Method of forming multi-specific antibodies from homodimer antibodies and pharmaceutical product including multi-specific antibodies thus obtained

Abstract

The present invention relates an in-vitro method of forming multi-specific antibodies from first and second homodimer antibodies. The invention also relates to multi-specific antibodies produced from the method.

Description

[0001] METHOD OF FORMING MULTI-SPECIFIC ANTIBODIES FROM HOMODIMER ANTIBODIES AND PHARMACEUTICAL PRODUCT INCLUDING MULTI-SPECIFIC ANTIBODIES THUS OBTAINED

[0002] Technical field

[0003] The present invention relates to processes of manufacturing multi-specific antibodies and more specifically to in-vitro methods of forming multi-specific antibodies from first and second homodimer antibodies.

[0004] Background of the invention

[0005] Multi-specific antibodies are important and novel class of therapeutics that have the potential to demonstrate improved potency and efficacy compared to current monoclonal / monospecific based therapies.

[0006] Several in vitro methods for generating multispecific antibodies have been explored, that utilize amino acid point mutations to promote the formation of heterodimerized antibodies from two unique homodimers. For example, US 9,527,926 discloses a method wherein point mutations of opposite charges are introduced into the hinge regions of two homodimeric antibodies. With this method, a high ratio of heterodimerization during a redox reaction to produce a heterodimerized antibody can be achieved.

[0007] Such method can be implemented in a purification process wherein each homodimer is produced in a respective bioreactor for cell culture and subjected to initial purification steps, including an affinity capture step, and then jointly pooled in a tank for heterodimerization by a redox reaction. A method of this type is illustrated on FIG.1 .

[0008] Such methods are relatively complex in that they involve two separate bioreactor feed streams, two separate affinity column purification steps to capture the two individual homodimers, a dedicated tank for the redox reaction, and an intermediate ultrafiltration / diafiltration operation to remove excess reductant. There is therefore a need for an improved method for generating multispecific antibodies that eliminates the need for separate bioreactors, intermediate steps and / or equipment such as tanks. It is also desirable to provide a method that achieves a high degree of heterodimerization, that do not damage, or significantly reduce, the biological activity of the protein.

[0009] Summary of the Invention

[0010] The inventors have found that the redox reaction may be advantageously achieved by performing the redox reaction in cell culture fluid or clarified media containing first and second homodimer antibodies. This method addresses the aforementioned problem and eliminates the need for separate bioreactors, intermediate steps and / or equipment such as tanks.

[0011] Certain enumerated non-limiting embodiments (E) of the inventions of the disclosure are set forth below. These and related embodiments are described in further detail in the Detailed Description, including the Examples and Figures. Those skilled in the art will recognize or will be able to ascertain, using no more than routine experimentation, equivalents to the specific embodiments described herein.

[0012] E1. A method of forming multispecific antibodies from first and second homodimer antibodies comprising contacting a solution comprising the first and second homodimer antibodies with a reductant, thereby forming a reductant solution, whereby multispecific antibodies are formed from the first and second homodimer antibodies and wherein the solution comprising the first and second homodimer antibodies is contacted with the reductant before a first chromatographic step.

[0013] E2. The method of E1 , wherein the antibody of the IgG, IgA, or IgM class.

[0014] E3. The method of E2, wherein the antibody is of the IgG class.

[0015] E4. The method of any preceding embodiment, wherein the formation of the multispecific antibody is enhanced by altering or engineering an interface between a first and a second Fc chain the multispecific antibody, with one Fc chain derived from a first homodimer antibody and the second Fc chain derived from a second homodimer antibody, such that opposing residues of the first and a second Fc chain carry the opposite charge. In one embodiment, one or more amino acid residues that make up the CH3-CH3 and hinge region interface are replaced with charged amino acid pairs for promoting the heterodimer formation.

[0016] E5. The method of any preceding embodiment, wherein the formed multispecific antibody comprises a first Fc chain comprising charged amino acid modifications at positions 221 , 228 and 409 (EU numbering scheme) and a second Fc chain comprising charged amino acid modifications at positions 221 , 228 and 368 (EU numbering scheme) wherein the charged amino acid modification of the second Fc chain are oppositely charged to those of the modifications of the first Fc chain.

[0017] E6. The method of any of E1 to E4, wherein the formed multispecific antibody comprises a first Fc chain comprising charged amino acid modifications at positions 220, 224, 228 and 409 (EU numbering scheme) and a second Fc chain comprising charged amino acid modifications at positions 220, 228 and 368 (EU numbering scheme) that are oppositely charged to those of the modifications of the first Fc chain.

[0018] E7. The method of any preceding embodiment wherein the solution comprising the first and second homodimer antibodies to which the reductant is added is a cell culture medium or a harvested cell culture solution.

[0019] E8. The method of E7, wherein the harvested cell culture solution is a clarified cell culture media comprising the first and second homodimer antibodies.

[0020] E9. The method of any preceding embodiment, wherein the reductant is a weak reductant.

[0021] E10. The method of E9, wherein the weak reductant is selected from cysteine, Glutathione (GSH) and Dithioerythritol (DTT).

[0022] E11 . The method of E10, wherein the weak reductant is cysteine.

[0023] E12. The method of E10, wherein the weak reductant is GSH.

[0024] E13. The method of E10, wherein the weak reductant is DTT.

[0025] E14. The method of any one of E9 to E13, wherein the weak reductant is present in the solution at between 1 mM and 500mM.

[0026] E15. The method of E14, wherein the weak reductant is present in the solution at between 10mM and 100mM.

[0027] E16. The method of any preceding embodiment, wherein the pH of the reductant solution is between 7.0 and 8.5.

[0028] E17. The method of E16, wherein the pH of the reductant solution is between 7.5 and 8.0. E18. The method of E16, wherein the pH of the reductant solution is between 7.0 and 7.5.

[0029] E19. The method of any preceding embodiment, wherein the reductant solution comprising the first and second homodimer antibodies is further contacted with an oxidant.

[0030] E20. The method of any preceding embodiment, wherein the reductant solution further comprises a chelator.

[0031] E21. The method of E20, wherein the chelator is Ethylenediaminetetraacetic acid (EDTA) or diethylenetriaminepentaacetic acid (DTPA).

[0032] E22. The method of either E20 or E21 wherein the chelator is present between 5mM and 50mM in the reductant solution.

[0033] E23. The method of any preceding embodiment, wherein the reductant solution further comprises at least one stabilizing excipient.

[0034] E24. The method of E23, wherein the stabilizing excipient is beta cyclodextrin or an amino acid.

[0035] E25. The method of E24, wherein the stabilizing excipient is an amino acid and the amino acid is arginine, lysine or a mixture thereof.

[0036] E26 The method of E25, wherein the stabilizing excipient is arginine.

[0037] E27. The method of E25, wherein the stabilizing excipient is lysine.

[0038] E28. The method of any one of E23 to E27, wherein the stabilizing excipient is present between 25mmM to 100mM in the reductant solution.

[0039] E29. The method of E28, wherein the stabilizing excipient is present at about 50mM in the reductant solution.

[0040] E30. The method of any preceding embodiment, wherein the redox reaction is performed at a temperature between 4C (Celsius) and 37C.

[0041] E31. The method of any preceding embodiment, wherein the solution comprising the first and second homodimer antibodies is contacted with a reductant for at least 1 hour.

[0042] E32. The method of any preceding embodiment, wherein the method further comprises performing a first chromatographic step comprising loading the produced multispecific antibodies onto an adsorbent chromatography medium, whereby the multispecific antibodies bind to the medium and eluting the bound antibodies with an elution buffer thereby obtaining an eluate pool comprising the multispecific antibodies.

[0043] E33. A multispecific antibody obtained by a method according to any of the preceding embodiments. E34. A pharmaceutical product comprising a multi-specific antibody obtained by a method according to any one of E1 to E32.

[0044] Detailed Description of the Invention

[0045] The following discussion is directed to various embodiments of the invention. The term “invention” is not intended to refer to any particular embodiment or otherwise limit the scope of the disclosure. Although one or more of these embodiments may be preferred, the embodiments disclosed should not be interpreted, or otherwise used, as limiting the scope of the disclosure, including the claims. In addition, one skilled in the art will understand that the following description has broad application, and the discussion of any embodiment is meant only to be an example of that embodiment, and not intended to intimate that the scope of the disclosure, including the claims, is limited to that embodiment.

[0046] According to a first aspect of the present invention, there is provided the method of forming multispecific antibodies from first and second homodimer antibodies comprising contacting a solution comprising the first and second homodimer antibodies with a reductant, thereby forming a reductant solution, whereby multispecific antibodies are formed from the first and second homodimer antibodies and wherein the solution comprising the first and second homodimer antibodies is contacted with the reductant before a first chromatographic step.

[0047] The method advantagouesly eliminates the need for two seperate protein A purification steps to capture the two individual homodimers. The process enables the use of just a single protein A step to capture the heterodimer which is formed in solution, reducing the number of protein A capture steps by half. It also negates the need for a separate redox tank step. Thus, the formation of multispecific antibodies is simplified, the manufacturing costs are decreased and the speed of manufacturing is increased.

[0048] Antibodies

[0049] A multispecific antibody comprises at least two antigen-binding sites with differing specificities. For example, the multi-specific antibody may be bispecific antibody or a trispecific antibody. In one embodiment the multispecific is a bispecific antibody. The antibody of the invention may comprise a hinge region. The antibody of the invention may comprise an Fc region. In one embodiment, the antibody comprises a hinge region and an Fc region. In one embodiment, the antibody is a full-length antibody.

[0050] The antibody of the invention includes an antibody of any class, such as IgG, IgA, or IgM (or sub-class thereof).

