binding protein (e.g., modifications that affect FcyR binding, FcRn binding and thus half-life, and/or CDC ty). Such modifications include, but are not limited to, the following modifications and combinations thereof, with reference to EU numbering of an immunoglobulin constant region: 238, 239, 248, 249, 250, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 297, 298, 301, 303, 305, 307, 308, 309, 311, 312, 315, 318, 320, 322, 324, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 337, 338, 339, 340, 342, 344, 356, 358, 359, 360, 361, 362, 373, 375, 376, 378, 380, 382, 383, 384, 386, 388, 389, 398, 414, 416, 419, 428, 430, 433, 434, 435, 437, 438, and For example, and not by way of limitation, the g protein is an Fc-containing protein and exhibits enhanced serum half-life (as compared with the same Fc-containing protein without the recited modi?cation(s)) and have a modi?cation at position 250 (e. g., E or Q); 250 and 428 (e.g., L or F); 252 (e.g., L/Y/F/W or T), 254 (e.g., S or T), and 256 (e.g., S/lVQ/E/D or T); or a ation at 428 and/or 433 (e.g., L/lUSI/P/Q or K) and/or 434 (e.g., H/F or Y); or a modi?cation at 250 and/or 428; or a modi?cation at 307 or 308 (e.g., 308F, , and 434. In r example, the modi?cation can comprise a 428L (e. g., M428L) and 434S (e.g., N434S) modi?cation; a 428L, 2591 (e.g., V2591), and a 308F (e.g., V308F) modi?cation; a 433K (e.g., H433K) and a 434 (e.g., 434Y) modi?cation; a 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modi?cation; a 250Q and 428L modi?cation (e.g., T250Q and M428L); a 307 and/or 308 modi?cation (e.g., 308F or 308P).

The term "star tution", "Fc*", and "HC*" includes any molecule, immunoglobulin heavy chain, Fc fragment, Fc—containing le and the like which contain a sequence within the CH3 domain that abrogates binding to Protein A. It has been previously noted (Lindhofer, H. et ai (1995) J. Immunol. 9—225)) that because human lgG3 does not to bind to Protein A, it can potentially be used together with any of the other three human IgG subclasses in a puri?cation strategy similar to the one used for mouse-rat hybrids. However, although the sequences of all four human IgG subclasses are highly homologous, it is not known how readily the Fc portions of IgG1 and lgG4 form , lgG2, heterodimers with lgG3; even merely preferential formation of homodimers would have a negative impact on total yields of the desired heterodimers under certain circumstances (e. g., isolation from quadromas). It has been reported (Jendeberg, L. et al. (1997) J.

Immunological Meth. 201 )) that the inability of lgG3 to bind Protein A is determined by a single amino acid residue, Arg435 (EU numbering; Arg95 by IMGT), which ponding position in the other IgG subclasses is occupied by a histidine residue. It is therefore possible, instead of lgG3, to use an IgGl sequence in which His435 is mutated to Arg. Thus, a single point mutation in IgGl should be suf?cient to create the different binding af?nities le to a new puri?cation scheme. This modi?cation will be referred to as IgGI AA, to denote its inability to bind Protein A (and, similarly, lgGZAA and lgG4AA — or more generally, FcAA).

However, the speci?ed point mutation introduces a novel peptide ce across the mutation, which could potentially be immunogenic. The point mutation could, in theory, be loaded onto an MHC class 11 molecule and presented to T cells, and consequently elicit an immune response. To avoid this l, a dipeptide mutation, H43 51VY43 6F (EU numbering; H95R/Y96F by IMGT) can be used. The resulting sequence in the Vicinity of the tion is identical to that of 1gG3, and would therefore be expected to be immunologically "invisible," because there would be no non-native short es available for presentation to T cells. It has been reported that this double mutant still does not bind Protein A berg, L. et al. (1997) J. Immunological Meth. 201 :25-34). Finally, the ide mutation does not include any of the residues that form the Fc dimer interface, so it is unlikely to interfere with the ion of heterodimers. This ide mutation is designated as the "star substitution".

The term "cell" includes any cell that is suitable for expressing a recombinant nucleic acid sequence. Cells include those of yotes and eukaryotes (single-cell or multiple- cell), bacterial cells (e.g., strains of E. coli, Bacillus spp., Streptomyces spp., etc.), mycobacteria cells, fungal cells, yeast cells (e.g., S. cerevisiae, S. pombe, P. pastoris, P. methanolica, etc.), plant cells, insect cells (e. g., SF—9, SF—21, baculovirus—infected insect cells, Trichoplusia ni, etc.), non—human animal cells, human cells, or cell fusions such as, for example, hybridomas or quadromas. In some embodiments, the cell is a human, monkey, ape, hamster, rat, or mouse cell. In some embodiments, the cell is eukaryotic and is selected from the following cells: CHO (e.g., CHO Kl, DXB—ll CHO, Veggie—CHO), COS (e. g., COS—7), retinal cell, Vero, CV1, kidney (e.g., HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK), HeLa, HepG2, W13 8, MRC 5, C010205, HB 8065, HL—60, (e.g., BHK21), Jurkat, Daudi, A431 (epidermal), CV-l, U937, 3T3, L cell, C127 cell, SP2/0, NS—0, MMT 060562, Sertoli cell, BRL 3A cell, HT1080 cell, myeloma cell, tumor cell, and a cell line derived from an aforementioned cell. In some embodiments, the cell comprises one or more Viral genes, e. g. a retinal cell that expresses a Viral gene (e.g., a TM cell).