[0051] In one embodiment the antibody is of the IgG class.

[0052] Where the antibody is an IgG, the antibody may be of the subclass lgG1 , lgG2, lgG3, lgG4. In one embodiment the antibody is lgG1 or lgG2.

[0053] The formation of the desired multispecific antibody may be enhanced by altering or engineering an interface between a first and a second Fc chain of the multispecific antibody.

[0054] Accordingly, the formation of the desired multispecific antibody (e.g., bispecific antibody) may be enhanced by altering or engineering an interface between a first and a second Fc chain of the multispecific antibody, with one Fc chain derived from a first homodimer antibody and the second Fc chain derived from a second homodimer antibody, such that opposing residues of the first and a second Fc chain carry the opposite charge. In one embodiment, one or more amino acid residues that make up the CH3-CH3 and hinge region interface are replaced with charged amino acid pairs for promoting the heterodimer formation.

[0055] Where the homodimer antibodies are lgG1 , the first homodimer antibody Fc chain may comprise charged amino acid modifications at positions 221 , 228 and 409 (Ell numbering scheme) and the second homodimer antibody Fc chain may comprise charged amino acid modifications at positions 221 , 228 and 368 (Ell numbering scheme) that are oppositely charged to those of the modifications of the first homodimer antibody.

[0056] Thus, the formed multispecific antibody may comprise a first Fc chain comprising charged amino acid modifications at positions 221 , 228 and 409 (Ell numbering scheme) and a second Fc chain comprising charged amino acid modifications at positions 221 , 228 and 368 (EU numbering scheme) wherein the charged amino acid modification of the second Fc chain are oppositely charged to those of the modifications of the first Fc chain.

[0057] In some embodiments, the Fc chain of the first homodimer antibody comprises amino acid modifications at positions 221 and 228 (e.g., (D221 R or D221 E) and (P228R or P228E)) in the hinge region and at position 409 (e.g., K409R) in the CH3 region and the Fc chain of the second homodimer antibody comprises amino acid modifications at positions 221 and 228 (e.g., (D221 R or D221 E) and (P228R or P228E)) in the hinge region and 368 (e.g., L368E (EU numbering scheme)) in the CH3 region of human lgG1 .

[0058] In one embodiment, the first homodimer antibody Fc chain comprises the amino acid modifications at position 221 of D221 R, the amid modification as position 228 of P228R and the amino acid modification at position 409 of K409R, and the second homodimer antibody Fc chain comprises the amino acid modification at position 221 of D221 E, the amid modification at position 228 of P228E and the amino acid modification at position 368 is L368E in the CH3 region of human IgG 1 .

[0059] Where the homodimer antibodies are lgG2, the first homodimer antibody may comprise charged amino acid modifications at positions 220, 224, 228 and 409 (EU numbering scheme) and the second homodimer antibody may comprise charged amino acid modifications at positions 220, 228 and 368 (EU numbering scheme) that are oppositely charged to those of the modifications of the first homodimer antibody.

[0060] Thus, the formed multispecific antibody may comprise a first Fc chain comprising charged amino acid modifications at positions 220, 224, 228 and 409 (EU numbering scheme) and a second Fc chain comprising charged amino acid modifications at positions 220, 228 and 368 (EU numbering scheme) that are oppositely charged to those of the modifications of the first Fc chain.

[0061] Where the homodimer antibodies are lgG2, the first homodimer antibody may comprise charged amino acid modifications at positions 220, 228 and 409 (EU numbering scheme) and the second homodimer antibody may comprise charged amino acid modifications at positions 220, 224, 228 and 368 (EU numbering scheme) that are oppositely charged to those of the modifications of the first homodimer antibody.

[0062] Thus, the formed multispecific antibody may comprise a first Fc chain comprising charged amino acid modifications at positions 220, 228 and 409 (EU numbering scheme) and a second Fc chain comprising charged amino acid modifications at positions 220, 224, 228 and 368 (EU numbering scheme) that are oppositely charged to those of the modifications of the first Fc chain.

[0063] In one embodiment, the Fc chain of the first homodimer antibody comprises amino acid modifications at positions 220 and 228 (e.g., (C220E or C220R), and (P228E or P228R)) in the hinge region and at position 409 (e.g., K409R (EU numbering scheme)) in the CH3 region and the Fc chain of the second homodimer antibody comprises amino acid modifications at positions 220, 224 and 228 (e.g., (C220E or C220R), (E224R), and (P228E or P228R)) in the hinge region and at position 368 (e.g., L368E (EU numbering scheme)) in the CH3 region of human lgG2.

[0064] In one embodiment, the first homodimer antibody Fc chain comprises the amino acid modifications at position 220 of C220R, the amid modification as position 228 of P228R and the amino acid modification at position 409 of K409R, and the second homodimer antibody Fc chain comprises the amino acid modification at position 220 of C220E, , the amid modification at position 224 of E224R, the amid modification at position 228 of P228E and the amino acid modification at position 368 is L368E in the CH3 region of human lgG2.

[0065] In other embodiments, the Fc chain of the first homodimer antibody comprises amino acid modifications at positions 220 and 228 (e.g., (C220E or C220R) and (P228E or P228R)) in the hinge region and at position 368 (e.g., L368E (EU numbering scheme)) in the CH3 region of human lgG2, and the Fc chain of the second homodimer antibody comprises amino acid modifications at positions 220, 224 and 228 (e.g., (C220E or C220R), (E224R), and (P228E or P228R)) in the hinge region and at position 409 (e.g., K409R (EU numbering scheme)) in the CH3 region of human lgG2. In one embodiment, the first homodimer antibody Fc chain comprises the amino acid modifications at position 220 of C220R, the amid modification at position 224 of E224R, the amid modification as position 228 of P228R and the amino acid modification at position 409 of K409R, and the second homodimer antibody Fc chain comprises the amino acid modification at position 220 of C220E, the amid modification at position 228 of P228E and the amino acid modification at position 368 is L368E in the CH3 region of human lgG2.

[0066] The term “interface,” as used herein typically refers to any amino acid residue present in the domain that can be involved in first polypeptide and second polypeptide contacts. An “original amino acid” residue is one which is replaced by an “import amino acid” residue which can have a smaller or larger side chain volume than the original residue. The import amino acid residue can be a naturally occurring or non-naturally occurring amino acid residue, but preferably is the former. “Naturally occurring” amino acid residues are those residues encoded by the genetic code. By “non-naturally occurring” amino acid residue is meant a residue which is not encoded by the genetic code, but which is able to covalently bind adjacent amino acid residue(s) in the polypeptide chain. Examples of non- naturally occurring amino acid residues are norleucine, ornithine, norvaline, homoserine and other amino acid residue analogues such as those described in Ellman et al., Meth. Enzym. 202:301 -336 (1991 ).

[0067] Solution comprising the first and second homodimer antibodies

[0068] The reductant may be added to a pre- or post-harvest cell culture solution. Thus, the solution comprising the first and second homodimer antibodies to which the reductant is added may be a cell culture medium or a harvested cell culture solution.

[0069] The reductant may be added to a cell culture medium, whereby cells expressing each of the homodimer antibodies are present in the same cell culture medium to which the reductant is added. Thus, in one embodiment a cell culture medium comprising the first and second homodimer antibodies is contacted with the reductant.

[0070] In one embodiment, the reductant is added to the cell culture medium after at least 5 days from the start of the cell culture. In one embodiment, the reductant is added to the cell culture medium after at least 10 days from the start of the cell culture. In one embodiment, the reductant is added to the cell culture medium between 5 days and 20 days from the start of cell culture. In one embodiment, the reductant is added to the cell culture medium between 10 days and 15 days from the start of the cell culture.

[0071] In one embodiment, the reductant is added to the cell culture medium about 12 days from the start of the cell culture.

[0072] Alternatively, a harvested cell culture solution may be contacted with the reductant, which may be a combined harvested cell culture fluid, derived from separate cultures of cells expressing the first and second homodimer antibodies or a harvested cell culture solution from a co-culture of cells expressing the first and second homodimer antibody.

[0073] The harvested cell culture solution may be clarified. Thus, in one embodiment the reductant is contacted with clarified cell culture media comprising the first and second homodimer antibodies.

[0074] The homodimer antibodies may be present in the reductant solution at equimolar concentrations. The homodimer antibodies may be present in the reductant solution at a concentration of between IpM and lOOpM.

[0075] Cells

[0076] Any type of cell grown in typical biotechnology settings may be utilized in the method of the invention. For example, the cells used in the method of the invention may be selected from mammalian cells, bacteria, yeast, and insect cells. However, preferably, the cells used in the method of the invention expressing the antibodies are mammalian cells. In one embodiment, the mammalian cells are selected from Chinese hamster ovary (CHO), baby hamster kidney or human embryo kidney (HEK) cells. In one embodiment, CHO cells are used in the method of the invention.

[0077] Reductant solution

[0078] The reductant is preferably a weak reductant. A weak reductant may be a reductant with a redox potential of less than about -200mV. In one embodiment the weak reductant is selected from cysteine, glutathione (GSH) and dithioerythritol (DTT).

[0079] In one embodiment the reductant is cysteine. In one embodiment the reductant is GSH. In one embodiment the reductant is DTT.

[0080] The reductant may be present in the reductant solution at a concentration of between 1 mM and 500mM. In one embodiment the reductant is present in the reductant solution at between 5 and 50m M. In one embodiment the reductant is present in the reductant solution at between 10 and 40mM. In one embodiment the reductant is present in the reductant solution at between 10 and 25mM.