The phrase e phase modi?er" includes es that reduce the effect of, or disrupt, non-specific (i.e., non-af?nity) ionic and other non—covalent interactions between proteins. e phase modifiers" include, for e, salts, ionic combinations of Group 1 and Group 11 metals with acetate, bicarbonate, carbonate, a halogen (e.g., chloride or ?uoride), nitrate, phosphate, or sulfate. A non—limiting illustrative list of "mobile phase ers" includes beryllium, lithium, sodium, and potassium salts of acetate; sodium and potassium bicarbonates; lithium, sodium, potassium, and cesium carbonates; lithium, sodium, potassium, cesium, and magnesium chlorides; sodium and potassium ?uorides; sodium, potassium, and calcium nitrates; sodium and potassium phosphates; and calcium and ium sulfates.

"Mobile phase modifiers" also include chaotropic agents, which weaken or otherwise interfere with non—covalent forces and increase entropy within biomolecular systems. Non— limiting es of chaotropic agents include butanol, calcium chloride, ethanol, guanidinium de, lithium perchlorate, m acetate, magnesium chloride, phenol, ol, sodium dodecyl sulfate, thiourea, and urea. Chaotropic agents include salts that affect the solubility of proteins. The more chaotropic anions include for example chloride, e, bromide, chlorate, iodide, perchlorate, and thiocyanate. The more chaotropic s include for example lithium, magnesium, calcium, and guanidinium.

"Mobile phase modi?ers" include those moieties that affect ionic or other non- covalent interactions that, upon addition to a pH nt or step, or upon equilibration of a Protein A support in a "mobile phase modi?er" and application of a pH step or gradient, results in a broadening of pH unit ce between elution of a homodimeric IgG and a heterodimeric IgG (e. g., a wild-type human IgG and the same IgG but bearing one or more modi?cations of its CH3 domain as described herein). A le concentration of a "mobile phase modi?er" can be determined by its concentration employing the same , pH step or gradient, with sing tration of "mobile phase modi?er" until a maximal pH distance is reached at a given pH step or pH gradient. "Mobile phase modifiers" may also include non-polar modi?ers, including for example propylene , ethylene glycol, and the like.

As used herein, "af?nity chromatography" is a chromatographic method that makes use of the speci?c, reversible interactions between biomolecules rather than l properties of the biomolecule such as isoelectric point, hydrophobicity, or size, to effect chromatographic separation. "Protein A af?nity tography" or "Protein A chromatography" refers to a speci?c af?nity chromatographic method that makes use of the af?nity of the IgG binding domains of Protein A for the Fc portion of an immunoglobulin molecule. This Fc portion comprises human or animal immunoglobulin constant domains CH2 and CH3 or immunoglobulin domains substantially similar to these. Protein A encompasses native protein from the cell wall of Staphylococcus aureus, Protein A produced by recombinant or synthetic methods, and variants that retain the ability to bind to an Fc . In practice, Protein A chromatography involves using Protein A immobilized to a solid support. See Gagnon, Protein A Af?nity Chromotography, Puri?cation Tools for Monoclonal Antibodies, pp. 155-198, ted Biosystems, 1996. n G and n L may also be used for af?nity chromotography. The solid support is a non-aqueous matrix onto which n A adheres. Such supports include agarose, sepharose, glass, silica, polystyrene, nitrocellulose, charcoal, sand, cellulose and any other suitable material. Such materials are well known in the art. Any suitable method can be used to affix the second n to the solid support. Methods for affixing proteins to suitable solid supports are well known in the art. See e. g. Ostrove, in Guide to Protein Purification, Methods in Enzymology, 182: 357—371, 1990. Such solid supports, with and t immobilized Protein A, are readily available from many commercial sources including such as Vector Laboratory (Burlingame, Calif), Santa Cruz Biotechnology (Santa Cruz, Calif), BioRad (Hercules, Calif), Amersham ences (part of GE Healthcare, Uppsala, Sweden), Pall (Port gton, NY) and EMD—Millipore rica, Mass.) Protein A immobilized to a pore glass matrix is commercially available as PROSEP®-A pore). The solid phase may also be an agarose-based matrix. Protein A lized on an agarose matrix is commercially available as MABSELECTTM (Amersham Biosciences).

Affinity chromatography also includes media that can be used to ively bind and thus purify antibodies, fragments of dies, or chimeric fusion proteins that contain immunoglobulin domains and/or sequences. Antibodies include IgG, IgA, IgM, IgY, IgD and IgE types. Antibodies also include single chain antibodies such as camelid antibodies, engineered camelid antibodies, single chain antibodies, single-domain antibodies, nanobodies, and the like. Antibody nts include VH, VL, CL, CH sequences.

Antibody fragments and fusion proteins containing antibody sequences include for example F(ab’)3, F(ab’)2, Fab, Fc, Fv, dst, (scFv)2, scFv, scAb, minibody, diabody, triabody, tetrabody, Fc-fusion ns, trap molecules, and the like (see Ayyar et al., Methods 56 (2012): 116-129). Such affinity tography media may contain ligands that selectively bind antibodies, their fragments, and fusion proteins contains those fragments. Such ligands e antibody binding proteins, bacterially derived receptors, antigens, lectins or anti- antibodies directed to the target molecule. the antibody requiring puri?cation. For example, camelid-derived af?nity ligands directed against any one or more of IgG-CHl, , IgG- CH3, IgGl, LC-kappa, LC-lambda, IgG3/4, IgA, IgM, and the like may be used as af?nity ligands (commercially available as CAPTURESELECT tography resins, Life logies, Inc., Carlsbad, Calif.) EXAMPLE 1: PEAK RESOLUTION PROCESS Bispeci?c antibodies were separated from the contaminating homodimers via Protein A tography utilizing the star substitution as follows. Since Fc*Fc* homodimer has both Protein A binding sites deleted from the Fc region, this product-related ty was expected to ?ow though the column and be removed while the i?c and Fch homodimer was expected to be retained on the column. A series of washes was applied to remove s—related contaminants such as CHO DNA or host cell n (HCP). The bispeci?c was then selectively eluted via a pH gradient or step while the Fch contaminant was retained due to its stronger binding relative to the bispeci?c.

Wide variability in separation performance from bispeci?c to i?c was observed when initial experiments were med using a recombinant Staphylococcal Protein A (SpA) chromatography resin. Whereas baseline resolution between the g Fch homodimers and the bispeci?c product was obtained in some cases, a subgroup of bispeci?c antibodies ("bsAbs") exhibited very poor resolution. For these molecules, good resolution was attainable when performing the separation on an af?nity resin engineered to exhibit improved base stability. One e of this is bsAb E, for which harvested clari?ed cell culture ?uid was loaded to 5 g/L on either a SpA resin (MABSELECT XTRATM, Figure 1A) or an engineered Protein A-based resin (MABSELECT SURETM, Figure 1B). After a series of (e.g., pH 6 — 8) buffer wash steps, a 40 column volume ("CV") gradient from pH 5 to 3 in 40mM acetate 500mM NaCl was applied to elute the bound species. On MABSELECT XTRATM (Figure 1A) an n peak between 25 — 35 CV contained both the bispecific and the Fch homodimer with no resolution. Furthermore, despite the lack of Protein A binding in the Fc region of the Fc*Fc* contaminant, a leading shallow elution peak (0 — 25 CV) consisted of the Fc*Fc* homodimer Protein. However, the same bsAb E load applied to ECT SURETM yielded two well resolved peaks ning the bispecific and Fch homodimer, respectively (Figure 1B), with all the Fc*Fc* homodimer ?owing through the column during the load. This may be caused by the different ctions between IgG and native Protein A ) and the engineered SuRe ligand.

In addition to the classical binding site, some antibodies have been shown to contain an alternative SpA binding site on the variable region of the heavy chain ("VH"). In particular, some IgGs that contain heavy chains from the human VH3 gene family have been shown to exhibit this behavior, with nearly half of human VH germline genes belonging to the VH3 subfamily. (See Sasso et al., Journal of Immunology 1989; 142:2778—83; Sasso et al., Journal of Immunology 1991; 147:1877—83; Schroeder et al., International immunology 1990; 2:41-50; and Walter, M.A., and D.W. Cox, American Journal of Human Genetics 1988; 42:446—51.) Therefore it appears likely that on sed resins, bispeciflc dies such as bsAb E t poor resolution of the two binding species and retention of the "non— binding" Fc*Fc* homodimer resins due to VH g. The VH binding is thus thought to reduce the y difference between the bispeci?c, and the Fch homodimer, and the low affinity binding of Fc*Fc*.

This hypothesis is supported by the improved purification observed with MABSELECT SURETM. Binding studies between SpA and antibodies have shown that while all five domains of SpA (E, D, A, B and C) bind IgG via the Fc-region, only domains D and E exhibit cant Fab binding. (See e.g., Starovasnik et al., Protein Science 1999; 8:1423-31). The engineered MABSELECT SURETM affinity ligand is a er of the Z- domain, a protein-engineered version of the native, non-Fab binding SpA B domain. The Z- domain is known to have negligible binding to the antibody variable region (Starovasnik, supra). Therefore when this resin carrying the MABSELECT SURETM ligand is used, the increased ence in avididty between the bispeci?c and the Fch homodimer allows for improved resolution of those peaks; and the Fc*Fc* mer is not retained.