[0081] In one embodiment the reductant is present at a concentration of between 1 mM and 500mM. In one embodiment the reductant is present at a concentration of between 20mM and 500mM. In one embodiment the reductant is present at a concentration of between 5mM and 250mM. In one embodiment the reductant is present at a concentration of between 10mM and 250mM. In one embodiment the reductant is present at a concentration of between 5mM and 100mM. In one embodiment the reductant is present at a concentration of between 1mM and 50mM.ln one embodiment the reductant is present at a concentration of between 10mM and 50mM

[0082] Where the reductant is cysteine, the concentration of cysteine present in the reductant solution may be between 1 mM and 500mM. In one embodiment, the concentration of cysteine present in the reductant solution may be between 10mM and 500mM. In one embodiment, the concentration of cysteine present in the reductant solution may be between 10mM and 250mM. In one embodiment, the concentration of cysteine present in the reductant solution may be between 20mM and 250mM. In one embodiment, the concentration of cysteine in the reductant solution is between 10mM and 100mM. In one embodiment, the concentration of cysteine in the reductant solution is between 10mM and 50mM.

[0083] In one embodiment, the reductant is DTT and is present in the reductant solution at a concentration of between 1 mM and 100mM. In one embodiment, the concentration of DTT present in the reductant solution is between 5mM and 20mM. In one embodiment, the reductant is GSH present in the reductant solution at a concentration of between 1 mM and 100mM. In one embodiment, the concentration of GSH is between 1 mM and 20mM.

[0084] The reductant may be present in the reductant solution between 20 and 500 molar excess. In one embodiment, the reductant may be present in the reductant solution between 50 and 200 molar excess.

[0085] The solution comprising the first and second homodimer antibodies may be contacted with a reductant for at least 1 hour. In one embodiment, the solution comprising the first and second homodimer antibodies may be contacted with a reductant for at least 5 hours. In one embodiment, the solution comprising the first and second homodimer antibodies may be contacted with a reductant for at least 10 hours.

[0086] The solution comprising the first and second homodimer antibodies may be contacted with a reductant for at most 48 hours. In one embodiment, the solution comprising the first and second homodimer antibodies may be contacted with a reductant for at most 24 hours. In one embodiment, the solution comprising the first and second homodimer antibodies may be contacted with a reductant for at most 12 hours.

[0087] The solution comprising the first and second homodimer antibodies may be contacted with a reductant for between 1 and 48 hours. In one embodiment, the first and second homodimer antibodies are contacted with a reductant for between 4 and 24 hours.

[0088] In one embodiment, the first and second homodimer antibodies are contacted with a reductant for between 4 and 12 hours.

[0089] The redox reaction may be performed at a temperature between 4C (Celsius) and 37C.

[0090] In one embodiment, the temperature may be between 4C and 25C. In one embodiment, the temperature may be between 20C and 37C. In one embodiment, the temperature may be between 10C and 30C. In one embodiment, the temperature may be between 20C and 25C. In one embodiment, the temperature may be between 4C and 10C. In one embodiment, the temperature may be between 30C and 37C.

[0091] In one embodiment when the reductant is contacted with a cell culture media, the temperature is between 20C and 37C. In one embodiment when the reductant is contacted with a cell culture media, the temperature is between 30C and 37C.

[0092] In one embodiment when the reductant is contacted with a harvested cell culture solution the temperature is between 4C and 25C. In one embodiment when the reductant is contacted with a harvested cell culture solution the temperature is between 4C and 10C.

[0093] The pH of the reductant solution may be between 7.0 and 8.5. In one embodiment the pH of the solution is between 7.5 and 8.5. In one embodiment the pH of the reductant solution is between 7.5 and 8.1.

[0094] In another embodiment the pH of the reductant solution is between 7.6 and 8.1. In another embodiment the pH of the reductant solution is between 7.7 and 8.1. In another embodiment the pH of the reductant solution is between 7.8 and 8.1. In another embodiment the pH of the reductant solution is between 7.9 and 8.1.

[0095] In another embodiment the pH of the solution is between 7.0 and 8.0. In another embodiment the pH of the solution is between 7.0 and 7.9. In another embodiment the pH of the solution is between 7.0 and 7.8 In another embodiment the pH of the solution is between 7.0 and 7.7. In another embodiment the pH of the solution is between 7.0 and 7.6.

[0096] In one embodiment the pH of the solution is between 7.0 and 7.5.

[0097] In another embodiment the pH of the reductant solution is at least 7.0. In another embodiment the pH of the reductant solution is at least 7.1 . In another embodiment the pH of the reductant solution is at least 7.2. In another embodiment the pH of the reductant solution is at least 7.3. In another embodiment the pH of the reductant solution is at least 7.4. In another embodiment the pH of the reductant solution is at least 7.5. In another embodiment the pH is at least 7.6. In another embodiment the pH of the reductant solution is at least 7.7. In another embodiment the pH is at least 7.8. In another embodiment the pH is at least 7.9. In another embodiment the pH is at least 8.0.

[0098] In one embodiment the pH is about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0 or about 8.1 . In one embodiment the pH is about 8.1 .

[0099] In one embodiment, when the reducing agent is contacted with a cell culture medium, the pH may be 8.1. In one embodiment, when the reducing agent is contacted with a harvested cell culture solution, the pH may be between 7.0 and 8.5.

[0100] The reductant solution comprising the first and second homodimer antibodies may be further contacted with an oxidant. In one embodiment the oxidant is Glutathione Disulfide (GSSG). In one embodiment the oxidant is present at a concentration of between 1 mM and 200mM in the reductant solution. In one embodiment the oxidant is added at a concentration of between 5mM and 100mM. In one embodiment the oxidant is added at a concentration of between 15mM and 20mM.

[0101] The inventors have found that adding further additional agents to the reductant solution provides additional advantages which are described below.

[0102] Accordingly, the reductant solution may further comprise a chelator. The chelator may advantageously lead to greater recombination efficiency at an optimal pH in the presence of reducing agent and stabilizing excipients. Further, the chelator may act to stabilize the two homodimers and orientate them for the heterodimerization reaction.

[0103] The chelator may be Ethylenediaminetetraacetic acid (EDTA) or diethylenetriaminepentaacetic acid (DTPA). In one embodiment the chelator is EDTA. In one embodiment the chelator is DTPA.

[0104] The chelator maybe be present between 1 mM and 50mM in the reductant solution. In one embodiment, the chelator is present between 10mM and 50mM in the reductant solution. In one embodiment, the chelator is present at about 10mM in the reductant solution. The chelator may be present between 1 and 10% (v / v) in the reductant solution. In one embodiment, the chelator is present between 3-7 % (v / v) in the reductant solution.

[0105] In addition to the reductant, the reductant solution may further comprise at least one stabilizing excipient.

[0106] The stabilizing excipients advantageously stabilize the two homodimers and orient them optimally for the exchange (heterodimer formation). Further, the ability to perform the method at a lower pH advantageously obviates the need to change the pH downstream in the purification process providing further streamlining of the method.

[0107] The stabilizing excipient may be beta cyclodextrin or an amino acid.

[0108] In one embodiment the stabilizing excipient is beta cyclodextrin.

[0109] In one embodiment the stabilizing excipient is an amino acid. The amino acid may be arginine, Lysine or a mixture thereof. In one embodiment, the amino acid may be arginine.

[0110] In one embodiment the at least one stabilizing excipient is present between 10mmM to 500mM in the reductant solution.

[0111] In one embodiment the at least one stabilizing excipient is present between 25mmM to 100mM in the reductant solution. In one embodiment the at least one stabilizing excipient is present between 25mmM and 50mM in the reductant solution. In one embodiment the at least one stabilizing excipient is present between 50mmM and 100mM in the reductant solution.

[0112] In one embodiment the at least one stabilizing excipient is present at about 50mmM in the reductant solution.

[0113] In one embodiment, the stabilizing excipient is beta cyclodextrin present between 25mM to 150mM in the reductant solution. In one embodiment, the stabilizing excipient is beta cyclodextrin present at about 50mmM in the reductant solution. In one embodiment, the stabilizing excipient is an amino acid present between 25mmM to 100mM in the reductant solution. In one embodiment, the stabilizing excipient is an amino acid present at about 50mmM in the reductant solution.

[0114] In one embodiment, the stabilizing excipient is arginine present between 25mmM to 100mM in the reductant solution. In one embodiment, the stabilizing excipient is arginine present at about 50mmM in the reductant solution.

[0115] Thus, in one embodiment, the reductant solution comprises a chelator and at least one stabilizer as defined above.

[0116] As demonstrated in the examples, the percentage of multi-specific antibodies formed after contact with the reductant may be at least 50% of the total population of antibodies. The percentage of multi-specific antibodies formed after contact with the reductant may be at least 60% of the total population of antibodies. The percentage of multi-specific antibodies formed after contact with the reductant may be at least 70% of the total population of antibodies. The percentage of multi-specific antibodies formed after contact with the reductant may be at least 80% of the total population of antibodies. The percentage of multi-specific antibodies formed after contact with the reductant may be at least 90% of the total population of antibodies. The percentage of multi-specific antibodies formed after contact with the reductant may be at least 91 % of the total population of antibodies. The percentage of multi-specific antibodies formed after contact with the reductant may be at least 92% of the total population of antibodies. The percentage of multi-specific antibodies formed after contact with the reductant may be at least 93% of the total population of antibodies. The percentage of multi-specific antibodies formed after contact with the reductant may be at least 94% of the total population of antibodies. The percentage of multi-specific antibodies formed after contact with the reductant may be at least 95% of the total population of antibodies.

[0117] In one particular embodiment, the percentage of multi-specific antibodies formed after contact with the reductant may be at least 90% of the total population of antibodies.

[0118] In one particular embodiment, the percentage of multi-specific antibodies formed after contact with the reductant may be at least 95% of the total population of antibodies. The percentage of multi-specific antibodies formed after contact with the reductant may be between 60% and 100% of the total population of antibodies. The percentage of multispecific antibodies formed after contact with the reductant may be between 70% and 100% of the total population of antibodies. The percentage of multi-specific antibodies formed after contact with the reductant may be between 80% and 100% of the total population of antibodies. The percentage of multi-specific antibodies formed after contact with the reductant may be between 90% and 100% of the total population of antibodies.

[0119] The percentage of multi-specific antibodies formed after contact with the reductant may be between 60% and 95% of the total population of antibodies. The percentage of multispecific antibodies formed after contact with the reductant may be between 70% and 95% of the total population of antibodies. The percentage of multi-specific antibodies formed after contact with the reductant may be between 80% and 95% of the total population of antibodies. The percentage of multi-specific antibodies formed after contact with the reductant may be between 90% and 95% of the total population of antibodies.

[0120] The method may further comprise performing a first chromatographic step comprising loading the produced multispecific antibodies onto an adsorbent chromatography medium, whereby the multispecific antibodies bind to the medium and eluting the bound antibodies with an elution buffer thereby obtaining an eluate pool comprising the multispecific antibodies.

[0121] In one embodiment the first chromatographic step is protein A chromatography, such that the method comprises loading the loading the produced multispecific antibody onto a protein A column, whereby the multispecific antibody binds to the medium eluting the bound antibodies with an elution buffer thereby obtaining an eluate pool.

[0122] The method may further comprise, before the elution step, a step of washing the medium with an initial wash buffer for impurities to flow through with the wash buffer while the multispecific antibody remains bound to the medium.

[0123] The method may further comprise additional purification steps after the first chromatographic step. The method may further comprise a second chromatographic step. The second chromatographic step may be an anion exchange chromatography step (AEX).

[0124] The method may further comprise a third chromatographic step. The third chromatographic step may be a cation exchange chromatography step.

[0125] In one embodiment, the purification steps performed after the first chromatographic step are a diafiltration step (DF), an anion exchange chromatography step (AEX), a cation exchange chromatography step, and / or a virus filtration step and a final ultra-f iltration / dia- filtration step (IIFDF). In one embodiment, the method comprises a diafiltration step (DF), an anion exchange chromatography step (AEX), a cation exchange chromatography step, and a virus filtration step and a final ultra-filtration / dia-filtration step (UFDF).

[0126] The first aspect of the invention may be further defined by the following embodiments.

[0127] In one embodiment, the method of forming multispecific antibodies from first and second homodimer antibodies comprises contacting a solution comprising the first and second homodimer antibodies with a reductant, thereby forming a reductant solution, whereby multispecific antibodies are formed from first and second homodimer antibodies, wherein the solution comprising the first and second homodimer antibodies is contacted with the reductant prior to any chromatographic step, wherein the reductant is a weak reductant present at a concentration of between 10mM and 250mM in the reductant solution and wherein the solution comprising the first and second homodimer antibodies to which the reductant is added is a cell culture solution or a harvested cell culture solution and wherein the reductant solution further comprises a chelator and at least one stabilizer, wherein the chelator is present at a concentration of between 5mM and 50m M and the at least one stabilizer is present at a concentration of between 25mmM to 100mM in the reductant solution.

[0128] In one embodiment, the method of forming multispecific antibodies from first and second homodimer antibodies comprises contacting a solution comprising the first and second homodimer antibodies with a reductant, thereby forming a reductant solution, whereby multispecific antibodies are formed from first and second homodimer antibodies, wherein the solution comprising the first and second homodimer antibodies is contacted with the reductant prior to any chromatographic step, wherein the reductant is a weak reductant present at a concentration of between 1 mM and 50mM in the reductant solution and wherein the solution comprising the first and second homodimer antibodies to which the reductant is added is a cell culture solution or a harvested cell culture solution and wherein the reductant solution further comprises at least one stabilizer, wherein the at least one stabilizer is present at a concentration of between 25mmM to 100mM in the reductant solution.

[0129] In one embodiment, the method of forming multispecific antibodies from first and second homodimer antibodies comprises contacting a solution comprising the first and second homodimer antibodies with a reductant, thereby forming a reductant solution, whereby multispecific antibodies are formed from first and second homodimer antibodies, wherein the solution comprising the first and second homodimer antibodies is contacted with the reductant prior to any chromatographic step, wherein the reductant is a weak reductant present at a concentration of between 10mM and 250mM in the reductant solution and wherein the solution comprising the first and second homodimer antibodies to which the reductant is added is a cell culture solution or a harvested cell culture solution and wherein the reductant solution further comprises a chelator, wherein the chelator is present at a concentration of between 5mM and 50mM.

[0130] In one embodiment, the method of forming multispecific antibodies from first and second homodimer antibodies comprises contacting a solution comprising the first and second homodimer antibodies with a reductant, thereby forming a reductant solution, whereby multispecific antibodies are formed from first and second homodimer antibodies, wherein the solution comprising the first and second homodimer antibodies is contacted with the reductant prior to any chromatographic step, wherein the reductant is a weak reductant present at a concentration of between 10mM and 250mM in the reductant solution and wherein the solution comprising the first and second homodimer antibodies to which the reductant is added is a cell culture solution or a harvested cell culture solution and wherein the reductant solution further comprises a chelator and at least one stabilizer, wherein the chelator is present at a concentration of between 5mM and 50m M and the at least one stabilizer is present at a concentration of between 25mmM to 100mM in the reductant solution and wherein the reductant solution comprising the first and second homodimer antibodies is further contacted with an oxidant, wherein the oxidant is present in the reductant solution at a concentration of between 1mM and 200mM.

[0131] In one embodiment, the method of forming multispecific antibodies from first and second homodimer antibodies comprises contacting a solution comprising the first and second homodimer antibodies with a reductant, thereby forming a reductant solution, whereby multispecific antibodies are formed from first and second homodimer antibodies, wherein the solution comprising the first and second homodimer antibodies is contacted with the reductant prior to any chromatographic step, wherein the reductant is a weak reductant present at a concentration of between 10mM and 250mM in the reductant solution and wherein the solution comprising the first and second homodimer antibodies to which the reductant is added is a cell culture solution or a harvested cell culture solution and wherein the reductant solution further comprises a chelator and at least one stabilizer, wherein the chelator is present at a concentration of between 5mM and 50m M and the at least one stabilizer is present at a concentration of between 25mmM to 100mM in the reductant solution and wherein the redox reaction is performed at a temperature of between 4C and 10C.

[0132] In one embodiment, the method of forming multispecific antibodies from first and second homodimer antibodies comprises contacting a solution comprising the first and second homodimer antibodies with a reductant, thereby forming a reductant solution, whereby multispecific antibodies are formed from first and second homodimer antibodies, wherein the solution comprising the first and second homodimer antibodies is contacted with the reductant prior to any chromatographic step, wherein the reductant is a weak reductant present at a concentration of between 10mM and 250mM in the reductant solution and wherein the solution comprising the first and second homodimer antibodies to which the reductant is added is a cell culture solution or a harvested cell culture solution and wherein the reductant solution further comprises a chelator and at least one stabilizer, wherein the chelator is present at a concentration of between 5mM and 50m M and the at least one stabilizer is present at a concentration of between 25mmM to 100mM in the reductant solution and wherein the redox reaction is performed at a temperature of between 20C and 37C. In one embodiment, the method of forming multispecific antibodies from first and second homodimer antibodies comprises contacting a solution comprising the first and second homodimer antibodies with a reductant, thereby forming a reductant solution, whereby multispecific antibodies are formed from first and second homodimer antibodies, wherein the solution comprising the first and second homodimer antibodies is contacted with the reductant prior to any chromatographic step, wherein amino acid residues at the interface between the first and a second Fc chain of the multispecific antibody, with one Fc chain derived from the first homodimer antibody and the second Fc chain derived from the second homodimer antibody opposing residues of the first and a second Fc chain are modified to carry the opposite charge, wherein the reductant is cysteine present at a concentration of between 10mM and 250mM in the reductant solution, and wherein the solution comprising the first and second homodimer antibodies to which the reductant is added is a cell culture solution or a harvested cell culture solution and wherein the reductant solution further comprises a chelator and at least one stabilizer, wherein the chelator is EDTA present at a concentration of between 5mM and 50mM in the reductant solution and the at least one stabilizer is an amino acid present between 25mmM to 100mM in the reductant solution.

[0133] In one embodiment, the method of forming multispecific antibodies from first and second homodimer antibodies comprises contacting a solution comprising the first and second homodimer antibodies with a reductant, thereby forming a reductant solution, whereby multispecific antibodies are formed from first and second homodimer antibodies, wherein the solution comprising the first and second homodimer antibodies is contacted with the reductant prior to any chromatographic step, wherein amino acid residues at the interface between the first and a second Fc chain of the multispecific antibody, with one Fc chain derived from the first homodimer antibody and the second Fc chain derived from the second homodimer antibody opposing residues of the first and a second Fc chain are modified to carry the opposite charge, wherein the reductant is cysteine present at a concentration of between 10mM and 250mM in the reductant solution, and wherein the solution comprising the first and second homodimer antibodies to which the reductant is added is a cell culture solution or a harvested cell culture solution and wherein the reductant solution further comprises a chelator and at least one stabilizer, wherein the chelator is EDTA present at a concentration of between 5mM and 50mM in the reductant solution and the at least one stabilizer is arginine present between 25mmM to 100mM in the reductant solution.