Multiple n A-based chromatographic resins were screened in order to identify a resin for bispeci?c clinical and cial production. Two bsAbs were chosen for evaluation, one usly observed to bind SpA via the VH region (bsAb A) and one lacking this lity (bsAb B). The load material was previously subjected to standard positive mode affinity chromatography to remove the Fc*Fc* impurity. Starting bispecif1c purities (Fc*Fc/[Fc*Fc + Fc*Fc* + Fch]) were 84% and 76% for bsAb A and bsAb B respectively. All resins were loaded to 10 g total protein/L resin. After a series of washes the antibodies were eluted using a 30 CV gradient from pH 6 to 3 with either 500 mM NaCl or 500 mM CaClz as mobile phase modifiers.

The six selected commercially available Protein A resins exhibit a variety of base matrices, bead sizes and ligand types. Four of the resins used SpA, one was a tetramer of the Z-domain (MABSELECT SURETM), and one was a er of a base stabilized version of the non-Fab binding C domain, termed the Y-domain (TOYOPEARL AF-rProtein A-650F).

These data and the peak resolution (Rs = 1.18([tR2 — tR1]/[W1/22 + Wl/zl]; wherein Rs is peak resolution, Wl/z peak width at half height, and tR is retention time) obtained between the bispeciflc product and the binding Fch homodimer are detailed in Table l.

Table 1: ison of separation ef?ciency of bispeci?c from binding impurity obtained using a range of Protein A media with two antibodies: VH-Protein A binding (bsAb A) and non—VH binding (bsAb B). a Resolution calculated using width at half . If peak width at half height could not be calculated due to peak convergence, resolution marked as "no resolution".

Resolution eda (Rs) Average Particle n A Resin Base Matrix bsAb A bsAb A bsAb B Size Origin (modi?er: (modi?er: (modi?er: NaCl) Caclz) NaCl) Tetramer of z- MABSELECT domain Agarose SURE (modi?ed B domain) POROS Poly(styrene di— MABCAPTURE Recombinant Vinylbenzene) er of y- TOYOPEARL domain AF-rProtein A- Polymethacrylate (modi?ed C domain) ProSep Ultra Plus Controlled Pore No inant Af?nity Glass resolution Spherical Silica, te High modi?ed for high Recombinant 0.49 pH ance MabSelect Xtra 75 Agarose Recombinant N/M N/M resolution When using NaCl as a mobile phase modi?er in the elution buffer, increased resolution was noted in inverse proportion to bead size, with no resolution observed for SpA resins with a mean particle greater than 45 um. Interestingly MABSELECT SURETM (Rs: 0.92) showed comparable performance to TOYOPEARL AF-rProtein A-650F (Rs=0.87) with bsAb A. This was not expected due to (i) the smaller average bead size for TOYOPEARL AF-rProtein A-650F (45 cf. 85 um) and (ii) the similarity of the af?nity ligand, which is based on the Y-domain (derived from the C domain) and thus expected to lack VH binding (Starovasnik, supra). For bsAb B, POROS MABCAPTURE ATM exhibited superior resolution as compared to TOYOPEARL AF-rProtein A—650F and ABSOLUTE HICAPTM (2.50 ed to 2.37 and 2.41, respectively), despite not having the smaller particle size. This was hypothesized to be due to the element of perfusive ?ow in this base , facilitated by the large h pores, and mean pore diameter of 1100 Angstroms, aiding mass transfer. POROS MABCAPTURE ATM also exhibited better resolution of bsAb A than did any other SpA resin, with comparable tion to the non—VH binding resins of ARL AF—rProtein A—650F and MABSELECT SURETM (0.70 compared to 0.87 and 0.92 respectively).

When NaCl was replaced with CaClz as the mobile phase modi?er, POROS MABCAPTURE ATM was observed to greatly improve ing power, outperforming MABSELECT SURETM by a considerable margin (Rs of 1.83 c.f. 1.08, respectively). Based on the totality of this data, POROS MABCAPTURE ATM and ECT SURETM were evaluated further as possible resolving chromatographic resins for isocratic n.

Since the resin ison was performed at a relatively fast linear velocity of 400 cm/h, the MABSELECT SURE resin could have been reduced in efficacy relative to POROS MABCAPTURE A because of (i) the larger bead size and (ii) lack of perfusive flow. The resins were therefore compared at a production relevant range of residence times.

BsAb A was selected as the model molecule due to its observed binding to SpA via its VH region (thereby giving a greater avidity difference between the bispecific and Fch impurity to the non—VH binding MABSELECT SURE). Note a VH binding antibody was chosen for this evaluation, as without this avidity advantage MABSELECT SURE would be expected to be inferior due to the smaller bead size of MABCAPTURE A. The same ty captured bsAb A load material as used for the resin evaluation study was used, at a 10 g total n/L resin challenge. All chromatographic steps were performed at a 3 minute residence time with the exception of the elution, which was varied from 2—8 minutes (600—150 cm/h).

Calculation of the resolution of the i?c peak from the Fch mer peak showed that although the resolution of the resins increased with nce time, the effect was more pronounced for MABSELECT SURE than POROS MABCAPTURE A (Rs increase of 0.7 and 0.3, tively, Figure 2). onally, the POROS MABCAPTURE A resin showed superior resolution to MABSELECT SURE at all tested conditions, despite the disadvantage of VH binding for this resin, con?rming the overall superiority of the resin with regards to resolving power of the two binding species.

The inclusion of mobile phase modi?ers in the elution buffer altered and potentially improved the resolution of the bispeci?c product from the Fch homodimer (see Table 1).

Thus, the use of salts of varying position on the Hofmeister series was hypothesized to e resin selectivity by moderation of hydrophobic interactions between the antibody species and the Protein A ligand. The ding bsAb A was loaded at 10 g/L on MABCAPTURE A resin. Following a series of washes the antibodies were eluted using a 30 CV gradient from pH 6 to 3 with the following elution mobile phase modi?ers: sodium citrate, sodium chloride, magnesium chloride, and calcium chloride, ranked in order from kosmotroph to chaotroph in the ster series. A salt level of 500 mM was used for all salts but sodium citrate, where 250 mM was used due to protein precipitation in the load al when spiked to concentrations above 300 mM. Superior resolution between the bispeci?c product and the binding Fch homodimer was obtained with more the chaotropic salts (Figure 3). Bispeci?c peaks were collected from ?rst peak liftoff, to peak valley inflexion as detailed in Figure 3. Percent bispeci?c yield, percent bispeci?c purity, peak resolution (Rs) and percent soluble aggregate were measured (Table 2). The pH at bispeci?c pH apex was also calculated from the chromatograms. The use of the more chaotropic salts (calcium chloride and magnesium chloride) exhibited sed yield and bispeci?c purity.

Protein was also eluted at a higher pH with magnesium chloride and calcium chloride.

Neither the most chaotropic nor the most kosmotropic salts used induced signi?cant aggregation during elution of the bispecific. Therefore the use of chaotropic salts such as calcium chloride as mobile phase modi?ers in the elution buffer was shown to enhance the peak tion and the uent puri?cation of bsAb.

Table 2: Yield, soluble aggregate, peak apex pH, peak resolution and bispeciflc purity measured in bispeci?c fractions collected during gradient elution of bsAb A from POROS MABCAPTURE ATM using a variety of elution mobile phase modi?ers.

Bispeci?c Peak B1spec1fc Yield ific Purity Pool Soluble Resolution Aggregate (%) Obtained (Rs) Sodium Citrate No resolution Sodium de 4.2 N/M 0.54 Chloride ' N/M 1.66 Calcium Chloride E 2: DEVELOPMENT OF A COMMERCIAL PROCESS In order to determine feasibility of a star substitution based platform for purification of bispecific antibodies, a scalable process for the puri?cation of bsAb C was developed.

This protein was chosen as a worst-case test for the platform as it was found to display signi?cant VH binding to SpA. The key developmental goal were: (i) achieve isocratic (step) n to simplify plant fit and technology transfer while also reducing buffer consumption and sing time, (ii) identify polishing step interfacing with affinity resolving step with little or no load conditioning.

Initial factor screening and design space evaluation was performed using high throughput screening in the 96-well plate format . Elution pH, column loading (g/L total n) and mobile phase modifier concentration were identified as key process inputs (see Figure 2). Elution nce time was also considered. The load al for this study had been previously subjected to standard positive mode affinity chromatography to remove the Fc*Fc* impurity, ing in a 64% bispeci?c . Two 18—run central—composite design—of—experiments studies (CCD DoE) were performed in order to evaluate both MABSELECT SURE and POROS MABCAPTURE A in an isocratic elution mode. Factors studied for POROS MABCAPTURE A resin were column loading (range 10-25 g total protein/L), elution pH (4.5—5.5) and concentration of calcium chloride in the elution buffer (250—500 mM). Residence time was held constant at 3 min (400 cm/h). MABSELECT SURE was then evaluated in terms of column loading (range 10-25 g total protein/L), elution pH (3.8—5.0) and elution residence time (5—11 min). Calcium chloride concentration in the elution buffer was held constant at 500 mM.

For MABCAPTURE A, good models were obtained for both iflc yield (R2 = 0.97) and bispeciflc purity (R2 = 0.92) using a standard least squares fit algorithm. At all conditions, increasing m chloride levels from 250 - 500 mM in the elution buffer were observed to increase bispecific yields by 10 — 20% without ng bispecif1c purity (data not shown). Using the model, a sweet spot analysis was ted at 500 mM CaClz. The analysis illustrated that an elution pH of 5.0 — 5.1 would allow purity goals of > 95% and yield of > 80% to be met with a resin challenge of 17-25 g/L. Similar chromatographic conditions have been used at up to 2kL production scale, and bispeciflc purities in excess of 99% have been obtained after further optimization. ting MABSELECT SURE data, good model ?ts were attained for bispecif1c yield (R2 = 0.99) and bispecific purity (R2 = 1.00). Residence time was not shown to be a signi?cant factor for either response. As for MABCAPTURE A, constraints were imposed on contour plots at an 8 minute residence time to exclude regions where bispecif1c yield < 80% and bispeciflc purities < 95% were obtained. The desirable operating window or sweet spot is much smaller than observed with MABCAPTURE A. Therefore the use of MABSELECT SURE may result in a process exhibiting insufficient robustness at production scale, as small pH or loading changes may result in ptable ific purities or yields.