[0134] In one embodiment, the method of forming multispecific antibodies from first and second homodimer antibodies comprises contacting a solution comprising the first and second homodimer antibodies with a reductant, thereby forming a reductant solution, whereby multispecific antibodies are formed from first and second homodimer antibodies, wherein the solution comprising the first and second homodimer antibodies is contacted with the reductant prior to any chromatographic step, wherein amino acid residues at the interface between the first and a second Fc chain of the multispecific antibody, with one Fc chain derived from the first homodimer antibody and the second Fc chain derived from the second homodimer antibody opposing residues of the first and a second Fc chain are modified to carry the opposite charge, wherein the reductant is cysteine present at a concentration of between 10mM and 250mM in the reductant solution, and wherein the solution comprising the first and second homodimer antibodies to which the reductant is added is a cell culture solution or a harvested cell culture solution and wherein the reductant solution further comprises a chelator and at least one stabilizer, wherein the chelator is DTPA present at a concentration of between 5mM and 50mM in the reductant solution and the at least one stabilizer is arginine present between 25mmM to 100mM in the reductant solution.

[0135] In one embodiment, the method of forming multispecific antibodies from first and second homodimer antibodies comprises contacting a solution comprising the first and second homodimer antibodies with a reductant, thereby forming a reductant solution, whereby multispecific antibodies are formed from first and second homodimer antibodies, wherein the solution comprising the first and second homodimer antibodies is contacted with the reductant prior to any chromatographic step, wherein amino acid residues at the interface between the first and a second Fc chain of the multispecific antibody, with one Fc chain derived from the first homodimer antibody and the second Fc chain derived from the second homodimer antibody opposing residues of the first and a second Fc chain are modified to carry the opposite charge, wherein the reductant is GSH present at a concentration of between 1 and 100m M in the reductant solution, and wherein the solution comprising the first and second homodimer antibodies to which the reductant is added is a cell culture solution or a harvested cell culture solution and wherein the reductant solution further comprises a chelator and at least one stabilizer, wherein the chelator is EDTA present at a concentration of between 5mM and 50mM in the reductant solution and the at least one stabilizer is arginine present between 25mmM to 100mM in the reductant solution.

[0136] In one embodiment, the method of forming multispecific antibodies from first and second homodimer antibodies comprises contacting a solution comprising the first and second homodimer antibodies with a reductant, thereby forming a reductant solution, whereby multispecific antibodies are formed from first and second homodimer antibodies, wherein the solution comprising the first and second homodimer antibodies is contacted with the reductant prior to any chromatographic step, wherein amino acid residues at the interface between the first and a second Fc chain of the multispecific antibody, with one Fc chain derived from the first homodimer antibody and the second Fc chain derived from the second homodimer antibody opposing residues of the first and a second Fc chain are modified to carry the opposite charge, wherein the reductant is GSH present at a concentration of between 1 and 100m M in the reductant solution, and wherein the solution comprising the first and second homodimer antibodies to which the reductant is added is a cell culture solution or a harvested cell culture solution and wherein the reductant solution further comprises a chelator and at least one stabilizer, wherein the chelator is DTPA present at a concentration of between 5m M and 50m M in the reductant solution and the at least one stabilizer is arginine present between 25mmM to 100mM in the reductant solution.

[0137] In one embodiment, the method of forming multispecific antibodies from first and second homodimer antibodies comprises contacting a solution comprising the first and second homodimer antibodies with a reductant, thereby forming a reductant solution, whereby multispecific antibodies are formed from first and second homodimer antibodies, wherein the solution comprising the first and second homodimer antibodies is contacted with the reductant prior to any chromatographic step, wherein amino acid residues at the interface between the first and a second Fc chain of the multispecific antibody, with one Fc chain derived from the first homodimer antibody and the second Fc chain derived from the second homodimer antibody opposing residues of the first and a second Fc chain are modified to carry the opposite charge, wherein the reductant is DTT present at a concentration of between 1 and 100m M in the reductant solution, and wherein the solution comprising the first and second homodimer antibodies to which the reductant is added is a cell culture solution or a harvested cell culture solution and wherein the reductant solution further comprises a chelator and at least one stabilizer, wherein the chelator is EDTA present at a concentration of between 5mM and 50mM in the reductant solution and the at least one stabilizer is arginine present between 25mmM to 100mM in the reductant solution.

[0138] In one embodiment, the method of forming multispecific antibodies from first and second homodimer antibodies comprises contacting a solution comprising the first and second homodimer antibodies with a reductant, thereby forming a reductant solution, whereby multispecific antibodies are formed from first and second homodimer antibodies, wherein the solution comprising the first and second homodimer antibodies is contacted with the reductant prior to any chromatographic step, wherein amino acid residues at the interface between the first and a second Fc chain of the multispecific antibody, with one Fc chain derived from the first homodimer antibody and the second Fc chain derived from the second homodimer antibody opposing residues of the first and a second Fc chain are modified to carry the opposite charge, wherein the reductant is DTT present at a concentration of between 1 and 100m M in the reductant solution, and wherein the solution comprising the first and second homodimer antibodies to which the reductant is added is a cell culture solution or a harvested cell culture solution and wherein the reductant solution DTPA comprises a chelator and at least one stabilizer, wherein the chelator is EDTA present at a concentration of between 5mM and 50mM in the reductant solution and the at least one stabilizer is arginine present between 25mmM to 100mM in the reductant solution.

[0139] In one embodiment, the method of forming multispecific antibodies from first and second homodimer antibodies comprises contacting a cell culture medium comprising the first and second homodimer antibodies with a reductant, thereby forming a reductant solution, whereby multispecific antibodies are formed from first and second homodimer antibodies, wherein the solution comprising the first and second homodimer antibodies is contacted with the reductant prior to any chromatographic step, wherein the reductant is a weak reductant present at a concentration of between 10mM and 250mM in the reductant solution and wherein the solution comprising the first and second homodimer antibodies to which the reductant is added is a cell culture solution or a harvested cell culture solution and wherein the reductant solution further comprises a chelator and / or at least one stabilizer, wherein the chelator is present at a concentration of between 5mM and 50mM and the at least one stabilizer is present at a concentration of between 25mmM to 100mM in the reductant solution, wherein the temperature is between 30C and 37C.

[0140] In one embodiment, the method of forming multispecific antibodies from first and second homodimer antibodies comprises contacting a harvested cell culture solution comprising the first and second homodimer antibodies with a reductant, thereby forming a reductant solution, whereby multispecific antibodies are formed from first and second homodimer antibodies, wherein the solution comprising the first and second homodimer antibodies is contacted with the reductant prior to any chromatographic step, wherein the reductant is a weak reductant present at a concentration of between 10mM and 250mM in the reductant solution and wherein the solution comprising the first and second homodimer antibodies to which the reductant is added is a cell culture solution or a harvested cell culture solution and wherein the reductant solution further comprises a chelator and / or at least one stabilizer, wherein the chelator is present at a concentration of between 5mM and 50mM and the at least one stabilizer is present at a concentration of between 25mmM to 100mM in the reductant solution, wherein the temperature is between 4C and 37C.

[0141] In one embodiment, the method of forming multispecific antibodies from first and second homodimer antibodies comprises contacting a solution comprising the first and second homodimer antibodies with a reductant, thereby forming a reductant solution, whereby multispecific antibodies are formed from first and second homodimer antibodies, wherein the solution comprising the first and second homodimer antibodies is contacted with the reductant prior to any chromatographic step, wherein the reductant is a weak reductant (such as cysteine) present at a concentration of between 10mM and 250mM in the reductant solution and wherein the solution comprising the first and second homodimer antibodies to which the reductant is added is a cell culture solution or a harvested cell culture solution and wherein the reductant solution further comprises at least one stabilizer (such as arginine), wherein the stabilizer is present at a concentration of between 25mmM to 100mM in the reductant solution, wherein the pH of the reductant solution is between 7.0 and 8.5. In one embodiment, the method of forming multispecific antibodies from first and second homodimer antibodies comprises contacting a solution comprising the first and second homodimer antibodies with a reductant, thereby forming a reductant solution, whereby multispecific antibodies are formed from first and second homodimer antibodies, wherein the solution comprising the first and second homodimer antibodies is contacted with the reductant prior to any chromatographic step, wherein the reductant is a weak reductant (such as cysteine) present at a concentration of between 10mM and 250mM in the reductant solution and wherein the solution comprising the first and second homodimer antibodies to which the reductant is added is a cell culture solution or a harvested cell culture solution and wherein the reductant solution further comprises at least one stabilizer (such as arginine), wherein the stabilizer is present at a concentration of between 25mmM to 100mM in the reductant solution, wherein the pH of the reductant solution is between 7.0 and 8.1.

[0142] In one embodiment, the method of forming multispecific antibodies from first and second homodimer antibodies comprises contacting a solution comprising the first and second homodimer antibodies with a reductant, thereby forming a reductant solution, whereby multispecific antibodies are formed from first and second homodimer antibodies, wherein the solution comprising the first and second homodimer antibodies is contacted with the reductant prior to any chromatographic step, wherein the reductant is a weak reductant (such as cysteine) present at a concentration of between 10mM and 250mM in the reductant solution and wherein the solution comprising the first and second homodimer antibodies to which the reductant is added is a cell culture solution or a harvested cell culture solution and wherein the reductant solution further comprises at least one stabilizer (such as arginine), wherein the stabilizer is present at a concentration of between 25mmM to 100mM in the reductant solution, wherein the pH of the reductant solution is between 7.0 and 7.5.