EXAMPLE 3: POST—AFFINITY POLISHING Following the ty resolving step, the bispeci?c antibody may be subjected to polishing steps ed to remove process and product related impurities in a fashion highly similar to standard monoclonal antibodies. However, the use of a high concentration of calcium chloride in the elution buffer of the resolving affinity step might potentially complicate ream unit operations. An ultrafiltration/diafiltration (UF/DF) could be used to reduce the conductivity of the pool in order to facilitate ional mAb polishing steps, such as cation exchange or anion exchange chromatography. However, identification of a salt tolerant positive mode chromatographic step could avoid introduction of this additional unit operation. Multimodal or mixed mode chromatography combines various types of interactions such as hydrophobic interaction, hydrogen bonding and ionic interaction with a single resin. It has been noted that this can facilitate salt tolerant adsorption.

A variety of odal resins were ered as polishing steps: (i) CAPTO ADHERE and (ii) CAPTO ADHERE IMPRES (the N—benzl-methyl ethanol amine ligand of both of these contains anion exchange, hobic and hydrogen bonding interaction groups), (iii) ceramic hydroxyapatite and (iv) CAPTO MMC, a multimodal cation exchange resin with hydrophobic interaction and hydrogen bonding potential. Ultimately, CAPTO MMC was developed to simultaneously remove process— and product—related impurities while lowering process stream conductivity. ing process development, of— concept has been con?rmed at manufacturing scale utilizing positive mode CAPTO MMC tography with bsAb C and bsAb D (see Table 3). In both ces reasonable (19—25 g/L) dynamic binding capacity was obtained with yields 2 85%, up to 3—fold reduction in soluble aggregate, and moderate (0.2 — 0.7 log l) CHO host cell protein clearance.

Table 3: Yield and contaminant removal obtained by positive mode multimodal tography using CAPTOMMC media loaded with ty resolving pool ning 250mM CaClz.

Column Load Pool Bispecific Loading (g Soluble Soluble Yield (%) Molecule total Aggregate Aggregate protein/L) (%) (%) EXAMPLE 4: PURIFICATION STRATEGIES Based on the discoveries made and described above, two downstream bispeciflc process platforms are envisioned (Figure 4). If the bispecific antibody is derived from the VH3 gene fragment family and is capable of binding SpA through the VH region, an additional affinity chromatography unit operation, termed affinity capture chromatography may be employed e 4A). After removal of cells and debris by harvest, affinity capture chromatography is performed using MABSELECT SURE resin, since the lack ofVH binding via the Z-domain s removal of the Fc*Fc* parental antibody ty. This step can be performed using Protein A binding, wash and elution conditions standard to commercial onal antibodies and also acts to increase protein concentration and remove process— and product—related impurities. As the protein is eluted at low pH it is also convenient to perform a low pH hold for viral inactivation with the product pool. Following this, removal of the Fch impurity is achieved by a second ve mode Protein A step termed "affinity resolving tography". The use of POROS MABCAPTURE A resin coupled with chaotropic modi?ers in the elution buffer can result in pools of > 95% bispeci?c purity. After af?nity resolving chromatography, the use of positive mode salt tolerant multimodal chromatography facilitates direct interfacing with the af?nity resolving step thus obviating the need for an ening UF/DF operation to remove the chaotropic salt from the s stream. Coupled with an additional polishing step such as anion exchange chromatography and virus retentive filtration, aggregates, HCP, DNA, viruses and other impurities can be removed to acceptable levels. Finally, the purified product is concentrated into the final formulation buffer by standard ultrafiltration/diafiltration methodologies. This purification train can be simpli?ed if the bispeciflc molecule does not exhibit VH binding to SpA by removal of the affinity capture step (Figure 4B). In this case, removal of both the Fch and the Fc*Fc* impurities can be performed by affinity resolving chromatography.

E 5: MATERIALS AND METHODS All ific antibodies and cell culture ?uid used in these examples were expressed in CHO cells. tographic resins were acquired from their manufacturers: ECT SURE, MABSELECT XTRA, CAPTO MMC (GE Healthcare), POROS MABCAPTURE A (Life Technologies), TOYOPEARL AF-rProtein A-650F (TOSOH Biosciences), TE HIGH CAP (Novasep Inc.), PROSEP ULTRA PLUS (EMD Millipore). All chemicals used were supplied by J.T. Baker.

Lab scale chromatographic separations were performed using an AKTA AVANT chromatographic system from GE Healthcare and 1.0 cm inner diameter (I.D.) T BENCHMARK chromatography columns (Omni?t Ltd). Pilot scale tography applied AKTA PILOT chromatographic systems and 73.0 cm I.D. INdEX chromatography columns from GE Healthcare. Production scale chromatography was conducted on an OCESS chromatography skid and 40 cm I.D. CHROMOFLOW columns (GE care). UPLC analysis ged an ACQUITY UPLC system from Waters Corporation. Cell culture was performed using either a 2L T B—DCU bench top bioreactor (Sartorius), a 50, 250, or 2000L HYCLONE single use bioreactor (Thermo Scienti?c), or a l60L stainless steel bioreactor (ABEC Inc.).

When clari?ed cell culture ?uid was not used directly, load material for y resolving development was produced by af?nity capture chromatography using 20 :: 1 cm bed height MABSELECT SURE columns. After equilibration with two column volumes (CVs) of 20 mM sodium phosphate pH 7.2 the columns were loaded to 10 — 40 g binding antibody/L with clari?ed cell culture ?uid. Binding antibody concentration was determined by summation of the bispeci?c and Fch titers. Columns were washed and protein eluted with a proprietary buffer system before a 2 CV column strip. The entire elution peak was collected and neutralized to pH 7.5 :: 0.5 with 2M Tris base.

All af?nity resolving tography was performed using 20 :: 1 cm bed height columns. The Protein A columns studied were equilibrated with two CVs 20 mM sodium phosphate pH 7.2 before load application. Following loading, columns were washed with a proprietary wash buffer system and eluted with either a gradient or isocratic elution as ed. For isocratic runs a four CV elution volume was used, collecting pool from 0.5 — 4 CVs after initiation of the elution step. Both gradient and isocratic elution buffers contained 40 mM e as the buffering species. Following elution columns were stripped with 2 CV of buffer. Unless ise stated all steps were performed at a linear velocity of 400 cm/h.

UNICORN 6.1 re (GE Healthcare) was used for chromatographic analysis, including calculation of peak resolution (Rs) assuming an peaks using the width at half height method. When automated fractionation was performed, peak liftoff was defined by > 50 mAu se in baseline UV280. Statistical design of experiments, is and modeling was performed using JMP 11.1.1 (SAS Institute Inc.).

CAPTO MMC chromatography was performed using 25.1 L columns (20 cm bed height; 40 cm ID.) with all steps performed at a 4 min residence time (linear velocity 300 cm/h). Af?nity resolving pools were diluted 50% with water and adjusted to pH 5.0 :: 0.1.

The columns were pre—equilibrated with 2 CV of 2 M NaCl before a 2 CV equilibration in 40 mM sodium acetate, 250 mM calcium chloride, pH 5.0 :: 0.1. After load application the columns were washed with 3 CV of 40 mM Tris, 40 mM acetate, pH 5.0 :: 0.1 and product was then eluted with 8 CV of either 20 mM Tris, 60 mM acetate, pH 8.0 :: 0.1 (bsAb C) or mM Tris, 40 mM acetate, pH 8.0 :: 0.1 (bsAb D). Pools were collected from UVngI1m lift off to the end of the elution step. Following elution, columns were cleaned with 2 CV of 2 M NaCl followed by 2 CV of 1 M NaOH.

] Host cell protein ) quantification was performed using a commercially available ELISA kit Cat#F550 (Cygnus Technologies). Soluble aggregate quantification by two Y UPLC PrST SEC Columns, 200A, 1.7 pm, 4.6 mm x 150 mm cat#186005225 in series in a 10 mM sodium phosphate, 500 mM sodium chloride, pH 7.0 mobile phase. Bispeci?c purity was measured using three prepacked POROS A 20 um columns (2.1 mm x 30 mm, 0.1mL) cat#2—lOOl-00 in series and an tic elution buffer system. Bispeci?c and Fch titers were measured using a POROS A 20 um column (2.1 mm x 30 mm, 0.1mL) cat#2-lOOl-00, and Fc*Fc* titers were measured by loading the ?owthrough over a POROS G 20 um column (2.1 mm x 30 mm, 0.1 mL).

Claims (20)

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. A method of making a protein comprising: (a) loading an affinity matrix with a mixture of multimeric proteins sing (i) a first homodimer sing two copies of a first polypeptide, (ii) a second homodimer comprising two copies of a second polypeptide, and (iii) a heterodimer comprising the first ptide and the second polypeptide, wherein the first polypeptide has ty for Protein A ligand, and has greater affinity for the affinity matrix than does the second polypeptide, wherein the affinity matrix comprises a Protein A ligand affixed to a substrate comprising a multiplicity of particles having a mean diameter of 45 µm and comprising pores having a mean diameter of 1100 Å; and (b) eluting and collecting the heterodimer from the affinity matrix in a buffer comprising CaCl2 or MgCl2 and having a first pH range, wherein the first homodimer elutes from the affinity matrix in the buffer at a second pH range and the second mer elutes from the affinity matrix in the buffer at a third pH range, wherein the third pH range comprises a higher pH than the first pH range, which comprises a higher pH than the second pH range.

2. A method of purifying a bispecific antibody comprising: a. loading an ty matrix with a e of multimeric proteins comprising (i) a first mer comprising two copies of a first heavy chain ptide and two copies of a light chain polypeptide, (ii) a second homodimer comprising two copies of a second heavy chain polypeptide and two copies of the light chain polypeptide, and (iii) a heterodimeric bispecific antibody comprising the first heavy chain polypeptide and the second heavy chain polypeptide and two copies of the light chain polypeptide, wherein the first heavy chain polypeptide has greater affinity for the affinity matrix than does the second heavy chain polypeptide, wherein the affinity matrix comprises a Protein A ligand affixed to a substrate comprising a multiplicity of particles having a mean diameter of 45 µm and comprising pores having a mean diameter of 1100 Å; and b. eluting and collecting the heterodimeric bispecific antibody from the affinity matrix in a buffer comprising CaCl2 or MgCl2 and having a first pH range, wherein the first homodimer elutes from the ty matrix in the buffer at a second pH range and the second homodimer elutes from the affinity matrix in the buffer at a third pH range, wherein the third pH range comprises a higher pH than the first pH range, which comprises a higher pH than the second pH range.