[0143] In one embodiment, the method of forming multispecific antibodies from first and second homodimer antibodies comprises contacting a solution comprising the first and second homodimer antibodies with a reductant, thereby forming a reductant solution, whereby multispecific antibodies are formed from first and second homodimer antibodies, wherein the solution comprising the first and second homodimer antibodies is contacted with the reductant prior to any chromatographic step, wherein the reductant is a weak reductant (such as cysteine) present at a concentration of between 10mM and 250mM in the reductant solution and wherein the solution comprising the first and second homodimer antibodies to which the reductant is added is a cell culture solution or a harvested cell culture solution and wherein the reductant solution further comprises at least one stabilizer (such as arginine), wherein the stabilizer is present at a concentration of between 25mmM to 100mM in the reductant solution, wherein the pH of the reductant solution is between 7.5 and 8.1.

[0144] In one embodiment, the method of forming multispecific antibodies from first and second homodimer antibodies comprises contacting a solution comprising the first and second homodimer antibodies with a reductant, thereby forming a reductant solution, whereby multispecific antibodies are formed from first and second homodimer antibodies, wherein the solution comprising the first and second homodimer antibodies is contacted with the reductant prior to any chromatographic step, wherein the reductant is cysteine present at a concentration of between 10mM and 50mM in the reductant solution and wherein the solution comprising the first and second homodimer antibodies to which the reductant is added is a cell culture solution or a harvested cell culture solution and wherein the reductant solution further comprises at least one stabilizer, wherein the stabilizer is arginine present at a concentration of between 25mmM to 100mM in the reductant solution, wherein the pH of the reductant solution is between 7.0 and 8.1.

[0145] In one embodiment, the method of forming multispecific antibodies from first and second homodimer antibodies comprises contacting a solution comprising the first and second homodimer antibodies with a reductant, thereby forming a reductant solution, whereby multispecific antibodies are formed from first and second homodimer antibodies, wherein the solution comprising the first and second homodimer antibodies is contacted with the reductant prior to any chromatographic step, wherein the reductant is cysteine present at a concentration of between 10mM and 50mM in the reductant solution and wherein the solution comprising the first and second homodimer antibodies to which the reductant is added is a cell culture solution or a harvested cell culture solution and wherein the reductant solution further comprises at least one stabilizer, wherein the stabilizer is arginine present at a concentration of between 25mmM to 100mM in the reductant solution, wherein the pH of the reductant solution is between 7.0 and 7.5. In one embodiment, the method of forming multispecific antibodies from first and second homodimer antibodies comprises contacting a solution comprising the first and second homodimer antibodies with a reductant, thereby forming a reductant solution, whereby multispecific antibodies are formed from first and second homodimer antibodies, wherein the solution comprising the first and second homodimer antibodies is contacted with the reductant prior to any chromatographic step, wherein the reductant is cysteine present at a concentration of between 10mM and 50mM in the reductant solution and wherein the solution comprising the first and second homodimer antibodies to which the reductant is added is a cell culture solution or a harvested cell culture solution and wherein the reductant solution further comprises at least one stabilizer, wherein the stabilizer is arginine present at a concentration of between 25mmM to 100mM in the reductant solution, wherein the pH of the reductant solution is between 7.5 and 8.1.

[0146] In one embodiment, the method of forming multispecific antibodies from first and second homodimer antibodies comprises contacting a solution comprising the first and second homodimer antibodies with a reductant, thereby forming a reductant solution, whereby multispecific antibodies are formed from first and second homodimer antibodies, wherein the solution comprising the first and second homodimer antibodies is contacted with the reductant prior to any chromatographic step, wherein the reductant is a weak reductant present at a concentration of between 10mM and 250mM in the reductant solution and wherein the solution comprising the first and second homodimer antibodies to which the reductant is added is a cell culture solution or a harvested cell culture solution and wherein the reductant solution further comprises a chelator and at least one stabilizer, wherein the chelator is present at a concentration of between 5mM and 50m M and the at least one stabilizer is present at a concentration of between 25mmM to 100mM in the reductant solution and wherein the reductant solution comprising the first and second homodimer antibodies is further contacted with an oxidant, wherein the oxidant is present in the reductant solution at a concentration of between 1 mM and 200mM, wherein the pH of the reductant solution is between 7.0 and 7.5.

[0147] In one embodiment, the method of forming multispecific antibodies from first and second homodimer antibodies comprises contacting a solution comprising the first and second homodimer antibodies with a reductant, thereby forming a reductant solution, whereby multispecific antibodies are formed from first and second homodimer antibodies, wherein the solution comprising the first and second homodimer antibodies is contacted with the reductant prior to any chromatographic step, wherein the reductant is a weak reductant present at a concentration of between 10mM and 250mM in the reductant solution and wherein the solution comprising the first and second homodimer antibodies to which the reductant is added is a cell culture solution or a harvested cell culture solution and wherein the reductant solution further comprises a chelator and at least one stabilizer, wherein the chelator is present at a concentration of between 5mM and 50m M and the at least one stabilizer is present at a concentration of between 25mmM to 100mM in the reductant solution and wherein the reductant solution comprising the first and second homodimer antibodies is further contacted with an oxidant, wherein the oxidant is present in the reductant solution at a concentration of between 1 mM and 200mM, wherein the pH of the reductant solution is between 7.5 and 8.1 .

[0148] In a further aspect of the invention, there is provided a multispecific antibody obtained by a method according to the first aspect of the invention.

[0149] In a further aspect of the invention, there is provided a pharmaceutical product including multi-specific antibodies obtained by a method according to the first aspect of the invention.

[0150] Certain Definitions

[0151] The following definitions will be used in the present description and claims:

[0152] - an “antibody” refers to an immunoglobulin molecule capable of specific binding to a target, such as a polypeptide, carbohydrate, polynucleotide, lipid, etc., through at least one antigen binding site, located in the variable region of the immunoglobulin molecule. As used herein, the term “antibody” can encompass any type of antibody (e.g. monospecific, bispecific, trispecific, multispecific), and includes portions of intact antibodies that retain the ability to bind to a given antigen (e.g. an “antigen-binding fragment”), and any other modified configuration of an immunoglobulin molecule that comprises an antigen binding site. Examples of antibody antigen-binding fragments and modified configurations include (i) a Fab fragment (a monovalent fragment consisting of the VL, VH, CL and CH1 domains); (ii) a F(ab’)2 fragment (a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region); and (iii) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody. Furthermore, although the two domains of an Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv)); see e.g., Bird et al., Science 1988; 242:423-426 and Huston et al., Proc. Natl. Acad. Sci. 1988 USA 85:5879-5883;

[0153] - a “Fc domain” refers to the portion of an immunoglobulin (Ig) molecule that correlates to a crystallizable fragment obtained by papain digestion of an Ig molecule. As used herein, the term relates to the 2-chained constant region of an antibody, each chain excluding the first constant region immunoglobulin domain. Within an Fc domain, there are two “Fc chains” (e.g. a “first Fc chain” and a “second Fc chain”). “Fc chain” generally refers to the C-terminal portion of an antibody heavy chain. Thus, Fc chain refers to the last two constant region immunoglobulin domains (CH2 and CH3) of IgA, IgD, and IgG heavy chains, and the last three constant region immunoglobulin domains of IgE and IgM heavy chains, and optionally the flexible hinge Nterminal to these domains. Although the boundaries of the Fc chain may vary, the human IgG heavy chain Fc chain is usually defined to comprise residues C226 or P230 to its carboxyl-term inus, wherein the numbering is according to the EU index of Edelman et al., Proc. Natl. Acad. Sci. USA 1969; 20 63(1 ):78-85 and as described in Kabat et al., 1991. Typically, the Fc chain comprises from about amino acid residue 236 to about 447 of the human lgG1 heavy chain constant region. “Fc chain” may refer to this polypeptide in isolation, or in the context of a larger molecule (e.g. in an antibody heavy chain or Fc fusion protein);

[0154] - a “heterodimer, “heterodimeric complex”, or “heterodimeric polypeptide” is a molecule comprising at least a first polypeptide and a second polypeptide, wherein the second polypeptide differs in amino acid sequence from the first polypeptide by at least one amino acid residue. The heteromultimer can comprise a “heterodimer” formed by the first and second polypeptide or can form higher order tertiary structures where polypeptides in addition to the first and second polypeptide are present; - a “bispecific antibody” refers to a molecule that has binding specificity for at least two different epitopes. In some embodiments, bispecific antibodies can bind simultaneously two different antigens. In other embodiments, the two different epitopes may reside on the same antigen;

[0155] - as used herein, a “trispecific antibody” is an antibody that has binding specificity for three different epitopes. In some embodiments, trispecific antibodies can bind simultaneously three different antigens. In other embodiments, the three different epitopes may reside on the same antigen;

[0156] - as used herein, a “multispecific antibody” is an antibody that has binding specificity for at least two different epitopes. In some embodiments, multispecific antibodies can bind simultaneously at least two different antigens. In other embodiments, the at least two different epitopes may reside on the same antigen;

[0157] - the term “harvested cell culture fluid” (“harvested cell culture solution”, “harvested CCF” or “HCCF”) refers to a solution containing at least one target substance which is sought to be purified from other substances also present. The harvested CCFs are often complex mixtures containing many biological molecules (such as proteins, antibodies, hormones, and viruses), small molecules (such as salts, sugars, lipids, etc.) and even particulate matter. While a typical harvested CCF of biological origin may be an aqueous solution or suspension, it may also contain organic solvents used in earlier separation steps such as solvent precipitations, extractions, and the like. Examples of harvested CCFs that may contain valuable biological substances amenable to the purification by various embodiments of the present invention include, but are not limited to, a culture supernatant from a bioreactor, a homogenized cell suspension, plasma, plasma fractions, and milk;

[0158] - the term “load” refers to any material containing the target substance or an intermediate constituent thereof, either derived from the cell culture (the harvested CCF) or from a chromatography step (thus partially purified), and loaded onto a chromatography medium;

[0159] - the term “load challenge” refers to the total mass of substance loaded onto the chromatography medium in the load cycle of a chromatography step, measured in units of mass of substance per unit volume of medium;

[0160] - the term “clarified culture medium” refers to media that was used to culture cells containing the secreted antibodies of interest that has been subjected to a process to remove the cells from the culture medium, such as filtration and / or centrifugation.

[0161] - the term “impurities” refers to materials in the harvested CCF or clarified culture medium that are different from the protein of interest (or target protein) and are desirably excluded from the final therapeutic protein formulation. Typical impurities include nucleic acids, proteins (including HCPs and high and low molecular weight species, peptides, endotoxins, viruses) and small molecules;

[0162] - the term “excipients” means the constituents of the final therapeutic protein formulation, which are not the therapeutic protein. The excipients typically include protein stabilizers, surfactants, amino-acids e.g. contributing to protein stabilization, etc;

[0163] - unless stated otherwise, the term “about” associated with a numeral value means within a range of ± 10% of said value.

[0164] Other objects, features and advantages of the present invention will be apparent from the foregoing detailed description. It should be understood, however, that the detailed description and the specific examples that follow, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes, modifications and equivalents within the spirit and scope of the invention will be apparent from the detailed description and examples to those of ordinary skill in the art and fall within the scope of the appended claims.

[0165] All publications and references, including but not limited to articles, abstracts, patents, patent applications (whether published or unpublished), and biological sequences (including, but not limited to those identified by specific database reference numbers) cited herein are hereby incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication or reference were specifically and individually indicated to be so incorporated by reference. Any patent application to which this application claims priority directly or indirectly is also incorporated herein by reference in its entirety.

[0166] Brief description of the Figures The invention will now be further illustrated by the following Example, with reference to the Figures listed below, wherein:

[0167] - FIG.1 is a schematic illustration of an example of prior art process steps for the formation and the purification of a multi-specific antibody.

[0168] - FIG.2 is a schematic illustration of an example of process steps for the formation and the purification of a multi-specific antibody according to the invention.

[0169] FIG.1 illustrates a conventional process for the formation and purification of multi-specific antibodies, in particular bi-specific antibodies, from parent homodimers of two different types. In the illustrated method, each parent homodimer is produced in a respective bioreactor for cell culture. The first steps of purification are also achieved by centrifugation of the HCCF from the respective bioreactor, clarification and capture chromatography (more specifically, in the illustrated example, protein A chromatography) steps. Those steps are performed in parallel with respective equipment dedicated to each parent homodimer.

[0170] Each clarified CCF is loaded into the respective protein A chromatography column (or more generally onto a protein A chromatography medium), in conditions such that both parent homodimer antibodies bind to the protein A medium. The medium is then washed with one or more wash buffers such that impurities bound to the medium are removed. The parent homodimers are then eluted from the medium at low pH conditions by means of an elution buffer, whereby the eluate from each protein A column contains the respective parent homodimers, which are then loaded into a redox tank.

[0171] This tank is dedicated to the redox reaction, whereby the heterodimers are formed from the parent homodimers.

[0172] The solution containing the heterodimers is then subjected to further purification steps, namely in the illustrated example: a diafiltration step (DF), an anion exchange chromatography step (AEX), an additional chromatography step (e.g. a cation exchange chromatography step), a virus filtration step and a final ultra-filtration / dia-filtration step (UFDF). As represented on FIG.2, in a method according to the invention both parent homodimers (first and second homodimer antibodies) may be produced in a single bioreactor, or alternatively in two separate bioreactors (not shown), and jointly pooled in a single HCCF to which the reductant is added to form the reductant solution. As for the prior art example, those initial steps include centrifugation and clarification that can be achieved in a single line, with pieces of equipment which are common to both parent homodimers.

[0173] The reductant may be added to the cell culture, whereby both parent homodimers (first and second homodimer antibodies) are produced in a single bioreactor, such that the heterodimer is formed in the co-culture, this is denoted by the arrow labelled “a” in the figure. The cell culture may then be harvested in a conventional fashion, i.e. the cell culture fluid is clarified. Alternatively, the reductant may be added to a harvested cell culture medium. For example, to a clarified conditioned media, such that both parent homodimers may have been produced in a single bioreactor, or alternatively in two separate bioreactors, the cell culture is then harvested in a conventional fashion forming the clarified conditioned media to which the reductant is added to form the reductant solution, this is denoted by the arrow labelled “b” in the figure.

[0174] The clarified solution containing the heterodimer (multispecific antibody) produced in either the cell culture (which is subsequently clarified) or the harvested cell culture media may then be loaded into a single protein A chromatography column, or more generally onto a protein A chromatography medium, or still more generally onto an adsorbent chromatography medium, in conditions such that the heterodimer binds to the medium.

[0175] The medium is then washed with one or more wash buffers such that impurities bound to the medium flow through with the wash buffer(s) and are therefore removed, while the heterodimer remains bound to the medium.

[0176] The bound antibodies are then eluted from the medium at low pH conditions by means of an elution buffer. The eluate pool collected from the protein A column contains the multispecific antibodies (heterodimers).

[0177] Like in the conventional process of FIG.1 , the solution (or eluate pool) containing the heterodimers may then be subjected to further purification steps, namely in the illustrated example: a diafiltration step (DF), an anion exchange chromatography step (AEX), an additional chromatography step (e.g. a cation exchange chromatography step), a virus filtration step and a final ultra-filtration / dia-filtration step (UFDF).

[0178] Unless otherwise indicated, the examples below describe experiments that were or are performed using standard techniques well known and routine to those of ordinary skill in the art. The examples are illustrative, but do not limit the invention.

[0179] Examples

[0180] Example 1 - production of bispecific antibody from homodimer antibodies A and B

[0181] In Example 1 , which is illustrative of the invention, bispecific antibody Z was formed with the method of the invention from pairs of homodimer antibodies A and B, which are of the lgG1 subtype, where the homodimer antibody A Fc chain contained charged amino acid modifications at positions 221 , 228 and 409 (EU numbering scheme) and the homodimer antibody B Fc chain contained charged amino acid modifications at positions 221 , 228 and 368 (EU numbering scheme) that are oppositely charged to those of the modifications of the homodimer antibody A. In this example, the inventors demonstrated the effectiveness of performing the method in a clarified cell culture media.

[0182] The inventors directly combined the clarified conditioned media containing the two homodimers at two distinct pH conditions in the presence of a weak reducing agent (Cysteine), a chelator (Ethylenediaminetetraacetic acid; EDTA) and other stabilizers / excipients (Arginine). They then captured the reaction product (in the conditioned media) by MabSelect SuRe™ Protein A resin to interrogate the extent of the reaction.

[0183] An equimolar amount of the two homodimer antibodies A and B (corresponding volumes of conditioned media) was mixed with a 5% (by volume) EDTA such that the final concentration of EDTA in the solution was 10mM. 500mM cysteine was added to this mixture to a concentration of 40mM cysteine in the solution. This was followed by the addition of Arginine to provide a concentration of 50mM in the solution. Finally, the pH of the mixture was adjusted to 7.5 or 8 (as shown in Table 1). The reaction was carried out for 12 hours at ambient temperature.

[0184] The conditioned media containing the reaction mixture was captured on MabSelect Sure LX resin on an Akta Avant (GE Healthcare Life Sciences). The evaluation of % conversion was carried out by HP-HIC (High Performance - Hydrophobic Interaction Chromatography ) - the method leveraged the hydrophobicity differences of the two homodimers / components (antibody A and antibody B) and the target protein. Samples were ran using a gradient of 1 M sodium sulfate, 50 mM Sodium phosphate, pH 7.2 on a ProPac HIC-10 column (Thermo Fisher).

[0185] To the inventors’ surprise, they found multiple conditions where the reaction went to completion (>90% recombination efficiency) with both pH and stabilizing excipients playing a role in delineating the process.

[0186] A reducing agent was shown to be necessary as the reactions did not proceed (0% conversion; controls) in the absence of Cysteine. pH was also found to affect recombination - while the reaction went to near conversion at a pH of 8.0 (94.69%), although the reaction was still advantageously able to proceed to 56.83% conversion at a pH of 7.5.

[0187] Further, addition of a stabilizing excipient (Arginine) improved the efficacy at pH of 8.0 (increasing the recombination from 94.69% to 96.45%) while the inventors’ found that it drastically increased the recombination efficiency to near completion at a pH of 7.5 (to 94.34%).

[0188] The use of a weak reducing agent that is compatible with biologies advantageously does not affect the structure / activity of the individual proteins in a deleterious manner.

[0189] The results demonstrate that the stabilizing excipients stabilize the two homodimers and orient them optimally for the exchange (heterodimer formation).

[0190] Thus, the at stabilized protein components lead to greater recombination efficiency at an optimal pH in the presence of reducing agent and stabilizing excipients. Further, the ability to perform the method at a lower pH advantageously obviates the need to change the pH downstream in the purification process providing further streamlining of the method.

[0191] The results are summarized in Table 1 below.

[0192] Table 1: Summary of the assembly of heterodimeric antibodies in clarified conditioned media.

[0193] Concentrations and nomenclature referred to for buffers / components are follows: a : ‘Cys” refers to the presence of reducing agent 500mM Cysteine in the reaction. b : ‘Arg’ refers to the presence of a stabilizing amino acid excipient like 50mM Arginine in the reaction. c : '% recombination’ refers to fraction (%) of recombined product formed as measure by aHIC (HPLC) following Protein A chromatography.