3. The method according to claim 1 or 2 sing the steps of: (c) ng the collected heterodimer or heterodimeric bispecific antibody of step (b) to a multimodal chromatography resin in a buffer having an acidic pH; (d) eluting the heterodimer or the dimeric ific antibody from the multimodal tography resin in a buffer having a more alkaline pH; and (e) collecting the heterodimer or the heterodimeric bispecific antibody.

4. The method according to any one of claims 1-3, wherein the substrate comprises any one or more of agarose, poly(styrene divinylbenzene), polymethacrylate, cellulose, controlled pore glass, and spherical silica.

5. The method according to any one of claims 1-4, wherein 5 to 50 grams of protein are loaded per liter of affinity matrix.

6. The method ing to any one of claims 1-5 comprising applying a pH gradient to the loaded affinity matrix of step (a).

7. The method according to claim 6 comprising washing the loaded affinity matrix of step (a) with a solution at pH 6-8 prior applying the pH gradient.

8. The method according to claim 6, wherein the pH gradient is run between pH 6 and pH 3.

9. The method according to any one of claims 6-8, wherein the first pH range is selected from a range within pH 5.5 and pH 3.6.

10. The method according to claim 9, wherein the buffer comprises acetate.

11. The method according to claim 10, wherein the buffer comprises 40 mM acetate.

12. The method according to any one of claims 1-11, wherein the buffer comprises CaCl2.

13. The method ing to any one of claims 1-11, wherein the buffer ses MgCl2.

14. The method according to claim 12, wherein the buffer comprises 250-500 mM CaCl2.

15. The method according to claim 13, wherein the buffer comprises 250-500 mM MgCl2.

16. The method according to claim 1, wherein the heterodimer is an antibody, and the first polypeptide comprises a CH3 domain that is capable of binding to the Protein A ligand and the second polypeptide comprises a CH3 domain that is not capable of binding to the Protein A ligand.

17. The method according to claim 16, wherein the heterodimer is a bispecific antibody.

18. The method according to claim 2, wherein the first heavy chain ptide comprises a CH3 domain that is capable of binding to Protein A and the second heavy chain ptide comprises a CH3 domain that is not capable of binding to Protein A.

19. The method according to any one of claims 16-18, wherein the second polypeptide or second heavy chain polypeptide ses a H435R substitution and a Y436F substitution (EU numbering) in its CH3 .

20. The method ing to claim 1 or 2, wherein the mixture of multimeric proteins is produced by a plurality of eukaryotic cells in a cell culture, optionally wherein the eukaryotic cells comprise Chinese hamster ovary (CHO) cells or derivatives thereof. FIGURE 1A A .2538 8' it) 350 _ 3 B1spec1?EXFch E; a s N m 42 m c3 3 3 5i} 2 Q 3 0 10 20 30 40 Column s (CV) SUBSTITUTE SHEET (RULE 26) FIGURE 1B 8 3m.) 3 3 15;} g} § 1:33 ,3 E ‘5 3 f3 . o 3‘3 .2 4 i 0 10 20 30 40 Column Volumes (CV) SUBSTITUTE SHEET (RULE 26) FIGURE 2 Resolution Residence Time (Min) SUBSTITUTE SHEET (RULE 26) FIGURE 3A A Rm g i} a ? <: $38 4 2m: Column Volumes (CV) SUBSTITUTE SHEET (RULE 26) FIGURE 3B 8 13m (mAU) 280nm Absorbance é?? Column s (CV) SUBSTITUTE SHEET (RULE 26) FIGURE 3C :Iti‘ Q 26 (mAU) '3‘ yttiié 280nm ’9- ance 3% Column Volumes (CV) SUBSTITUTE SHEET (RULE 26) FIGURE 3D {3 was (mAU) 280nm 8&3 Absorbance £5733 Column Volumes (CV) SUBSTITUTE SHEET (RULE 26) FIGURE 4 A B HARVEST HARVEST AFFINITY CAPTURE CHROMATOGRAPHY AFFINITY RESOLVING CHROMATOGRAPHY VIRAL VATION VIRAL INACTIVATION Q G AFFINITY RESOLVING CHROMATOGRAPHY SALT TOLERANT MU LTIMODAL POSITIVE MODE CHROMATOGRAPHY SALT TOLERANT ODAL POSITIVE ' MODE CHROMATOGRAPHY ADD'L POLISHING CHROMATOGRAPHY | VIRAL FILTRATION ULTRAFILTRATION/DIAFILTRATION ULTRAFILTRATION/DIAFILTRATION SUBSTITUTE SHEET (RULE 26)