[0194] Example 2 - Production of bispecific from homodimer antibodies C and D

[0195] In Example 2, which is further illustrative of the invention, bispecific antibody Y was formed with the method of the invention from pairs of homodimer antibodies C and D which are of the lgG2 serotype whereby homodimer antibody C contained charged amino acid modifications at positions 220, 224, 228 and 409 (EU numbering scheme) and the homodimer antibody D contained charged amino acid modifications at positions 220, 228 and 368 (EU numbering scheme) that are oppositely charged to those of the modifications of homodimer antibody C.]. In this example, the inventors demonstrate the effectiveness of performing the method in a cell culture medium.

[0196] The co-culture process involved separate cell banks for each CHO cell line. The cell lines were then scaled up in the conventional manner until the appropriate combination point. The cell lines can be combined at the production bioreactor step or earlier in the seed train. The seed train cell culture conditions were within standard cell culture processes appropriate for scale-up, such as: nutrient media agitated in flasks held at a temperature around 36.5C with pH ranging from 6.5- 7.5.

[0197] The cell culture process was optimized for growth and titer in a 12-day fed batch process. Optimization required fine-tuning feed rate, controlling lactate production, and consideration of best pH ranges. Typically, within a process lactate can be controlled through high-end pH-controlled delivery of glucose (HIPDOG) feeding (as disclosed in W02004104186, Gagnon et al, Biotechnology and Bioengineering, Vol. 108, No. 6, June, 2011 (Gagnon et Al) or W02004048556), pH ranges are typically between 6.9-7.3, feed rates are unique to each process, and temperature is generally about 36.5C.

[0198] After an optimized 12-day cell culture process, wherein both cell lines were grown simultaneously, the redox reaction was then initiated in the bioreactor on Day 12. The pH of the cell culture medium was increased to pH 8.1 by sodium bicarbonate to facilitate redox reaction. The temperature was kept constant at 37C and the dissolved oxygen was kept constant at 40%. A bolus of cysteine was added within a short time frame a total of 150 mL of 150 mM cysteine, for a final concentration of 15-20 mM cysteine in the reactor. The reactor was left at 260rpm agitation with the above conditions for 24 hrs. At the end of 24 hours, the cell culture was harvested in the conventional fashion, (centrifuge at 3000 rpm for 20 minutes), and filtered through 0.2 urn filters before analysis.

[0199] An assay was developed to measure both parents simultaneously after the co-culture production. The assay is a pH-gradient based AEX method relying on the differences in pl values between the parent homodimer molecules and the multispecific. This assay quantifies both parents in the proA pools.

[0200] Table 2: Summary of the assembly of heterodimeric antibodies in a cell culture medium

[0201] The results are shown in Table 2, which demonstrated that the bispecific formed from this coculture with the redox reaction, was found to be as high as 60%.

[0202] The data show that conducting the redox pre-column in the cell media is advantageous. Increased optimization of the amount and timing of reducing agent addition and oxidation of the reduced antibodies could increase the multispecific production further.

[0203] Example 3 - Production of bispecific antibody from homodimer antibodies E and F

[0204] In Example 3, which is further illustrative of the invention, bispecific antibody W was formed with the method of the invention from pairs of homodimer antibodies E and F, which are of the lgG1 subtype. In this example the homodimer antibody E Fc chain contained charged amino acid modifications at positions 221 , 228 and 409 (EU numbering scheme) and the homodimer antibody B Fc chain contained charged amino acid modifications at positions 221 , 228 and 368 (EU numbering scheme) that are oppositely charged to those of the modifications of the homodimer antibody F. In this example, the inventors further demonstrated the effectiveness of performing the method in a clarified cell culture media when using a higher concentration of weak reductant.

[0205] The inventors directly combined the clarified conditioned media containing the two homodimers at two distinct pH conditions in the presence of a weak reducing agent (cysteine). An equimolar amount of the two homodimer antibodies E and F were mixed in conditioned media. A bolus of ysteine was added into the mixture for a final concentration of 250mM. The reductant solution was left to incubate at ambient temperature for six hours at pH 8.0.

[0206] The reaction product was captured (in the conditioned media) by MabSelect SURE Lx on an AKTA XPRESS (GE Healthcare Life Sciences).

[0207] Samples were run using a gradient of 1 M sodium chloride, 50 mM MES, pH 5.6 on a ProPac WCX-10 4 x 100 mm (Thermo Fisher).

[0208] The evaluation of percent conversion to a heterodimer was performed by HP-WaCEX (High Performance - Weak Analytical Cation Exchange Chromatography). The percentage of heterodimer formed was 93.7 %, as shown in Table 3.

[0209] Table 3 - Summary of the assembly of heterodimeric antibodies from homodimer antibodies E and F in a cell culture medium.

[0210] Conclusion

[0211] The examples demonstrate that the method is highly effective and advantageously applicable to various types of homodimers, in particular to antibodies of the sub-classes lgG1 and lgG2 performing with both pre or post-harvest media. The method of the invention is suitable to achieve high conversion rates i.e. percentages of multi-specific antibodies of the total population of antibodies.

Claims

CLAIMS1 . A method of forming multispecific antibodies from first and second homodimer antibodies comprising contacting a solution comprising the first and second homodimer antibodies with a reductant, thereby forming a reductant solution, whereby multispecific antibodies are formed from the first and second homodimer antibodies and wherein the solution comprising the first and second homodimer antibodies is contacted with the reductant before a first chromatographic step.

2. The method of claim 1 , wherein the antibody of the IgG, IgA, or IgM class.

3. The method of claim 2, wherein the antibody is of the IgG class.

4. The method of any preceding claim, wherein the formation of the multispecific antibody is enhanced by altering or engineering an interface between a first and a second Fc chain the multispecific antibody, with one Fc chain derived from a first homodimer antibody and the second Fc chain derived from a second homodimer antibody, such that opposing residues of the first and a second Fc chain carry the opposite charge. In one embodiment, one or more amino acid residues that make up the CH3-CH3 and hinge region interface are replaced with charged amino acid pairs for promoting the heterodimer formation.

5. The method of any preceding claim, wherein the formed multispecific antibody comprises a first Fc chain comprising charged amino acid modifications at positions 221 , 228 and 409 (Ell numbering scheme) and a second Fc chain comprising charged amino acid modifications at positions 221 , 228 and 368 (Ell numbering scheme) wherein the charged amino acid modification of the second Fc chain are oppositely charged to those of the modifications of the first Fc chain.

6. The method of any of claims 1 to 4, wherein the formed multispecific antibody comprises a first Fc chain comprising charged amino acid modifications at positions 220, 224, 228 and 409 (Ell numbering scheme) and a second Fcchain comprising charged amino acid modifications at positions 220, 228 and 368 (EU numbering scheme) that are oppositely charged to those of the modifications of the first Fc chain.

7. The method of any preceding claim wherein the solution comprising the first and second homodimer antibodies to which the reductant is added is a cell culture medium or a harvested cell culture solution.

8. The method of claim 7, wherein the harvested cell culture solution is a clarified cell culture media comprising the first and second homodimer antibodies.

9. The method of any preceding claim, wherein the reductant is a weak reductant.

10. The method of claim 9, wherein the weak reductant is selected from cysteine, Glutathione (GSH) and Dithioerythritol (DTT).11 . The method of claim 10, wherein the weak reductant is cysteine.

12. The method of claim 10, wherein the weak reductant is GSH.

13. The method of claim 10, wherein the weak reductant is DTT.

14. The method of any one of claims 9 to 13, wherein the weak reductant is present in the solution at between 1 mM and 500mM.

15. The method of claim 14, wherein the weak reductant is present in the solution at between 10mM and 100mM.

16. The method of any preceding claim, wherein the pH of the reductant solution is between 7.0 and 8.5.

17. The method of claim 16, wherein the pH of the reductant solution is between 7.5 and 8.0.

18. The method of claim 16, wherein the pH of the reductant solution is between 7.0 and 7.5.

19. The method of any preceding claim, wherein the reductant solution comprising the first and second homodimer antibodies is further contacted with an oxidant.

20. The method of any preceding claim, wherein the reductant solution further comprises a chelator.

21. The method of claim 20, wherein the chelator is Ethylenediaminetetraacetic acid (EDTA) or diethylenetriaminepentaacetic acid (DTPA).

22. The method of either claim 20 or claim 21 wherein the chelator is present between 5mM and 50mM in the reductant solution.

23. The method of any preceding claim, wherein the reductant solution further comprises at least one stabilizing excipient.

24. The method of claim 23, wherein the stabilizing excipient is beta cyclodextrin or an amino acid.

25. The method of claim 24, wherein the stabilizing excipient is an amino acid and the amino acid is arginine, lysine or a mixture thereof.

26. The method of claim 25, wherein the stabilizing excipient is arginine.

27. The method of claim 25, wherein the stabilizing excipient is lysine.

28. The method of any one of claims 23 to 27, wherein the stabilizing excipient is present between 25mmM to 100mM in the reductant solution.

29. The method of claim 28, wherein the stabilizing excipient is present at about 50mM in the reductant solution.

30. The method of any preceding claim, wherein the redox reaction is performed at a temperature between 4C (Celsius) and 37C.

31. The method of any preceding claim, wherein the solution comprising the first and second homodimer antibodies is contacted with a reductant for at least 1 hour.

32. The method of any preceding claim, wherein the method further comprises performing a first chromatographic step comprising loading the produced multispecific antibodies onto an adsorbent chromatography medium, whereby the multispecific antibodies bind to the medium and eluting the bound antibodies with an elution buffer thereby obtaining an eluate pool comprising the multispecific antibodies.

33. A multispecific antibody obtained by a method according to any of the preceding claims.

34. A pharmaceutical product comprising a multi-specific antibody obtained by a method according to any one of claims 1 to 32.

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